Second Edition

WATER
POLLUTION
and fish
PHYSIOLOGY
Alan G. Heath
Departm ent of Biology
Virginia Polytechnic Institute and State University
Blacksburg, V irg inia

(c^
LEWIS PUBLISHERS
Boca Raton N ew York London Tokyo
Library of Congress Cataloging>in-Publication Data

Heath, Alan G.
Water pollution and fish physiology / Alan G. Heath. — 2nd ed.
p. cm.
Includes bibliographical references and index.
ISBN 0-87371-632-9 (permanent paper)
1. Fishes—Effect of water pollution on. 2. Fishes—Physiology.
I. Title.
SH174.H43 1995
597'.024—dc20 95-16292
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 Preface

The genesis of this book came from my having taught a postgraduate course to
biology and fisheries students on the physiological action of water pollutants in fish
for many years. The literature is scattered in numerous journals and books, and while
there have been several excellent symposia volumes published, most of the papers
therein have been presentations of primary research. As with many healthy disci­
plines, this one has experienced a tremendous growth over the past 20 years;
especially during the 1980s and early 1990s. Recently, there have been a number of
good reviews dealing with fairly specific aspects of the subject (e.g., effects of
pollutants on osmoregulation); however, there are no books which attempt to look at
the whole field. Therefore, the objective of this book is to provide a reasonably
concise synthesis of what is known about how pollutants affect physiological pro­
cesses in fish.
As in the first edition, this revised and updated second edition begins with a
discussion of some concepts that are important in understanding pollution biology
and fish physiology. These concepts are often implied though rarely mentioned
explicitly. Following this brief chapter, an analysis of the physiological responses to
environmental hypoxia is provided. This is discussed early in the book because
polluted waters are often lacking in dissolved oxygen and many toxic chemicals at
acute concentrations induce an hypoxic condition in the fish. Each of the subsequent
chapters is generally devoted to a specific physiological process (e.g., energetics,
uptake and accumulation of contaminants, reproduction). Each of these begins with
a review of some basic physiology applied to fish followed by a more detailed
discussion of how various pollutants affect these functions. This second edition has
had two additional chapters added: immunology and acid toxicity, which reflect the
large amount of past and present research conducted in these fields. Throughout, the
emphasis is on the mechanisms of sublethal effects, rather than lethal ones, for those
are what the fish will usually confront in contaminated waterways. The book closes
with a critical look at some physiological and biochemical measurements that are, or
could be, utilized in work on water pollution control.
The literature coverage is through 1993, but I have not attempted to be all-
inclusive, for one could easily get bogged down in details and therefore lose sight of
generalizations. Indeed, some of the individual chapters could easily be expanded
into a whole book unto themselves. Where the literature is extensive, I have tried to
cite examples of trends. The most recent studies and reviews have been emphasized
so that those who wish to pursue a topic in further depth can quickly get into the
literature.
In preparing an interpretive treatise of this sort, there is always the danger of
“over” interpreting the results of others. At times, I have been rather free with
speculations. I have tried to make it obvious where I am speculating and apologize
in advance to those whose ideas may have been inadvertently used without adequate
attribution.
Several people critically read specific chapters. For this I am grateful to Drs.
Douglas Anderson (National Fisheries Research Laboratory, Leetown, West
Virginia), Gary Atchison (Iowa State University), Joe Cech (University of Califor­
nia, Davis), Brian Eddy (University of Dundee, Scotland), Mark Greeley, Jr., (Oak
Ridge National Laboratory, Tennessee), Steven Koenig (West Virginia University),
James McKim (USEPA, Duluth), and Chris Wood (McMaster University). Their
perceptive suggestions and corrections have been extremely helpful as my level of
expertise in some of these areas is clearly limited. Of course, all blame for errors of
omission and commission must rest with me.

Alan G. Heath
Blacksburg, Virginia
 The Author

Alan G. Heath, Ph.D., is a professor in the Department

of Biology at Virginia Polytechnic Institute and State
University, Blacksburg, Virginia.
Dr. Heath received his B.S. degree from San Jose
State University in California in 1958 and M.S. and Ph.D.
degrees from Oregon State University in 1961 and 1963,
respectively. After a year as an National Institutes of
Health postdoctoral fellow at the University of Miami
Marine Laboratory, he moved to Virginia Tech, where he
has been ever since. Dr. Heath served as a Senior U.S.
Environmental Protection Agency Postdoctoral Fellow in
Bristol, England for a year and recently spent a year as a Visiting Scholar in the
Department of Wildlife and Fisheries Biology at the University of California, Davis.
Dr. Heath’s research, often in collaboration with his graduate students, has been
primarily devoted to laboratory investigations on the sublethal physiological re­
sponses of fish to environmental hypoxia or to the presence of waterborne chemical
toxicants often present in polluted waters. His professional affiliations include Soci­
ety of Environmental Toxicology and Chemistry, American Fisheries Society, Ameri­
can Society of Zoologists, American Institute of Biological Sciences, AAAS, and
Sigma Xi.
 Table of Contents

Chapter 1
Some Introductory Concepts
I. Types of Water Pollution..................................................................1
A. Putrecible Organic Materials.........................................................1
B. Excessive Nutrition........................................................................1
C. Suspended Solids..........................................................................2
D. Toxic Chemicals............................................................................ 2
1. M etals..................................................................................... 2
2. Chlorine.................................................................................. 2
3. Cyanides................................................................................. 3
4. Ammonia................................................................................ 3
5. D etergents.............................................................................. 3
6. A cids.......................................................................................3
7. Pesticides................................................................................ 3
8. PolychlorinatedBiphenyls...................................................... 4
9. Petroleum Hydrocarbons...................................................... 4
10. Pulpmill Effluents................................................................... 4
11. Miscellaneous........................................................................5
E. Thermal Pollution..........................................................................5
II. The Relationship Between Aquatic Toxicology and Fish
Physiology........................................................................................... 5
III. Levels of Biological Organization.....................................................6
IV. Importance of Dose and Duration of Exposure.............................. 7
V. Stress....................................................................................................9
VI. Toxic Mode of A ction..................................................................... 10
References................................................................................................ 11

Chapter 2
Environmental Hypoxia
I. Introduction...................................................................................... 15
II. Minimum Levels of Oxygen Requiredfor Fish Life..................... 16
III. Interaction of Hypoxia and Toxicity of Pollutant
Chemicals......................................................................................... 17
 IV. Gill vs. Cutaneous Respiration.........................................................18
V. Adjustments in Ventilation...............................................................20
VI. Adjustments by the Gills to Hypoxia............................................. 24
VII. Transport of Oxygen by the Blood.................................................24
VIII. Cardiovascular Changes During Hypoxia......................................26
IX. Respiratory Regulation and Conformity.........................................27
X. Anaerobic Metabolism..................................................................... 29
XI. Swimming Speed.............................................................................. 31
XII. Behavior............................................................................................ 33
XIII. Blood and Orine................................................................................ 34
XIV. Histopathology.................................................................................. 36
XV. Acclimation to Hypoxia................................................................... 36
References..................................................................................................37

Chapter 3
Respiratory and Cardiovascular Responses
I. Introduction........................................................................................47
II. Overview of Normal Respiratory Physiology................................ 47
III. Histopathology of Gill Lamellae Exposedto Pollutants................ 49
IV. Ventilation Changes in Response to Pollutants.............................52
V. Physiological Mechanisms of Changes inVentilation................... 55
VI. Circulatory Physiology..................................................................... 58
VII. Cardiac Responses to Pollutants....................................................59
References..................................................................................................62

Chapter 4
Hematology
I. Introduction........................................................................................ 67
II. Fish Blood Cells and their Measurement.......................................67
A. Hematocrit....................................................................................68
B. Blood Cell C ount.........................................................................68
C. Hemoglobin Concentration........................................................ 68
D. Blood Cell Differential Count..................................................... 68
III. Chemicals that Cause A nem ia....................................................... 69
A. Cadmium......................................................................................69
B. Lead...............................................................................................70
C. Mercury......................................................................................... 71
D. Chloramine...................................................................................71
E. Nitrite............................................................................................ 72
F. Pulpmill Effluent..........................................................................72
G. Miscellaneous.............................................................................. 72
H. Biological Significance of Anemia............................................ 73
IV. Chemicals Causing an Increase in Hematological Variables.... 73
A. A cid...............................................................................................73
B. Pesticides......................................................................................74
C. Copper.......................................................................................... 74
V. Concluding R em arks........................................................................74
References..................................................................................................75
Chapter 5
Uptake, Accumulation, Biotransformation, and Excretion of
Xenobiotics
I. Introduction........................................................................................ 79
II. Uptake from the Environment........................................................ 80
A. M etals........................................................................................... 81
B. O rganics.......................................................................................87
III. Transport Within the Fish of Metals and O rganics...................... 91
IV. Accumulation of Metals in Different Organs................................. 91
V. Regulation of Metal Concentration..................................................96
VI. Glutathione and Metal Detoxification............................................. 97
VII. Involvement of Metallothionein in Metal Accumulation and
Acclimation to Metals...................................................................... 99
VIII. Bioconcentration of Organic Pollutants..................................... 101
IX. Biotransformation of Organic Contam inants.............................. 106
X. Excretion of Organic Contam inants........................................... 110
References................................................................................................113

Chapter 6
Liver
I. Introduction...................................................................................... 125
II. Structure of the Liver.................................................................... 125
III. Alterations in Liver/Somatic Index...............................................126
IV. Histopathological Effects of Pollutants........................................ 127
V. Major Functions of the Liver........................................................ 128
A. Interconversion of Foodstuffs.................................................. 128
B. Storage of Glycogen................................................................. 128
C. Removal and Metabolism of Foreign Chemicals in
the Blood....................................................................................129
D. Formation of Bile.......................................................................129
E. Synthesis ofManyPlasma Proteins....................................... 129
F. Synthesis of Cholesterol for Use in Steroid Hormones
and Cell Membranes................................................................ 129
G. Exocrine Pancreatic Secretion.................................................129
H. Metabolism of Hormones..........................................................129
VI. Effects of Pollutants on Liver Function...................................... 129
A. Plasma Clearance by theLiver of Specific Dyes.................... 130
B. Plasma Enzymes of Liver O rigin............................................ 130
C. Changes in Plasma Concentration of Chemicals
Produced or Excreted by Liver................................................132
VII. Ascorbic Acid and Pollutant Exposure........................................ 134
References................................................................................................136

Chapter 7
Osmotic and Ionic Regulation
I. Overview of Some General Principles....................................... 141
II. Effects of Pollutants on Osmotic and Ionic Regulation..............144
A. Copper..................................................................................... 145
 B. Zinc............................................................................................ 150
C. Cadmium.................................................................................. 151
D. Mercury....................................................................................... 153
E. Chromium................................................................................. 155
F. Lead........................................................................................... 155
G. Aluminum...................................................................................156
H. T in ...............................................................................................157
I. Manganese and Iron................................................................. 157
J. D etergents................................................................................ 157
K. Phenol......................................................................................... 158
L. DDT.............................................................................................158
M. Fenvalerate.................................................................................158
M. Petroleum Hydrocarbons........................................................ 159
O. Ammonia....................................................................................160
P. Nitrite.......................................................................................... 160
Q. Chlorine and Ozone.................................................................. 161
III. M ucus...............................................................................................161
IV. Chloride Cell Proliferation.............................................................. 162
V. Some Summary Comments Regarding Osmoregulatory
and Electrolyte Alterations............................................................162
References.............................................................................................. 164

Chapter 8
Physiological Energetics
I. Introduction.................................................................................... 171
II. General C oncepts........................................................................... 171
III. Methods of Measuring Energy Expenditure in F ish................. 174
IV. Effects of Metals on Metabolic R ate............................................ 176
A. Copper........................................................................................ 176
B. Miscellaneous M etals................................................................ 180
V. Gill Tissue Metabolism: Effects of Metals and Possible
Relation of Gill Metabolism to Whole-Body Metabolic Rate .... 182
VI. Effects of Pesticides on Whole-Body and Individual
Tissue Respiration........................................................................ 184
VII. Effects of Petroleum Hydrocarbons on Energy Metabolism .... 189
VIII. Methods Applicable to Measurement of Energy Expenditure
in the F ield...................................................................................... 189
IX. Effects of Pollutants on Larval and Juvenile Growth................. 191
X. Swimming Performance................................................................. 195
XI. Changes in Carbohydrate, Lipid and Protein Energy Stores ... 200
A. Introduction................................................................................ 200
B. Effect of Pulpmill Effluent........................................................ 202
C. M etals......................................................................................... 203
D. Petroleum Hydrocarbons and Surfactants.............................205
E. Pesticides....................................................................................205
F. Miscellaneous Pollutants.......................................................... 207
G. Concluding Comment...............................................................207
References.............................................................................................. 208
Chapter 9
Alterations in Cellular Enzyme Activity, Antioxidants, Adenylates,
and Stress Proteins
I. Introduction......................................................................................217
II. Some Comments About Enzyme Methodology..........................217
III. Alterations in Cellular Enzyme Activity Resulting from
Metal Exposure............................................................................... 219
IV. Enzyme Effects from Organic Chemicals................................... 225
V. Concluding Comments on Enzyme Effects................................ 230
VI. Antioxidants.....................................................................................231
VII. Adenylates.......................................................................................232
VIII. Stress Proteins................................................................................ 233
References................................................................................................234

Chapter 10
Acid Pollution
I. Introduction......................................................................................239
II. Spawning......................................................................................... 240
III. Embryonic Development and Hatching.......................................242
IV. Larvae from Hatching Through Swim-Up................................... 242
V. Juvenile and Adult: Blood Acid-Base Balance and
Electrolyte Changes from Acute Exposures............................... 244
VI. Juvenile and Adult: Blood Changes from Chronic
Exposure ......................................................................................... 247
VII. Hormonal Responses..................................................................... 249
VIII. Ventilation and Blood G ases......................................................... 250
IX. Oxygen Consumption, Swimming Performance, and
Swim Bladder Inflation................................................................... 251
X. Behavior.......................................................................................... 252
XI. Concluding Comment.................................................................... 253
References................................................................................................253

Chapter 11
The Immune System
I. Overview of Fish Immunology...................................................... 259
II. Effects of Pollutants on Immune Function.................................. 260
A. M etals......................................................................................... 261
1. Cadmium............................................................................... 261
2. Copper....................................................................................262
3. Miscellaneous M etals............................................................262
B. Organic Pollutants.................................................................... 263
C. Miscellaneous Pollutants.......................................................... 264
D. Immune Effects in Fish from Contaminated
Natural W aters...........................................................................265
III. Hormonal Modulation of Immune Response............................... 266
References................................................................................................266
Chapter 12
Behavior and Nervous System Function
I. Introduction......................................................................................271
II. Locomotor Activity.........................................................................272
A. Methods for Measuring Locomotor Activity...........................272
B. M etals......................................................................................... 273
C. Miscellaneous Chemicals......................................................... 274
III. Avoidance of or Attractance to Waterborne Chem icals..........276
IV. Sensory Receptors..........................................................................279
V. Feeding and Predator-Prey Behavior........................................... 282
VI. Aggression.......................................................................................284
VII. Learning........................................................................................... 285
VIII. Optomotor Response..................................................................... 287
IX. Acetylcholinesterase...................................................................... 288
X. Concluding Com m ents.................................................................. 289
References.............................................................................................. 290

Chapter 13
Reproduction
I. Introduction......................................................................................299
II. Brief Overview of Fish Reproductive Physiology....................... 299
A. Reproductive Strategies............................................................300
B. Hormonal Control of Gametogenesis......................................300
C. Spawning....................................................................................301
D. Development............................................................................. 302
III. Action of Pollutants on Reproductive Processes........................ 303
A. Hormonal Controls.................................................................... 303
B. Egg Production..........................................................................308
C. Transfer of Contaminants into Eggs fromAdults.................. 309
D. Effects on Sperm and Fertilization.........................................310
E. Egg Activation...........................................................................310
F. Oocyte and Embryo Energy Metabolism............................... 311
G. Changes In Pollutant Sensitivity During Embryonic
Development............................................................................. 312
H. Hatching Time and Viable Hatch............................................ 313
I. Larvae......................................................................................... 314
J. Concluding Comment...............................................................317
References.............................................................................................. 317

Chapter 14
Ose of Physiological and Biochemical Measures in Pollution Biology
I. Introduction......................................................................................325
II. Water Quality Criteria.................................................................... 326
III. Biomonitoring of Fish in the Field and M esocosm s..................327
A. Cellular and Tissue Chemistry................................................ 329
B. Histopathology...........................................................................331
 C. Hematology, Immunology, and Blood Chemistry.................331
D. Challenge T ests.........................................................................334
E. Behavior T ests...........................................................................335
F. Conclusions............................................................................... 335
IV. Early Warning S ystem s............................................................... 336
References 337

Index.........................................................................................................339
 Chapter

Som e Introductory
Concepts
I. TYPES OF WATER POLLUTION
For all practical purposes, water pollution is the addition by humans of something to the
water that alters its chemical composition, temperature, or microbial composition to such
an extent that harm occurs to resident organisms or to humans (Lloyd, 1992). While
chemical pollution has implications for human health, both directly from toxic chemicals
in drinking water, and indirectly from the accumulation of toxic compounds by organisms
that are then eaten by people, this book will not deal with these types of pollution, nor
with the introduction of pathogenic microbes and carcinogenic chemicals into waterways.
Instead, an attempt will be made to look at pollution “through the eye of the fish” from
a functional standpoint.
The brief survey of pollutants presented here is not meant to be comprehensive, but
instead is meant as an introduction to the sorts of pollution that may have physiological
effects on fish.

A. PUTRECIBLE ORGANIC MATERIALS

Putrecible organic materials are characteristic of untreated or inadequately treated domes­
tic and industrial waste. Oxygen is required for the microbial decomposition of this
organic matter and the quantitative measure of this oxygen requirement is referred to as
the biochemical oxygen demand (BOD). As the BOD gets larger due to a greater organic
load, unless there is considerable mixing of the water, a condition of abnormally low
dissolved oxygen (hypoxia) occurs. It is this environmental hypoxia that is of primary
interest from the standpoint of fish physiology.

B. EXCESSIVE NUTRITION
In locations where there is agricultural runoff or non-biodegradable detergents being
added to the water, the growth of phytoplankton is stimulated due to excess amounts of
plant nutrients. This eutrophication process results in large daily changes in dissolved
oxygen from photosynthesis during the daylight hours and respiration at night. The
utilization of oxygen by phytoplanktonic respiration at night can produce conditions of
very low dissolved oxygen in the hours just before daybreak.
C. SUSPENDED SOLIDS
Silt suspended in the water column is probably the most prevêlant of the suspended solids.
It generally results from runoff where land has been disturbed by plowing or excavation.
Ground up wood fibers can also be a significant form of suspended solid pollution.

D. TOXIC CHEMICALS
The conditions producing a low dissolved oxygen concentration and toxic chemicals are
the most important types of water pollution that affect fish. There are some 65,000
industrial chemicals in use and 3-5 new ones enter the marketplace each day (World
Commission on Environment and Development, 1987). Fortunately, a very small percent­
age of those chemicals enter waterways, but the possibilities are immense. The major
classes of toxic chemicals of concern for fish are metals, chlorine, cyanides, ammonia,
detergents, acids, pesticides, polychlorinated biphenyls, petroleum hydrocarbons, pulpmill
effluents, and other miscellaneous chemicals.

1. Metals
There has been a tendency in the literature on water pollution to speak of nearly all metals
as “heavy metals”, although some have tried to avoid this designation (Nieboer and
Richardson, 1980). Here we will not attempt to separate heavy metals from any others.
(Metaloids such as selenium and arsenic are included with the metals.)
When speaking of metals the expression “trace metals” is often used (e.g., Leland and
Kuwabara, 1985). This reflects the important fact that many metals are required for
normal physiological function in animals but only at trace concentrations, and these
concentrations vary considerably between different species. Important trace metals in­
clude copper, iron, zinc, iodine, manganese, cobalt, selenium, tin, and chromium. Altered
physiological function results when one or more of these reach sufficiently high concen­
trations in cells.
Metals enter waterways from a wide variety of industrial effluents and old mines. Acid
precipitation also causes leaching of metals from surrounding soils (Norton, 1982; Spry
and Wiener, 1991). The metals of most concern for studies of the effect of pollution on
fish physiology include copper, zinc, tin (primarily the methylated form), cadmium,
mercury (both the methylated and non-methylated forms), chromium, lead, nickel, ar­
senic, and aluminum. An important problem when working with metals in water is that
they tend to complex with organic and inorganic chemicals and this may reduce their
bioavailability to resident organisms (Leland and Kuwabara, 1985). Thus a simple
analysis for total metal could actually overestimate the bioavailability. A comprehensive
review of the chemistry and biology of metals in natural waters has been published by
Moore and Ramamoorthy (1984).
In the past there has been some tendency to lump all the metals together when talking
about their “physiological mode of action”. We now know this is not valid, as will become
evident in later chapters, although there is considerable overlap in physiological effects
for many of them.

2. Chlorine
The concern here is not for the chloride ion, but rather with the chemicals formed when
chlorine gas is introduced into water either for antifouling in industrial cooling systems
or for disinfection of sewage effluents. The free gas does not exist in water for any
significant period of time, but quickly forms HOCl or OC1-, which are commonly called
“free chlorine”. In the presence of ammonia, some or all of the free chlorine is converted
into monochloramine (NH2CI) which is known as “combined chlorine”. Both free and
combined chlorine are oxidants with the former being the strongest. Total residual
chlorine is the sum of the free and combined concentrations. The relative stability and
toxicity of these forms of chlorine differs considerably. Free chlorine is more toxic but
the combined form is more stable and thus stays around longer (Hall et al., 1981).

3. Cyanides
The cyanide radical occurs in many industrial wastes, particularly those involved with the
manufacturing of synthetic fabrics and plastics and the processing of metals. “Free
cyanide” (CN ion and HCN) occurs mostly as molecular hydrogen cyanide, unless the pH
is above about 9. The toxicity of cyanide to fish and other organisms has been reviewed
by Leduc (1984) and Eisler (1991).

4. Ammonia
This compound not only occurs in many effluents, but also results from the natural
decomposition of organic matter. Ammonia gas forms ammonium hydroxide in water
which in turn can dissociate into ammonium and hydroxyl ions. It is the non-ionized form
of ammonia that is toxic to fish, but the toxicity is complicated by the fact that the degree
of dissociation depends on the pH and temperature of the water. Increasing the pH or
temperature increases the toxicity because more of the ammonia will be in the non-
ionized form. The pH and temperature in natural waters often change rapidly during the
day so ammonia toxicity becomes difficult to predict. In addition, the gill surface of fish
will have a much higher carbon dioxide concentration than the surrounding water and the
carbonic anhydrase in mucus will catalyze the formation of carbonic acid from that
carbon dioxide. The resulting pH decrease at the surface will then affect the ammonia
toxicity (Lloyd, 1992).
Oxidation of ammonia by bacteria produces nitrite which is further oxidized into
nitrate. Nitrite is far more toxic than nitrate (which has almost no toxicity), but it is present
usually in only trace amounts in natural waters. In aquaculture facilities which use
nitrification to convert ammonia to nitrate, however, the process may become impeded
and nitrite may accumulate (Russo, 1985).

5. Detergents
In 1965, there was a shift by the detergent industry from the alkylbenzene sulfonates
(ABS) to the more biodegradable linear alklylate sulfonates (LAS). This commendable
attempt at reducing their environmental impact is not unequivocally a good thing. The
LAS is four times as toxic to fish as is ABS (Pickering, 1966), but fortunately, the toxicity
is lost upon biodegradation.

6. Acids
The main effects on aquatic biota of acids are due to a simple change in the pH of the
water. In addition, however, there may also be an indirect effect due to altered toxicity
of certain pollutants (e.g., metals) (Baker, 1982). Acid pollution from mine drainage and
acid rain is an increasing problem in many parts of both developed and developing
countries. A tremendous body of information on the effects of acid on all forms of aquatic
life is rapidly accumulating (e.g., Morris et al., 1989). Because of that, we devote a whole
chapter in this book to the subject.
7. Pesticides
The pesticides of interest here are primarily the insecticides, herbicides, and wood
preservatives. Tributyltin and chlorine are also used as antifouling agents, a type of
pesticidal activity, but are taken up elsewhere. Briggs (1992) provides a good general
guide to all types of pesticides including both technical and common names.
Insecticides fall into four general types: organochlorine, organophosphate, carbamate,
and botanicals. The organochlorine insecticides include DDT, aldrin, chlordane, dieldrin,
endrin, heptachlor, lindane, methoxychlor, and toxaphene. Because of their environmen­
tal persistence and high toxicity, most are no longer legally used in the U.S. but are still
extensively used in some other countries.
The organophosphates include diazinon, malathion, parathion, methyl parathion, dichlor-
vos, Dursban®, etc. This is a steadily expanding list. Important carbamate insecticides are
Sevin® and carbofuran.
Botanical insecticides include rotenone, pyrethrum, and allethrin. The term “botani­
cal” refers to the fact they are derived from plants, although there has been considerable
development of synthetic forms.
The herbicides and fungicides include among others amitrol, diquat, endothall, molinate,
silvex, and paraquat.
Wood preservatives include pentachlorophenol and 2-(thiocyanomethylthio)
benzothiazole (TCMTB).
The acute toxicity of many of these pesticides to fish and other aquatic life has been
reviewed by Livingston (1977) and Murty (1986). As a group, the acute toxicity of the
organochlorine insecticides tends to be considerably greater than for the organophos­
phates. The 96-h LC50s for organochlorine compounds are generally in the low micro­
gram per liter range whereas the LC50s for the organophosphate compounds are in the
range of low milligram per liter. Pyrethroids are similar to organochlorine insecticides in
toxicity to fish, and the herbicides have, with a few exceptions, relatively low toxicides
for fish, but when used for weed control, they can cause depletion of oxygen (Murty,
1986). As we shall see in later chapters, nearly all pesticides can have some subtle and
not so subtle physiological effects under conditions of chronic exposure.

8. Polychlorinated Biphenyls
These compounds, commonly called PCBs, have generated considerable interest prima­
rily due to their toxicity to humans. They are also quite toxic to fish and other aquatic life.
The term “Aroclor” with a four-digit number after it refers to a specific PCB formulation.

9. Petroleum Hydrocarbons
The composition of crude oil is complex and varies from region to region. The major
components are aliphatic hydrocarbons, cyclic paraffin hydrocarbons, aromatic hydrocar­
bons, naphtheno-aromatic hydrocarbons, resins, asphaltenes, heteroatomic compounds,
and metallic compounds. The aromatic and naphtheno-aromatic hydrocarbons are consid­
ered to be the most toxic components in oil (Anderson, 1979), and they increase in percent
of the total content of the oil during the refining process. Extensive discussions of the
sources, fates, and biological effects of petroleum hydrocarbons are found in Neff and
Anderson (1981).

10. Pulpmill Effluents

In the U.S., pulp and paper mills are the largest dischargers of conventional pollutants
subject to national effluent standards (General Accounting Office, 1987). This waste is
often called kraft mill effluent (KME) and it results from the digestion of wood in an
alkaline mixture which may be followed by bleaching. The resulting effluent possesses
a complex mixture of organic and inorganic salts which has a considerable BOD. It also
has several toxic substances such as resin acids and chlorinated phenolics (Lindstrom-
Seppa and Oikari, 1990).

11. Miscellaneous
With the huge number of chemicals that are introduced into the industrial stream every
year, there are those that do not fall into any group listed above. Fortunately, most of these
currently do not appear in waterways to a great extent, but potentially they could.

E. THERMAL POLLUTION
Elevated temperatures occur from clearing of cover over streams and heated effluents
from steam power generating plants. The available literature on the effects of temperature
on fish is huge. Raney et al. (1972) compiled a bibliography on this subject that included
over 4000 references, and the number of papers has expanded exponentially since then.
A good, concise summary of this extremely large topic is that by Houston (1982).
Because he is a fish physiologist, his treatise has a physiological “flavor” to it. Other good
reviews of the effects of temperature on various physiological functions in fish include
Crawshaw and Hazel (1984) and Hazel (1993). Space limitations here will permit only
a consideration of temperature in relation to its interaction with hypoxia and with toxic
chemicals on the fish.

II. THE RELATIONSHIP BETWEEN AQUATIC TOXICOLOGY AND

FISH PHYSIOLOGY
Toxicology is the study of poisons, their identification, chemistry, degree of toxicity, and
physiological actions. The major aim of the aquatic toxicologist, as with other toxicolo­
gists, is to protect the organisms that potentially may be the recepients of some harmful
chemical in the environment. Mammalian (classical) toxicology has a long and distin­
guished history while aquatic toxicology is a much younger discipline. Its history has
been briefly reviewed by Macek (1980) who traces the early development in the 1930s
through the stimulus that occurred from water quality legislation in the 1960s and finally
in the 1970s when several parallel branches evolved. One of these involves the use of
aquatic organisms as animal models for human toxicological problems, which is a
merging of biomedical research and aquatic toxicology.
Aquatic toxicology has adopted many of the techniques of its classical predecessor.
One of the major techniques is the acute bioassay whereby the concentration lethal to 50%
of a population of fish or invertebrate in a given exposure time is determined (LC50). The
procedures for carrying out aquatic bioassays have become rather well standardized
(APHA, 1992), but the excellent short paper by Sprague (1973) is still useful.
It has been said many times, but needs emphasis here: in a bioassay, the organisms are
actually acting as a sort of chemical measuring device which also integrates other
conditions into the measure such as temperature, disease, dissolved oxygen, etc. The
bioassay then is useful for comparing the gross effects of various chemicals on different
species and populations. These acute tests, along with chronic bioassays in which a
population is exposed for weeks, months, or even through one or more generations to
different concentrations of a toxicant, have been used as tools in establishing water
quality criteria (Alabaster and Lloyd, 1980; EPA, 1986). Such criteria are then used by
government regulators to formulate legal water quality standards. In a nutshell then, much
of aquatic toxicology has primarily been aimed at the important function of determining
the maximum amount of some toxicant that can be permitted in the environment without
causing significant harm to the resident biota.
From approximately the mid-1970s aquatic toxicology has increasingly used the
tools of the physiologists. This is partly to understand why a fish or invertebrate is
debilitated, but it is also because of a realization that there are many sublethal effects
that may occur without necessarily resulting in death of the individual organism; or as
Jan Prager was quoted (Sindermann, 1979, p. 438) as saying: “Death is too extreme a
criterion for determining whether a substance is harmful to marine biota.” While
bioassays extending over several generations are useful, they are also extremely time
consuming and expensive to carry out. Thus, there has been considerable interest in
developing physiological and biochemical tests, or more commonly known as biomarkers,
to assess the “health” of aquatic animals (Adams, 1990; McCarthy and Shugart, 1990;
Huggett et al., 1992).
Fish physiologists traditionally have had little interest in aquatic toxicology. Their
concern has been the understanding of the organ systems of various fish species and their
physiological adaptations to environmental variables such as temperature and dissolved
oxygen. In the multivolume work entitled Fish Physiology (Hoar and Randall, 1969-
1992) the effects that pollution has on these organisms rarely is mentioned. The chapter
by von Westernhagen (1988) is a notable exception. Such seeming neglect may be due
to at least three factors: (1) the authors are, with a few exceptions, not professionally
involved with work on pollution, (2) the database has, until recently, been severely
limited and the quality of some of the work was not especially high, and (3) it is customary
in physiology books (whether on humans or other animals) to not spend much if any time
on the effects of toxicants.
Fish physiology is now becoming an integral part of aquatic toxicology. From a purely
physiological standpoint, the presence of pollutants in the environment at sublethal
concentrations can be considered as another extremely interesting environmental variable
to which a fish will physiologically respond.

III. LEVELS OF BIOLOGICAL ORGANIZATION

When investigating the effects of pollution on fish (or other organisims) it is useful to
keep in mind the spectrum shown in Figure 1. (The expressions “levels of complexity”
or “levels of integration” are often used to designate this topic.) Starting from the left,
foreign chemicals, or other environmental stressors, such as elevated temperatures, exert
their primary effects at the enzyme level, or they may alter some other cell function such
as permeability of membranes. These changes affect cell integrity, ultramicroscopic
structure, and grosser functions such as energy expenditure or secretion rate of a hormone.
If these changes are severe enough, many cells may die resulting in histological lesions
which are visible using light-microscopic techniques. Because organs are composed of
many types of cells, effects on one or more of these types will be reflected in changes in
organ function. For example, many pollutants cause a thickening and even necrosis (ceil
death) of the gill epithelium. This in turn produces a reduction in permeability of the gill
to oxygen which thereby affects respiratory function for the whole animal. A failure of
homeostasis may then be seen. Some organs show compensatory changes (e.g., increased
breathing rate) when homeostasis is altered as an attempt to bring the internal condition
back toward normal. In this example, the initial gill damage is a pathological effect which
then causes one or more physiological responses.
 Gene Function Cell Integrity Histological Organ Homeostasis Growth Ecology
Enzyme Activity and Lesions Function and and
Membrane Perm. Metabolism Reproduction Behavior

Figure 1 Levels of biological complexity in the study of the effect of some environmental
factor, Including pollution. The extent of complexity increases as one progresses from left to
right.

Moving further across the levels of organization spectrum (Figure 1), chronic expo­
sure to a pollutant may depress growth. Reproduction is one of the processes of fish that
is most sensitive to pollution, particularly the larval stages. Anything that effects the
nervous system will alter behavior, and many substances directly cause alterations in the
functions of the nervous system. They may affect behavior indirectly as well by affecting
other organ functions such as osmoregulation and metabolism of sex hormones. Finally,
changes in the function of a group of organisms in an ecosystem cause effects on other
organisms, whether they be predators or prey.
In the levels of organization spectrum it is important to realize that no level is more
important than another. As Bartholomew (1964, p. 8) said so well: “...each level offers
unique problems and insights; each level finds its explanation of mechanism in the levels
below, and its significance in the levels above.” (Also see Jorgensen, 1983.) As a rule,
the higher the level, the more generalized the response. So if one wishes to assess the
general “health” of an organism, higher levels are appropriate; however, if one is
interested in studying more specific actions of various things and wishes to understand
mechanisms, lower levels are investigated.

IV. IMPORTANCE OF DOSE AND DURATION OF EXPOSURE

Figure 2 illustrates the general effect of environmental concentration of a chemical or
altered physical parameter such as dissolved oxygen on some measurable response in
the organism. Concentrations below the sublethal response threshold are best called a
“no effect” level rather than a “safe” level as has unfortunately been done in some
studies. The sublethal threshold will vary with the response that is being measured, and
due to the fact that only small changes are being measured, random (sometimes called
stochastic) processes will make it difficult to specify with precision (Dinman, 1972).
Within the sublethal range a wide variety of reversible and irreversible processes take
place. This is the area of most interest to physiologists and many pollution biologists.
Prolonged exposure within the upper end of the sublethal zone may cause death through
a general weakening of the animal so it becomes more susceptible to disease and/or
predation.
The lethal concentration (LC50) is defined as that concentration which causes death
to half the test animals within a specified period of time (frequently 96 h). The higher one
goes into the lethal range, the more rapidly death occurs. This resistance time can be
quantified as the median lethal time (LT50) which refers to the time required for 50% of
the test population to succumb to the experimental condition. Exposure to concentrations
that produce death in 96 h or less are usually called acute exposures, whereas the
concentrations in the sublethal zone are referred to as sublethal, or chronic if the time of
exposure exceeds 96 h.
In physiological studies, one measures either rate functions (e.g., breathing rate,
swimming speed, oxygen consumption) or the concentrations of something (e.g..
Figure 2 Idealized diagram of the effect of dose on the response as measured by some
physiological change (including percent dead). (Modified from Waldichuck, M., The Assessment
ofSublethal Effects of Pollutants in the Sea, Cole, H. A., Ed., Philos. Trans. R. Soc. London B,
286, 397, 1979.)

serum electrolytes, serum glucose, liver glutathione). A physiological response then

is a change in one or more of these measures caused by the altered environmental
condition. The response may be initiated by the fish as a means to maintain homeo­
stasis, or the response may reflect a breakdown of some physiolgical function. In that
case, it may be better to designate it as an effect, rather than a response. Thus, a
physiological effect of a pollutant (e.g., increased loss of blood electrolytes) may
initiate a physiological response (e.g., increased cortisol) to correct the altered
internal state of the animal.
The duration of exposure to an experimental condition may have a considerable
impact on both the qualitative character of a physiological change and its quantitative
aspects. Figure 3 shows a generalized view of the major sorts of changes that may occur
in a physiological measure (e.g., blood glucose or breathing rate) during the period of
experimental exposure. If the concentration of pollutant is sufficiently high, death may
ensue and be reflected in the physiological variable going rapidly one way or the other.
This does not necessarily mean this was the physiological mode of death, as some workers
have claimed, for other even more important things may have taken place but were not
measured.
If the exposure is to sublethal levels, then either an increase or decrease in the variable
may occur, usually over a period of hours or days. This may be followed by a return
toward normal which we can call recovery, even though the exposure continues. Another
variation shown that is not uncommon, is the phenomenon of initial acceleration followed
by inhibition. This is generally observed where there is an initial physical irritation to
sensitive tissues, or where there is a “psychological stress” (Schreck, 1981) followed by
toxicological effects.
In general, the effect of increasing exposure concentration is to move the curves shown
in Figure 3 to the left and to increase the amplitude of the change. Clearly then, attention
Figure 3 Generalized illustration of the types of changes that can occur in some physiological
variable as a result of pollution exposure. See text for explanation.

must be given to when one measures a particular physiological variable during the period
of exposure as it will have a very profound effect on the data obtained both quantitatively
and qualitatively. What this argues for is measurements taken at several time intervals
during a given exposure.

V. STRESS
The concept of stress in physiological systems has an extensive history. The “father” of
stress studies is generally considered to be Hans Selye, although others such as Cannon
(1929) set the stage. Selye (1950) developed the idea that a mammal when subjected to
almost any kind of stress exhibits a generalized group of physiological responses. There
is a rapid elevation in adrenalin and noradrenalin which mobilizes muscle glycogen into
blood sugar, causes blood pressure to rise, and in general causes the body to undergo the
“fight or flight” response. If the stressful condition continues, the adrenal cortex is
stimulated to release increased amounts of cortisol which sustain the changes caused by
the adrenalin and also cause a mobilization of some of the body protein into plasma amino
acids and an assortment of other physiological changes. The hormonal changes are
referred to as primary effects of the stress and the other physiological alterations produced
by them are called secondary effects. Collectively, they are called the general adaption
syndrome (GAS), because it is a group of stereotyped responses which do not differ with
the original cause. Similar changes have been observed in fish (Donaldson, 1981; Mazeaud
and Mazeaud, 1981; Schreck, 1990) and are further discussed in Chapter 8. A biblio­
graphic database for personal computers on the subject is available from Davis and
Schreck (1994).
The terminology used in the physiology of stress has been confusing because some
refer to stress as the cause of the responses (e.g., thermal stress). Other workers call the
responses themselves stress (i.e., the animal is showing physiological stress) and the
causes become known as stressors. Pickering (1981) gives a good short review of this
10

problem, particularly with reference to fish, pointing out that strong arguments can be
made for each of the various terminologies. In more recent years most who deal with
stress in fish consider the animal is under stress when it exhibits diminished function,
which implies potential death of the individual or depressed reproduction leading to a
declining population (Heath, 1990).
Determination of whether and how much an animal is under stress revolves partly
around the problem of defining normality. There is considerable variation in what might
be termed normal values for physiological measures. These variations may be caused by
time of day when measured, psychological disturbances, age, season, and that all-
encompassing blanket called biological variability. The problem is not at all insurmount­
able, but needs to be faced by all who work in this field.
Assuming one can define the “normal” range by using some animals as controls,
which are treated the same as the experimentáis except that they do not receive the
stressor, then the problem becomes both statistical and biological. Statistical tests, such
as the Student's / test, can tell if the experimentáis are doing something different from the
controls (assuming one is measuring a relevant variable). The statistical tests will not,
however, tell whether the change is important for the animals. A 10% increase in the
hemoglobin concentration, for example, may be statistically significant but have little
functional relevance to the organism. There are a great deal of data in the literature which
show statistically significant changes in some variable in the organisms subjected to a
stressor, but an evaluation of the biological significance is usually lacking. Admittedly,
such an evaluation may have a large subjective element to it but would give additional
perspective to the data.

VI. TOXIC MODE OF ACTION

There is a popular misconception that a given chemical will have a single mode of toxic
action or a single target organ in the organism. Such a conclusion is probably not true for
any chemical. While it is true that harmful chemicals may tend to act mostly on certain
organs or physiological functions, there is usually more than one being affected. This may
in part be due to the fact that when one function is affected, this can immediately bring
about a series of other changes that may reflect altered homeostasis or compensatory
responses to that altered homeostasis. Even at the level of individual cellular enzymes,
most foreign chemicals inhibit or stimulate several enzymes, although some are more
sensitive than others to a given chemical (see Chapter 9).
The concept of target organ was probably first developed from work on drugs where
the aim is to develop a drug to act on specific organ(s). Toxic chemicals in the environ­
ment tend to accumulate in particular organs but the organs most affected by that
chemical are not necessarily the ones with the highest concentration. For example, DDT
tends to accumulate in fat where it produces no known effect; DDT acts instead on nerves
and on the process of electrolyte and amino acid uptake in the gut. Note that if an
investigator had only looked for effects on the nervous system, the conclusion might have
been that this is a neurotoxin only, whereas the latter effects may be more important at
chronic levels of DDT where it might inhibit the uptake of necessary nutrients.
A foreign chemical will generally produce a group of physiological effects which is
referred to as a syndrome. This concept has been useful in grouping chemicals into, “fish
acute toxicity syndromes” (FATS), which can then be used for making predictions of
toxic action based on chemical and physical characteristics of the xenobiotic (McKim et
al., 1987). For example, at very high environmental concentrations, a respiratory irritant
 11

causes increased coughing and elevated arterial carbon dioxide (among other things),
whereas a respiratory uncoupler causes increased oxygen consumption and ventilation
volume (among other things).
One of the more interesting and important conclusions that is beginning to emerge
from work on fish is that modes of action may differ markedly depending on the exposure
concentration. For example, when fish are exposed to a high concentration of waterborne
zinc (1.5 mg/L), a rapid drop in blood oxygen and pH occurs over a period of less than
12 h, and they die of hypoxia. At a lower level of zinc (0.8 mg/L) exposure for 3 days,
they experience slight blood alkalosis and no change in blood oxygen, however, some
mortality still occurs from unknown mechanisms (Spry and Wood, 1984). Therefore, the
concentration and duration of exposure can greatly affect the syndrome observed in the
fish.

REFERENCES

Adams, S. M., Ed., Biological Indicators of Stress in Fish, American Fisheries Symposium 8, Bethesda,
MD, 1990.
Alabaster, J. S. and Lloyd, R., Water Quality Criteria for Freshwater Fish, Butterworths, London, 1980.
Anderson, J. W., An assessment of knowledge concerning the fate and effects of petroleum hydrocarbons
in the marine environment, in. Marine Pollution: Functional Responses, Vernberg, W. B., Thurberg,
F. P., Calabrese, A. and Vernberg, F. J., Eds., Academic Press, New York, 1979, 3.
APHA, Standard Methods for the Examination o f Water and Wastewater, 18th Edition, American Public
Health Association, Washington, D.C., 1992.
Baker, J. P., Effects on fish of metals associated with acidification, in. Acid Rain/Fisheries, Johnson, R.
E., Ed., American Fisheries Society, Bethesda, MD, 1982, 165.
Bartholomew, G. A., The roles of physiology and behavior in the maintenance of homeostasis in the
desert environment, in. Homeostasis and Feedback Mechanisms, Symposia Society Experimental
Biology, No. 18, Cambridge University Press, New York, 1964, 7.
Briggs, S. A., Basic Guide to Pesticides, Their Characteristics and Hazards, Hemisphere Publishing,
Washington, D.C., 1992.
Cannon, W. B., Bodily Changes in Pain, Hunger, Fear and Rage: An Account of Recent Researches Into
the Function of Emotional Excitement, Appleton, New York, 1929.
Crawshaw, L. E. and Hazel, J. R., Temperature effects in fish. Am. J. Physiol., 246, (Regulatory,
Integrative, Comparative Physiology 15), R439, 1984.
Davis, L. E. and Schreck, C. B., Annotated bibliography on stress and transportation of fishes, and
downstream passage of salmonids, Oregon Cooperative Fishery Research Unit, Oregon State Uni­
versity, Corvallis, OR, 97331, 1994.
Dinman, B. D., “Non-concept” of “no-threshold”: chemicals in the environment. Science, 175,495, 1972.
Donaldson, E. M., The pituitary-interrenal axis as an indicator of stress in fish, in. Stress and Fish,
Pickering, A. D., Ed., Academic Press, New York, 1981, chap. 2.
Eisler, R., Cyanide hazards to fish, wildlife and invertebrates: A synoptic review, U.S. Fish Wildl. Serv.
Biol. Rep., 85 (1.23), 55, 1991.
EPA, Quality Criteria for Water 1986, United States Environmental Protection Agency, Office of Water
Regulations and Standards, Wasnington, D.C., EPA 440/5-86-001.
General Accounting Office, Water Pollution: Application of National Cleanup Standards to the Pulp and
Paper Industry, National Technical Information Service PB87-193231, U.S. Department of Com­
merce, Washington, D.C., 36, 1987.
Hall, L. W., Burton, D. T. and Liden, L. H., An interpretative literature analysis evaluating the effects
of power plant chlorination on freshwater organisms, CRC Crit. Rev. Toxicol., 9, 1, 1981.
Hazel, J. R., Thermal biology, in. The Physiology o f Fishes, Evans, D. H., Ed., CRC Press, Boca Raton,
FL, 1993, chap. 14.
Heath, A. G., Summary and perspectives, American Fisheries Society Symposium, 8, 183, 1990.
12

Hoar, W. S. and Randall, D. J., Eds., Fish Physiology, Vols. 1-10, 1969-1985.
Houston, A. H., Thermal Effects Upon Fishes, National Research Council Canada, 1982.
Huggett, R. J., Kimerle, R. A., Mehrle, P. M. and Bergman, H. L., Eds., Biomarkers: Biochemical
Physiological, and Histological Markers of Anthropogenic Stress, Lewis Publishers, Boca Raton,
FL, 1992.
Jorgensen, C. B., Ecological physiology, background and perspectives. Comp. Biochem. Physiol, 75A,
5, 1983.
Leduc, G., Cyanides in water: toxicological significance, in. Aquatic Toxicology, Voi. 2, Weber, L. J., Ed.,
Raven Press, New York, 1984, 153.
Leland, H. V. and Kuwabara, J. S., Trace metals, in. Fundamentals o f Aquatic Toxicology, Methods and
Applications, Rand, G. M. and Petrocelli, S. R., Eds., Hemisphere Publishing, Washington, D.C.,
1985, chap. 13.
Lindstom-Seppa, P. and Oikari, A., Biotransformation and other toxicological and physiological re­
sponses in rainbow trout {Salmo gairdneri) caged in a lake receiving effluents of pulp and paper
industry, Toxicol 16, 187, 1990.
Livingston, R. J., Review of current literature concerning acute and chronic effects of pesticides on
aquatic organisms, CRC Crit. Rev. Environ. Control 1, 325, 1977.
Lloyd, R., Pollution and Freshwater Fish, Fishing News Books, Oxford, 1992.
Macek, K. J., Aquatic toxicology: fact or fiction?. Environ. Health Perspect., 34, 159, 1980.
Mazeaud, M. M. and Mazeaud, F., Adrenergic responses to stress in fish, in Stress and Fish, Pickering,
A. D., Ed., Academic Press, New York, 1981, chap. 3.
McCarthy, J. F. and Shugart, L. R., Eds., Biomarkers o f Environmental Contamination, Lewis Publish­
ers, Boca Raton, FL, 1990.
McKim, J. M., Bradbury, S. P. and Niemi, G. J., Fish acute toxicity syndromes and their use in the
QSAR approach to hazard assessment. Environ. Health Perspect., 71, 171, 1987.
Moore, J. W. and Ramamoorthy, S., Heavy Metals in Natural Waters: Applied Monitoring and Impact
Assessment, Springer-Verlag, New York, 1984.
Morris, R., Taylor, E., Brown, D. and Brown, D., Eds., Acid Toxicity and Aquatic Animals, Cambridge
University Press, New York, 1989.
Neff, J. M. and Anderson, J. W., Responses of Marine Animals to Petroleum and Specific Petroleum
Hydrocarbons, Applied Science, London, 1981.
Murty, A. S., Toxicity o f Pesticides to Fish, CRC Press, Boca Raton, FL, 1986.
Nieboer, E. and Richardson, D. H. S., The replacement of the nondescript term “heavy metals” by a
biologically and chemically significant classification of metal ions. Environ. Poll, 1, 3, 1980.
Norton, S. A., The effects of acidification on the chemistry of ground and surface waters, in. Acid Rain/
Fisheries, Johnson, R. E., Ed., American Fisheries Society, Bethesda, MD, 1982, 93.
Pickering, Q. H., Acute toxicity of alkylbenzene sulfonate and linear alkylate sulfonate to the eggs of the
fathead minnow, Pimephales promelas. Air Water Pollut., 10, 385, 1966.
Pickering, A. D., Introduction: the concept of biological stress, in. Stress and Fish, Pickering, A. D., Ed.,
Academic Press, New York, 1981, chap. 1.
Raney, E. C., Menzel, B. W. and Weller, E. C., Heated Effluents and Effects on Aquatic Life with
Emphasis on Fishes; a Bibliography, U.S. Atomic Energy Commission, TID-3918, 1972.
Russo, R. C., Ammonia, nitrite and nitrate, in. Fundamentals of Aquatic Toxicology, Methods and
Applications, Rand, G. M. and Petrocelli, S. R., Hemisphere Publishing, Washington, D.C., 1985,
chap. 15.
Schreck, C. B., Stress and compensation in teleostean fishes: response to social and physical factors, in.
Stress and Fish, Pickering, A. D., Ed., Academic Press, New York, 1981, chap. 13.
Schreck, C. B., Physiological, behavioral and performance indicators of stress. Am. Fish. Soc. Symp., 8,
29, 1990.
Selye, H., The Physiology and Pathology o f Exposure to Stress, a treatise based on The Concepts of The
General-Adaptation-Syndrome and the Diseases of Adaptation, Acta, Montreal, 1950.
Sindermann, C. J., An opinion about research activities and needs concerning physiological effects of
pollutants in the environment, in. Marine Pollution: Functional Responses, Vernberg, W. B.,
Thurberg, F. P., Calabrese, A. and Vernberg, F. J., Eds., Academic Press, New York, 1979, 437.
Sprague, J. B., The ABC’s of pollutant bioassay using fish. Biological Methods for the Assessment o f
Water Quality, ASTM STP 528, American Society for Testing and Materials, 6, 1973.
 13

Spry, D. J. and Wiener, J. G., Metal bioavailability and toxicity to fish in low-alkalinity lakes: a critical
review, Environ. Pollut., 71, 243, 1991.
Spry, D. J. and Wood, C. M., Acid-base, plasma ion and blood gas changes in rainbow trout during short
term toxic zinc exposure, J. Comp. Physiol. B, 154, 149, 1984.
von Westernhagen, H., Sublethal effects of pollutants on fish eggs and larvae, in. Fish Physiology, Vol.
XI, Part A, Hoar, W. S. and Randall, D. J., Eds., Academic Press, New York, 1988, chap. 4.
Waldichuk, M., Review of the problems, in. The Assessment of Sublethal Effects of Pollutants in the Sea,
Cole, H. A., Ed., Philos. Trans. R. Soc. London B, 286, 397, 1979.
World Commission on Environment and Development, Our Common Future, Oxford University Press,
Oxford, 1987.
 Chapter
2
Environmental Hypoxia
I. INTRODUCTION
Hypoxia refers to any condition in which the amount of oxygen is measurably below air
saturation levels. Anoxia means no oxygen and should be reserved for such conditions.
Aquatic biologists generally think in terms of dissolved oxygen expressed as milligrams
per liter (or the equivalent ppm) concentration of oxygen in the water. Physiologists
usually measure oxygen in the environment or body fluids as partial pressure (P02) which
is expressed in mmHg (= torr), or as pascals (1 mmHg = 133.32 pascals) (Bridges and
Butler, 1989). While it is reasonably safe to assume a direct relationship between
dissolved oxygen concentration and P02, this holds true only at a given temperature and
salinity. As either of these increases, the solubility, and thus concentration, of oxygen
decreases at any given P02.
Environmental hypoxia is taken up early in this book because (1) dissolved oxygen is
often low in polluted waters and (2) many of the physiological responses of fish to
chemical pollutants at acute concentrations, are similar to those produced in response to
enviromental hypoxia. Therefore, a treatment of this topic is a good introduction to the
types of actions pollutant chemicals may exert on the functions of various organs.
There are several potential causes of environmental hypoxia, some of which are not
due to human activities (i.e., they are “natural”) (Boutilier, 1990). For example, in
thermally stratified eutrophic lakes, the P02 of the hypolimnion is almost always hypoxic
(Barnes and Mann, 1991), and during the winter in lakes that are frozen over, respiration
can cause depletion of oxygen in the water trapped below the ice (Pennak, 1968).
In lakes and stagnant streams where the concentration of nutrients are high, algal
blooms may cause a considerable decrease in oxygen. The opposite of hypoxia, a
supersaturation of oxygen (hyperoxia), may occur during midday in some of these ponds
due to photosynthesis and warming of the water (Garey and Rahn, 1970). Also, wherever
there is a large amount of putrescible organic matter in the water from industrial or
domestic waste, microbial respiration utilizes a large percentage of the dissolved oxygen
(i.e., the biochemical oxygen demand is elevated) (Poppe, 1990; Warren, 1971).
Hypoxia is not limited to freshwater habitats. Oxygen levels in the ocean vary with
depth, temperature, salinity, and productivity (Bushnell et al., 1990). Nutrient enrichment
and unique meteorological conditions as well as phytoplankton blooms can produce

15
16

severely hypoxic conditions in marine habitats (Boesch, 1983; Swanson and Sinderman,
1979). Conditions of near zero dissolved oxygen have been observed due to pulpmill
wastes confined in a partially enclosed saltwater bay (Swanson and Sinderman, 1979),
and diurnal changes in oxygen can occur in intertidal areas with high nutrient loads
(Truchot and Duhamel-Jowe, 1980).

II. MINIMUM LEVELS OF OXYGEN REQUIRED FOR FISH LIFE

Many of the earlier studies of fish and hypoxia were devoted to determining the minimum
levels of oxygen required by fish. Doudoroff and Shumway (1970) reviewed much of this
and provided extensive tabular data on lethal levels of oxygen for a wide variety of
species. They also discussed some of the physiological effects of low oxygen. Davis
(1975) provides a somewhat less extensive but still very useful review of this topic. His
primary aim was the formulation of criteria for dissolved oxygen for Canadian fish and
invertebrates. The approach used by Davis was to examine the literature looking for
threshold levels of dissolved oxygen that caused changes in some physiological param­
eter such as reduced swimming stamina, increases or decreases in metabolic rate, reduced
blood oxygen saturation, etc. The presumption is that at any level of oxygen below that
threshold, the organisms will be expending excess energy to maintain homeostasis and
thus experience some physiological stress.
Frequently, freshwater fish have been grouped into salmonids and non-salmonids with
regard to their minimum oxygen requirements. The former are considered to be less
tolerant of hypoxia than the latter. This broadly held assumption is borne out in the data
summarized in the above-mentioned reviews, especially for the levels that are lethal.
According to Davis (1975), the average physiological threshold for salmonids is a P02 of
120 mmHg and for non-salmonds it is 95 mmHg. Assuming temperatures of 15 and 25°C,
respectively, those translate into dissolved oxygen concentrations of 7.8 and 5.2 ppm. The
two groups do not often cohabitate but oxygen may not always be the reason. Tempera­
ture may in many areas be a more limiting factor for salmonid distribution than oxygen.
The minimum oxygen requirements of pelagic marine species has been little studied.
There is little reason to presume they are especially well-adapted for low oxygen,
although some certainly are quite tolerant of hypoxia (Wu and Woo, 1984). In the deeper
oceanic regions there are large eutrophic oxygen-minimum zones where oxygen levels
can even approach zero; fish from these areas are generally quite tolerant of low oxygen
(Douglas et al., 1976; Yang et al., 1992).
Some species of freshwater fish are extremely resistant to low levels of oxygen, or
even anoxia. The cyprinids are especially notable as they include the crucian carp
(Cyprinus carpio), which can survive up to 6 months in cold water in the absence of
oxygen (Blazka, 1958; Holopainen et al., 1986) and the common goldfish {Carassius
auratus), which survives total anoxia for up to 22 h at 20°C (Van den Thillart et al., 1983).
Another species that exhibits remarkable tolerance of anoxia is the toadfish (Opsanus
tau), a marine species. These fish can survive an average of 20 h in oxygen-free water at
22°C (Ultsch et al., 1981).
In general, relatively low temperatures are required for high tolerance to anoxia, but
there are exceptions. For example, Mathur (1967) reported that Rasbora daniconius
(another cyprinid) can survive about 3 months of anoxia at 33°C.
Among freshwater fish, sensitivity of eggs and larvae to low oxygen varies with the
particular stage of development. The early embryo is relatively resistant to low concen­
trations of oxygen, but as the embryo grows, its sensitivity to hypoxia increases to a
maximum at hatching (Doudoroff and Shumway, 1970). The early fish embryo obtains
its energy mostly by means of anaerobic glycolysis, and then as development proceeds.
 17

aerobic respiration becomes more important (Boulekbache, 1981). Thus, this ontogenetic
change in sensitivity to hypoxia by the fish embryo appears to be related at least in part
to the dominant mode of energy metabolism that it uses at a particular developmental
stage. It should be noted here that although early fish embryos may tolerate a lack of
oxygen rather well, it is often the most sensitive stage in the whole life cycle to the
“insult” of a chemical pollutant (see Chapter 13).
Doudoroff and Shumway (1970) summarize a considerable amount of work on salmo-
nids of several species and conclude that any reduction of oxygen below air saturation
may produce delays in hatching and smaller than normal fry. These fry, however, are
usually viable and not deformed unless the oxygen levels are below 2-3 mg/L. The
measurement of oxygen levels in the water of a stream will not indicate the true oxygen
availability to the salmonid embryos as these are buried in the streambed gravels where
oxygen concentrations are often considerably less than that of the flowing water. Some
warmwater species seemingly require higher oxygen concentrations for normal develop­
ment than do salmonids (Doudoroff and Shumway, 1970).
The larvae of fish are far less able to tolerate hypoxia than the adults of the same
species (Davis, 1975). As the larvae develop, marked changes in hypoxia tolerance can
occur over just a few days. Spoor (1984) found that newly hatched smallmouth bass
(Micropterus dolomieui) larvae are comparatively resistant to hypoxia (90% survived 6 h
at 1 mg O2/L at 20°). However, starting with the second day posthatch, they became
increasingly sensitive to the lack of oxygen and this reached a maximum at the fourth day
(none survived 3 h at 1 mg/L dissolved oxygen.). This high sensitivity continues until day
10; thereafter there occurs a rather sudden decrease in sensitivity to hypoxic conditions.
The larvae do not start to breathe until the fourth day when they are least able to tolerate
hypoxia (Spoor, 1984).

III. INTERACTION OF HYPOXIA AND TOXICITY OF

POLLUTANT CHEMICALS
As a general rule, a given chemical becomes more toxic at lower levels of dissolved
oxygen, but for most chemicals, the effect appears to be modest (Sprague, 1984; Rattner
and Heath, 1994). Because the number of chemicals tested is quite small, however, and
data on sublethal effects are rare, such a generalization should be treated as quite
tentative. Because our interest here is primarily sublethal effects of chemicals, the
following four studies are relevant.
In 32-day tests on the effects of 1,2,3-trichlorobenzene on larval fathead minnows,
Carlson (1987) reported that when the dissolved oxygen (DO) was lowered to 4.5 ppm,
the chemical caused a much greater effect on growth and survival of the larvae than when
tested at near saturation for oxygen, but the threshold acute toxic concentration was
unchanged. From an environmental standpoint, the sublethal effect is probably more
important and it should be noted that a DO of 4.5 ppm is not very low.
Paraquat (an herbicide) causes accumulation of free radicals in cells as a result of its
metabolism. This in turn induces formation of more of the enzyme superoxide dismutase
as a mechanism for free radical removal. Severe hypoxia alone in carp induced superox­
ide dismutase in gill, liver, and brain tissue. Paraquat alone stimulated dismutase activity
in only the gill tissue. Combining hypoxia and paraquat caused an additive effect in gill
tissue but no further stimulation in the other two tissues (Vig and Nemcsok, 1989).
Perhaps the most interesting finding was the induction of superoxide dismutase by
hypoxia alone, a phenomenon that deserves further investigation.
Acetylcholinesterase in the nervous system of fish and other animals is greatly
inhibited by organophosphorus pesticide poisoning (Mayer et al., 1992). There was also
18

a report (Malyarevskaya, 1979) that hypoxia caused an inhibition of this enzyme in perch,
however, we were unable to confirm this finding in my laboratory using trout (unpub­
lished observations). Hoy et al. (1991) also found no effect of hypoxia on acetylcholines­
terase activity in trout, but if the fish were exposed to the organophosphorus compound
dichlorovos and hypoxia, a greater degree of inhibition occurred than if the exposures
took place in water saturated with oxygen. This may have been due to a more rapid uptake
of the poison by the fish because of hyperventilation in the hypoxic water, a mechanism
that undoubtedly applies in many hypoxia-toxicity interactions.
Finally, we examine the complex relationship between acute toxicity of anthracene
and dissolved oxygen. The toxicity of this polycyclic aromatic hydrocarbon is induced by
UV light, a process that is also proportional to the amount of oxygen present. In an
apparent contradiction of this physical phenomenon, maximum toxicity to fish occurs at
an intermediate DO. McCloskey and Oris (1991) hypothesize that at intermediate DO
levels, both hyperventilation due to mild hypoxia and photoinduced toxicity combine to
cause the greatest effect on the fish. At a high DO, ventilation is reduced so less poison
is brought to the gills and at low DO, the lack of oxygen in the water suppresses toxicity.
Exposure to a sublethal concentration of a toxic chemical can affect subsequent
responses of a fish to hypoxia. Phenol causes histopathological changes in gill tissues and
thereby reduces the ability of fish to tolerate hypoxia (Hlohowskyj and Chagnon, 1992).
Within the gill, if transport of oxygen by the blood is compromised by some chemicals
such as nitrate, there is a reduced ability to tolerate hypoxia (Watenpaugh and Beitinger,
1986). Finally, sublethal exposure to copper for a week results in an amplified stress
response of bluegill (Lepomis macrochirus) to a rapid hypoxia exposure (Heath, 1991).
The remainder of this chapter will be devoted to the physiological responses of fish
to hypoxia or anoxia. We will move from the processes of respiratory gas exchange and
transport during hypoxia to the biochemical changes invoved in anaerobic metabolism
during anoxia. Some emphasis will be placed on the adaptations that enable certain
species to be better able than others to function under conditions of oxygen lack.

IV. GILL VS. CUTANEOUS RESPIRATION

The site of oxygen uptake in adult fish is primarily the gills. The skin, however, can be
an important oxygen exchanger in some species. For example, in the buried plaice
(Pleuronectes platessa), a significant amount of the oxygen uptake takes place through
the skin under normoxic conditions. If the oxygen tension is lowered, the cutaneous
oxygen uptake stays relatively constant while the gill, and thus total, oxygen uptake
declines (Steffensen et al., 1981). Such an increased utilization of skin for oxygen uptake
in the hypoxic plaice can be contrasted with the common carp (C. carpio) in which the
opposite occurs. Cutaneous oxygen uptake is directly related to ambient oxygen over both
hypoxic and hyperoxic conditions (Figure 1). Under normoxia Takeda (1989) found that
this species obtains about 10% of its total oxygen uptake through the skin. Most if not all
of that, however, is utilized directly by the cutaneous tissues (Nonnotte, 1981), which
appears to also be true for a variety of teleosts including the plaice (Steffensen et al.,
1985). Teleost skin has a very high rate of weight-specific metabolism, 1.7-1.9 times that
of the intact fish (Steffensen et al., 1985). The energy released may be utilized in mucus
production or osmoregulation, although Steffensen and Lomholt (1985) found that chang­
ing the salinity of the surrounding water had little effect on skin respiratory rate so it is
not currently clear why the skin has such a high energy requirement.
Cutaneous oxygen uptake might be important for those species that occasionally go
out on land because gill tissue typically collapses in air. Such amphibious species include
some of the catfish (Ictalurus) (Nonnotte, 1984) and European eels, although in the latter,
cutaneous tissue utilization of oxygen was equal to or greater than that taken from the air;
 19

Figure 1 Respiratory frequency (fR), total O2 uptake (V0 2 total), gill O2 uptake (V0 2 gill), and
cutaneous O2 uptake (V0 2 skin) as a function of ambient P0 2 (PWO2 in) in the carp. Significance
of difference from normoxic levels: **p <0.01; ‘p <0.05. (From Takeda, T., Comp. Biochem.
Physiol., 94A, 205, 1989. With permission from Elsevier Publishers.)

the skin therefore cannot be considered as an oxygen exchanger under these conditions
(Nonnotte, 1984).
Larval fish rely exclusively on cutaneous respiration immediately after hatching. The
rate of development of gill respiration varies greatly between different species (Rombough,
1988); in the cyprinids, at least, this development is a gradual process wherein the
cutaneous respiration may remain important well into the juvenile stages (El-Fiky and
Weiser, 1988).
Rombough (1992), measured intravascular and skin-water interface P02 with micro­
electrodes in these larvae and found the intravascular P02Sof the larvae to be considerably
lower than those of adults. He attributed this difference to the relatively large diffusional
boundary layer in the larvae which is not ventilated as would occur with gills, so it
becomes stagnant.
20

V. ADJUSTMENTS IN VENTILATION
As oxygen moves from the water which is passing over the gills to the site of utilization
in the cells, it encounters a series of resistances (Figure 2). Environmental hypoxia lowers
the starting point on the left of this curve, but by increasing the ventilation, the boundary
layer next to the lamellae is more rapidly replaced with fresh water and the drop in oxygen
tension due to interlamellar water convection is presumably reduced (i.e., the first
resistance is lowered). Therefore, under mildly hypoxic conditions, the P02 of the arterial
blood may remain relatively unchanged.
As the oxygen level in the water decreases, most species start to increase their gill
ventilation volume. The threshold P02 varies with the species; limited data suggest that
species better adapted for low oxygen (e.g., carp) tend to have a lower threshold, which
is probably related to the oxygen-binding characteristics of their hemoglobin (Figure 3).
The bottom part of Figure 3 illustrates the point that various species alter gill ventilation
volume in different ways. Since ventilation volume is the product of stroke volume and
breathing frequency (the latter sometimes called the opercular beat), changes in either one
or both may be utilized. Trout (Smith and Jones, 1982), channel catfish (/. punctatus)
(Burggren and Cameron, 1980), and the marine dragonet {Callionymus) (Hughes and
Umezawa, 1968) alter ventilation largely by changes in stroke volume. The dragonet
actually decreases its ventilation frequency in response to hypoxia, but the stroke volume
increases to such an extent that ventilation volume rises (Hughes and Umezawa, 1968).
Some species, such as the bluegill (L. macrochirus) (Heath, 1973) and carp (Lomholt and
Johansen, 1979) rely more on changes in breathing frequency than stroke volume when
responding to changes in level of oxygen or metabolic demand.
The sturgeon is a somewhat interesting case in that two studies have reported com­
pletely contradictory findings. Burggren and Randall (1978) claimed that gill ventilation

EXTERNAL RESPIRATORY INTERLAMEUAR PL ASA« VENOUS

MITOCHONORU
WATER CAVITIES WATER CONVEaiON d iffusion REAaiON ADMIXTURE
CONVECTION

ENVIRONMENT

D E

Figure 2 Diagram showing approximate changes in Posfrom the environment to the mitochon­
dria of the cells. (From Hughes, G. M., Am. Zool., 13, 475, 1973. With permission.)
 21

C. carpio S. gairdneri

Figure 3 Relationship between gill ventilation and partial pressures of O2 of inhaled water
(PIO2 ) in carp (C. carpio) and rainbow trout (O. mykiss). The lower panels indicate the relative
contribution of breathing rate (dotted lines) and stroke volume (solid lines) to change in gill
ventilation. (Redrawn from Shelton, G., Jones, D. and Milsom, W., Handbook of Physiology,
Sect. 3, Vol. II, Part 2, Fishman, A. et al., Eds., American Physiology Society, Bethesda, MD,
1986.)

volume in this species declined in hypoxia. They measured it utilizing a plastic membrane
sutured around the mouth to separate the buccal water from opercular water. Recently,
Nonnotte et al. (1993) performed essentially the same experiments wherein the fish were
exposed to a gradual lowering of DO, but ventilation amplitude was estimated using
pressure transducers rather than the membrane system of the Burggren and Randall
(1978) study. Nonnotte et al. (1993) found that the ventilation increased both in amplitude
and frequency in an almost linear relationship to ambient DO. They suggest that the
presence of the membrane in the earlier study reduced the ability of the fish to respond
to the hypoxia. Decreases in ventilation in response to hypoxia have been observed (Wu
and Woo, 1984) but it would appear to be a rare phenomenon. We will return to the
question of oxyconformity later in this chapter.
Control of ventilation volume in fish has been much investigated, but the mechanisms
are only beginning to be understood. Randall concluded in his 1982 review (based mostly
from work on trout) that the ventilation changes seen in response to environmental
hypoxia are based on the oxygen content of the arterial blood, rather than its P02. The
receptor(s) that detect the blood oxygen content were believed to lie in the post-gill
arterial complex. In is now believed (Burleson et al., 1993) that there are two sets of
oxygen-sensitive receptors involved in ventilatory reflexes in trout: one set measures the
water oxygen and the other the arterial blood and both groups are located in the first gill
arch.
22

In trout, the arterial P02 tracks that of the environment as the latter changes (Holeton
and Randall, 1967), whereas the oxygen content of the blood at any given environmental
P02 will depend on the oxygen dissociation characteristics of the blood (to be discussed
below). Thus, the relationship between ventilation and environmental oxygen may de­
pend largely on the affinity of the blood for oxygen in a given species if the ventilation
is controlled by blood oxygen concentration.
Randall’s (1982) conclusion that ventilation is controlled by arterial oxygen content,
rather than P02, does not appear to apply to all species. More recent work on carp (Glass
et al., 1990; Williams et al., 1992) has shown this species regulates its breathing in
response to changes in P02 of the water independent of the oxygen content of the blood.
Whether a given species controls ventilation based on arterial P02, arterial oxygen
content, or water P02 may depend greatly on the oxygen affinity of the blood. Shelton et
al. (1986), in their review of respiratory control in fish, note that those species with blood
that has rather low oxygen affinity (e.g., trout), maintain high arterial P02S and that this
oxygen tension is rather dependent on that of the water. Those species with high oxygen
affinities (e.g., carp and Silurus) maintain low arterial P02 and this is little influenced by
the water P02 (Figure 4). A point that seemingly has not been considered is that a species
such as the carp, which monitors water P02, might increase ventilation even when there
was no need for such a response because its hemoglobin was still >80% saturated (Figure
5), and thereby wastes considerable amounts of energy. Intuitively, it seems like it would
make more homeostatic sense to monitor the variable that needs to be kept as constant as
possible, namely the oxygen content of the arterial blood.
In recent years there has been considerable interest in the involvement of catechola­
mines in respiratory control. Aota et al. (1990) reported that plasma adrenalin and
noradrenalin rose during hypoxia in trout and that part of the hyperventilation in response
to that hypoxia could be eliminated by adrenergic blocking agents. In seeming contrast
to this finding. Perry and Kinkead (1990) reported hyperventilation in trout under mild
hypoxia with little change in plasma catecholamines. In a follow-up study (Kinkead and
Perry, 1991) they administered boluses of catecholamines into the blood of trout while
the fish were experiencing hypoxia and observed an actual inhibition of ventilation, rather
than a stimulation. Perry and Kinkead (1990; Kinkead et al., 1991) also found that
Atlantic cod (Gadus morhud) respond to severe hypoxia with a strong catecholamine rise
but this seemed to have no influence on ventilation because adrenergic blocking agents
had no effect on the hypoxic hyperventilation. Randall and Perry, in their 1992 review
(p. 280) note that “...there is evidence that catecholamine infusion has an effect on
breathing in fish, causing either an increase or a decrease in rate depending on the species.

Figure 4 Relationship between inhaled

and arterial P0 2 in trout and Silurus (a
cyprinid). (Data from Porgue, J. et al.,
J. Exp. Biol., 143, 305, 1989, and
Boutilier, R. G. et al., Respir. Physiol.,
71, 69, 1988.) Inspired P02 (mm Hg)
 23

Po2 (mm Hg)

Figure 5 Comparison of oxygen dissociation curves for the hemoglobin from carp (C. carpio)
and rainbow trout (O. mykiss). Carp data at pH 7.9 and 20°C, from Weber, R. E. and Lykkeboe,
G., J. Comp. Physiol., 128, 127, 1978. Trout at 3 mmHg COg and 15°C, from Cameron, J. N.,
Comp. Biochem. Physiol., 38A, 699, 1971.

the time of year, and the physiological state of the animal.” Thus, the physiological
relevance of catecholamines for ventilatory control remains uncertain in spite of consid­
erable work. Indeed, Randall and Taylor (1991) argue that it does have relevance while
Perry et al. (1992) argue against such a conclusion. So, while the involvement of
catecholamines in the control of ventilation during hypoxia may or may not be important,
it will be seen below that catecholamines have a considerable influence on respiratory gas
exchange both at the gill and in the process of oxygen transport by the blood.
It has generally been assumed that arterial PCO2 or pH has little influence on fish
respiration. When elevations in respiration have been seen associated with elevated
carbon dioxide, this was attributed to reduced oxygen loading due to the Bohr or Root
effects. Perry and Wood (1989, p. 2962), however, argue that “...there now exists
sufficient evidence to indicate that CO2 and (or) pH also can stimulate Vw through
mechanisms independent of O2.” Obviously, with the extreme diversity of fish species
adapted to quite different sorts of habitats, it perhaps is not surprising that they would
exhibit a diversity of approaches to respiratory control.
Some fish species, such as members of the Scombridae family, and some salmon
exhibit a transition from active branchial ventilation to ram gill ventilation during
swimming at high speeds. As they reach a threshold swimming velocity, ventilatory
movements cease and the mouth is held open so the water is forced over the gills by the
forward movement of the fish (Roberts, 1975). This shift in ventilatory mode provides a
significant energetic savings while swimming (Steffensen, 1985). Roberts (1975) re­
ported that hypoxia had little effect on the transition velocity for Scombrids and con­
cluded that it was controlled by mechanoreceptors. More recently, Steffensen (1985)
explored this relationship between transition velocity and ambient P02 in rainbow trout
and sharksuckers (Echeneis naucrates). Some populations of rainbow trout exhibit ram
ventilation while others do not, so there is probably a genetic component to the behavior
in this species. He used a group that exhibited the ventilation mode and found that both
it and the sharksucker shifted to ram ventilation at a higher velocity as the ambient P02
was lowered. Clearly, there is thus a chemoreceptor, perhaps along with a mechanorecep-
tor, controlling this mode of ventilation.
In work on two species of tuna which exhibit obligate ram ventilation, Bushnell and
Brill (1991) found that swimming speed and mouth gape increased with hypoxia. While
24

the increased swimming speed might appear to be a mechanism to increase ventilation

volume, the authors maintain, based on their indirect measurements of ventilation vol­
ume, that it is instead, an escape behavior aimed at removing the fish from the hypoxic
water.

VI, ADJUSTMENTS BY THE GILLS TO HYPOXIA

The respiratory surface area of the gills of fishes appears to be related to the normal
activity of the species. Those that are active and therefore require high levels of oxygen
uptake have the largest gill surface areas (Hughes, 1966). As was discussed above, some
species of fish are far more tolerant of low oxygen than others. It might be expected that
they would have larger gill surface areas, but this does not appear to be the case. For
example, the trout, which is an active oxyphilic species, has a larger gill surface area than
the more hypoxia-resistant catfish. It also has a thinner epithelial diffusion distance
between the water and blood (Laurent and Perry, 1991).
While there may not be a morphological difference in gill structure that aids hypoxia-
resistant species, fish have been shown to enhance both lamellar surface area and reduce
the diffusion distance from water to blood in response to both short- and long-term
hypoxia. The lamellae of resting normoxic fish are partially buried in the filament thereby
reducing the respiratory gas exchange surface. Hypoxia causes them to become more
erect and project further out of the filament. This may be due to a combination of
hemodynamic and intrafilamental smooth muscle contractions (Laurent and Perry, 1991).
An environmental factor that can alter the gill surface area is the salt content of the
water. Trout in freshwater have less lamellar surface area than those in water with more
NaCl. This is due to a greater number of chloride cells in the freshwater-adapted trout.
Thomas et al. (1988) found that those in natural waters having NaCl at only 0.1 mM/L
were less tolerant of hypoxia (presumably due to the greater number of chloride cells) in
that their arterial P02 was more sensitive to ambient P02 than those in water with higher
salt concentrations (1 mM/L).
The control of blood circulation through the gill of fish is complex involving local
effects of the oxygen content of the water as well as hormonal and neural mechanisms.
In normoxic rainbow trout, only 60% of the gill lamellae (the structures where gas
exchange occurs) are perfused with blood (Booth, 1978), so any mechanism to increase
this during hypoxia would appear to be helpful, as it would increase the surface area for
gas exchange. Environmental hypoxia appears to produce a vasoconstrictor effect in
specific lamellae, and since the point of vasoconstriction is efferent to the lamellae, it can
cause a dilation of previously unperfused lamellae and thereby increase the overall
number of these structures receiving blood (Butler and Metcalf, 1983). The catechola­
mines, adrenaline and noradrenaline, reduce the resistance to blood flow through the gills
and also cause a recruitment of unperfused lamellae. Adrenaline, in addition to its effects
on gill circulation, may enhance the permeability of the gill epithelium to oxygen (Butler
and Metcalf, 1983).
Another possible mechanism for microcirculatory adjustments in the gills involves
prostaglandins. Mustafa and Jensen (1992) recorded elevated levels of the unstable
prostaglandin thromboxane A2 in blood downstream from the gills of trout following
hypoxia. They note that these hormones are involved in mammalian microcirculatory
adjustments to stress.

VII. TRANSPORT OF OXYGEN BY THE BLOOD

An important adaptation for taking up oxygen that all fish, except Antarctic ice fish
(family Chaenichthyidae), show is the presence of hemoglobin in their erythrocytes. The
 25

amount of oxygen carried by the hemoglobin in relation to the P02 is not linear (Figure 5).
This means that as the arterial P02 decreases, as during hypoxia, the oxygen content of
the blood remains virtually unchanged until it reaches a P02 where the curve starts
downward. Thus, under mild hypoxia, assuming no change in circulation, the number of
molecules of oxygen carried to the tissues should remain nearly unchanged.
As is illustrated in Figure 5, the oxygen dissociation curves are different for different
species of fish. The hemoglobin of carp shows a higher affinity for oxygen than does that
of trout, because it achieves a higher level of saturation (and thereby content of oxygen)
at any given P02. This is clearly a key adaptation enabling the carp to function better than
the trout in conditions of hypoxia. It does mean, however, that the tissues must function
at a lower P02 in order for the hemoglobin to give up oxygen to them. Garey and Rahn
(1970) recorded a tissue P02 for carp of approximately 12 mmHg and for trout between
30 and 40 mmHg. These measurements are probably on the high side of the true values
as they estimated the tissue P02 by measuring the gas tensions in a bubble of air injected
into the peritoneal cavity and allowed it to equilibrate with the surrounding tissues. The
P02 of mixed venous blood taken from indwelling catheters was 3 mmHg for the carp
(Garey, 1967) and 19 mmHg for trout (Stevens and Randall, 1967). Notice in Figure 2
that if the tissues were at these oxygen tensions, that would allow a somewhat better than
50% unloading of the oxygen from the hemoglobin, which probably comes close to
approximating the true situation in the resting fish.
Several factors, both environmental and internal, cause the oxygen dissociation curves
to shift to the right or left (lowering or raising the oxygen affinity, respectively). As the
pH is lowered, or the CO2 is raised, the curve shifts to the right and sometimes downward
due to the well-known Bohr and Root effects, respectively. Increasing the ambient
temperature lowers the oxygen affinity of the blood in most fish; so warm, acidic, hypoxic
conditions are especially difficult for fish to cope with.
It is common to see blood acidosis in fish exposed to severe hypoxia (Thomas and
Hughes, 1982; Tetens and Lykkeboe, 1985), although the opposite can also occur (Perry
and Thomas, 1991). The source of plasma acidity may be muscle lactic acid and protons
released from erythrocytes in exchange for plasma sodium. The sodium/proton exchange
in erythrocytes is apparently stimulated by catecholamines, especially noradrenaline
(Fievet et al., 1988).
The effect of extrusion of protons from the erythrocytes into the plasma is to raise the
intracellular pH above that of the extracellular pH within minutes and this causes an
increase in oxygen affinity of the hemoglobin (Fievet et al., 1988; Tetens and Lykkeboe,
1985). The increase in catecholamines also causes erythrocyte swelling and a decreased
concentration of intracellular organic phosphates which further improves oxygen affinity
of the hemoglobin (Perry and Thomas, 1991). The cellular swelling is mediated by the
second messenger cAMP and the effect, at least in carp, is potentiated by lower arterial
oxygen tensions (Salama and Nikinmaa, 1990; Thomas and Perry, 1992). Thus, these
“stress hormones” not only enhance the flow of blood through the gills and the perme­
ability of gill tissue to oxygen, they also aid the blood in removing oxygen from water
where it is in low concentration.
The percentage of the available dissolved oxygen that is removed from the water
during its passage over the gills is referred to as the percentage utilization (%U) or
extraction efficiency. In normoxic water, this figure ranges from 23% in the lamprey to
over 80% in the carp (see Campagna and Cech, 1981, for review). Increasing the
ventilation volume has generally been reported to decrease the %U, however, Campagna
and Cech (1981) have noted that the extent of this decrease is less in those species well
adapted for living in hypoxic conditions, such as carp and Sacramento blackfish {Orthodon
microlepidotis). Using a quantitative measure of this decrease, plus the extent of hyper­
ventilation during hypoxia, and the blood affinity for oxygen, they have calculated a
26

“respiratory efficiency index” that can be used to compare species as to their tolerance
for environmental hypoxia. On this basis, ranging from most tolerant to least are the white
sturgeon, carp. Pacific lamprey, striped mullet, channel catfish, rainbow trout, and starry
flounder. A comparative evaluation of the importance of catecholamines in this adapta­
tion would be interesting.

VIII. CARDIOVASCULAR CHANGES DURING HYPOXIA

The classical response of the fish heart to environmental hypoxia is a distinct slowing of
the heart rate (bradycardia) (reviewed in Randall, 1982; Fritsch, 1990). There appears
from recent work, however, to be a few exceptions to this generalization. Glass et al.
(1991), using a fairly slow rate of imposition of the hypoxia, found a tachycardia in carp
at P02Sdown to 50 mmHg. Below 30 mmHg, bradycardia occurred but the authors present
evidence that this may have involved some myocardial dysfunction from hypoxemia.
Fritsch (1990), using an abrupt lowering of water P02, observed bradycardia in two
species of marine teleosts, but not in a third one. An important factor that ought to be
considered is the rate at which the oxygen level is lowered in the test chambers. A rapid
decrease in oxygen tension, as has been used by many workers, is likely to initiate
excitement and cause release of catecholamines. The importance of this is well exempli­
fied in the study by Borch et al. (1993). Contrary to what most others have observed, they
recorded a tachycardia in rainbow trout during hypoxia (60 mmHg) which was imposed
very slowly. At lower levels of oxygen, bradycardia eventually occurred, possibly due to
hypoxemia acting directly on the heart muscle.
When bradycardia is observed it is initially caused by the cardiac branch of the vagus
nerve (Randall and Smith, 1967; Wood and Shelton, 1980), so it is not a direct effect of
low oxygen on the heart itself except at extremely low levels of oxygen. During brady­
cardia, the cardiac output (amount of blood pumped per minute) may go down (Farrell,
1982), however, due to an increase in stroke volume, the cardiac output can remain
unchanged (Wood and Shelton, 1980) or it may even increase (Itazawa and Takeda,
1978), depending on the species and level of hypoxia.
In teleosts, the receptors for oxygen tension that initiate the hypoxic slowing of the
heart are located on the first gill arch in the region of the efferent vessel and are distinct
from the receptors that initiate ventilatory responses to hypoxia (Daxboek and Holeton,
1978; Burleson and Milsom, 1993). Burleson and Milsom (1993) claim the receptors are
externally oriented so they measure water P02 rather than that of blood.
Because bradycardia in response to hypoxia is such a widespread phenomenon in
fishes, the question of its physiological value has repeatedly come up, especially when it
is observed that cardiac output sometimes remains unchanged or increases. Vagotomy in
the dogfish (Scyliorhynus canícula) eliminated the response but did not affect oxygen
transfer under hypoxic conditions (Short et al., 1979), so the bradycardia was no direct
benefit in this species. With increases in mean systemic blood pressures and enhanced
stroke volume due to Starling’s Law of the heart, the pulse pressure will increase. Butler
and Metcalf (1983) suggest this change in pulse pressure may aid lamellar recruitment in
the gills of some species. Finally, many fish species do not have a separate coronary
circulation, therefore, the heart muscle must obtain oxygen from the venous blood within
the heart chambers (Davie and Farrell, 1991). Slowing the heartbeat during hypoxia
would permit more time for this diffusion of oxygen to take place; so the bradycardia
response might be a mechanism to maintain heart function rather than to facilitate oxygen
uptake from the water.
During hypoxia, a rise in blood pressure is often seen even with a slowing of the heart.
This is caused by peripheral vasoconstriction mediated possibly by a mixture of neural
stimulation and circulating catecholamines (Kinkead et al., 1991; Satchell, 1991). As is
 27

discussed below, this peripheral vasoconstriction can influence kidney function in the
hypoxic fish.

IX, RESPIRATORY REGULATION AND CONFORMITY

The main reason for adjusting the various processes involved in taking up oxygen from
the water is to increase respiratory independence (or regulation). When the resting oxygen
consumption of a fish species that is an oxyregulator is measured at several different
environmental P02S, there is usually observed a range of oxygen levels where the
consumption of oxygen is unaffected, or it may go up somewhat due to the increased
energy demand of hyperventilation and overall restlessness (e.g., Petersen and Petersen,
1990). At P02S below some value referred to as the critical oxygen tension (PC or TC),
the animal is unable to maintain normal aerobic respiration and the oxygen consumption
begins to decline. The position of the PC is a function of ventilatory and circulatory
changes and on the oxygen affinity of the hemoglobin. It will be emphasized below that
it is also very much a function of the amount of physical activity the fish engages in during
hypoxic exposure and the amount of time given for adjusting to the lowered oxygen
content.
It has been taken for granted for some time that the PC is lower for fish species that
are better adapted for hypoxia (Beamish, 1964; Jones et al., 1970). Thus, Beamish (1964)
reported a PC of 90 mmHg for trout and 60 mmHg for carp. However, Ott et al. (1980)
reexamined the question by measuring oxygen consumption at a given P02 only after the
fish had become “adjusted” to the new P02. Under these conditions, the trout and carp did
not differ and both exhibited a seemingly low PC of 20 mmHg. This suggests that when
fish are exposed to hypoxia rather slowly, it may allow enough time for changes in the
efficiency of oxygen extraction from the water to take place.
Within a species, the PC depends on whether one is measuring the resting or active
consumption of oxygen. When fish are forced to swim at a maximum cruising speed,
oxygen consumption may increase 8- to 10-fold or more (Brett and Groves, 1979), and
the PC moves to higher environmental levels of oxygen. This may actually exceed the
point of saturation of oxygen in the water so, in essence, the fish becomes a conformer
(Beamish, 1964; Kaufmann and Wieser, 1992).
Several workers (reviewed by Ott et al., 1980) have reported a rise in the PC with an
increase in temperature. Carefully conducted studies are not in agreement as to the effect
of temperature on PC. Ott et al. (1980) found little effect on the rainbow trout and carp,
whereas Cech et al. (1979a,b) report a raising of the PC with increased temperature in
largemouth bass and Sacramento blackfish. Ott et al. (1980) attribute their seemingly
contradictory findings to the possibility that others were actually measuring a level of
metabolism well above the “standard” rate, perhaps more of a “routine” rate of metabo­
lism. Assuming that explanation to be valid, the argument can be made that “routine”
rates of metabolism probably come closer to representing that actually seen in nature. A
further factor that may be important is that some species exhibit a lowering of the PC in
the summer.
Rombough (1988b) determined the PC for steelhead (Salmo gairdrieri) embryos and
larvae and found that it changed dramatically with stage of development (Figure 6). The
early embryo had a very low PC but this rose to a maximum at hatching apparently due
to increasing metabolic demand of the growing embryo. The resistance to oxygen flow
then drops abruptly at hatching as the animal escapes the confines of the egg capsule. The
PC continued to decline during subsequent development of the larvae even though growth
continued. This improvement in ability to remove dissolved oxygen is related to the
expansion of gill area (Rombough, 1988a). Note also in Figure 6 that the PC at a given
stage of development increased with temperature. Other than Rombough’s study, there
28

Figure 6 Critical dissolved oxygen levels (P^) for steelhead embryos and alevins Incubated at
four temperatures; h = hatch; mtw = maximum tissue weight. Error bars give 95% confidence
limits for Pc- Horizontal arrows indicate oxygen concentration at 100% saturated. (From Rombough,
P. J., Can. J. ZooL, 66, 651, 1988b. With permission.)

has been very little work on the ontogenetic changes in respiratory capability in fish.
Given that there are so many species with quite diverse developmental patterns (Balón,
1984) and ecologies, this appears to be a fruitful area for further investigation.
The opposite of oxyregulation is conformity. A metabolic conformer would be an
animal in which resting oxygen consumption was directly proportional to P02. There have
been occasional examples of this observed, starting with an early (and often cited) report
(Hall, 1929) that the toadfish (O. tau) is a conformer. However, Ultsch et al. (1981)
reexamined this question and found that the toadfish is actually a rather good regulator
(i.e., has a wide range of respiratory independence) providing it is given time to adjust
to the lowered oxygen levels. The channel catfish (/. punctatus) has been reported to be
a regulator (Burggren and Cameron, 1980) and a conformer (Gerald and Cech, 1970).
Marvin and Heath (1968) observed respiratory conformity in a different species of catfish
(/. nebulosis) that tolerates hypoxia much better than does the channel catfish, and Hughes
et al. (1983) found conformity in the carp at 10°C, but it showed fairly good respiratory
regulation at 20°C.
Several marine and estuarine species have been claimed to exhibit respiratory confor­
mity (Courtois, 1976; Subrahmanyam, 1980; Wu and Woou, 1984). However, in all of
these studies, the P02 was lowered rapidly over a few minutes, or hours at the most. In
a series of experiments where it was lowered more slowly on the lingcod {Ophiodon
elongatus), Farrell and Daxboeck (1981) demonstrated that this species can exhibit a wide
range of respiratory independence (i.e., it is a regulator), and Heath (1964) found “good”
regulation in the marine grouper (Epinephelus).
When all is said and done, the extent of conformity or regulation seen with a given
species undoubtedly depends on a combination of factors including the species, tempera­
ture, duration of hypoxic exposure, physical activity of the fish, and on how confined the
fish are in the respirometer chambers when the measurements are made. Ham (1993) has
demonstrated how stepwise discriminant analysis can be used to sort out the influence of
 29

things like feeding level, body mass, temperature, and pH on the relationship between
oxygen consumption and oxygen availability. While it might seem logical to carry out a
comparative study using several species in exactly the same experimental design, “appro­
priate” temperatures and respirometer design would have to be different for each species,
which would make comparisons challenging. Perhaps more attention should be paid to
the actual ecology of the animals so laboratory simulations can have more environmental
realism.
The foregoing account of the debate on respiratory regulation and conformity may
sound like a trivial academic exercise. It actually has important physiological and eco­
logical implications for fish in water with low dissolved oxygen. Fish are, of course,
basically aerobic organisms and the essence of much of this discussion is that they do all
they can to avoid hypoxic conditions in their tissues. Some do this better than others but
all may, on occasion, encounter conditions where the transport of oxygen to the tissues
does not meet their requirements. Assuming the PC is based on a truly resting fish, at any
environmental P02 below that, energy demand must be partially met by anaerobic metabo­
lism, or the energy demand must be reduced.
Metabolic depression would be a good strategy if the animal has the opportunity to
become quiescent, as during low-temperature dormancy (Crawshaw, 1984) and is “prac­
ticed” even at higher temperatures by at least some of those species that are well adapted
for living in conditions of low or zero oxygen, such as the goldfish and crucian carp (Van
Waversveld et al., 1989; Nilsson et al.,1993). Recent findings on crucian carp (Nilsson,
1990) suggest elevations during anoxia in the brain of inhibitory neurotransmitters along
with a concomitant decline in excitatory amino acids. These changes provide a mecha­
nism for reducing muscular activity.

X. ANAEROBIC METABOLISM
Adenosine triphosphate (ATP) is the energy currency in cells and this can be produced
by either aerobic or anaerobic metabolism, or a combination of the two. Aerobic metabo­
lism, which is measured as oxygen consumption, is about 18 times as efficient as
anaerobic metabolism in generating ATPs. This means that fish subjected to a period of
severe hypoxia often show a rapid depletion of glycogen (Heath and Pritchard, 1965;
Jogensen and Mustafa, 1980) a major, but not the only, (van Waversveld et al., 1989)
anaerobic fuel. Concurrent with the reduction in glycogen there occurs a rise in tissue
lactic acid, a major endproduct of this type of metabolism.
Measures of the concentration of lactic acid in tissues or blood gives a rough indication
of the extent of anaerobiosis that is occurring. When fish are exposed to a gradually lowered
oxygen tension (over a period of hours) a threshold is encountered where a rise in lactic acid
begins to take place (Burton and Heath, 1980; Boutilier et al., 1988) This threshold oxygen
tension correlates reasonably well with the relative oxygen affinity of the blood for each of
the species in question. The threshold may or may not correspond to the PC, because as was
discussed in the preceding section, different authors have reported markedly different PC
values for the same species. As was found with attempts to define a PC for various fish
species, the anaerobic threshold is greatly influenced by the rapidity with which the hypoxic
condition is imposed (Heath et al., 1980). By giving the fish a longer time to adjust to a given
oxygen level, the threshold is effectively moved to a lower P02. Most of the more recent
studies have used exposure periods at a given P02 of 8-24 h which is much longer than the
duration of exposures used by earlier investigators.
The environmental threshold for anaerobic metabolism is clearly temperature depen­
dent (Burton and Heath, 1980) in that it moves to a lower P02 at colder temperatures of
acclimation. This is probably a reflection of a lesser energy demand as well as an effect
of temperature on the oxygen affinity of the blood. Although the anaerobic threshold is
30

shifted downward by a lower temperature, Van den Thillart et al. (1983) have shown that
the ability to generate ATP anaerobically in goldfish muscles is much less at 5°C than at
either 10 or 20°C. This change in anaerobic capacity with temperature was estimated, in
part, from the median survival times of the fish in anoxia which were 45, 65, and 22 h,
respectively. Thus, lO^^C would appear to be a sort of optimum temperature for survival
of this species in anoxic conditions.
The energy status of a tissue can be assessed by measuring the concentration of the
adenylates (ATP, ADP, and AMP) and then using these values to calculate a dimension­
less number called the energy charge, which ranges from zero to one (Atkinson, 1977).
Skeletal muscle (Boutilier et al., 1988) and heart muscle (Koke and Anderson, 1986) from
resting fish show little change in energy charge when the animal is exposed to hypoxia,
but liver from the same animal exhibits large decreases, almost entirely due to a precipi­
tous drop in concentration of ATP (Van Waarde et al., 1983; Vetter and Hodson, 1982).
This difference between muscle and liver is probably due to a greater capacity for
anaerobic metabolism in muscle, which usually “uses” this ability to obtain energy during
bursts of swimming, rather than hypoxia. Teleost heart muscle evidently also has a high
anaerobic capacity (Koke and Anderson, 1986).
Brain tissue of vertebrate animals is traditionally considered to be very sensitive to a
lack of oxygen. In mammals, the enzymes for anaerobic glycolysis are present, but the
levels of glycogen are quite low and are quickly depleted during oxygen deprivation
(Dunn and Bondy, 1974). However, the bullhead catfish, which is quite resistant to
anoxia, has four times the glycogen levels of the rainbow trout, which is sensitive to
anoxia. Thus, the catfish is endowed with considerably more fuel in the brain to support
anaerobiosis there (DiAngelo and Heath, 1987). During anoxia, the brain glycogen
becomes depleted in both species of fish but only the catfish exhibits much of an increase
of lactic acid in its brain. This suggests that the catfish brain tissue has a greater anaerobic
capacity than does the trout. Surprisingly, the trout showed almost no decrease in brain
ATP during anoxia, whereas the catfish exhibited a significant loss of this adenylate even
though it survives anoxia five times longer than the trout (DiAngelo and Heath, 1987).
Evidently, the trout dies even while its brain energy status is near normal, so the cause
of anoxic death (i.e., they stop breathing) in this species remains unknown.
In a follow-up study (Heath, 1988), in vitro energy metabolism of brain and liver tissue
from the same two fish species was investigated to examine the hypothesis that the
Pasteur effect would be less in the species more tolerant of anoxia (Hochachka and
Guppy, 1987). The Pasteur effect refers to an enhanced rate of glycolysis when oxygen
is in inadequate supply either due to exercise or lack of environmental oxygen. A reduced
Pasteur effect would imply metabolic depression in that tissue and is presumably adaptive
for anoxic conditions (Hochachka and Guppy, 1987). However, anoxic catfish brain
tissue exhibited a slightly greater rate of anaerobiosis than did that from trout although
the Pasteur effect was eliminated at cold temperatures in both species. It was also absent
in liver tissue from both species at all temperatures. Based on enzyme activity measure­
ments in goldfish brain, this species may also exhibit a large Pasteur effect (Storey, 1987).
It therefore appears that whole-animal metabolic depression can occur during hypoxia or
anoxia, but brain tissue of fish may not necessarily experience such a depression except
in cold temperatures.
Typical anaerobic metabolism (glycolysis) which yields lactic acid as an endproduct
is obviously too inefficient for long-term use. Yet there are invertebrates that may live for
many days buried in anoxic mud. Biochemical studies have shown they use modified
metabolic pathways in their cells which produce succinate, alanine, propionate, and some
other endproducts with little or no accumulation of lactic acid (Hochachka, 1980). More
importantly, these alternate metabolic pathways produce two to four times as many ATPs
per gram of fuel over the traditional glycolysis.
 31

The finding of more efficient metabolic pathways for the generation of ATP in some
invertebrates stimulated a similar search for them in fish. The results were exciting but
not in quite the expected way. While some species (e.g., flounder and crucian carp)
accumulate succinate and/or alanine in selected tissues under severe hypoxia (Jorgensen
and Mustafa, 1980; Johnston, 1975), for the most part, fish appear to do things differently
than the invertebrates. The goldfish (Carassius auratus) and crucian carp (C. carassins)
have received by far the most attention, primarily because they are very good at tolerating
anoxia. Goldfish exposed to anoxia initially accumulate lactic acid and experience a
severe reduction in muscle glycogen, a classic Pasteur effect, but this does not persist
under continued anoxia. Instead, a very large amount of metabolic CO2 is produced.
Indeed, CO2 production is greater in anoxic goldfish than in controls breathing well-
aerated water (Van den Thiellart and Van Waarde, 1985).
In animal cells in which metabolic pathways are well known, carbon dioxide is a
byproduct of the Kreb’s cycle which requires oxygen as an ultimate hydrogen acceptor.
Therefore, in these fish there must be another hydrogen acceptor which can be used in the
absence of oxygen. Mourik et al. (1982) present evidence that this acceptor is acetalde­
hyde which the anaerobic mitochondria produces from pyruvate. Part of the CO2 produc­
tion is coupled with the formation of ethanol in the same manner as done by yeast
(Shoubridge and Hochachka, 1980). However, much more ethanol is produced than can
be accounted for by breakdown of glycogen alone, and a variety of pieces of biochemical
evidence indicate that amino acids are serving as substrates for ethanol and CO2 produc­
tion during anoxia in this species (Van Waarde, 1988). Ammonia is also produced in the
anoxic goldfish muscle by the deamination of adenosine monophosphate. A further
peculiar aspect of anaerobic metabolism in goldfish is that found by Shoubridge (1980)
in which the heart and brain produce lactic acid in the process of obtaining energy while
the muscle actually uses lactic acid for energy. In summary, the goldfish tissues (particu­
larly muscle) have the ability to utilize under anoxic conditions both traditional glycolysis
and Kreb’s cycle but with the production of ethanol and CO2 as endproducts, instead of
lactate or in addition to ethanol.
The ethanol metabolic pathway has also been confirmed in crucian carp, but was not
found in common carp (C. carpio) or a wide variety of Northern European and tropical
teleosts (Wissing and Zebe, 1988). Wissing and Zebe did, however, discover the ethanol
pathway in the bitterling {Rhodeus amarus), a European cyprinid. The bitterling, how­
ever, survives anoxia no better than does the common carp. Thus, the presence of the
ethanol pathway does not automatically permit long anoxia survival.
The ethanol pathway does not actually yield a more efficient energy metabolism.
Rather, based on in vivo ^^P-NMR measurements in crucian compared with common carp,
its main advantage appears to be that it retards acid buildup in both muscle cells and blood
during anoxia (Van den Thillart and Van Waarde, 1991).

XL SWIMMING SPEED
It was pointed out earlier in this chapter that hypoxia (if severe enough) reduces the
active oxygen uptake rate of fish, so an effect of low oxygen on sustained swimming
would also be predicted. In a relatively early study it was found that reducing the
concentration of oxygen to about 3 ppm did not prevent salmon and largemouth bass from
swimming for several hours at submaximal speeds (Dahlberg et al., 1968). However,
maximum sustained swimming speed is another matter. This is measured by forcing the
fish to swim in a water tunnel where the speed of the current can be increased incremen­
tally. The fish is allowed to swim at a particular speed for a fixed interval (frequently 10
or 30 min) then the speed is increased to the next step. The maximum sustained swimming
speed is determined as that speed where the fish fails to maintain position in the current.
32

Figure 7 The effect of dissolved oxygen concentration on final (maximum) swimming speed of
coho salmon (O. kisutch) at 20°C and largemouth bass (Micropterus salmoides) at 25°C.
Velocity increments were at 10-mln intervals. (Redrawn from Dahiberg, M. L. et al., J. Fish. Res.
Bd. Can., 25, 49, 1968.)

Figure 7 shows data from coho salmon and largemouth bass, the latter a species more
resistant to hypoxia than the salmon. The shape of the curves are similar to those obtained
from oxygen consumption studies of resting fish in that there is a threshold concentration
of oxygen below which the maximum swimming speed becomes depressed. However, the
point of inflection in the swimming speed curve seems low compared to that obtained
from active oxygen consumption data which tends to approach or exceed saturation for
oxygen (Beamish, 1964). In other words, the maximum swimming speed may be less
sensitive than active oxygen consumption to the effects of hypoxia. If so, the utilization
of some anaerobic metabolism to supplement the aerobic metabolism may be a possible
mechanism here.
The contribution of anaerobic metabolism toward the energy requirement for sustained
swimming in oxygen-saturated water is somewhat unclear and undoubtedly varies between
species (Jones and Randall, 1978). When the oxygen in the water is limiting (i.e., at levels
below the PC for active oxygen uptake), then the fish may compensate for a reduction in
aerobic metabolism by increased anaerobiosis. This could tend to push the inflection point
for maximum swimming speed to a lower concentration of oxygen than would be reflected
in the curves for active oxygen uptake. Still because of the low efficiency of anaerobiosis,
such a finding would only be evident where the time intervals for swimming were relatively
short, otherwise, the fuel reserves would be rapidly depleted.
The actual reason for a reduced sustained maximum swimming speed under low
oxygen tensions is unknown. Simple fatigue, whereby muscle glycogen becomes de­
pleted and alterations in cellular pH occur, generally results in a cessation of swimming
(Kutty, 1968), whereas Smit (1971) and Fry (1971) have hypothesized a depressing effect
of low oxygen on the nervous system as a reason for the slowed swimming. Because
nervous tissue is classically considered to be the tissue most sensitive to oxygen lack, such
a hypothesis may have merit.
Recent studies on cyprinid larvae (Kaufmann and Wieser, 1992) reveal an interesting
phenomenon; as expected, critical swimming speed was reduced under hypoxia but at the
same time the net metabolic cost of swimming was about 30% lower. Evidently, when
 33

the ambient oxygen is low, maintenance functions not related to swimming are greatly
reduced which gives the effect of lowering the cost of swimming. The logical follow-up
question is: To what extent does this apply to adult fish?
Because reductions in dissolved oxygen can reduce maximum swimming ability,
supersaturation of oxygen might be expected to enhance it. However, several studies on
adult fish (reviewed in Jones and Randall, 1978) and cyprinid larvae (Kaufmann and
Wieser, 1992) have not supported this hypothesis, nor does supersaturation affect growth
or feed conversion in rainbow or cutthroat trout (Edsall and Smith, 1990).

XII. BEHAVIOR
Measurements on the effects of environmental hypoxia on behavior of fish fall into five
groups: (1) changes in spontaneous muscular activity, (2) increased use of aquatic surface
respiration, (3) increased use of air breathing (in those capable of doing it), (4) avoidance
of areas of low dissolved oxygen, and (5) selection of cooler temperatures. Overall, there
has not been a great deal of systematic study of behavioral effects.
It was mentioned above that some fish will decrease muscular activity when in water
low in oxygen as a way to decrease the oxygen demand, although the increased demand
from ventilation may cause the oxygen consumption to actually go up slightly. Fish
almost always feed less when under hypoxia (Kramer, 1987) and this may partially
account for a decreased level of activity as well as a decline in growth rate.
Some fish may show increased activity in hypoxia which could be a non-specific
“fear” response to a changed environment (Schreck, 1981). They may also be attempting
to find water with higher levels of oxygen (Kramer, 1987; Petersen and Petersen, 1990).
If guppies are given access to the surface, they increase activity under hypoxia, but
activity is decreased if access to the surface is prevented (Weber and Kramer, 1983). A
rather specific reason for increased activity occurs with three-spined sticklebacks during
reproduction. The males guard the nest and ventilate the eggs by fanning water over them.
This fanning behavior increases during periods of low oxygen (Reebs et al., 1984), which
indicates a well-developed sense of the level of dissolved oxygen.
Aquatic surface respiration takes advantage of the fact that the upper few millimeters
of water often have much higher levels of oxygen than the deeper layers due to the fact
that oxygen diffuses through water very slowly. It can permit survival in waters that
otherwise would be lethal (Kramer, 1987). It is, however, largely limited to rather small
species and thus makes them more vulnerable to predation both from birds above and
larger fish and reptiles from below.
Air breathing is found in what are referred to as bimodal fish (i.e., they use both gills
and some air-breathing organ). Most of these are in tropical freshwater environments
where hypoxia or anoxia is common. The morphological arrangements which have been
evolved to permit air breathing in fish are quite diverse. They include modifications of
a variety of organs including the pharynx, swim bladder, intestine, and skin (Johansen,
1970). Most bimodal fish use water respiration if the levels of dissolved oxygen are high,
presumably because it is less energetically expensive for them than is air breathing, but
as dissolved oxygen gets lower, the increased costs of water ventilation makes air
breathing more efficient.
Because dissolved oxygen is so variable in aquatic habitats and spatial heterogeneity
is considerable, it might be expected that fish would be able to detect and avoid areas of
low dissolved oxygen. Earlier studies (e.g., Hoglund, 1961) suggested that the P02 had to
be so low as to cause respiratory distress before this occurred, however, later studies
showed that many fish can clearly detect and avoid low oxygen (reviewed in Kramer,
1987). This ability has been shown both in field studies (Suthers and Gee, 1986; Phil et
al., 1991) and the laboratory (Kramer, 1987; Spoor, 1990). Spoor (1990) observed that
34

brook trout (Salvelinus) tested in a gradient avoided oxygen concentrations below 4 mg/L
and preferred 5 mg/L or higher. Interestingly, Hallock et al. (1970) present circumstantial
evidence that migrating salmon would not move through an area until the dissolved
oxygen rose above 4.5-5.0 mg/L. This apparent threshold of around 4-5 mg/L for
avoidance of hypoxic waters by salmonids deserves further study.
The avoidance of areas of low P02 may be beneficial under some circumstances, but
Phil et al. (1991) point out that dimersal fish leave the deeper areas on a seasonal basis
which correlates with levels of dissolved oxygen. As increased eutrophication occurs due
to organic pollution, this may cause the duration of hypoxia in these areas to be extended.
Because these same areas are important nursery grounds for some species, it could have
a considerable negative impact on the fisheries.
One final behavioral aspect that has only recently received any attention is the effect
of hypoxia on selected temperature. Nearly all fish have preferred temperatures that vary
with the species and thermal acclimation history (Reynolds, 1977). A wide variety of
animals, both invertebrate and vertebrate (even protozoa!) behaviorally select cooler
temperatures when subjected to low oxygen availability (Wood, 1991). This includes fish
although only a very few species have been investigated. For example, the goldfish begins
to choose a cooler temperature at a P02 threshold of approximately 35 torr and as the P02
is lowered still further, the selected temperature can drop as much as lO'^C (Wood, 1991).
This should be beneficial as it would reduce the oxygen demand due to the well-known
QIO effect on metabolism. Rausch and Crawshaw (1990) found that goldfish exposed to
anoxia initially chose a cooler temperature, but then moved to a temperature of around
19°C. This is probably related to the effect of temperature on the anaerobic capacity of
this species (Van den Thillart et al., 1983). This capacity drops rapidly at temperatures
below 10°C.
It should be recalled that the goldfish is rather unique in that it produces ethanol during
anaerobic respiration. It is therefore interesting to note that this species responds to the
presence of ethanol in the water with a reduction in selected temperature (O’Connor et
al., 1988). The authors are apparently unaware of the unique metabolic capability of this
species, but it is tempting to hypothesize that the goldfish is responding to ethanol as if
it were hypoxia.

XIII. BLOOD AND URINE

Increases in tissue metabolites such as lactic acid will be reflected in increased levels of
these in the blood. Severe hypoxia in trout can result in increases of 18-fold or more in
the level of blood lactic acid (Heath and Pritchard, 1965; Dunn and Hochachka, 1986).
While this is only half as high as that reached in trout exercised to exhaustion (Black et al.,
1962), it still raises the possibility of acid-base alterations. In studies utilizing an extra­
corporeal circulation which permitted continuous measurements of blood gases and pH
in trout, Thomas and associates (1986, 1988) found that exposure to severe hypoxia (P02
= 40 torr) produces an initial blood alkalosis presumably due to hyperventilation. Within
5 min this is followed by an acidosis which initially precedes the rise in blood lactic acid.
Further blood acidosis is clearly caused by diffusion of lactic acid from white muscle and
adrenergic stimulation of H'^/Na'^ exchange in erythrocytes (Fievet et al., 1987). The latter
mechanism serves to remove hydrogen ions from erythrocytes and thereby maintain the
intracellular pH in these cells. This helps prevent any compromise in oxygen transport by
the hemoglobin, as was discussed earlier. There is also a marked rise in plasma sodium
from the tissues which serves to balance the lactate ions. Most of the hydrogen ions
produced by the dissociation of lactic acid are buffered intracellularly (Thomas et al.,
1986).
 35

It has been known for a long time that fish require several hours for blood lactic acid
levels to return to normal following exercise (Black et al., 1962) or hypoxia (Heath and
Pritchard, 1965). However, the acid-base status of the blood following hypoxia returns to
control levels much more rapidly. This is seemingly achieved by redistribution of ions
between extracellular and intracellular compartments rather than by branchial or renal
mechanisms (Thomas et al., 1986). It should also be mentioned here that trout that are
exposed to deep hypoxia for even longer times (i.e., several hours) experience a compen­
sation that restores plasma pH within 2 h after the initial metabolic acidosis (Thomas and
Hughes, 1982), even while the animal is continuing to experience the hypoxia.
The lactic acid that appears in the blood is metabolized by some tissues for energy, but
this capacity varies with different fish species and tissue types (e.g., Shoubridge, 1980).
Apparently, very little of the lactate is converted to liver glycogen (Dando, 1969) as is
common in mammals. Some lactic acid is excreted via the urine but this seems to not
account for much of the loss from the blood (Hunn, 1969; Thomas et al., 1986).
Blood glucose may or may not be elevated during hypoxia (Heath and Pritchard, 1965;
Swift, 1981; Dunn and Hochachka, 1986). To a large extent, elevations in blood glucose
may reflect a generalized stress response to a variety of environmental conditions and is
brought about by increased levels of adrenalin and cortisol (Thomas, 1990). Thus, the rate
of change in environmental oxygen could have a considerable effect on any changes in
blood glucose; a rapid hypoxic stress would be more likely to induce hyperglycemia
because it is “perceived” by the fish as a sudden change in its environment (Schreck,
1981).
In trout, the urine flow rate increases immediately upon exposure to hypoxia. This
initial increase seems to be a non-specific, stress-induced diuresis which may cause a
hemoconcentration of the blood (Swift and Lloyd, 1974). If the hypoxia is prolonged
(e.g., 24 h) there appears to be an increase in fish permeability to water. Because hypoxia
can cause physical damage to the gill tissue (see Section XIV), the increased permeability
may be a result of that damage. The increased urine flow is accompanied by an elevation
of electrolytes in the urine (Hunn, 1969), but this appears to have little effect on the
regulation of blood electrolytes (Thomas et al., 1986).
Urine flow rate in carp changes in a very different manner than in trout. Kakuta and
Murachi (1992) exposed carp to a gradually induced hypoxia over 6 h and noted a decline
in glomerular filtrate and urine volume. The urine decline paralleled the DO level in the
water very closely. During the hypoxia exposure, concentrations of various ions rose in
the urine; even urinary lactic acid rose threefold. During hypoxia in the carp, plasma
catecholamines rose several orders of magnitude. The authors point out that these hor­
monal increases probably caused peripheral vasoconstriction and reduced renal blood
flow, and thus a reduced glomerular filtration rate.
The final aspects of blood chemistry to be considered are the hemoglobin concentra­
tion, hematocrit, and blood cell count. While these are obviously related to each other, it
is possible to see larger changes in one than the other (e.g., the animal may produce a large
number of cells lacking in hemoglobin). Increases in one or more of these factors have
been reported many times in hypoxic fish, although not all species exhibit this (Weber and
Jensen, 1988). The cause of the increase during acute hypoxic exposure is water loss from
the blood and/or release of cells from storage points in the liver and spleen (Swift, 1981;
Wells and Weber, 1990). Splenic contraction occurs in trout in response to acute hypoxia
but not chronic exposure (Wells and Weber, 1990). The effect of these responses is to
increase the oxygen-carrying capacity of the blood, but it does not change the affinity for
oxygen. Under acclimation to hypoxia, changes in both capacity and affinity occur (see
below).
36

XIV. HISTOPATHOLOGY
Histopathological lesions in various tissues have frequently been reported following a
chemical exposure, but fish that have experienced hypoxia alone have not generally been
examined for these. When channel catfish were exposed to 1.5 ppm dissolved oxygen for
up to 72 h necrosis, hemorrhage, hyperplasia, hypertrophy, and hyperanemia were
observed in gills, liver, kidney and spleen (Scott and Rogers, 1980). Somewhat less severe
damage was reported by Drewett and Abel (1983) in brown trout sampled after death in
acutely hypoxic water (0.3-1.5 ppm). Among other things, they noted numerous small
breaks in the gill epithelium which were evident only under electron microscopic obser­
vation.
The significance of the histological effects produced by hypoxia remains to be eluci­
dated but does raise the question as to what degree are the changes in various physiologi­
cal factors (e.g., urine flow rate) due to these lesions. Also, hypoxia may produce effects
on gill histopathology which are synergistic with chemical exposure (see Chapter 3).

XV. ACCLIMATION TO HYPOXIA

It is well known that acclimation to high altitudes enhances the ability of terrestrial
animals, including humans, to function under those conditions of low oxygen (West and
Lahiri, 1984). Acclimation to hypoxia would seem to be a very important ability for those
fish that experience chronic conditions of low oxygen.
Shepard (1955) elegantly showed that acclimation of young speckled trout to low
dissolved oxygen made them able to tolerate a lower level of oxygen in their environment.
This was indirectly shown to be due to an enhanced ability to extract oxygen from the
water. The acclimation process required several days, or if it was done gradually, 20-33
h were required to acclimate to a change of 1.0 ppm oxygen. He further found that
acclimated trout were able to resist a lethal level of hypoxia longer than controls, which
suggests the anaerobic capacity of the acclimated fish was also increased. This latter
compensation may depend on the level of hypoxia to which they were acclimated as more
severe levels appear to weaken trout (Smith and Heath, 1980).
Studies of the physiological mechanisms of acclimation to hypoxia at the “whole
animal” level have largely been devoted to the eel, carp, goldfish, killifish, and flounders
(Greaney et al, 1980; Lomholt and Johansen, 1979; Kerstens et al., 1979; Wood and
Johansen, 1973; Wood et al., 1975; Jensen and Weber, 1985). A common observation is
that the acclimated fish have a lower resting oxygen uptake rate and the critical oxygen
tension (PC) is shifted to a lower P02. The lower oxygen uptake of the resting fish after
acclimation seems to reflect less spontaneous activity in these animals.
Extraction efficiency (percent of oxygen removed from water as it passes over the
gills) is generally enhanced with acclimation to low oxygen. This is achieved through an
increase in the effective gill surface and an increased affinity and capacity of the blood
for oxygen. The changes in the blood are brought about through increases in hematocrit
(presumably from more red blood cell production) and lowered erythrocyte nucleoside
triphosphate (NTP) levels (Weber and Lykkeboe, 1978; Wood et al., 1975; Smit and
Hattingh, 1981). The latter causes the increased affinity for oxygen and requires a variable
period of time (hours to days, depending on species) to come into play. The NTP in trout
erythrocytes is ATP whereas the main modulator in fish species more tolerant of hypoxia
is GTP, which has a greater effect on affinity than does ATP (Weber and Jensen, 1988).
The increased ability of the blood to carry oxygen results in a large decrease in cardiac
output in acclimated eels (Wood and Johansen, 1973).
Although acclimation to hypoxia has been shown to enhance the ability of fish to
extract oxygen from hypoxic water, two studies indicate this provides no improvement
 37

in swimming performance of either goldfish or rainbow trout in hypoxic waters (Kutty,

1968; Bushnell et al., 1984). Johnston and Bernard (1982) found that acclimation of tench
to hypoxia caused a reduction in the number of capillaries per fiber for both slow and fast
muscles, thus the lack of an effect on aerobic swimming could rest with a reduced
diffusion surface area in the muscle. Johnston and Bernard associated this loss of
capillaries with the decreased resting oxygen consumption rate of fish acclimated to
hypoxia, which raises the question of cause and effect. Does the decreased oxygen
availability cause the loss of capillaries which then reduces oxygen uptake rate, or does
the lowered oxygen uptake from reduced spontaneous activity cause the reduction in
capillaries?
It has long been known that fish growth is quite dependent on adequate levels of
oxygen (Weatherley and Gill, 1987). In part, this is due to a shift in metabolism away
from anabolism which results in declines in free amino acids and protein in plasma and
free histidine in muscle (Medale et al., 1987).
Two relevant studies show there are changes in certain cellular enzyme activities
during acclimation to hypoxia. Greaney et al. (1980) found in Fundulus that acclimation
to hypoxia enhanced the glycolytic and biosynthetic capacity of the liver (but there were
no changes in white muscle). Most of these changes returned to control levels sometime
between 28 and 35 days of continued low oxygen exposure. Smith and Heath (1980)
acclimated trout and carp to hypoxia for 7-10 days and found increased succinate
anaerobiosis in carp red muscle but not in the trout. This alternate form of anaerobic
metabolism which is found among facultative anerobic invertebrates (see above) is more
efficient than the usual glycolysis so it is clearly adaptive.
The two studies above suggest that acclimation to low oxygen improves the anaerobic
metabolism abilities of some species of fish. It will be interesting to learn how widespread
among fish species this is and how important the dynamics are (see Greaney et al., 1980).
For example, a chronic exposure to a mild hypoxia might be expected to affect aerobic
processes such as respiratory gas exchange mechanisms, whereas repeated brief acute
exposures to severe hypoxia might induce a greater anaerobic capacity.

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 43

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 45

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 Respiratory and
Chapter
3
Cardiovascular Responses
L INTRODUCTION
The respiratory system provides the most extensive interface of a fish with the aquatic
environment. Because of this and its delicate epithelium, it is frequently the first system
to be affected by pollutants. When death occurs as a result of acute exposure, it is often
due to failure of respiratory homeostasis. The circulatory system provides the means of
transport for essentially everything in an animal’s body. Because control of the rate of
blood flow and its distribution in a fish is based primarily on homeostasis of respiratory
gases, the cardiovascular responses to pollution are included in this chapter with the
respiratory system.

II. OVERVIEW OF NORMAL RESPIRATORY PHYSIOLOGY

The typical teleost fish respiratory system consists of the buccal and paired opercular
cavities and the gills suspended between them (Figure 1). Ventilation is provided by the
coordinated expansion and contraction of the buccal and opercular cavities which, by
their movements, work as a “double pump” providing a nearly constant flow of water over
the gills throughout a single respiratory cycle. Ventilation volume is adjusted primarily
in response to changes in arterial oxygen (discussed in Chapter 2).
The gills consist of horizontal flat filaments which are supported in the water stream
by the bony gill arches. On the filaments are found the secondary lamellae which range
in frequency along a given filament from 10-60/mm; the higher numbers are found in the
more active species (Hughes, 1982). Because of the structure of the gills, they provide a
large surface area for the movement of oxygen, carbon dioxide, electrolytes, water,
ammonia, and hydrogen ions between the blood and water. Thus, the gills are really a
multipurpose organ directly involved in a variety of functions including respiratory gas
exchange, osmoregulation, acid-base balance, and nitrogenous waste excretion. The
majority of these fluxes of chemicals take place across the epithelial surface of the
lamellae, which because of its multiple function, has been referred to as the “renaissance
epithelium” (Bentley, 1980).
The cross-sectional structure of a “typical” lamellum is shown in Figure 2a. The blood
is separated from the water by two outer layers of epithelial cells, a basement membrane.

47
48

GILL ARCH

-OPERCULUM
~(oreo shown in B)

LATERAL V lE ^ P HEAD

DIAGRAMMATIC HORIZONTAL
SECTION THROUGH HEAD
(A)

CONSTRICTOR
GILL SKELETON
MUSCLE SECONDARY
> ^ LAMELLAE
BUCCAL CAVITY
Figure 1 Major features of the AFFERENT AND EFFERENT
respiratory system of a teleost fish. BLOOD VESSELS

(A) The general arrangement of the

buccal and opercular cavities with
the associated gill structures. Ar­
rows indicate the direction of water
flow. (B) Enlarged view of segments
of two gill arches showing the two
rows of filaments on each arch and
the secondary lamellae projecting GILL ARCH WATER CURRENT
dorsally and ventrally from each
filament. (Redrawn from Hughes,
G. M., New Sci., 11, 346, 1961.)

and the flanges of the pillar cells. The total thickness of these layers ranges from 1-10 pm
depending on the species of fish (Hughes, 1980). The outer epithelial cells have microridges
and microvillae when viewed with a scanning electron microscope; these may help to
anchor mucus and increase the surface area for exchange of gases (Hughes, 1979). Blood
flows in the lamellum in a direction opposite to that of the water through a plexus formed
by the pillar cells. This countercurrent flow aids in the achievement of a high utilization
efficiency of oxygen (i.e., percent of the dissolved oxygen removed from the water in one
passage over the gills).
Recent studies have revealed that the fluxes across the gill membranes alter the
chemical characteristics of the water in the branchial boundary layer. For example, under
most conditions, due to the excretion of protons, the water next to the lamellar epithelium
is more acid than that midstream between the lamellae and this can affect the flux of other
ions such as ammonia (Randall et al., 1991). This localized microenvironment also has
implications for modeling of toxicant uptake from the water via the gills (Chapter 5).
Several comprehensive reviews of the normal physiology and histology of the fish
respiratory system have been published (Cameron, 1989; Perry and Wood, 1989; Randall,
1990; Perry and McDonald, 1993). Additionally, the proceedings of a symposium on
 49

Figure 2 Composite diagram of the common

gill lesions induced by pollutants. Six lamellae
are shown (a-f), the top one of which is char­
acteristic of a normal rainbow trout. The le­
sions are numbered as follows: (1) epithelial
lifting; (2) necrosis; (3) lamellar fusion; (4) hy­
pertrophy; (5) hyperplasia; (6) epithelial rup­
ture; (7) mucus secretion; (8) lamellar aneurism;
(9) vascular congestion; (10) mucus cell prolif­
eration; (11) chloride cells damaged early; (12)
chloride cell proliferation; (13) leukocyte infil­
tration of epithelium; (14A) lamellar blood si­
nus dilates; and (14B) lamellar sinus constricts.
Abbreviations; bl = basal lamina; cc = chloride
cell; e = typical lamellar epithelial cells; lbs =
lamellar blood sinus; pi = pillar cell; rbc = eryth­
rocyte. (From Mallatt, J., Can. J. Fish. Aquat.
Sci., 42, 630, 1985. With permission.)

environmental effects (excluding for the most part, pollutants) on gills have been pub­
lished in Physiological Zoology, Vol. 64 (1), 1991.
A wide variety of pollutants cause impaired regulation of plasma electrolytes and this
is due mostly to effects on the gills, a topic considered at length in Chapters 7 and 10.
Some aspects of the effects of pollutants on fish respiratory physiology are reviewed by
Rankin et al. (1982) and Satchell (1984).

III. HISTOPATHOLOGY OF GILL LAMELLAE

EXPOSED TO POLLUTANTS
Because respiratory gases must pass through the lamellar epithelium by diffusion alone,
this surface is quite delicate compared with the rest of the surface of the fish. Moreover,
there is a large flow of water over the lamellae even in a resting fish [e.g., approximately
70 ml/min in a 0.5-kg trout (McKim and Goeden, 1982)]. There is thus ample opportunity
for dissolved or suspended materials to come in contact with the delicate lamellae. In the
1920s and 1930s there were reports that fish exposed to heavy metals (e.g., zinc) in the
water suffocated due to damage to the gills and/or clogging of the lamellae with mucus
[see Jones (1964) for review of this earlier work]. A variety of histological studies since
then have in part confirmed that view and revealed that many pollutants, both inorganic
and organic, affect gill tissue (Table 1). This list is not meant to be all inclusive, nor does
it include all the references in which damage has been reported for a given chemical. It
is included here to give an indication of the wide variety of substances that can cause gill
histopathology. (See Mallatt, 1985, for a more comprehensive listing.) It is important to
realize that there are marked quantitative differences between the chemicals. For ex­
ample, chromium is not nearly as harmful to gill tissue as is nickel, when fish are
immersed in solutions of comparable toxicity (Hughes et al., 1979).
50

Table 1 Some Substances Reported to

Cause Gill Histopathology in Fish
Substance Ref.
Acid Daye and Garside (1977)
Ammonia Kirk and Lewis (1993)
Arsenic Sorenson et al. (1979)
Beryllium Jagoe et al. (1993)
Cadmium Stromberg et al. (1983)
Chlorine Bass et al. (1977)
Chlorothalonil Davies (1987)
Copper Wilson and Taylor (1993)
Detergent Abel and Skidmore (1975)
Formalin Smith and Piper (1972)
Iron Cruz (1969)
Lead Sippel et al. (1983)
Malathion Richmonds and Dutta (1987)
Mercury Olson et al. (1973)
Nickel Hughes et al. (1979)
Ozone Wedemeyer et al. (1979)
Paraquat Meyers and Hendricks (1985)
Permethrin Kumaraguru et al. (1982)
Petroleum Prasad (1991)
Phenol Kirk and Lewis (1993)
Zinc Tuurala and Soivio (1982)

Nearly all studies of gill histopathology resulting from exposure to some pollutant
involved the fish receiving the test chemical in the water. However, gill damage can also
be caused by the fish eating contaminated food. The insecticide permethrin caused essen­
tially equivalent histopathology in trout which had either eaten contaminated food or been
immersed in solutions of the chemical (Kumaraguru et al., 1982). Thus, this toxicant at least,
can reach the gills by way of the circulation in sufficient concentration to cause damage.
Perhaps others have a similar potential but this seemingly has not been investigated.
Obviously, from the list in Table 1, a broad spectrum of substances causes alterations in gill
structure. The lesions include necrosis, hyperplasia, inflammation, epithelial lifting, cell
swelling, and hypersecretion of mucus (Figure 2). A typical chronology of damage from
acute exposure to the test chemical is first a lifting of the outer layer of the lamellar
epithelium (desquamation), usually starting in the area of the chloride cells. Edematous
spaces are formed between the layers of epithelium (Figure 2) and these may become
infiltrated with leukocytes. Eventually the whole epithelium sloughs off and the lamellum
loses rigidity. On the blood side of the lamellum the central spaces collapse, but the marginal
channel often remains normal until the rest of the lamellum is essentially destroyed. Acute
exposure to some chemicals can cause rapid destruction of the gill lamellae within a few
hours. Death then may follow as a probable result of blood hypoxia (see below). However,
exposure with some substances such as ammonia causes relatively little gill damage unless
concentrations are quite high, but death still takes place, obviously from other causes
(Smart, 1978). Thus, gill histopathology may or may not indicate “cause of death”, depend­
ing on the extent of the lesions encountered.
Some authors have reported clubbing of the ends of the lamellae and a tendency of
adjacent lamellae to stick together. Increased discharge of mucus has been observed by
some but not by others. Examination with scanning electron microscopy of lamellae from
trout exposed to acid revealed loss of microridge patterns on the epithelium (Jagoe and
Haines, 1983). All of these changes will reduce diffusion of oxygen (and possibly carbon
dioxide) so blood gases will be affected (discussed below).
 51

It would be nice to find there are certain histological changes diagnostic for particular
toxicants, and this has been attempted (Abel and Skidmore, 1975; Wobeser, 1975).
Mallatt (1985) has done an extensive quantitative and qualitative review of 133 published
studies of fish gill pathologies involving over two dozen chemicals. He concludes that the
structural alterations are a stereotyped physiological reaction to environmental stressors.
Indeed, it may be that nearly any pollutant can cause gill damage if in sufficient
concentration in the water. Furthermore, there seem to be far more similarities than
differences in the gill pathologies from acute exposures, and even non-chemical stressors,
such as hypoxia and temperature change, can produce some of the same tissue changes.
More recently, Kirk and Lewis (1993) have used scanning electron microscopy of gills
from fish exposed to three different pollutants and claim some distinct differences. They
tested copper, phenol and ammonia at two different concentrations and time intervals of
either 2 or 24 h. All three chemicals caused disorganization of lamellae and proliferation
of mucus cells. High concentrations of copper (1.0 mg/L) caused lamellar fusion and
swelling in the distal tips of filaments. Lamellar epithelium also exhibited hypertrophy.
Phenol at 10 mg/L stripped the epithelium away from lamellae and ammonia caused the
formation of distinctive circular depression in the epithelium. All these changes were
after rather high doses; effects of chronic exposures may be quite different.
The histological lesions induced by chronic exposures to chemicals are much less
known than those from acute exposures and may be more pollutant specific, especially
if they are studied at the level of transmission or scanning electron microscopy; and at the
level of light microscopy, there is a need for more quantification of the changes seen.
Sublethal exposure to various chemicals can cause swelling of the epithelium which
increases the diffusion distance for oxygen transfer. Hughes and Perry (1976) proposed
a light-microscopic method for quantifying this and Hughes et al. (1979) subsequently
applied it with limited success to a study of rainbow trout exposed to nickel, chromium,
and cadmium. Unfortunately, the method is tedious and quite labor intensive. Newer
computerized image analysis equipment greatly improves this procedure, however, it has
rarely been utilized.
Niki and Farrell (1993) demonstrated well how gill morphometric data can be used to
understand the relationship between histopathological changes and physiological capac­
ity. Juvenile salmonids were exposed to a wood preservative at several concentrations for
96 h after which their maximum aerobic swimming speed was measured in a water tunnel.
Figure 3 summarizes the data obtained showing a fairly good association between
interlamellar distance or the blood-water diffusion distance and the percentage decrease
in swim speed.
It should be pointed out that there are at least two potential problems that may be
encountered in studies of histopathology in gill tissue of freshwater fish following
chronic exposure to a pollutant. One is the susceptibility of the fish to bacterial gill
disease. This disease is frequently brought on by environmental stress and is character­
ized by proliferation of the gill epithelium and clubbing and fusing of the lamellae
(Snieszko, 1980). These very same things are frequently observed in work on toxicants
and could be incorrectly interpreted as a direct effect of some test chemical. A second
source of error has been noted by Mitchell and Cech (1983) who found that exposure
to ammonia alone did not cause histopathology of the gills of channel catfish, but when
this was combined with a low level of monochloramine (a form of residual chlorine)
in the water, acute tissue hyperplasia occurred. The levels of monochloramine used
(which alone did not cause gill damage) were the same as those often present in
charcoal-filtered domestic tap water, a technique widely used in laboratories for re­
moval of chlorine in flowing water systems for experimental aquaria. This same
synergistic effect might take place in tests with other pollutants in laboratories where
charcoal filtering of chlorinated tap water is practiced.
52

% Decrease in ILD % Increase in BWDD

Figure 3 Comparison of percentage reductions in interlamellar distance (ILD) and percentage

increases in water blood-water diffusion distance (BWDD) associated with a given reduction in
Chinook salmon swimming speed after exposure to TCMTB, a wood preservative. Measured
histological changes relating to a 20% reduction in critical swimming speed are indicated by the
dashed line. (From Niki, D. and Farrell, A., Aquat. Toxicol., 27, 245, 1993. With permission.).

IV. VENTILATION CHANGES IN RESPONSE TO POLLUTANTS

Ventilation refers to the movement of water over the gills. It is synonymous with
breathing; the use of the term “respiration” for this process should be avoided since it is
a general term that means different things to different people (e.g., many equate it with
rate of oxygen consumption).
As discussed earlier, the fish respiratory system moves water over the gills by the
coordinated expansion and contraction of the buccal and opercular cavities. In order to
measure this, one would ideally determine the rate of water flow through the operculae,
but this is technically difficult or impossible to do with most species; so other more
indirect means are usually used which measure some aspect of the ventilatory movements
(Heath, 1972). The simplest method is to merely count visually the opercular beat,
providing one can see the fish in the experimental apparatus. However, the “subject” often
will react negatively to the presence of an observer and visual counting is quite labor
intensive. Because movement of the water is accomplished by pressure changes in the
opercular and buccal cavities, the process of ventilation can be studied by detecting these
pressure changes with suitable transducers and displaying them on a strip-chart recorder.
This requires cannulation of at least one of the cavities with a thin catheter tube and
subsequent partial restraint of the fish so it will not get the tube(s) tangled (Sellers et al.,
1975). The advantage of this method is that it gives an indication of relative changes in
stroke volume (depth of breathing) as well as breathing frequency. The actual ventilation
 53

volume is the product of these two measures and some fish species, such as the rainbow
trout, may change stroke volume more than frequency when subjected to some stress
(Shelton et al., 1986). Because the fish must be partially restrained, however, it does
impose an additional factor that may concern some workers, and for long-term experi­
ments (i.e., more than a week), erosion of the tissue around the catheter can be trouble­
some.
In studies on the effect of pollutants on ventilation, a method that has been used
extensively, especially for biomonitoring of water quality, is one referred to as “dual
external electrodes” (Heath, 1972). For this purpose electrodes are placed at both ends of
a small aquarium containing a single fish. The breathing movements of the fish cause a
cyclic potential change between the electrodes which is amplified and displayed on a
suitable oscillographic recorder. Carlson (1982) has analyzed this method in considerable
detail and coined the term “electrobranchiogram” (EBG) to refer to the oscillographic
record obtained (Figure 4). Both the frequency of ventilation and that of coughing can be
detected in this manner, and occasionally, the electrocardiogram is superimposed on the
record. Even the breathing of fry can be detected with this technique (Thomas and Rice,
1979). A further advantage of this method is that it lends itself to automation whereby a
computer can be used to count the ventilation frequency (but generally not the coughs).
Such an application has considerable potential for early warning systems in biological
monitoring (Cairns and van der Schalie, 1980; Gruber et al., 1991).
Figure 4 shows the record of a cough, a common occurrence in fish exposed to a wide
variety of pollutants (Table 2). Hughes (1975) has studied coughing in rainbow trout in
some detail including electromyographic analyses of the muscles involved. A cough
occurs at a random position during a respiratory cycle and is characterized as a rapid
expansion and contraction of the buccal and opercular cavities. These movements pro­
duce a two- to fivefold increase in the reversal phase of the differential pressure across

Figure 4 Simultaneous recordings of bioelectric potentials and buccal water pressure changes
associated with ventilatory movements in the bluegill. (a) Fish in non-contaminated water and
(b) fish after 1 h exposure to 10 mg/L waterborne zinc. Note frequency of breathing and coughing
was increased in the exposed fish. (From Carlson, R. W., Environ. Pollut Ser. A, 29, 35, 1982.
With permission.)
54

Table 2 Chemicals Reported to Cause Changes in Ventilation and/or Coughing

Chemical Cough or ventilation Species Ref.
Acrolein C Bluegill Carlson (1990)
Aluminum c,v Rainbow trout Make and Weber (1988)
Ammonia V Rainbow trout Smart (1978)
Anthracene c Bluegill McCloskey and Oris (1991)
Benzaldehyde c,v Bluegill Carlson (1990)
Cadmium c,v Bluegill Bishop and McIntosh (1981)
Carbaryl V Catfish Arunachlam et al. (1980)
Clay V Sunfish Horkel and Pearson (1976)
Chlorine c,v Rainbow trout Bass and Heath (1977)
Chlorothalonil V Rainbow trout Davies (1987)
Coal dust c Rainbow trout Hughes (1975)
Copper c Brook trout Drummond et al. (1973)
Chromium c,v Rainbow trout van der Putte et al. (1982)
Crude oil c,v Atlantic salmon Barnett and Toews (1978)
Cyanide V Rainbow trout Sawyer and Heath (1988)
Cypermethrin c Rainbow trout Bradbury et al. (1991)
DDT c Rainbow trout Lunn et al. (1976)
Endosulfan c Rainbow trout Bradbury et al. (1991)
Fenitrothion c,v Rainbow trout Klaverkamp (1982)
Fenvalerate c Rainbow trout Bradbury et al. (1991)
Oil + dispersant c,v Atlantic salmon Barnett and Toews (1978)
Mercury c Brook trout Drummond et al. (1974)
Methyl parathion V Tilapia Bashamohideen et al. (1987)
Naphthalene V Pink salmon fry Thomas and Rice (1979)
Paraquat V Catfish fry Tortorelli et al. (1990)
Pulpmill effluent c,v Sockeye salmon Davis (1973)
Toluene V Pink salmon Thomas and Rice (1979)
Surfactants c,v Bluegill Maki (1979)
Wood pulp c Rainbow trout Hughes(1975)
Zinc c,v Rainbow trout Hugh and Tort (1985)

the gills. This phase normally occupies only about 10% of the normal cycle and is of low
amplitude. Thus, the cough produces a quick reversal of water flow that presumably aids
in freeing the gills of suspended matter. Backward coughs can also be observed whereby
the water exits via the opercular openings (Hughes and Adney, 1977a). Some fish show
a low frequency of coughing, even in presumably clean water. The interval between
individual coughs can be quite regular and when the frequency increases from chemical
irritation, this regularity is maintained (Bass and Heath, 1977). In rainbow trout, there
appears to be a maximum cough frequency of around 15-20 per minute, no matter what
the extent of stimulus (Bass and Heath, 1977).
Table 2 lists several of the substances that have been observed to affect ventilation
frequency, coughing, or both. The table does not include all of the reports for a given
chemical; in most cases the most recent study was cited. In addition, while acid effects
on ventilation have been examined by a number of workers, the results are very dependent
on the level of carbon dioxide in the water (see Chapter 10). As with histological damage
to the sensitive gill tissues, nearly any pollutant can probably cause changes in ventilation
if the test concentration is high enough. It is of more interest to deal with incipient lethal
and sublethal concentrations as these will probably have more relevance to what may be
encountered in nature. In most cases, those studies cited in Table 2 included a dose that
was sublethal.
 55

There are several aspects of the typical ventilatory response to a pollutant: (1) the
percent change in coughing frequency, if present, is usually greater than the percent
change in ventilation frequency, although some chemicals such as cyanide or ammonia
do not cause coughing, even at lethal levels; (2) at low concentrations, an initial irritation
response may be followed by a sort of acclimation to the toxicant; (3) conversely, if the
chemical affects the nervous system, but only after accumulation, the frequency changes
may not take place until some level of accumulation has taken place; and (4) the change
in frequency of ventilation or coughing is usually an increase, rather than a decrease from
control rates, although when concentrations are sublethal, there have been several ex­
amples of decreases in ventilation seen (e.g., Bashamohideen et al., 1987; Tortorelli et al.,
1990). Also, the character of the response may depend to some extent on the fish species.
While most people have observed hyperventilation in the presence of cadmium, Moffitt
(1990) reported a decreasing ventilation frequency in brown bullhead catfish {Ictalurus
nebulosus) experiencing cadmium stress. Because this species may show a slowing of
ventilation under environmental hypoxia (Marvin and Heath, 1968), the response to
cadmium may not be unexpected.
The ventilatory change may be proportional to the dose even at very low levels. Bishop
and McIntosh (1981) report a good correlation between the threshold for an increase in
cough frequency in water containing cadmium and the maximum acceptable toxicant
concentration (MATC) obtained by growth and reproduction studies. A similar type of
correlation with the MATC was seen with coughing of rainbow trout in water containing
aluminum (Ogilvie and Stechey, 1983) and ventilation frequency of bluegill immersed in
surfactant solutions (Maki, 1979). Thus, these responses could be a useful method for
estimating water quality criteria, providing it is recognized by the investigators that not
all substances cause changes in ventilation. For example, in tests on DDT, dieldrin, and
methyl carbamate with rainbow trout, only DDT gave useful results with the ventilatory
response. The other two pesticides produced no response until clearly lethal concentra­
tions were used (Lunn et al., 1976). More recently, Carlson (1990) found that malathion
caused no ventilatory response in bluegill whereas carbaryl, another acetylcholinesterase
inhibitor like malathion, caused hyperventilation but only at fairly high concentrations.
It should also be realized that the initial response to some chemicals may be lost after
about a day of continuous exposure so measurements taken at only one time interval of
exposure could be misleading.

V, PHYSIOLOGICAL MECHANISMS OF CHANGES IN VENTILATION

There is more than one potential physiological cause for the ventilatory responses
observed in fish emersed in various chemicals:
1. Simple irritation of the sensitive epithelial tissues of the gills and buccal cavity can
cause coughing and hyperventilation. The latter could be explained as a sort of psycho­
logical alarm response (Schreck, 1981). Under more prolonged exposure, the alarm
component, and maybe even the irritation effects may be lost, so the frequency of the
two ventilatory factors may return toward the control level.
2. Changes in demand for metabolic energy by the fish will be reflected in increased or
decreased ventilation. If this is the sole cause of an observed response, then coughing
will not necessarily occur. Of course, irritation from the chemical can cause more
spontaneous activity (see Chapter 8) which would elevate the demand for energy, but,
in general, changes in ventilation should not be interpreted as being due to altered
metabolic demand without supporting data on oxygen consumption. This is because
reason number three (below) may be a more important cause of the observed changes.
56

3. Impaired gas exchange in the lamellae will cause hyperventilation because this is
interpreted by the arterial oxygen receptors in the first gill arch (Burleson and Milsom,
1993) as a hypoxic condition. The histopathology seen in gills that have been exposed
to a pollutant strongly suggests this is a common mechanism by which hyperventilation
is induced because most of these histological changes increase the diffusion distance for
oxygen from the water to the blood. A reduction in the flux of oxygen from the water
into the blood from fish with damaged gills can be confirmed by taking serial samples
for P02 measurement of arterial blood from trout with catheters in their dorsal aortas.
Skidmore (1970) was the first to do this using an extremely high concentration of zinc
(40 ppm) and found the oxygen tension of the blood dropped to less than 20 mmHg at
death. Using lower concentrations of zinc, which approximated that of the 48-h LC50,
Sellers et al. (1975) and Spry and Wood (1984) also observed a very low arterial blood
P02 following 12-24 h of exposure. Under these acute exposures, accumulation of zinc
in the blood is negligible (Spry and Wood, 1984) but lactic acid levels rise, which
indicates anaerobic glycolysis. Anaerobic metabolism is to be expected if there is an
impairment of oxygen flux from the water into the blood, whether this is produced by
environmental hypoxia (Chapter 2) or a diffusion barrier at the gill surface.
In fish with gills damaged by zinc, accumulation of lactic acid and carbon dioxide in
the blood produced a combined metabolic and respiratory acidosis (Spry and Wood,
1984). Even though blood pH decreased in the trout exposed to zinc, the extent of this
change was no greater than that observed in trout that swam to exhaustion (Turner et al.,
1983), a condition from which they readily recover. Thus, acidosis is not the cause of
death. All these findings are consistent with the hypothesis originally formulated long ago
by Carpenter (1927) that hypoxemia (low blood oxygen) is the cause of death from acute
exposure to zinc. A similar conclusion is appropriate for high doses of free chlorine (Bass
and Heath, 1977), but not for monochloramine (combined chlorine) at the same concen­
tration (Travis and Heath, 1981). Based on numerous findings of gill histopathology
(discussed previously), this hypoxemia is undoubtedly a major contributing factor to
mortality from acute concentrations of a large number of substances, but other physi­
ological “failures”, such as in blood electrolyte homeostasis or in nervous system func­
tion, may be of equal or greater importance in the etiology of death, especially at lower,
but still lethal environmental levels of some pollutant (e.g., Wilson and Taylor, 1993).
Some idea of how fast the oxygen tension in the blood can drop during exposure to
a pollutant, and then recover following it, was determined by Bass and Heath (1977).
Rainbow trout were subjected to “pulses” of chlorine which rose to a peak concentration
in 30 min and then declined to 0 in about 45 min after the peak. These were repeated at
8-h intervals. (This simulates the mode of effluent release from many steam-powered
electric generating plants.) At the peak of the chlorine pulse, arterial P02 had dropped to
40 mmHg from a control level of 102 mmHg, but then just before the second pulse, P02
had nearly recovered to that of the controls. The extent of recovery of blood oxygen
between chlorine pulses, however, became less and less with each subsequent pulse until
death occurred at an arterial P02 below 20 mmHg.
Recovery of gill function following a chemical “insult” has been studied little. It can
require days (Skidmore and Tovell, 1972) or weeks (Hughes et al., 1979; Scheier and
Cairns, 1966) for histological morphology to completely return to normal when the fish
is removed from contaminated water. The findings mentioned above on the effects of
chlorine suggest that if the exposure is of short enough duration, at least a partial
restoration of capacity to diffuse oxygen can be achieved within a matter of hours. In
order to determine if the same thing would hold true for a very different type of chemical,
in a preliminary study in my laboratory, rainbow trout with catheters in their dorsal aortas
 57

were exposed to 2 ppm of zinc for 11 h at which time the arterial P02 averaged 40 mmHg.
The changes in arterial P02 during recovery were then followed. These fish exhibited
almost complete recovery of arterial oxygen within 10 h after this rather acute exposure
to a toxicant. This implies that there was no actual sloughing off of the respiratory
epithelia in the gills with this short-term exposure, but rather a swelling of the epithelial
layer and/or an accumulation and subsequent loss of mucus (Ultsch and Gros, 1979),
which increased the diffusion distance for oxygen. Using a 10-fold higher zinc concen­
tration (Hughes and Tort, 1985) reported that following exposures of 120 min to the
dissolved metal it required several days for ventilation and coughing to return to the
control level. Such findings as these may have relevance to industrial spills where the
situation is corrected quickly.
Lower concentrations of zinc (equivalent to the 96-h LC50) produce only a slight
reduction in arterial P02 after 3 days of exposure but a significant accumulation of zinc
in the blood. Spry and Wood (1984) attribute death at this concentration to some internal
actions of zinc, independent of the damage to the respiratory system. They also noted a
slight blood alkalosis at the lower level of zinc exposure (recall that acute exposure
caused acidosis). It might be assumed that hyperventilation produced this alkalosis by
“blowing o ff’ blood CO2. However, fish are not like terrestrial vertebrates in that they are
unable to regulate blood pH and carbon dioxide to much of an extent by respiratory means
(Cameron, 1979). Spry and Wood (1984) propose several alternative reasons for the
alkalosis including accumulation of ammonia, alterations in the chloride-bicarbonate, and
sodium-hydrogen electroneutral exchanges at the gill surface and/or an inhibition of
carbonic anhydrase activity in gill tissue. The work of Spry and Wood (1984) on fish
exposed to two different lethal concentrations of a toxicant points up what may be an
important generalization; the mechanisms of toxic death can be very different depending
on the concentration of the chemical. Very high doses of zinc, acid (Milligan and Wood,
1982), and probably many other chemicals, cause death by hypoxemia due to impaired
oxygen uptake by the gills, whereas somewhat lower doses produce mortality more
slowly by the chemical getting inside and causing alterations in other physiological
machinery directly.
Of course, a combination of hypoxemia and other dysfunctions, such as ionoregulatory,
may cause death as has been recently demonstrated for acute copper exposure. Wilson
and Taylor (1993) exposed trout to a high dose of copper and found declines in plasma
electrolytes and arterial P02. These results are similar to what occurs with acute acid
exposure where death results from a secondary hemoconcentration, rapid elevation in
arterial blood pressure, and probable heart failure (see Chapter 10).
One of the significant questions in work on pollution is to what extent does a long-term
exposure to some chemical affect the ability of the organism to handle a subsequent
environmental stressor of a different sort, such as low dissolved oxygen. Majewski and
Giles (1981) investigated this in trout which had been exposed to 6.4 mg/L cadmium for
30 days. After the exposure, the fish were fitted with dorsal aortic catheters and allowed
to recover in non-contaminated water for 3 days. Then, they were subjected to a gradually
lowered environmental oxygen tension while the arterial P02 was monitored. The fish that
had received the cadmium treatment exhibited a slightly lower arterial oxygen tension
(6-10 mmHg) than controls at most of the environmental levels of oxygen. This
difference is slight, but statistically significant, indicating that even an exposure at 40%
of the incipient lethal level produced a measurable reduction in oxygen diffusion
capacity across the gills. It seems doubtful that this amount of respiratory impairment
would affect the fish’s ability to function in the environment at rest, but it certainly
could have an inhibitory effect on a fish swimming actively in hypoxic waters.
58

VI. CIRCULATORY PHYSIOLOGY

The function of the circulatory system is intimately related to the process of respiratory
gas exchange and the control mechanisms for this system are largely based on the
detection of changes in blood oxygen. Thus, any environmental factor that alters the
process of oxygen uptake can be expected to affect circulation. Of course, this is not to
exclude the possibility of some chemical acting directly on the heart and/or blood vessels.
Further, adrenergic excitation, as a stereotyped response to stress (Mazeaud and Mazeaud,
1981) , will have marked cardiovascular results.
The teleost heart has four chambers arranged in series. The sequence by which blood
passes through the heart is sinus venosus, atrium, ventricle, and bulbus arteriosus. The
ventricle provides the arterial blood pressure while the bulbus dampens the rise and fall
of blood pressure caused by the individual heartbeats and thereby probably protects the
delicate gill capillaries from damage. It also helps maintain a constant blood flow
throughout a large portion of the cardiac cycle which probably aids in the uptake of
oxygen by the gills. The pacemaker tissue in the typical teleost heart is diffuse rather than
being nodal as in mammals. Also, because most fish are poikilothermic, the intrinsic beat
is temperature dependent. The intrinsic beat is altered to meet varying demands by
changes in autonomic neural activity. All fish hearts, except hagfishes, have extensive
vagal (parasympathetic cholinergic) innervation and most teleost hearts apparently have
sympathetic (adrenergic) innervation. Many species of fish appear to have hearts that are
under the influence of considerable parasympathetic tone which slows the intrinsic beat.
Thus, alterations in rate of heartbeat could, theoretically, be achieved largely by changes
in the level of this activity alone.
The ventral aorta, which is an extension of the bulbus, has approximately twice the
blood pressure of the dorsal aorta which is on the efferent side of the gills. This pressure
drop across the gills is variable depending on autonomic nervous activity and the action
of hormones such as catecholamines and acetylcholine. Working out the pathways for
blood flow through the gill filaments and lamellae has proven to be difficult in part
because the gills possess a highly variable network of vessels (Laurent, 1984). A major
problem is in understanding how the number of lamellae perfused with blood is increased
or decreased in response to the rise and fall of internal demand for oxygen or changes in
availability of environmental oxygen.
Blood flows from the ventral aorta to the afferent filament arteries, through the
lamellae to the efferent arteries, and then to the dorsal aorta. Blood can, however,
effectively by-pass a lamellum by going through its basal channel rather than through the
lamellum proper. At rest, only 60% of the lamellae are perfused and these are the ones
located more proximally on the filaments (Booth, 1978). Catecholamines (adrenaline and
noradrenaline) increase the number of lamellae perfused and cause a reduction in vascular
resistance to blood flow through the gills (Booth, 1979). Because these changes increase
the permeability of the gills to water, it is considered likely that they also produce an
enhancement in oxygen permeability. Acetylcholine, as would be released by parasym­
pathetic activity, has the opposite effect of the catecholamines. The respiratory surface
area can also be changed by non-neurohumoral mechanisms as well, because an increase
in blood pressure, as occurs during exercise, causes recruitment of more lamellae (Randall,
1982) . Further details on the fish circulatory system can be found in reviews by Klaverkamp
(1982), Butler and Metcalf (1983), Satchell (1991), Hoar et al. (1992), and Farrell (1993).
This brief overview of the circulatory physiology of the fish emphasizes the close
relationship between the processes of circulation and respiration. It also shows how there
are points where pollutants could have a considerable effect on gill function via alter­
ations in circulation through the gill but this is an area of research that has received little
attention, perhaps in part because the techniques are difficult to master.
 59

An example of the sort of study that can be revealing is that of Bolis and Rankin (1980)
in which they found that the detergent linear alkylate sulfonate (LAS) caused a vasodi­
lation of gill vasculature that was apparently mediated through the beta adrenergic
receptors. The detergent also, however, inhibited the normal vasodilatory action of
catecholamines in the gills of eels (Anguilla anguilla) and brown trout (Salmo trutta). It
is possible that such an effect could ieke place with other chemicals as well and would
reduce the ability of the gill vasculature to respond to changes in oxygen demand or
supply.

VII. CARDIAC RESPONSES TO POLLUTANTS

There has been some interest in the effect of pollutants on heart rate in fish. By implanting
thin electrode wires in the ventral side of the body and allowing the fish to remain awake
in a chamber which prevents extensive swimming movements, reasonably “normal”
electrocardiographic records can be obtained. From these, the heart rate is counted. With
suitable electrodes or pressure transducers, the ventilatory activity can be simultaneously
recorded along with the ECG (Bass and Heath, 1977; Hughes and Adney, 1977b;
Bradbury et al., 1991). When fish are exposed to a variety of different chemicals in acute
concentrations, a slowing (bradycardia) of the heart usually occurs simultaneously with
an increase in ventilation. These chemicals include residual chlorine (Bass and Heath,
1977), cyanide (Figure 5A), DDT and dieldrin (Lunn et al., 1976), organophosphorus
insecticides (Klaverkamp, 1982), the herbicide paraquat (Tortorelli et al., 1990), oil spill
dispersant (Kiceniuk et al., 1978), and zinc (Hughes and Adney, 1977a). Dose depen­
dency was shown with paraquat and catfish fry heart rate (Tortorelli et al., 1990) and
cyanide (Sawyer and Heath, 1988) (Figure 5A); it is not known if a similar dependency
would be found with other chemicals. Most of the chemicals that induce a bradycardia
also produce acute internal hypoxia. In a sense, cyanide is an exception in that blood
oxygen probably remains unchanged, but the effect on the animal is similar to hypoxia
and the receptors that bring about the hypoxic reflexes are affected similarly by cyanide
(Burleson and Smatresk, 1990). Where internal hypoxia occurs as a result of the pollutant
exposure, the bradycardia seen may be a result of vagus nerve inhibition of the heart, as
occurs with exposure to environmental hypoxia (see Chapter 2). This neurogenic origin
of the bradycardia in response to a chemical in the water was confirmed in the study on
cyanide (Sawyer and Heath, 1988). There it was shown that the initial cardiac slowing
could be blocked by injecting atropine (an acetylcholine antagonist) through a catheter
into the pericardium during cyanide exposure. At higher doses of cyanide, reductions in
heart rate occurred apparently due to a direct effect of cyanide on the heart. At least some
pollutants can have a direct effect on heart contractility. This has been shown for both
cadmium and zinc, metals that block calcium channels in excitable tissues such as heart
muscle (Tort and Madsen, 1991).
The neurotoxin from the organism that causes red tide (Chattonella marina) causes
fish death apparently by inducing a severe bradycardia which results in anoxia because
of reduced gill blood circulation. Endo et al. (1992) further showed that the bradycardia
is due to vagal stimulation because it was blocked by atropine injection. In vitro studies
showed that fractions of the neurotoxin depolarized the vagus nerve directly rather than
acting as an acetylcholine agonist.
Note that the data displayed in Figure 5B on brown bullhead catfish (7. nebulosus) is
considerably different than that seen with rainbow trout shown in Figure 5A. Instead of
a bradycardia, the catfish responded to cyanide with an increased heart rate. The tachycardia
seen in the catfish may have been due to a gustatory stimulus. When an extract of fish food
was added to the inflowing water it caused tachycardia in the catfish but not the trout.
60

Figure 5 Changes in ventilation and heart rates of rainbow trout (A) and brown bullhead catfish
(B) exposed to a linearly Increasing cyanide concentration for 3.5 h and then held at a constant
level throughout the remainder of the experiment. Points are the means (±1 SE) of four fish.
Cyanide concentration is indicated by the solid line without points. Note that the concentration
used with the catfish is an order of magnitude higher than that In the trout experiments. (From
Sawyer, P. L. and Heath, A. G., Fish Physiol. Biochem., 4, 203, 1988. With permission.)

Catfish have acute gustatory sensitivity (Atema, 1971) and because cyanide has a bitter
taste and odor, it may have produced an adrenergic stress response in this species. These
data also illustrate how different species may respond to the same environmental stimulus
in very different ways.
Very high concentrations of pollutants can produce tachycardia in trout, at least
initially. This has been shown for ammonia (Smart, 1978) where trout exposed to very
high doses (death occurred between 1 and 3 h) experienced elevated heart rates even
though blood oxygen was reduced to less than half that of controls. The fish struggled
considerably and this caused an elevated rate of oxygen consumption, which was mea­
sured. Arillo et al. (1981) found elevated renin activity in plasma of trout which had been
 61

exposed to sublethal levels of ammonia. This enzyme catalyzes the formation of angio­
tensin, a powerful vasoconstrictor. They speculated that the elevated blood pressures that
Smart observed upon ammonia exposure were due to this renin-angiotensin mechanism,
although a larger part of this response was probably due to simple struggling of the fish.
One might also speculate that angiotensin could cause vasoconstriction in the gills and
thereby reduce respiratory gas exchange. This hypothesis gains some support from the
observation that acute levels of ammonia apparently cause a severe lowering of dorsal
aortic P02 in the absence of gill damage (Smart, 1978).
Increased heart rate and blood pressure can certainly result from stress-induced
catecholamine release from autonomic nerves and/or interrenal cells. This was nicely
shown by Milligan and Wood (1982) in trout exposed for several days to water of pH 4-
4.5, but the cardiovascular effects caused by acid were also found to not be exclusively
a result of catecholamines as adrenergic blocking agents only partially blocked them.
During acid exposure, whole blood viscosity increased greatly due to a loss of plasma
water and a consequent rise in plasma protein concentration and hematocrit. This cascade
of effects was originally triggered by a loss of blood electrolytes through the gills (see
Chapter 10). According to Milligan and Wood (1982), death of adult trout from acid water
(pH 4-6) may be due to a combination of increasing blood pressure and decreasing
plasma volume, which ultimately leads to circulatory failure. (The cause of death at a pH
below 4 may be hypoxemia because of severe gill damage.)
Recently, Wilson and Taylor (1993) have reported that trout exposed to a dose of
waterborne copper sufficient to cause death in 24 h experienced physiological distur­
bances quite similar to those seen with acute acid exposure. This included an immediate
tachycardia (78% increase) that was seemingly due to loss of vagal tone rather than
catecholamines, because there was no initial change in blood pressure. With continued
exposure, the blood pressure rose due to hemoconcentration and probable catecholamine
vasoconstriction. Ultimately, death resulted from circulatory failure brought on primarily
from inoregulatory collapse.
In an investigation of a more chronic type of toxicant exposure on circulation,
Majewski and Giles (1981) found that trout in water with a cadmium dose 40% of the
incipient lethal level exhibited a 25% increase in heart rate after 3 days exposure. This
persisted for several months of continuous cadmium exposure. Because ventilation was
also elevated throughout this time, the rate of oxygen consumption was probably in­
creased, although this was not measured. The increased heart rate would be explained by
this mechanism.
The generality can probably be made that chemical-dose combinations that cause
internal hypoxia (e.g., acute doses of zinc) will result in bradycardia, while those com­
binations that cause elevations in oxygen consumption (see Chapter 8) and/or catechola­
mines will result in tachycardia. Small transient changes in heart rate mean little because
fish respond to mild disturbances such as noises usually with a short-term bradycardia
followed by a tachycardia (personal observations). In the future, slow changes in concen­
tration of a test pollutant may be more revealing of action than abrupt changes, and would
probably have greater environmental realism.
For some time there has been interest in the benefit, if any, of the well-known
bradycardia response of a fish to environmental hypoxia (Chapter 2). It would appear that
such a response would be clearly beneficial to a fish experiencing internal hypoxia that
had been produced by a reduction in the transfer of oxygen in the gills. Blood normally
spends about 1 second in the lamellum in which time it must become oxygenated (Hughes
et al., 1981). If a gill was suffering from a reduced capacity for oxygen transfer, the time
needed for oxygenation might be prolonged (Figure 6), thus by slowing the blood flow
rate through the gill, better oxygenation of the blood could occur.
62

Figure 6 Hypothetical schematic of the effect pollutant damage to a fish gill has on the P0 2 of
blood as it passes through a lamellum. In the normal gill, the 1-s interval in which the blood Is
in the lamellum permits the blood to essentially reach equilibrium with the water by the time it
reaches the dorsal aorta. In the damaged gill, diffusion is slowed so equilibrium Is not reached
before the blood enters the dorsal aorta. (Values for the normal gill are from Holeton, G. F. and
Randall, D. J., J. Exp. Biol., 46, 317,1967.) A decrease in venous P0 2 is assumed based on an
Increased rate of oxygen consumption in fish exposed to substances that cause gill damage (see
Chapter 8).

REFERENCES

Abel, P. D. and Skidmore, F., Toxic effects of an anionic detergent on the gills of rainbow trout. Water
Res., 9, 759, 1975.
Arillo, A., Uva, B. and Vallarino, M., Renin activity in rainbow trout (Salmo gairdneri) and effects of
environmental ammonia. Comp. Biochem. Physiol, 68A, 307, 1981.
Arunachlam, S. et aL, Toxic and sublethal effects of carbaryl on a freshwater fish, Mystus vattatus, Arch.
Environ. Contam. Toxicol, 9, 307, 1980.
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 63

Booth, J. H., The distribution of blood flow in gills of fish: application of a new technique to rainbow trout
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Majewski, H. S. and Giles, M. A., Cardiovascular-respiratory responses of rainbow trout {Salmo
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Mazeaud, M. M. and Mazeaud, F., Adrenergic responses to stress in fish, in. Stress and Fish, Pickering,
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 65

McKim, J. M. and Goeden, H. M., A direct measure of the uptake efficiency of a xenobiotic chemical
across the gills of brook trout (Salvelinus fontinalis) under normoxic and hypoxic conditions, Comp.
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function associated with low environmental pH in the rainbow trout, (Salmo gairdneri), J. Exp. Biol,
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66

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 Chapter
4
Hematology
L INTRODUCTION
Hematology is defined as the study of blood and the blood-forming tissues. Blood is
composed of the liquid plasma and the blood cells (sometimes called formed elements).
Serum is the fluid left after blood has clotted; it has essentially the same chemical makeup
as plasma, except for the absence of some of the clotting factors. While many workers
include the chemistry of plasma as a part of hematology (Hawkins and Mawdesley-
Thomas, 1972; Folmar, 1993), they will be kept separate here, because the composition
of plasma is a reflection of the function of numerous organs (e.g., gills, liver, kidney,
muscle, etc.) that are considered in other parts of this book. Thus, this chapter will be
limited to a review of the “toxicophysiology” of blood cells only.

II. FISH BLOOD CELLS AND THEIR MEASUREMENT

The blood cells of teleost fish are produced in the hematopoietic tissue which is located
primarily in the spleen and kidney (Satchell, 1991; Fange, 1992). In contrast to mammals,
there is no bone marrow in fish, nor do they have lymph nodes. Within the blood cell­
forming tissues, the hemocytoblast is the cell that gives rise to all the others, both
erythrocytes and leukocytes. The latter are divided into the lymphocytes, granulocytes (of
which the neutrophils and eosinophils are the most common), and thrombocytes. Lym­
phocytes are the most numerous of all the leukocytes. Fish do not have platelets as in
mammals, instead the thrombocytes contain the clotting factors that are normally found
in platelets.
An overall reduction in leukocytes (leukocytopenia) in response to stress is character­
istic of all sorts of vertebrates. It is a non-specific response to any type of inner or
environmental stressor and is mediated by the corticosteroid hormones. It is a fundamen­
tal mechanism in the increased susceptibility of fish to disease organisms when they are
suffering from the stress of pollution (see Chapter 11).
The erythrocytes (red blood cells) of fish are nucleated and similar in size to the
leukocytes, in contrast to mammals in which the latter are the much larger cell. Erythro­
cytes contain hemoglobin, which enables the blood to hold a far greater amount of oxygen
than it otherwise could (50- to 100-fold, depending on the hemoglobin concentration).

67
68

The term anemia is applied to any condition where the concentration of the hemoglobin
in the blood is abnormally low, whether this is due to a reduction in number of erythro­
cytes or to an inadequate amount of hemoglobin in the cells.
For the measurement of hematological factors in fish, techniques similar to those used
in human and veterinary clinical laboratories are generally used, with minor modifica­
tions. Blaxhall and Daisley (1973), Wedemeyer and Yasutake (1977), Ellis (1977), and
especially Houston (1990) provide practical guides to these methods adapted for use on
fish blood. The following are the principle factors measured in work on the action of
pollutants.

A. HEMATOCRIT
Hematrocrit is sometimes called the “packed cell volume” and is determined by spinning
a sample of whole blood (usually contained in a sealed capillary tube) in a centrifuge and
the relative volume of the packed cells is then measured. Hypoxia (in vitro) causes the
cells to swell, thereby increasing the hematocrit. Because erythrocytes of fish have a high
intrinsic rate of oxygen consumption (Eddy, 1977), delays in spinning down the hema­
tocrit tubes can yield excessively high values due to cell swelling induced by their
utilization of the available oxygen in the microhematocrit tube.

B. BLOOD CELL COUNT

Fish erythrocytes are easier to count than mammalian ones because they are larger and
fewer in number. Both these and the leukocytes can be counted in the same hemocytometers
used for human blood or with the aid of automatic electronic counters found in many
clinical laboratories. (The latter may not distinguish leukocytes because in fish they are
approximately the same size as the erythrocytes.)

C. HEMOGLOBIN CONCENTRATION
For this purpose, whole blood is analyzed after the cells have been hemolyzed to release the
hemoglobin into solution. This requires less blood than does a hematocrit so when working
with small fish where volumes are severely limited, this may be preferable to use.
There is generally a good correlation between the hematocrit, hemoglobin concentra­
tion, and RBC count, but if all three are measured, it is useful to calculate the following:

MCV = Mean corpuscular volume in |im^ /cell

= Hematocrit ratio x 1000/RBC count

MCH = Mean corpuscular hemoglobin in jiig/cell

_ Hb cone, in g/1000 ml
RBC count

In both cases, the RBC count is expressed in the unit millions/mm^. With these calculated
data, one may be able to detect the presence of a physiological lesion in the hemoglobin­
forming process as opposed to the rate of blood cell mitosis.

D. BLOOD CELL DIFFERENTIAL COUNT

In order to perform the counts necessary for calculating these, fresh blood smears are
made and stained for microscopic examination. The relative numbers of lymphocytes,
granulocytes, and thrombocytes are then enumerated. Some workers also count different
developmental stages of the erythrocytes.
 69

Houston et al. (1993) use the term “erythron organization” to refer to the relative
numbers of karyorrhetic cells (those with disintegrated nuclei), dividing blood cells,
and changes in cytomorphology. They maintain this is more sensitive to pollutant
stressors than the more traditional hematological measures. Moreover, the blood smears
can be prepared in the field, an obvious advantage where natural populations are being
sampled.
The location in the fish from which blood is sampled can influence the hematological
values recorded. Soivio et al. (1981) have discovered that blood coming from the gills
(i.e., in the dorsal aorta) has a higher hematocrit than blood going into the gills (i.e., in
the ventral aorta). This is due to “plasma skimming” by the gills. The skimmed off
plasma is recycled back to the heart via the venous system so blood taken from the
severed caudal peduncle (a common practice) will have a slightly higher hematocrit and
hemoglobin concentration than that taken from the heart (another common practice).
The estimation of normal values for hematocrit, hemoglobin, and RBC count can be
a challenging problem (Miller et al., 1983). Variables that can influence one or more of
the factors include season (Denton and Yousef, 1975; Bidwell and Heath, 1993), disease
(Barham et al., 1980), stress of capture (Lowe-Jinde and Niimi, 1983), and chemical
pollutants, which are the topic of this chapter.
Increases or decreases in hematological factors in response to environmental stressors,
chemical or otherwise, may be due to water loss or gain in the blood (see Chapter 7). Thus
osmoregulatory dysfunction may cause an apparent anemia or its opposite, polycythemia,
even though the blood-forming machinery is unaffected by the pollutant. Consequently,
it is useful to have measures of electrolytes, osmolality, and/or plasma protein concentra­
tion as additional information when doing hematological studies of fish. Finally, if
repeated blood sampling from the same specimen is contemplated, whether from a
catheter or by heart puncture, the matter of blood loss needs to be considered (Hoffman
and Lommel, 1984).
Exposure to chemical pollutants or environmental hypoxia can induce either increases
or decreases in the different hematological measures; the trend is almost always the same
in all three traditional measures. We will discuss first the conditions that produce anemia
and then consider the opposite physiological response. Within groups, the mechanisms
responsible for the changes differ somewhat with the chemical.

III. CHEMICALS THAT CAUSE ANEMIA

A. CADMIUM
Cadmium has been reported to cause anemia in a variety of fish species at low and high
concentrations (e.g., Larsson et al., 1976; Newman and McLean, 1974; Larsson et al.,
1985; Gill and Pant, 1985; Houston et al., 1993). It is also well known for its ability to
cause anemia in mammals (Friberg et al., 1974). The mechanism involved in these
animals is reportedly to be due, largely, to a reduction in absorption of iron from the gut
for the synthesis of hemoglobin. It is not known if this mechanism also occurs in fish. If
so, it is certainly not the only one, as an anemic condition is produced in flounders
exposed to cadmium at 1 ppm for 15 days during which time they were not fed (Larsson
et al., 1976), thus, there was no opportunity for iron uptake from the gut in either control
or experimental animals.
Cadmium also causes an abnormally large number of malformed erythrocytes (Newman
and McLean, 1974; Houston et al., 1993) which implies a lesion in the machinery for
forming blood cells. Measurements of the enzyme delta-aminolevulinic acid dehydratase
(ALA-D; see section on lead below) in the kidney have shown a compensatory increase
70

as a result of exposure to cadmium (Johansson-Sjobeck and Larsson, 1978). This indi­

cates that the first steps of hemoglobin synthesis are not blocked by cadmium, but in spite
of the increased enzyme activity, hemoglobin synthesis still goes down. The net effect is
a slowing of erythrocyte maturation (Houston et al., 1993). In addition to the reduction
in capacity to produce normal blood cells, some workers have also noted increased rates
of erythrocyte breakdown (Houston et al., 1993). Palace et al., (1993) reported that
erythrocytes from rainbow trout exposed to cadmium for 181 days had a tendency for
lysis. This increased cell fragility was attributed to lipid peroxidation of the erythrocyte
membranes which affects membrane fluidity. They present other evidence of stimulated
antioxidant defenses which are mobilized to counteract increased peroxidation.
After an induction of anemia by cadmium in flounder, Larsson et al. (1985) found that
a year’s recovery in non-contaminated water abolished the anemia. This recovery is
interesting in light of the finding by the same research team that carbohydrate metabolism
was still disturbed, possibly due to permanent damage to the insulin-secreting cells in the
pancreas (see Chapter 8). From this, it appears that some cadmium-induced lesions are
far more permanent than others.
Cadmium has also been shown to alter the leukocyte differential count, but the
direction of change has not been consistent between different studies. Newman and
MacLean (1974) reported a dose-dependent increase (threefold) in neutrophils and a
dose-dependent decrease (also nearly threefold) in lymphocytes in the cunner
{Tautogolabrus adspersus). This contrasts with the “markedly enhanced lymphocyte
count” observed by Larsson et al. (1985) in perch from a river polluted with cadmium.
The contradictory results could be due to a different pattern of disease organisms
present in the two studies. Differential leukocyte counts are especially susceptible to
this variable.

B. LEAD
Chronic exposure of the cyprinid Barbus to lead for up to 60 days at 47 \igih resulted in
severe reductions (12 to 31%) in erythrocyte count, hematocrit, hemoglobin concentra­
tion, and MCV (Tewari et al., 1987). This element has been shown to cause anemia in
mammals by inhibiting hemoglobin synthesis and shortening the lifespan of circulating
erythrocytes (Hemberg, 1976). Lead inhibits the enzyme ALA-D which is required in the
early stages of hemoglobin synthesis in the hemopoietic tissue. The inhibition of this
enzyme by lead is quite specific; other metals such as cadmium have no effect on it in
mammals. Such specificity of inhibition is rare, so measurement of ALA-D activity in
erythrocytes of humans has been useful for diagnosing lead poisoning (Hemberg et al.,
1970), and, whereas it is stimulated by cadmium in the kidney of fish (discussed above),
erythrocytic ALA-D is unaffected by this element (Hodson et al., 1984).
Trout, exposed to waterborne lead at concentrations of 10, 75, and 300 |Llg/L for 30
days, exhibited anemia only at the highest concentration. On the other hand, there was a
dose-dependent inhibition of ALA-D in the erythrocytes and spleen (Johansson-Sjobeck
and Larsson, 1979). At the highest dose, an 86% inhibition of erythrocytic ALA-D was
observed while the lower lead concentrations induced an inhibition of 74 and 21%,
respectively. No effect on hemoglobin synthesis was noted at these lower levels of lead
implying the enzyme has a large reserve capacity, because essentially normal function
was possible with only 25% of the usual enzymatic activity.
The recovery of ALA-D activity is very slow when fish are allowed to reside in lead-
free water following an exposure. Johansson-Sjobeck and Larsson (1979) found that after
7 weeks of recovery there was only a slight improvement in enzyme activity. It is not clear
whether this is due to the continued presence of lead in the tissues (it was not measured),
or a slow rate of enzyme synthesis. Intuitively, the former seems far more likely as lead
is retained in the kidney of fish for at least a month following its uptake from the
 71

environment (Reichert et al., 1979). From a practical standpoint, the continued inhibition
of ALA-D following exposure to lead may add to its usefulness for diagnosing lead
poisoning in fish taken from polluted waterways. The assay of ALA-D activity is
presumably easier than measuring lead in the tissues.

C. MERCURY
Both winter flounder and striped bass exhibited a distinct anemia when exposed to
mercury (inorganic) for 60 days (Dawson, 1979,1982). The magnitude of the change was
smaller in the flounder even though a higher mercury concentration was used with that
species. Anemia was also reported in the plaice with higher and shorter duration expo­
sures (Fletcher and White, 1986). Mercury tends to concentrate in the kidney of teleosts
(Penreath, 1976) where it probably inhibits uroporphyrinogen I synthetase, a heme
biosynthetic enzyme (Tephly et al., 1978). It has also been shown to reduce the deformability
of the erythrocyte membranes which could contribute to their early destruction (Brouwer
and Brower-Hoexum, 1985).
Methylmercury appears to be less “effective” at producing anemia than is inorganic
mercury even though the former is more toxic (Lock et al., 1981). When rainbow trout
were exposed to 15 jitg/L waterborne methylmercury for 75-119 days, they accumulated
whole-body residue levels of 3-12 mg/kg, but no discernible hematological effects were
found (Niimi and Lowe-Jinde, 1984). In addition, the effect on the hematocrit from
methylmercury in the food is equivocal; there have been increases and decreases reported
(Rogers and Beamish, 1982; Bidwell and Heath, 1993).
One final point about mercury in fish should be mentioned. Methylmercury binds
reversibly with the hemoglobin in erythrocytes. This has been shown both in vitro and in
vivo; the reaction is with the -SH groups of the hemoglobin molecule (Massaro, 1974).
Thus, it could have an effect on the binding of hemoglobin with oxygen, although this
seemingly is not known.

D. CHLORAMINE
When chlorine (as a gas or as hypochlorite) is added to water as a disinfectant or for
antifouling purposes, it immediately forms hypochlorous acid, which is commonly called
“free chlorine”. If ammonia is present, the chlorine will unite with it and become a
chloramine (sometimes called “combined chlorine”). There are similarities and differ­
ences in the physiological actions of free and combined chlorine (Travis and Heath,
1981). Buckley et al. (1976) found hemolytic anemia in salmon exposed for 12 weeks to
municipal wastewater containing chloramines. Free chlorine, on the other hand, has been
shown to cause an increase in hemoglobin concentration (Bass and Heath, 1977). This
increase is probably an adaptation to the internal hypoxia induced by the damage to the
gill tissue. Internal hypoxia does not occur with chloramines (Travis and Heath, 1981)
except perhaps at unrealistically high doses.
Exposure of fish to chloramine, in addition to causing anemia, results in oxidation of
part of the hemoglobin to methemoglobin, which does not transport oxygen (Grothe and
Eaton, 1975). This is in contrast to free chlorine which causes little methemoglobin
formation in trout (Bass and Heath, 1977) or even when the erythrocytes are exposed to
the chemical in vitro (Buckley, 1981). Thus, both forms of chlorine reduce the available
oxygen to the tissue. Chloramine lowers the oxygen-carrying capacity of the blood while
free chlorine lowers the oxygen tension in the arterial blood by causing an impairment of
oxygen uptake by the gills (Chapter 3). From the standpoint of the fish, chloramine will
probably be more debilitating because, due to the oxygen dissociation characteristics of
the blood, a considerable decrease in arterial oxygen tension is required before a reduction
in actual oxygen availability occurs. Free chlorine affects only the tension whereas
chloramine reduces the amount of oxygen on the hemoglobin molecule.
72

E. NITRITE
Nitrite, from the bacterial reduction of nitrate, enters the gills of fish via the chloride­
transporting mechanism. It can accumulate in the blood plasma against a concentration
gradient (Williams and Eddy, 1988). As with chloramine discussed above, nitrite oxidizes
hemoglobin to methemoglobin; the extent of this conversion is directly proportional to the
plasma nitrite concentration which, in turn, is proportional to environmental concentra­
tion (Tomasso, 1986). Along with reducing the oxygen-carrying capacity of the blood by
oxidizing the hemoglobin, nitrite also seems to reduce oxygen affinity, as was shown with
in vitro studies of carp blood where the P50was increased (indicating reduced affinity) by
almost threefold (Williams et al., 1993). This could have a considerable effect on the
ability of the fish to tolerate low dissolved oxygen (see Chapter 2). The conversion of
hemoglobin to methemoglobin causes hemolytic anemia which can cause the blood
plasma to acquire a reddish-brown color (Williams and Eddy, 1988).
Nitrite poisoning of hemoglobin also affects the temperature tolerance. Watenpaugh
et al. (1985) exposed channel catfish to several sublethal concentrations of nitrite for 24
h and then tested the critical thermal maximum. This was inversely related to the nitrite
concentration with mean lethal temperatures ranging from 38°C for controls to 35.9°C for
those exposed to 1.4 mg/L nitrite.

F. PULPMILL EFFLUENT
Long-term (25 days) exposure to the effluent from kraft pulpmills has been found to cause
anemia in salmon (McLeay, 1973). The composition of this effluent is extremely variable
between different mills and at various times at a single one (Davis, 1976). There are
several toxic components in the effluent, one of which is dehydroabietic acid, one of the
naturally occurring resin acids extracted from softwood trees. When coho salmon were
exposed for up to 4 days to a concentration of this chemical approximating half the 96-
h LC50, there were no changes found in the hematology. However, there was a significant
increase in clotting time in blood from exposed animals (Iwama et al., 1976). Because
there was also a decrease in total white cell count, it is probable that thrombocytes were
down, although they were not counted separately. Such a decrease in leukocytes is a
typical response to virtually any environmental stress (Ellis, 1981). However, a decrease
in clotting time is the usual response to stress, instead of an increase as was seen here.
Thus, this effect on clotting time could be a rather specific action of dehydroabietic acid.
In an in vitro study using rainbow trout erythrocytes, Bushnell et al. (1985) found the
resin acid caused a decrease in cellular ATP and oxygen consumption and marked
increase in hemolysis. In other work they cite, jaundice has been noted in fish exposed
to some pulpmill effluents; the hemolytic anemia could be the primary reason for that
condition.
Lehtinen et al. (1990) used hematological measurements (along with those for osmo­
regulation and mixed function oxidase) to compare different bleaching processes in kraft
mills. The exposures were for 7 weeks at concentrations of 400 and 2000 times dilution.
Little effect was seen on hematology except with the effluent from conventional chlorine
bleaching which caused a considerable anemia. This approach of comparing different
industrial processes using several biomarkers in fish is one that should be utilized more
extensively.

G. MISCELLANEOUS
A variety of chemicals are probably capable of causing varying degrees of anemia in fish.
These include organochlorine pesticides (Venkateshwarlu et al., 1990), the fungicide
chlorothalonil (Davies, 1987), and cobalt (Pamila et al., 1991). Cobalt is somewhat
interesting in that it inhibits the enzyme 5-aminolevulinate synthetase, one of the enzymes
involved in heme synthesis. When Sarotherodon mossambicus, an Indian freshwater
 73

teleost, was exposed to cobalt for up to 15 days, a marked increase in erythrocyte count
occurred within 5 days, although total hemoglobin content of the blood was declining. It
appeared the fish were overcompensating for the inhibited hemoglobin synthesis.

H. BIOLOGICAL SIGNIFICANCE OF ANEMIA

In evaluating the chemicals listed above and others that may be found, it must be kept in
mind that a mild condition of anemia, such as was found in most of the studies mentioned
here, is probably not particularly debilitating. There is some evidence to suggest that fish
are able to tolerate a far greater degree of anemia than can mammals. Holeton (1971)
subjected rainbow trout to carbon monoxide thereby completely inactivating their hemo­
globin and found the fish were able to survive this (at rest), providing temperatures did
not exceed 12°C. Anthony (1961) did the same thing with goldfish and reported that they
survived over 24 h at 30°C and indefinitely at lower temperatures. More importantly,
routine activity in the goldfish did not appear to depend on the presence of hemoglobin;
and, of course, there are ice fish living around Antarctica which lack hemoglobin, but
have compensatory adaptations, such as a large cardiac output (Holeton, 1970).
Where a low hemoglobin level may be most important is in fish subjected to severe
environmental hypoxia (Holeton, 1972) (also see Chapter 2), or forced to swim at a high
speed for long distances. Jones (1971) induced a condition of hemolytic anemia in rainbow
trout by injection of phenylhydrazine and then tested their ability to swim in a water tunnel.
By this means, he determined that a reduction of the hematocrit to one third of normal
caused a 34 to 40% reduction in maximum sustained swimming speed. Thus, this could be
an especially important debilitation for salmon during the upstream spawning migration.

IV. CHEMICALS CAUSING AN INCREASE IN

HEMATOLOGICAL VARIABLES
An increase in the hematocrit alone has often been observed in fish subjected to a non­
specific stressor (such as being lifted out of the water), and this can occur within minutes
of the onset of stress (Casillas and Smith, 1977). Part, if not all, of this change is due to
swelling of the erythrocytes which occurs whenever fish blood cells are exposed to a
hypoxic environment (Soivio and Nikinmaa, 1981). It apparently does not matter whether
the blood cells are exposed to the hypoxia in vitro or in vivo (Soivio and Nikinmaa, 1981;
Swift and Lloyd, 1974). Thus, virtually any dose of pollutant that results in gill damage,
and a subsequent internal hypoxia (this includes many chemicals, see Chapter 3) can be
expected to also cause an increase in hematocrit.
Casillas and Smith (1977) further found that the hematocrit may return to normal
within 60 min of cessation of the stress (in their case it was handling). These findings
argue for the measuring of more than just the hematocrit when doing hematological
studies; and transient increases in response to an acute exposure dose mean very little
from a hematological standpoint.

A. ACID
It is quite common to have an increase in hematocrit, hemoglobin, and/or RBC count in
fish residing in acidic water (see Wood and McDonald, 1982 for review). At an acutely
low pH, this is largely due to hemoconcentration and swelling of blood cells brought on
by a failure to regulate plasma electrolytes (see Chapter 10). There is also a release of
erythrocytes from the spleen (Milligan and Wood, 1982). When the exposure to a low pH
is of a more chronic type, the hematological increases could be a response to an impaired
oxygen transport by the blood. Due to the well-known Bohr effect, the blood will have
less affinity for oxygen at a lower pH so stimulation of erythrocyte production would then
increase the oxygen capacity and thus partially compensate for this.
74

B. PESTICIDES
Among organic pesticides, it appears that some chlorinated hydrocarbons stimulate
erythropoiesis; aldrin, chlordane, and pentachlorophenol are all in this group (Dhillon and
Gupta, 1983; Davies, 1987; Iwama et al., 1986). Aldrin caused a dose-dependent increase
in RBC count and total hemoglobin concentration, but the mean corpuscular hemoglobin
actually went down (Dhillon and Gupta, 1983). This suggests a large increase in cell
formation; so much so that hemoglobin synthesis did not keep up with it. The mechanism
of this stimulatory effect of chlorinated hydrocarbons insecticides is unclear. In part, it
may be due to an impairment in oxygen transfer at the gills, but that is only a speculation.
Organophosphate insecticides mostly cause increases in hematological variables
(Natarajan, 1984; Lai et al., 1986). Evidence is beginning to accumulate supporting the
idea that the effect is due to histological damage to gill tissues which produces an internal
hypoxia and subsequent stimulation of erythropoiesis (Areechon and Plumb, 1990).

C. COPPER
McKim et al. (1970) observed that copper increased the hematocrit, hemoglobin, and
RBC count in trout when exposed for 6 days to a concentration far below the LC50. By
21 days of continued exposure the hematocrit had returned to normal and by the 11th
month, hemoglobin and blood cell counts had also returned to normal. Waiwood (1980)
also reported increased hematocrits in trout exposed to copper but he was able to account
for the entire change as being due to a shift of water from the plasma to the muscle cells,
thereby producing hemoconcentration, as has also been observed by Wilson and Taylor
(1993) in trout exposed to lethal copper concentrations. The McKim et al. (1970) chronic
study found just the opposite; a slight increase in plasma water. Because copper is
required for hemoglobin synthesis, a mild excess may be stimulatory, particularly if the
fish were marginally deficient in copper at the start of an experiment.
In mammals, excess copper can cause hemolytic anemia by inhibiting glycolysis in the
erythrocytes, denaturing the hemoglobin, and oxidizing glutathione (Fairbanks, 1967).
Because fish erythrocytes have a greater aerobic capacity than mammalian ones (Eddy,
1977), perhaps they are not as sensitive to this element.
Other chemicals that stimulate blood cell/hemoglobin production, such as ozone
(Wedemeyer et al., 1979), nickel (Ghazaly, 1992), zinc (Mishra and Srivastava, 1979;
Hilmy et al., 1987), and hexavalent chromium (van der Putte et al., 1982) generally induce
a hypoxic condition in the fish. Pentachlorophenol, on the other hand, may increase the
metabolic demand by the organism for oxygen (Holmberg et al., 1972). Thus, these
observed increases in oxygen capacity of the blood can be viewed as an adaptation to
an altered respiratory homeostasis caused by the pollutant and not a toxic or direct
stimulatory action of the chemical on the blood cell-forming tissues. A similar thing
happens during acclimation to environmental hypoxia (see Chapter 2), and, in any
situation where acute stress is imposed on the animal, adrenergic stimulation of the
spleen can cause it to contract and release stored erythrocytes into the circulation
Nilsson and Grove, 1974.

V. CONCLUDING REMARKS
Some final caveats are in order regarding the presumed stimulatory or inhibitory effect
of some chemical on hematological variables in teleosts. It is not unheard of for a
substance to produce one type of effect when exposure is long term but cause an exact
opposite effect if the exposure is highly acute (e.g., Dhillon and Gupta, 1983). Where
histological damage to gills reduces the transfer of oxygen into the blood, this can
stimulate erythropoiesis while the chemical may, at the same time, be inhibiting some step
in the formation of hemoglobin. The net effect on hematological variables will then be
 75

some kind of algebraic sum of these opposite effects. Finally, recent work (Bollard et al.,
1993) has shown that artificially elevating the cortisol level can cause a drop in mean
cellular hemoglobin content. Cortisol is frequently elevated in fish under a variety of
stressors, physical, chemical or psychological, so this might be a further mechanism
producing an anemia. Thus, considerable caution must be exercised when designating a
chemical as being anemia-causing, or the reverse.

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Biochem. Physiol., 21, 194, 1984.
Newman, M. W. and MacLean, S. A., Physiological response of the cunner, Tautogolabrus adspersus,
to cadmium. VI. Histopathology, National Oceanic and Atmospheric Administration Technical
Report, NMFS SSRF-681, 27, 1974.
Niimi, A. J. and Lowe-Jinde, L. L., Differential blood cell ratios of rainbow trout (Salmo gairdneri)
exposed to methylmercury and chlorobenzenes. Arch. Environ. Contam. Toxicol, 13, 303,1984.
Nilsson, S. and Grove, D. J., Adrenergic and cholinergic innervation of the spleen of the cod: Gadus
morhua, Eur. J. Pharmacol, 28, 135, 1974.
Palace, V. P., Majewski, H. S. and Klaverkamp, J. F., Interactions among antioxidant defenses in liver
of rainbow trout {Oncorhynchus mykiss) exposed to cadmium. Can. J. Fish. Aquat. Scl, 50, 156,
1993.
Pamila, D., Subbiayan, P. and Ramashwamy, M., Toxic effects of chromium and cobalt on Sarotherodon
mossambicus, Indian J. Environ. Health, 33, 218, 1991.
Penreath, R. J., The accumulation of inorganic mercury from the seawater by the plaice, Pleuronectes
platessa, J. Exp. Mar. Biol Ecol, 24, 103, 1976.
Reichert, W. L., Federighi, D. A. and Malins, D. C., Uptake and metabolism of lead and cadmium in
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Rogers, D. W. and Beamish, F. W. H., Dynamics of dietary methylmercury in rainbow trout {Salmo
gairdneri), Aquat. Toxicol, 2,211, 1982.
Satchell, G., Physiology and Form of Fish Circulation, Cambridge University Press, New York, 1991.
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Swift, D. J. and Lloyd, R., Changes in urine flow rate and haematocrit value of rainbow trout {Salmo
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Tephly, T. R., Wagner, G., Sedman, R. and Piper, W., Effects of metals on heme biosynthesis and
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78

Tewari, H., Gill, T. and Pant. J., Impact of chronic lead poisoning on the hematological and biochemical
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Eds., University of South Carolina Press, Columbia, 1985, 63.
Tomasso, J. R., Comparative toxicity of nitrite to freshwater fishes, Aquat. Toxicol, 8, 129, 1986.
Travis, T. W. and Heath, A. G., Some physiological responses of rainbow trout (Salmo gairdneri) to
intermittent monochloramine exposure. Water Res., 15, 977, 1981.
van der Putte, L., Laurier, M. B. H. M. and van Eijk, G. J. M., Respiration and osmoregulation in
rainbow trout {Salmo gairdneri) exposed to hexavalent chromium at different pH values, Aquat.
Toxicol, 2, 99, 1982.
Venkateshwarlu, P., Rani, V., Janaiah, C. and Prasad, M., Effect of endosulfan and kelthane on
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Trans. Am. Fish. Soc., 114, 274, 1985.
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Wedemeyer, G. A., Nelson, N. C. and Yasutake, W. T., Physiological and biochemical aspects of ozone
toxicity to rainbow trout {Salmo gairdneri), J. Fish. Res. Bd. Can., 36, 605, 1979.
Williams, E. and Eddy. F. B., Anion transport, chloride cell number and nitrite-induced
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Toxicol, 13, 29, 1988.
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Fisheries, Johnson, R. E., Ed., American Fisheries Society, Bethesda, MD, 1982, 197.
 üptake, Accumulation,
Chapter
5
Biotransformation, and
Excretion of Xenobiotics
I. INTRODUCTION
Investigations of uptake and accumulation are fundamental in helping to understand the
effects these chemicals have on specific organ systems in fish. Figure 1 outlines in a
general way the possible fates of a contaminant coming in contact with a fish either by
way of the water or the food of the fish. The chemical, once it is absorbed via the gills
or gut, is usually bound to a protein and then transported by the blood to either a storage
point, such as fat, or to the liver for transformation and/or storage. If transformed by the
liver, it may be stored there, excreted in the bile, or passed back into the blood for possible
excretion by the kidney or gills, or stored in extrahepatic tissues such as fat. Thus, the
concentration found in different organs after environmental exposure for a particular time
depends on several simultaneous dynamic processes. The total body burden of a foreign
chemical will be a weighted average of all the tissues, which may differ from each other
in concentration of this chemical by orders of magnitude.
A word about terminology: the expressions “bioaccumulation” and “bioconcentration”
are used rather synonymously in the literature. They refer to the process of a fish
acquiring a body burden of some chemical that is at a higher concentration than that in
the water. The biological concentration factor (BCF) describes the extent to which
something accumulates in an aquatic organism. It is a unitless value obtained by dividing
the concentration in one or more of the tissues by the average concentration in the water.
Veith et al. (1979) suggest the term bioconcentration be limited to the accumulation of
a chemical directly from the water but excluding that obtained via the food, while
bioaccumulation would refer to accumulation from both food and water, a suggestion that
will be followed here.
“Biomagnification” is best reserved for the process of acquiring a greater body burden
from being at a higher trophic level. Of course, a fish may be “practicing” both
bioconcentration directly from the water and biomagnification from eating lower on the
food chain. It might be noted here that not all substances lend themselves to
biomagnification. For example, mercury biomagnifies easily whereas cadmium and lead
seemingly do not; nor do the body concentrations of the latter two metals increase with
age or size of the fish as does mercury (Spry and Wiener, 1991).

79
80

Chemical

C
O
Fot
O
E
w
O - Liver*- Liver 0>
</> O»
C
O
w
s
O
-K id n ey -^------- Kidney -
O (7)
m - M e ta b o lite s -

Muscle
-Mucous- -S k in -
L iv e r--------- B ile --------- In te s tin e - -A n u s -
-G ills -
------- Kidney---------- U rin e ----------►
Excretion

Figure 1 Schematic diagram of the possible directions of movement and fates of a pollutant
after it has been absorbed into the bloodstream of a fish.

In the literature, several workers have used the term “uptake” when referring to the
accumulation of some pollutant chemical in the fish. For clarity, it would be better to use
this term to describe the process(es) by which the substance is actually taken into the body
of the fish. The amount that is actually accumulated will depend on the balance between
uptake rate, metabolism of the chemical, and excretion rate.

II. UPTAKE FROM THE ENVIRONMENT

The routes by which a foreign chemical is taken into a terrestrial animal for the most part
do not apply to fish. This is because the aquatic environment imposes several constraints
on the animals in it. One of the most critical is the matter of obtaining sufficient oxygen.
A fish must breathe roughly 20 times more of its respiratory medium (i.e., water) than a
terrestrial animal in order to obtain an equivalent amount of oxygen. For example, a 250-g
trout at rest will pass approximately 48 L of water over its gills each hour (Reid and
McDonald, 1991). Therefore, the gill tissue (which is the main point of entry for dissolved
substances) will be exposed to a far greater amount of selected pollutants than a terrestrial
animal breathing air, even allowing for a lower oxygen consumption in the water­
dwelling poikilotherm. Furthermore, the gills possess a countercurrent blood/water flow
system, very thin epithelial membranes, and large surface areas; these are all features that
facilitate the uptake of materials from the water and their transfer to blood.
The matter of drinking water will be different in fish from mammals, but in a
somewhat peculiar way. Contrary to popular belief, only marine fish “drink like a fish”.
Freshwater fish drink almost nothing except what is swallowed with food.
There are four possible routes for a substance to enter a fish: gills, food, drinking
water, and skin. Gills and food vary greatly as routes of exposure, depending mostly on
the availability of the substance in question (i.e., whether it is concentrated in the food
or dissolved in the water). For example. Spry and Wiener (1991) conclude after reviewing
the literature that over 90% of the mercury accumulated in wild fish is acquired directly
from the food. The importance of skin varies with the size of the fish and toxicant. For
large fish (about 1 kg), it accounts for less than 10% of total absorbed dose (McKim and
Nichols, 1991), but for small fish of 4 g or less, due to the large surface to volume ratio,
skin can account for up to one half of the absorbed dose (Liem and McKim, 1993).
 81

The distribution into various organs is influenced by the route of uptake. This is
because xenobiotics that enter the circulation via the gills can be rapidly distributed
throughout the body to all organs. Xenobiotics that are absorbed via the intestine will, on
the other hand, pass first to the liver, via the hepatic portal system, where some metabo­
lism or sequestering may occur.
Assimilation by the intestine influences the relative importance of intestinal uptake vs.
gill. For example, at least some polycyclic aromatic hydrocarbons are not absorbed well
by the digestive tract and yet are taken up very readily by the gills (Kennedy and Law,
1990).

A. METALS
When metals enter natural waters there are several things that may happen to them which
can greatly affect their bioavailability (Figure 2). If there is a considerable amount of
organic material or suspended solids, the actual amount of dissolved metal available to
be absorbed by the fish will be greatly reduced. This tendency to form complexes with
organic and inorganic ligands (primarily chloride, carbonate, and hydroxide) varies with
the metal (Sprague, 1985). For example, copper binds to organics far more readily than
does either cadmium or silver (Engel et al., 1981). Consequently, it is important that the
dissolved metal be measured, rather than just the total concentration in the water, in order
to assess the amount of metal actually available for absorption by the fish.
It is assumed that most metals are absorbed by fish in the ionic form, although
methylmercury is certainly an exception, and studies of cultured gill epithelial cells
suggest that some cadmium chloride can be taken up along with the cadmium ion (Block
and Part, 1992). Because the outer surface of the gill tissue has a negative charge, it will
attract metallic ions (Reid and McDonald, 1991), but recently, it has been found that not
all metals bind to the surface equally well. Reid and McDonald (1991) report that affinity
of the gill for metals tested was La > Ca = Cd > Cu. The affinity of the gill for metals

MeCOs
Me(0H)2
MeCl2

Me
Adsorbed on
Cloy ond Detritus

Figure 2 Conceptual model of the different forms of metal (Me) present following its addition
to fresh, estuarine, or marine water. The double-ended arrows indicate that the metal Is in a
dynamic equilibrium with the various components of the water column, and that a change in any
one aspect of the system can shift the interrelationships. (Based on Engel et al.. Biological
Monitoring of Marine Pollutants, Vernberg, J., Calabrese, A., Thurberg, F. and Vernberg, W. B.,
Academic Press, New York, 1981, 127.)
82

is determined by the microenvironment of the gill surface which is complex, because it

includes the epithelial membranes as well as the mucus layer with its mixture of glyco­
proteins, mucopolysaccharides, assorted low molecular weight compounds, and water.
Contrary to what might first be expected, the absorption of a metal into the cells of the
gills is inversely proportional to the surface-binding affinity. Thus, copper, which has a
low affinity, is absorbed into the cells much more readily than either calcium or cadmium
(Reid and McDonald, 1991).
The fact that binding to the outer surface of the gill epithelium does not facilitate
further uptake is especially noteworthy with aluminum. In acidic soft water (where
aluminum contamination is most prevalent), much of it precipitates due to a rise in pH
from excreted ammonia as the water passes over the gill and this is sloughed off with the
mucus. The rest remains as a charged form bound to the gill surface (Playle and Wood,
1991). As a result of these effects of the gill microenvironment, relatively little dissolved
aluminum actually gets into the fish (Spry and Wiener, 1991; Handy and Eddy, 1990).
The mechanism of metal uptake through the gills has been assumed to be simple
diffusion (Bryan, 1979), although carrier mediation or endocytosis have not been elimi­
nated as possibilities (Luoma, 1983). Using an isolated perfused head preparation. Spry
and Wood (1988) showed that zinc is not absorbed in a simple concentration-dependent
manner. Rather, the kinetics suggest a saturable uptake mechanism, which they point out
is not necessarily active or carrier mediated but could be. They also present interesting
indirect evidence that much of the zinc is actually absorbed via the chloride cells in the
lamellar epithelium.
Mucus on the surface of the gill has a considerable influence on the accumulation by
the gills of metals (Handy and Eddy, 1990). Starved fish accumulate zinc on their gill
surface more rapidly than those fed due to a change in composition of the mucus during
starvation (Handy and Eddy, 1990). Apparently, there are more metal-binding ligands in
the mucus from starved fish.
Part and Lock (1983) reported that trout mucus in an in vitro preparation slowed the
diffusion rate of mercury and cadmium, but had no effect on the diffusion of calcium. This
difference was probably due to the relative binding characteristics of the metals with the
mucus. Overall, it appears that mucus reduces the uptake of metals into fish.
The rate of metal uptake into the gill tissue correlates with the weight-specific
metabolic rate, thus small fish accumulate it more rapidly than large ones (Anderson and
Spear, 1980). Because the higher flow rate of water over the gills of small fish apparently
results in greater uptake, the question arises as to what effect environmental hypoxia
would have on this process, as it also causes a greater rate of ventilatory water flow.
Exposure to mild hypoxia does cause a greater accumulation by the gills of cadmium,
chromium, or lead (Freeman, 1980), but Hughes and Flos (1978) found that the rate of
zinc uptake into gill tissue was actually slower in hypoxic fish compared to normoxic
controls. Just why this is remains unclear. Hughes and Flos (1978) suggest the possibility
that the histological damage to the gills caused by the zinc may be greater under hypoxia
than under more normal conditions. This may have the effect of making the gills less
permeable to zinc, as it does to oxygen. A point not mentioned by them is that hypoxia
alone has been shown to cause histological damage to fish gills (see Chapter 2), so this
could be a sort of synergism, or at least, an additive effect.
The expression “uptake efficiency” has been used to refer to how readily a fish
removes a contaminant from the water via the gills (Boddington et al., 1979; McKim and
Goeden, 1982). Fish respiratory physiologists have a long tradition of using the terms
“utilization efficiency” or “extraction coefficient” for the percent of the oxygen in the
inhalant water that is removed as it passes over the gills (Truchot and DeJours, 1989). The
parallel with uptake efficiency for pollutants is obvious. For these measures, one must
determine the concentration of some chemical (or oxygen) in the exhalant water, which
 83

is not an easy thing to do, for it must be kept from mixing with the inhalant water. The
fish must be restrained in a chamber that is divided by a flexible rubber membrane into
front and back. The fish’s head is inserted part way through the membrane and sutured
just in front of the operculae. In this way, the fish in the process of breathing pumps the
water from the front to the back chamber, both of which are arranged to avoid either a
positive or negative pressure gradiant for water flow. This design was originated by Van
Dam (1938) and such devices are frequently called “Van Dam chambers”.
Figure 3 illustrates a slightly more sophisticated form of Van Dam chamber. In this
design, there is a third chamber separating the back portion of the body from the gill
exhalant water. Analyses of the water from the rear chamber indicate the extent of
excretion or uptake of a chemical through the skin. Also by having a third chamber, the
size of the rear chamber is reduced so the turnover time is faster and a shorter sample
interval can be used. Note the urinary and fecal catheters enabling measures of volume
and composition of these excretory modes. This system also makes possible the determi­
nation of the volume of water passing over the gills in a given time interval.
Obviously, the process of obtaining data from a fish in this manner is quite tedious.
Unfortunately, the simpler method of cannulation of the opercular cavity in order to
obtain samples of the exhalant water yields misleading results, at least when it is applied
to the estimation of oxygen utilization efficiency (Davis and Waters, 1970).
Boddington et al. (1979) used an apparatus somewhat similar to that shown in Figure 3
(except that it was two-chambered) to measure the uptake efficiency of waterborne
methylmercury. Radiolabeled methylmercury was taken up with an average efficiency of
7.1%, which is about 0.13 of the extraction coefficient for oxygen in the same fish. It
would be interesting to compare different metals in this regard.
Rogers and Beamish (1983) use the term uptake efficiency in a slightly different
manner. For their work, they measured the rate of methylmercury uptake and related this
to oxygen consumption (not extraction efficiency). The equation of this is E = [AP/(P)/
Q/(02)] Delta P is the rate of methylmercury uptake in nanograms per hour. (P) is the
average mercury concentration in the water, Q is the rate of oxygen consumption (mg/h).

VENTRAL VIEW -ORAL

MEMBRANE ATTACHED.

SIDE VIEW-CHAMBER AND FISH WITH ORAL MEMBRANE ATTACHED.

Figure 3 Schematic view of respirometer-metabolism chamber containing a fish and empha­

sizing the design and placement of the oral membrane. (From McKim, J. M. and Goeden, H.,
Comp. Biochem. Physiol., 72C, 65, 1982. With permission.)
84

(mg L*^ os CoCO^

Control Control Hg CI 2 Hg Cl^ ZnSO^
(30/jg L-') (30>jg L-') (300>jg L*')

Figure 4 Efficiency of uptake of methylmercury relative to oxygen of rainbow trout, Salmo

gairdneri, and showing effects of water hardness, inorganic mercury, and zinc. Bars indicate
mean ± 95% confidence limits. Sample sizes in parentheses. (From Rogers, D. W. and Beamish,
F. W. H., Can. J. Fish. Aquat. Sci., 40, 824, 1983. With permission.)

and O2 is the mean oxygen concentration in milligrams per liter. The main questions
asked in their study were whether hardness and the presence of other pollutants affected
the uptake rate of methylmercury. The data in Figure 4 show that these factors do indeed
have a considerable effect. Increasing the hardness decreased the rate of mercury uptake.
A similar relationship between water calcium concentration and uptake of waterborne
lead has been noted (Varanasi and Gmur, 1978). These findings may help explain the
often repeated observation that acute toxicity of heavy metals is less in hard water
(Sprague, 1985).
Water hardness affects the gill permeability to water and ions so that the harder the
ambient water, the less permeable the tissue (Hunn, 1985). The calcium ion, which is the
major cation responsible for what we call “hardness”, also causes the electrical charge on
the outside of the gills to be more positive (McWilliams and Potts, 1978). This would
further tend to repel positively charged molecules such as toxic metals. It clearly reduces
the rate of uptake of these metals by the gills (Everall et al., 1989; Wicklund and Runn,
1988). The zinc flux rates in hard vs. soft water can differ by as much as 40-fold (Spry
and Wood, 1988). Spry and Wood (1988) have further shown that the effect of water
hardness is not due exclusively to the presence of external calcium, for fish that have been
acclimated to hard water retain their lower zinc uptake rate when later tested in soft water.
Thus, the hard water must cause some changes in the gill epithelial cells or the junctions
between them that inhibits the flux of metals.
In the aforementioned study on the uptake of mercury, the presence of inorganic
mercury actually increased the uptake of methylmercury (but canceled out the hardness
effect; Figure 4). Rogers and Beamish (1983) speculate the increased uptake of methyl­
mercury was due to an altered structure of the gill mucus caused by the inorganic
mercury. However, simply increasing the mucus as should occur with exposure to zinc,
actually decreased the uptake efficiency of methylmercury. The authors suggest this may
represent zinc competing for sites of methylmercury uptake on the surface of the gill.
 85

Ramamoorthy and Blumhagen (1984) found that zinc in soft water actually stimulated the
uptake of inorganic mercury by trout. They did not test hard water nor methylmercury,
so it is not possible to determine at this time the relative importance of these factors.
Finally, we must note that low levels of cadmium can inhibit the uptake of zinc from the
water, but zinc seems to have no effect on cadmium uptake (Bentley, 1992). Clearly, the
interactions of various metals and water hardness on metal uptake by fish is complex.
Two additional environmental factors that can influence the rate of metal uptake are
water pH and temperature. At a given concentration of calcium in the ambient water,
lowering the pH decreases the rate of zinc uptake into gill tissue of rainbow trout (Bradley
and Sprague, 1985). This generalization holds, however, only for high zinc concentra­
tions and gill tissue (the primary target of toxic action at high doses). Using much lower
concentrations and measuring total body burden, Bentley (1992) found that lowering the
pH increased zinc uptake, so sublethal effects might be greater at lower pH.
Copper uptake by the gills of fathead minnows was slower at lower pHs, but only in
hard water (Playle et al., 1992). In soft water, there was no difference in copper accumu­
lation by gills between pH 4.8 and 6.3. The authors conclude that calcium and hydrogen
ions compete with copper for gill-binding sites.
Based on toxicity data, cadmium appears to be taken up in direct correlation with pH.
However, lead exhibits exactly the opposite; lowering the pH increases uptake (Campbell
and Stokes, 1985).
The effects of water pH on gill function are made complicated by the fact that carbon
dioxide and ammonia are excreted by the gill which alters the microenvironment of the
gill lamellae. The net effect of these substances is to decrease the water pH if the inhalant
water has a pH of 6 or above while increasing the pH of the interlamellar water if the
inhalant water has a pH of less than 5 (Randall et al., 1991). Thus, the exhalant water will
tend not to vary in pH as much as the inhalant water (Playle et al., 1992). Still,
measurement of the exhalant water does not actually represent the true pH for the entire
length of a gill lamellum, because according to Randall et al. (1991) this changes in a
nearly linear fashion as it passes the lamellum. Thus, the epithelium of the lamellae is
exposed to a different pH at the two ends.
When rainbow trout were tested for aluminum accumulation on gill tissue at inspired
pHs of 5.1,4.7, or 4.1, Playle and Wood (1991) found accumulation greatest at an inspired
pH of 5.1 and lowest at 4.1. As expected, the expired pH was 0.2-0.7 pH units higher than
inspired pH. They also noted that only about 10% of the aluminum extracted from the
water by the gills remained bound there; most of the remainder was sloughed off by the
mucus.
Laboratory data on the effect of pH on the rate of uptake of methylmercury are not in
agreement. Drummond et al. (1974) found a faster rate of accumulation into the gills and
blood cells of brook trout at pH 6 than at pH 9. In contrast, Rogers et al. (1987) reported
that pH over the range of 5-7 had no effect on methylmercury accumulation by walleyes
and rainbow trout. Neither of these studies measured direct uptake by the gill so relative
rates of depuration and other factors such as food may have contributed to the differences.
Also, the pH range and concentration of mercury tested may be important variables.
Ponce and Bloom (1991) exposed rainbow trout fingerlings to a very low (1.38 ng/L)
concentration of methylmercury for 2 months. They used four different pH levels (5.8,
6.3, 7.0, 8.2) and found that uptake was the same in the upper three pHs. However, the
rate of uptake was approximately twice as fast at pH 5.8 as 6.3, which suggests a pH
threshold. Because many natural waters that are contaminated with methylmercury have
pH levels below 6, this finding may have considerable practical significance (Spry and
Wiener, 1991).
One pollutant may have a considerable effect on the uptake rate of another one. With
the aid of an isolated gill preparation. Part et al. (1985) tested the effect of the detergents
86

linear alkylaryl sulfonate (LAS) and nonylphenol ethoxy late (NP-IOEO) on the gill
uptake of cadmium. They found that LAS stimulated cadmium uptake, and because LAS
has a low affinity for the metal, the effect must be directly on the gill tissue instead of the
formation of a lipid soluble complex with LAS. The other detergent had no effect on
cadmium uptake, which indicates the mechanism is not merely due to a reduction of
surface tension. Instead, the authors suggest it involves an action of LAS on the proteins
of the gill epithelium.
In summary, we see that the particular metal in question and various environmental
factors can influence the rate of uptake of waterborne metals by the gills of fish. The
bioavailability will depend on the degree of complexation with ligands in the water (the
greater the tendency to form complexes the less the bioavailability), the microenviron­
ment of the gill lamellae of the gills, and changes in the gill epithelium.
Uptake of metals via the food can be quite important in nature. As a general rule,
invertebrates often accumulate higher levels of metals than do fish under similar condi­
tions (Waldichuck, 1974). Thus, predators on these invertebrates may obtain a consider­
able body burden from this source. Younger fish are reported to take up zinc via the food
more rapidly than older ones (Patrick and Loutit, 1978). This is to be expected as they
have higher rates of metabolism (per gram of tissue).
Phillips and Buhler (1978) exposed rainbow trout to methylmercury in the food, water,
or both and found the body burden increased linearly for at least 24 days irregardless of
the route of uptake, and methylmercury accumulated from one source had no effect on
the rate of uptake from the other. In other words, they were additive. The efficiency of
mercury removal from the medium was different, however. Approximately 70% of the
methylmercury ingested was absorbed whereas the gills took only 10% of the chemical
out of the water.
The concentration of mercury in the food can affect the assimilation efficiency by the
gut. Rogers and Beamish (1982) found a high assimilation efficiency for methylmercury
when the dose was low, but when the fish were fed a much higher dose of mercury in the
ration, the assimilation efficiency declined to less than 50%. This implied to the authors
a saturation of the uptake process in the gut and/or an induction of some sort of specific
block to the assimilation of methylmercury.
Handy (1993) recently tested the assimilation of aluminum in rainbow trout and found
a very low rate of less than 1%. In light of the study mentioned above on methylmercury
in which it was found that assimilation decreased with concentration in the food, it should
be noted that the aluminum study was done with very high concentrations. Thus, alumi­
num assimilation might be better at lower doses. Even so, it appears that aluminum is not
taken up by fish very well through either the gills or gut, but it does cause a considerable
effect on gill function (see Chapters 7 and 10).
Uptake via the drinking water effectively is no different than that via the food.
However, it must be realized that marine fish drink approximately 0.5% of their body
weight per hour, so a considerable amount of water enters the gut compared to the
freshwater fish, which drink very little water (see Chapter 7). Thus, dissolved substances
may enter by way of the gut to a much greater extent in marine fish, but careful studies
are needed to partition these routes of entry. Somero et al. (1977) reported that the
estuarine teleost Gillichthys mirabilis accumulates lead at a rate that is proportional to the
water salinity. This can be interpreted as being due to a greater drinking rate in saline
water.
There may be a distinct difference between marine and freshwater fish as to the
assimilation of dietary lead. According to Hodson et al. (1978) dietary lead is not
absorbed by rainbow trout, but more recently, Juedes and Thomas (1984) observed that
marine croakers can assimilate it.
 87

B. ORGANICS
As with metals, there is no evidence for active transport of organics across the gills into
the blood. In spite of this, some remarkably high uptake efficiencies have been found for
a few organics while for others, the gill tissue is nearly impermeable. There are several
physicochemical mechanisms that influence the movement of chemicals across biological
membranes. These include molecular weight, charge on the molecule, molecular volume,
concentration in the water, and lipid solubility. Furthermore, the bioavailability of organ­
ics is greatly reduced by the presence of various adsorbents (e.g., humic acids, sediments,
and suspended solids) (Spacie et al., 1982).
It has been shown (Veith et al., 1979) that the accumulation of organic chemicals is
correlated with their octanol/water partition coefficients (log K^^), which is a measure of
the lipid solubility of a chemical. However, more recent work indicates the relationship
is not linear because of the effect of blood and water flows. McKim and Erickson (1991)
summarized several studies of the direct uptake by gills of rainbow trout of 14 different
organic chemicals and found the relationship varies considerably with the log Kow
(Figure 5). The exchange coefficient represents uptake efficiency as was described above
for metals. Thus, the uptake efficiency was around 7% for those chemicals with log Kow
below 1. For those with coefficents between 1 and 3, there was an approximately direct
relationship between uptake efficiency and the log Kow, but then between log 3 and 6,
there was no further increase in uptake efficiency and a considerable decline in efficiency
above log Kow 6. Hansch and Clayton (1973) have suggested that the low fat solubility
of chemicals with low log Kow prevents their entry through membranes (except through
pores) while those with high lipid solubilities may become bound to the lipid membrane
and thus would be slow to move on into the blood.
It is interesting that the flux of organic chemicals in the opposite direction (i.e., from
blood to water) across the gill is also influenced by lipid solubility. However, those with
log Kow of 1-4 move more rapidly than those with log Kow of 4-6 (Thomas and Rice,
1981). This is, of course, the reverse of what was found with the inward movement.

CP

c
.S?
[o
%o
o
0)
CP
c
o
x:
oX
UJ

Log OctonoIrWoter Portition Coefficient

Figure 5 Relationship between exchange coefficient in the gill and log octanoliwater partition
coefficient of test chemicals. (Data from McKim, J. M. and Erickson, R. J., Physiol. Zoo!., 64, 39,
1991.)
88

s n E

Uu Mb
Figure 6 Diagram of gill rate-limiting bar­
riers to pollutant uptake. Vw = water flow
past lamellum; Vb = blood flow through
lamellum; S = aqueous stagnant layer; M
= mucus layer; and E = gill epithelium.
(Based on Hayton, W. L. and Barron,
W. G., Environ. Toxicol. Chem., 9, 151,
1990.)

Lipid solubility is only one of the factors that affect the transfer of chemicals across
the gill into the blood. Hayton and Barron (1990) have discussed several other barriers
that may be rate limiting. These include in addition to the epithelial cells the aqueous
stagnant layer next to the epithelium and the water and blood flows (Figure 6). Hayton
and Barron (1990) did not include the layer of mucus that usually covers the epithelium
(Hughes, 1979), which may have different diffusion properties than either the water or the
membranes. They make the point that, “In general, for any particular chemical, only one
of these barriers is operative with the resistance offered by the others being negligible”
(p. 151).
Hayton and Barron (1990) predicted that uptake efficiency of chemicals with low log
P would be limited by blood flow while those with high log P would be limited by water
flow over the gills. This was confirmed in an elegant experiment by Schmieder and Weber
(1992). Using a McKim and Goeden (1982) chamber they altered ventilation of the fish
and gill perfusion by changing the dissolved oxygen concentration. They found that the
uptake of a hydrophobic chemical (decanol) with high log P was ventilation limited
whereas the hydrophilic chemical butanol, with a low log P, was blood flow limited. The
mechanism for this distinction is probably that hydrophilic compounds will go primarily
into the aqueous phase of the blood and will thus quickly reach equilibrium with the blood
in the gill. Consequently, they become limited by blood flow. Compounds with higher log
P bind to blood proteins better so the limit then becomes the rate at which the water brings
the material to the “blood” at the gill epithelium (Schmieder and Weber, 1992).
It is interesting that endrin is taken up from the water at a higher efficiency than is
oxygen, and this is seemingly independent of the concentration of endrin in the ambient
water (Figure 7). McKim and Goeden (1982) further found the ventilation volume of their
fish was 65-75 ml/min for a 0.5-kg fish. Gill blood perfusion for a trout of that size is
approximately 8-9 ml/min (Daxboeck et al., 1982). Because the blood perfusion is
considerably less than the ventilation volume, the concentration of endrin in the blood
coming from the gills must be far greater than that in the water. This illustrates strikingly
the importance of blood proteins which maintain a large diffusion gradient by binding the
chemical and thereby taking it out of solution (Streit and Sire, 1993). This is, of course,
analogous to what hemoglobin does for oxygen. The binding of organic chemicals to the
blood proteins increases in proportion to Log Kow up to about 3 or 4. Above that point,
organic complexation in the water becomes progressively more important and this
reduces bioavailability of the chemical (McKim and Erickson, 1991).
Uptake efficiency of oxygen and endrin (an organochlorine insecticide) decreases with
increasing ventilation volume caused by hypoxia (Figure 7) (McKim and Goeden, 1982).
In spite of the decreased efficiency, the actual amount of endrin taken up per day rose as
 89

Figure 7 (a) Uptake efficiency of endrin and of oxygen in the gills of brook trout. Note that
endrin has a greater uptake efficiency than does oxygen, but efficiency of endrin uptake declines
more rapidly with increased ventilation volume produced by environmental hypoxia, (b) Effect of
dissolved oxygen concentration on the total endrin taken up by brook trout exposed to a high
(0.072 pg/L) and low (0.046 pg/L) endrin concentration. (Redrawn from McKim, J. M. and
Goeden, H., Comp. Biochem. Physiol., 72C, 65, 1982.)

the dissolved oxygen was decreased so the effective dose in the fish was higher during
hypoxia. This indicates that the uptake of endrin is water flow limited up to some
threshold, above which diffusion limitation becomes dominant.
The rate of oxygen consumption by a fish is profoundly affected by temperature with
QIO of 2 or higher common (Brett, 1972). Thus, the ventilation volume will also be
temperature dependent. So it is not surprising that Black et al. (1991) found that toxicant
uptake is directly related to temperature and correlates well with oxygen consumption.
The effect is not, however, explained by ventilation alone, because lower temperatures
resulted in declines in uptake efficiency. The direct effect of temperature on permeability
90

of gill membranes to hydrophobic compounds has been confirmed using isolated gill
preparations (Sijm et al., 1993).
Two other factors that can greatly affect uptake of organic xenobiotics are dissolved
organic matter (DOM) and pH. DOM complexes with metals (discussed above) and
organic pollutants. The binding affinity of an organic with high molecular weight DOM
greatly reduces the rate of passage through the gill tissues because of the large size of the
DOM molecules and the presence of polar groups on DOM. Thus, for some chemicals,
a DOM concentration of as little as 3 mg/L can nearly block uptake by the gills (Black
and McCarthy, 1988).
For most xenobiotic chemicals (if they are weak acids), increasing the pH decreases
the uptake because they become more ionized (McKim and Erickson, 1991). Ionized
substances have relatively little ability to cross epithelial membranes. The situation is
made more complex, however, by the changes in pH of the water as it passes through the
gills (discussed above) and shifts in spéciation of the xenobiotic compound in the
microenvironment of the gill lamellae. McKim and Erickson (1991) have rather success­
fully modeled the effects these various environmental and chemical factors have on
uptake rates.
Waterborne organics can certainly be taken up by the skin as well as the gills, but the
skin route is far more important in small fish (Liem and McKim, 1993). Tovell et al.
(1975) obtained evidence indicating that as much as 20% of the total detergent sodium
lauryl sulfate (SLS) accumulated from the water entered through the skin of adult
goldfish. Naphthalene accumulates in the skin of trout exposed to the compound for 24 h
(Varanasi et al., 1978). Part of this may have come from bloodbome naphthalene
absorbed via the gills, but the time course of changes in concentration suggests there may
be a significant amount of direct uptake by the skin from the environment. There is an
extensive microcirculation in the skin that is part of the fish’s secondary blood system
(Satchell, 1991), and these blood vessels might facilitate uptake there.
In general, chemicals up to a log Kow of 3 are mainly taken up by gills and skin. Those
with log Kow 3-6 are taken up by both gill and gut and those above log Kow 6 are probably
all gut uptake (James McKim, personal communication).
Because of the great diversity of morphology evident in fish gastrointestinal tracts,
there is a great deal to learn about the mechanisms involved in xenobiotic uptake across
the intestinal wall. For example, many fish lack a stomach and intestinal lengths vary
considerably in different species, as does the presence or absence of pyloric ceca (Fange
and Grove, 1979).
Van Veld (1990) has reviewed the processes involved in absorption of xenobiotics
from the digestive tracts of fish. The proximal portion of the intestine is where most of
the food absorption occurs and the pyloric ceca, if present, add absorptive surface area.
Lipophilic toxicants are assimilated in much the same way as dietary fat. The lipids (and
toxicants) are first digested by pancreatic lipase and bile salts yielding a fine suspension
of micelles which are colloidal particles of fatty elements clustered together with bile salts
in such a way as to facilitate their diffusion through the aqueous phase. The presence and
digestion of dietary lipids actually facilitates the accumulation of toxicants (Van Veld,
1990), perhaps by stimulation of bile and/or lipase secretion. Passage into the intestinal
enterocytes occurs presumably by diffusion of the fatty materials from the micelle, but
absorption of some materials such as fatty acids may be facilitated by a protein carrier.
Following absorption into the enterocyte, the fat digestion products are used to
resynthesize triglycerides. They then enter the circulation in the form of low-density
lipoproteins (Sire et al., 1981).
Compounds with molecular weights above 600 are poorly absorbed by the digestive
tract, but because most organic contaminants in waterways have molecular weights less
than this, that does not appear to be a problem. Octanol-water partition coefficients (log
 91

Kow) of chemicals do not appear to affect their absorption by the gut. Rather, the
triglyceride solubility seems to show a good correlation with absorption efficiency. The
absorption of hydrophobic organochlorines appears to be by simple diffusion rather than
lipid cotransport (Gobas et al., 1993). Overall, it is common for lipophilic toxicants to
have assimilation efficiencies in fish of 50% or more (Van Veld, 1990).
Assimilation efficiency can be influenced by the amount of food in the diet. When
guppies were fed four different chlorinated hydrocarbons in the food, the rate of accumu­
lation was proportional to the concentration of xenobiotic in the food (i.e., assimilation
efficiency was constant) (Clark and Mackay, 1991). However, if the “dose” was increased
by feeding more contaminated food (keeping the concentration in food constant), the rate
of uptake was not proportional to dose. This suggested the assimilation efficiency was
reduced by increasing the amount of food, some of which apparently was egested. The
authors note that changing the fat content of the food might also have interesting effects
on assimilation efficiency, a point well taken in light of the observations mentioned above
relative to the parallel digestion of lipophilic xenobiotics and lipids.

III. TRANSPORT WITHIN THE FISH OF METALS AND ORGANICS

Most if not all metals are probably carried by the blood bound to protein (Roesijadi and
Robinson, 1994). For example, the plasma protein ceruloplasmin binds copper in mam­
mals. Copper and zinc-binding proteins have also been found in fish (Fletcher and
Fletcher, 1980). There may be a different protein for each essential trace metal, and
presumably non-essential metals use one of the already existing proteins. Some metals
may also bind to amino acids (Stagg and Shuttle worth, 1982). Methylmercury is rather
unique in that it binds to the protein of hemoglobin inside the erythrocytes (Massaro,
1974). What effect, if any, the mercury has on hemoglobin function is apparently
unknown.
Insecticides such as dieldrin, carbaryl, and parathion bind to both albumin and lipo­
proteins in the plasma (Denison and Yarbrough, 1985). By having a chemical bind
loosely to a protein, it effectively removes it from the blood side of the gill, thus
maintaining a water-blood diffusion gradient even when the ambient concentration is
quite low. As was mentioned in Section II, this is a very important mechanism for
bioaccumulation of both inorganic and organic substances from the water environment.
The importance of blood proteins in transport and accumulation of lipophilic contami­
nants has recently been modeled by Streit and Sire (1993).
Organic contaminants that have been absorbed by the intestine are also largely
associated with plasma albumin and lipoproteins, however, the situation is a bit more
complex. The intestinal cells have the capability of metabolizing many organic sub­
stances so the products of that metabolism may be what are actually entering the
circulation. For example, hydroxylated metabolites of polyaromatic hydrocarbons (PAH)
bind primarily to albumin whereas the unmetabolized PAH associate more with plasma
lipoprotein (Van Veld, 1990). Based on work largely done in mammals, this partitioning
of xenobiotics in the plasma would appear to have an effect on the subsequent uptake by
tissues. Endothelial cells in certain organs (e.g., liver) have receptors for the albumin-
ligand complex so they are preferentially absorbed in these organs. Lipoproteins, on the
other hand, merely diffuse into endothelial cells of essentially all tissues and thus will
exhibit less specificity.

IV. ACCUMULATION OF METALS IN DIFFERENT ORGANS

As mentioned earlier, the biological concentration factor is the ratio of the concentration
of a chemical inside an organ (or whole animal) to the environmental concentration. For
92

Figure 8 Uptake of mercury in the muscle and liver of cod fed methylmercury and the effect
of a subsequent depletion period. (From Julshamm, K., Ringdal, O. and Braekkan, O. R., Bull.
Environ. Contam. Toxicol., 29, 544, 1982. With permission.)

nearly all metals, this is a positive number (Waldichuck, 1974) because of protein binding
in the tissues. The concentration factor in a given tissue changes over time of exposure
so it is not an absolute value for any metal or organ. For example, the data illustrated in
Figure 8 show how the methylmercury in the food of the fish moved first into the liver
and more gradually into the muscle. After day 32 in the study illustrated, the fish were
fed non-contaminated food, but note how the concentration continued to rise in the
muscle. The authors (Julshamm et al., 1982) suggest this was due to movement of the
mercury from the liver to the muscle. Similar time-course changes in the ratio of
liver:muscle mercury concentrations were reported by Olson et al. (1978) in rainbow
trout. Clearly, one would observe a different concentration factor for these two tissues
depending on when the measurements were made. Massaro (1974) reported that 50% of
the total mercury dose given intragastrically to trout was in the muscle when sampled at
100 days, even though higher concentrations were found in organs having large blood
volumes, such as the spleen and kidney. This points out the importance of the relatively
large muscle mass for sequestering some pollutant chemicals, such as methylmercury.
Mercury in tissues from wild-caught fish is mostly in the methylated form. Fish cannot
methylate mercury directly, although bacteria in the gut can (Rudd et al., 1980), and there
have been suggestions that slow déméthylation may occur (see Bryan, 1979 and Rogers
and Beamish, 1982 for review). While méthylation in the gut would make inorganic
mercury more bioavailable, according to Julshamm et al. (1982), dietary methylmercury
is accumulated about 10 times more readily than dietary inorganic mercury into the
tissues. Also, inorganic mercury, if taken up, is eliminated from the fish more rapidly than
is methylmercury (Spry and Wiener, 1991).
Histochemical observation of liver and kidney shows that methylmercury accumulates
mostly in the glomeruli of the kidney and in the nucleus and endoplasmic reticulum of liver
cells (Baatrup et al., 1986). Because a number of marine teleost species have kidneys which
lack glomeruli, it would be interesting to see where the mercury accumulates in them.
Mercury reaches fairly high concentrations in large predacious fish. This is due to a
combination of biomagnification up the food chain and old age (i.e., they have longer to
acquire a body burden). Moreover, fish eliminate methylmercury very slowly compared
 93

to uptake rate (McKim et al., 1976). Overt toxicity probably does not occur until whole-
body concentrations exceed 10-30 pg/g wet weight (Spry and Wiener, 1991).
Depending on the species, cadmium can differ considerably from mercury in its
distribution within the body of a fish. In contrast to mercury, neither the skeletal muscle
or brain of spot {Leiostomus xanthurus) accumulated cadmium (Hawkins et al., 1980).
Exposure to waterborne cadmium for 48 h yielded the highest accumulation in liver,
followed by gut, kidney, and gill in that order. The spot is a marine species so it is
interesting to compare it with rainbow trout which accumulated 99% of its total body
burden of cadmium in liver, kidney, and gills (Thomas et al., 1983). There was a virtual
absence of cadmium in the gut of the trout. Its high concentration in the gut of the spot
shows the importance of the marine environment, where the fish must drink rather large
volumes of water to replace that lost osmotically.
More recently, Harrison and Klaverkamp (1989) assessed the accumulation of cad­
mium in various tissues in rainbow trout and lake whitefish {Coregonus clupeaformis)
exposed to either waterborne or foodborne cadmium. Figure 9 illustrates the dynamics
that occurred during the 72-day exposures and subsequent 56-day depuration. Most
notable is the tendency for gills to accumulate the most cadmium, even in those exposed
to cadmium via the food. Gut and kidney also accumulate considerable amounts of the
metal when it is in the food, but not when in the water. Finally, depuration appears most
rapid for gut and gill. The kidney, however, shows little depuration of cadmium, perhaps
due to transfer of cadmium from other tissues to the kidney. The percentage of the dose
accumulated was almost 10-fold greater from the food than from the water.
Rainbow trout are considerably more sensitive to cadmium than are roach (Rutilus
rutilus) and stone loach {Noemacheilus barbatulus), as reflected in lower LC50 values for
the trout. Norey et al., (1990) showed that this difference is correlated with the relative
rate of accumulation of cadmium into tissues. As has been repeatedly emphasized in this
book, excretion could in part account for this difference. Thus, it is interesting that they
showed that there was no species difference in their depuration rates. Indeed, over a
period of 132-170 days in cadmium-free water, there was little loss of cadmium in any
of the three species tested, so the differences in toxicity must be related to uptake rate and/
or differences in sensitivity of target organ.
The metal chromium was not accumulated above the ambient concentration in trout
following exposure for 22 days (Buhler et al., 1977). However, when the exposure was
prolonged for months, some bioaccumulation of the element occurred. The authors
suggest there exists a “fast-turnover pool” (body fluids) for short-term exposures and a
“slow-turnover pool” for chronic exposures. The latter may involve synthesis of a
metallothionein-type of protein for binding. In spite of this, it appears that chromium has
relatively little bioaccumulation potental in trout. Other fish species, however, such as
bluegill (Lepomis macrochirus) can accumulate chromium quite well (Freeman, 1980).
The distribution of lead into various organs appears to vary greatly depending on the
fish species. According to Spry and Wiener, 1991), lead accumulates in freshwater fish
mostly in scales, bone, kidney, gill, and liver. The order of tendency (from greatest to
least) for accumulation of lead in the organs of an estuarine fish was spleen, gill, fin, and
intestine. Surprisingly, liver and muscle accumulated almost no lead (Somero, et al.,
1977). This can be compared with the order of lead residual concentration found in brook
trout after exposures of 2-38 weeks (Holcombe et al., 1976). In this species, the order was
kidney, gill, liver, and spleen with virtually none in the muscle (intestine, scales, and bone
were not measured). Binding to mucus may be an especially important mechanism for
lead accumulation and subsequent depuration (Somero et al., 1977) and gill and intestine
have an abundance of this material.
The above-mentioned studies all conclude that muscle does not accumulate lead, thus
the findings of Kumar and Mathur (1991) are interesting. They exposed the Indian
94

(a) Cd-109 IN GILL (b ) Cd-109 IN GUT

(C) Cd-109 IN LIVER (d) Cd-109 IN KIDNEY

DAYS

Figure 9 Cadmium concentrations (pg/g wet wt.) in gill, gut, liver, and kidney tissue on each
sample day for food (30.7 |ig/kg) and water (1.25 ng/L) exposed rainbow trout and whitefish.
Vertical dashed line marks end of exposure period. Note that the scale on the Y axis is different
for each tissue. (From Harrison, S. E. and Klaverkamp, J. F., Environ. Toxicol. Chem., 8, 87,
1989. With permission.)

freshwater teleost Colisa fasciatus to lead for up to 24 days and measured lead accumu­
lation in gill, liver, and muscle. Surprisingly, gill and muscle both exhibited approxi­
mately the same bioconcentration factor (7-8) while liver had a factor of 0.9. Unfortu­
nately, no other organs were measured nor do the authors seem to be aware of the
contradiction with earlier studies.
When bluegill were exposed to 0.5 ppm of waterborne lead, chromium, or cadmium
for 7 days the gills had higher concentrations of all three metals than did the liver
(Freeman, 1980). Moreover, the differences were large (i.e., order of magnitude greater).
In gills, the chromium and lead reached higher concentrations than did cadmium, whereas
all three metals were about the same in liver.
With copper, the liver has the highest concentration factor for almost any exposure
time (Brungs et al., 1973; Buckley et al., 1982; Felts and Heath, 1984; Stagg and
Shuttle worth, 1982). However, Felts and Heath (1984) observed that when bluegill
sunfish were exposed to a sublethal level of copper, only the gills exhibited significant
increases at 3 days. By 7 days of exposure, the liver exhibited large increases in copper
 95

concentration. At least some of the elevated copper concentration in the gills could be due
to the element complexing with the mucus, which is impossible to completely remove
from between the lamellae before the tissue is prepared for analysis. This, incidentally,
is a point that should be appreciated when doing analysis of any substance in the gill tissue
and may help explain seemingly high concentrations of various metals there.
In a time-course investigation involving chronic exposure of coho salmon to copper,
the plasma copper rose on day one, but when the fish were sampled at 2 weeks and longer
times, it was not elevated. During this time the liver copper was steadily increasing
(Buckley et al., 1982). Thus, it appears the copper was being rapidly removed from the
plasma by the liver and/or there was a reduction in the uptake rate by the gills during the
first few days of copper exposure. If this phenomenon can be verified, it could be quite
significant as it would provide a mechanism for regulation of copper accumulation other
than excretion.
Accumulation of copper by liver can be greatly influenced by the nutritional level of
the experimental fish. When fed and starved yearling roach (R. rutilus) were exposed to
waterborne copper for 7 days, only the starved fish accumulated copper in the liver
(Segner, 1987). Because one of the major functions of bile is digestion, it is suggested that
starved fish were unable to dump copper from the liver into the bile due to reduced bile
production.
In contrast to some other metals, present evidence suggests there is relatively little
tendency for copper to accumulate in the brain (Felts and Heath, 1984) or kidney (Brungs
et al., 1973; Buckley et al., 1982) of fish.
Zinc shows the greatest bioconcentration factor in skin and bone (Mount, 1964),
although liver, gill, and kidney also accumulate it to a considerable extent (Holcombe et
al., 1979). The ratio of the concentration of zinc in the gill to bone (actually opercular
flap) has been proposed as an autopsy technique for zinc-caused mortality (Mount, 1964).
Dietary zinc may show a different distribution pattern from the waterborne metal.
Hardy et al. (1987) gave rainbow trout a single feeding of isotopic zinc and measured
levels in various organs and tissues after 72 h. Blood concentrations of zinc were higher
than that of gill, liver, kidney, and spleen, whereas all other tissues (including bone) had
lower concentrations than did the blood.
When pregnant guppies {Foecilia reticulata) are exposed to waterborne zinc they take
it up and apparently actively transfer it to the developing embryo in utero. This results
in fry with high body zinc concentrations. As the fry grow, the levels of body zinc decline
(Pierson, 1981). Several other pollutants have been found to transfer from female fish into
developing oocytes and be deposited with the eggs (see Chapter 13).
The largest concentration factor for selenium is in the liver following chronic waterborne
exposure (Hodson et al., 1980). This element also accumulates to some extent in the
digestive organs and kidney. Dietary selenium is more toxic than the waterborne metal
and Hodson (1988) has suggested this is related to its metabolism by the fish. Waterborne
uptake and excretion is dependent exclusively on diffusion whereas dietary selenium is
accumulated independent of dietary concentration. Also, at high dietary concentrations,
the half-life for excretion decreases so more is retained. This perhaps illustrates a
saturation of excretory pathways.
Tributyltin, sometimes called organotin, accumulates rapidly from the water in
marine fish. Davies and McKie (1987) exposed Atlantic salmon to doses of tributyltin
for 26 days and reported that those receiving the 1.0 pg/L dose had organ concentra­
tions ranging from 0.24 mg/kg in ceca to 1.62 mg/kg in liver. Muscle, gonad, and gill
were only slightly higher in concentration than ceca, so this compound clearly shows
an affinity for liver.
A factor that can have a considerable impact on total body burden of a metal is the size
of the fish. Most metabolic processes in animals are inversely proportional to body size
96

so weight-specific rate functions generally exhibit an exponent of 0.75 (Schmidt-Nielsen,

1984) (i.e., small animals of the same species operate at a higher rate than larger ones).
Not surprisingly, mosquitofish (Gambusia) show decreasing zinc accumulation with
increasing body size. Small fish also depurated zinc more rapidly (Newman and Mitz,
1988).
All of the above observations on accumulation of metals actually are viewing the result
of both uptake and excretion. This is dramatically seen in the study of Eisler and Gardner
(1973) where they noted that dead fish accumulated more copper and zinc than live ones
when exposed to the waterborne elements for 48 h. The dead fish, of course, could not
excrete metals, and evidently the skin is remarkably permeable to them. (Another possible
explanation is that the metals adsorbed to the mucus on the dead fish, whereas live fish
would slough off this material.)

V. REGULATION OF METAL CONCENTRATION

By regulation, we are actually referring primarily to the ability to excrete a metal. The
rate of uptake of essential trace elements, such as iron, copper, and zinc may be
controlled, although the mechanisms involved in fish are largely unknown. Acute levels
of several different metals (e.g., cadmium, lead, zinc, copper) stimulate mucus secre­
tion. This could serve as a way of reducing uptake rate by chelation of metals and
inhibition of diffusion (Miller and MacKay, 1982; Handy and Eddy, 1990; Part and
Lock, 1983). During chronic exposures there is little or no mucus accumulation so this
variable declines in importance.
The term depuration is frequently used to refer to the ridding from the body of the fish
of some chemical after the animal is put in non-contaminated water, or after it has been
fed the test chemical. Different tissues depurate at different rates. For example. Figure 8
shows the slow rate at which the muscle lost mercury compared to liver. Whole-body
depuration rate is inversely proportional to body size so small fish are able to do this more
rapidly than large ones (Sharpe et al., 1977; Newman and Mitz, 1988).
Fish have different routes for possible excretion of harmful chemicals from mammals;
these include the gills, bile (via feces), kidney, and skin. When the metal is present in the
water, depending on the concentration, gills and skin may tend to take it up rather than
excrete it.
Fish can excrete, at least to some extent, copper, chromium, cadmium, mercury,
selenium, arsenic, lead, and zinc (Bryan, 1976; Hodson et al., 1980; Hardy et al., 1987;
Rogers and Beamish, 1982; Somero et al., 1977; Sorenson et al., 1979). Under continuous
exposure to a waterborne chemical, the excretory processes may take several hours or
days to become activated. Thus, the body burden can rise rapidly and then actually decline
somewhat with continued constant exposure.
The mechanisms of metal excretion in fish are only beginning to be understood. The
liver is the main organ for metal homeostasis in mammals (Klassen, 1976) and at least
to some extent seems to serve a similar function in fish. In mammals, many metals are
excreted via the bile which is formed by the liver (Klassen, 1976). Several metals have
been found at elevated concentrations in the bile of fish during or following their
ingestion or waterborne exposure. These include chromium (Buhler et al., 1977), arsenic
(Sorenson et al., 1979), triethyltin (Stroganov et al., 1973), and copper (Felts and Heath,
1984). In time-course measurements on bluegill exposed to low doses of copper (Felts
and Heath, 1984), it was found that the concentration of copper rose first in the bile (i.e.,
in 3 days) and then in the liver at 9 days. An interpretation of this is that the liver of the
bluegill picked up copper from the blood and immediately “dumped” it into the bile.
When the amount of copper exceeded the ability to empty it into the bile, it then began
to store it in the liver. It appears that mercury (Massaro, 1974) and zinc (Hardy et al..
 97

1987) are excreted to a small extent in fish bile. Evidence presented by Hardy et al. (1987)
for dietary zinc implicates the gill as the major excretory route for zinc.
Excretion of a metal in the bile does not necessarily rid the body of the metal in
question. The bile flows from the gallbladder through the bile duct into the anterior end
of the intestine where it then mixes with the foodstuffs that are being digested. In
mammals, there is often a significant uptake of the bile constituents, including harmful
chemicals, back into the blood from the intestine (Klassen, 1976). This process is referred
to as enterohepatic circulation because a chemical could, theoretically, be recycled more
than once. How important this enterohepatic circulation is in fish is unknown. The marked
diversity of anatomical and functional features of teleost digestive tracts (Fange and
Grove, 1979) suggests that it may vary considerably between different species.
The excretion of metals via the urine of teleost fish has received little attention,
although it is generally assumed that the kidney excretes some metals (Reichert et al.,
1979; Rogers and Beamish, 1982), but apparently not dietary zinc (Hardy et al., 1987).
As mentioned above, the kidney frequently accumulates metals to rather high concentra­
tions, but this may be merely a location for sequestering them and does not necessarily
indicate excretion. Reichert et al. (1979) report (p. 233) that “Salmonids exhibit a strong
tendency to retain lead and cadmium in the kidney for at least a month after the
termination of exposure.”
The structure and function of marine and freshwater fish kidneys differ considerably.
Many of the former lack glomeruli so must function exclusively as a secretory kidney
(Smith, 1982) and probably would have little ability to excrete metals via this route. Thus
the capability for excretion of metals in the urine may differ depending on whether it is
a marine or freshwater species.
There is some evidence that methylmercury must be demethylated before excretion,
and a major mode of exit from the body may be the gills (Olson et al., 1978), as has
already been mentioned for zinc. Rates of methylmercury elimination increase with
continued feeding of the material in the diet. Rogers and Beamish (1982) suggest this may
indicate an induction of enzymes involved in this process.
Exposure to waterborne zinc, copper, or cadmium has been shown to stimulate the
development of more chloride cells in the gills (Baker, 1969; Matthieson and Brafield,
1973; Oronsaye and Brafield, 1984). The response is quite rapid, requiring only a few
hours of exposure to the element. Oronsaye and Brafield (1984) speculate these cells are
excreting the metals, although they also recognize this could be an osmoregulatory
response, which seems more likely (see Chapter 7).
With the use of a Van Dam chamber for the test fish, Oladimeji et al. (1984) found that
40% of an ingested dose of arsenic was excreted via the gills in 7 days. Urinary excretion
in this time accounted for 15% of the dose. The authors suggest that with further clearance
time, the urine would account for only an additional 5% of the total taken in. Presumably
the remainder would be partitioned between gills and bile.
Loss of metals via the skin and gills probably involves the mucus. This proteinaceous
material is constantly secreted and sloughed off by these tissues. Significant amounts of
lead and cadmium which have been injected into the fish subsequently appear in the skin
mucus (Varanasi and Markey, 1978). This suggests that metals taken in by the food may
be excreted by this route, among others.

VI. GLUTATHIONE AND METAL DETOXIFICATION

The tripeptide glutathione performs a variety of critical functions in cells, including
metabolism of peroxides and free radicals. It plays a key role in the detoxification of
organic xenobiotics in a wide variety of animals (see Section IX). Because of its sulfhy-
dryl groups, it also binds to heavy metals (Luckey and Venugpal, 1976) and is involved
98

in excretion of these via the bile in mammals (Ellinder and Pannone, 1979). As will be
seen below, however, such a generalization cannot be made for fish.
Evidence now exists for the involvement of glutathione in the metabolism of metals
by fish. Work in Peter Thomas’ laboratory on two marine species has shown that chronic
exposures to lead, cadmium, and mercury cause increases in hepatic glutathione concen­
trations (Thomas and Juedes, 1992). Figure 10 illustrates this in mullet exposed to
cadmium. Clearly, there was a dose- and time-dependent relationship. Similar changes
occurred in the posterior kidney, but there were no significant alterations in glutathione
concentration in the brain of the fish, possibly because the blood-brain barrier prevented
much metal accumulation there. Measured concentrations of cadmium in the tissues of
the mullet correlated with those of glutathione. A similar relationship between metal
accumulation and glutathione increase was seen with lead exposure.

Expotur« TImt (wtPkt)

Figure 10 Hepatic glutathione concentration in mullett (Mugil cephalus) exposed to 0, 0.1, or

10 mg/L cadmium for up to six weeks. (Vertical bars = SEM, N = 6 for each point.) Asterisks
denote means significantly different from controls at alpha = 0.05. (From Thomas, P. and
Wofford, H., in Physiological Mechanisms of Marine Pollutant Toxicity, Vernberg, W. B., Calabrese,
A., Thurberg, F. P. and Vernberg, F. J., Eds., Academic Press, New York, 1982, 109. With
permission.)
 99

Thomas and Juedes (1992) have shown that for lead, at least, the increase in glu­
tathione is due to a stimulation of synthesis, rather than a decrease in utilization of the
tripeptide. Normally, glutathione acts in a negative feedback manner on its own synthesis,
so the elevations in rate of synthesis caused by xenobiotics may reflect a change in
setpoint (Thomas and Juedes, 1992).
Although lead and glutathione have been found to accumulate in the cytosol of liver
cells of fish, the lead did not bind to the glutathione, as it does in mammals. Instead, the
elevation in glutathione may help protect the cell from lipid peroxidation and aid in
maintenance of mitochondrial calcium content (see Thomas and Juedes, 1992, for re­
view).
A highly acute dose of waterborne cadmium can produce a depression in glutathione.
Chatterjee and Bhattacharya (1984) exposed climbing perch to a concentration of 166 mg/L
and found that within a day the liver glutathione in these fish dropped by 33% and stayed
at that level on day 2; but when allowed to recover in non-treated water, the glutathione
concentration was elevated by 50% above controls in 15 days. In this study, one of the
enzymes (glutathione-5-transferase) that utilizes glutathione as a substrate was assayed.
By day 2 of exposure the activity of this enzyme had increased 3.5-fold. This may help
explain the marked depression in glutathione observed as the transferase may be “using”
the glutathione in a conjugation reaction.
The involvement of glutathione in the metabolism of mercury is somewhat unclear.
Increases in kidney and liver glutathione from mercury exposure have been reported
(Thomas and Wofford, 1984; Heisinger and Wait, 1989). However, in a combined
laboratory and field study, Bidwell and Heath (1993) found a severe decrease in liver
glutathione in rock bass (a freshwater teleost) with exposure to either methylmercury or
inorganic mercury. A large amount of mercury was found in the bile which suggests the
glutathione may have been involved in that excretory process. Ballatori and Boyer (1986)
found that two different elasmobranchs handled mercury rather differently. The skate
excreted the mercury in the bile largely bound to glutathione whereas the dogfish shark
excreted it much more slowly and with less involvement of glutathione. Thus, there may
be some significant species differences in how various fish utilize glutathione in the
metabolism of mercury.
Finally, mention should be made of the distribution of glutathione in tissues other than
liver. Recently, it was found that levels of glutathione in gills and kidney of channel
catfish {Ictalurus punctatus) were about half that of the liver, indicating considerable
detoxification capacity in these extrahepatic tissues (Gallagher and DiGiulo, 1992). The
activity of the associated enzyme glutathione-5-transferase paralleled the levels of glu­
tathione. However, there may be considerable species differences in the distribution of
glutathione and its associated enzymes. Leaver et al. (1992) measured glutathione-5-
transferase activity in various tissues of the marine flatfish, the plaice (Pleuronectes
platessd). In this species, the gills had only 1% of the liver-specific activity of this enzyme
and the kidney had 6%. These low levels would suggest that the gills and kidney of this
species might be less able to handle contaminants than was seen in the catfish.

VII. INVOLVEMENT OF METALLOTHIONEIN IN METAL

ACCUMULATION AND ACCLIMATION TO METALS
Metallothioneins (MTs) are low molecular weight polypeptides (sometimes called pro­
teins) with many sulfhydryl groups due to the large amount of cysteine in the molecule.
MTs have been found in a very wide variety of organisms including prokaryotes, plants,
and animals. At least 34 species of elasmobranchs and teleosts have been reported to have
them (Roesijadi, 1992). Because of the numerous sulfhydryl groups in the molecule, MTs
100

bind a wide variety of metals, both essential and non-essential. For the essential metals,
copper and zinc, MTs sequester the metals or donate them for biochemical reactions
depending on demand for and concentration of a metal in the cell. Non-essential metals,
such as mercury, are sequestered by MTs thereby reducing toxic interactions with other
cellular proteins. The “spill over” hypothesis of Brown and Parsons (1978) proposed the
idea that if the capacity of MTs to sequester a toxic metal is exceeded, then the metal will
spill over and bind to other cellular proteins such as enzymes and produce toxic effects.
It is possibly an oversimplified view of the kinetics of cellular toxicity, but it still retains
considerable support, especially where exposures are chronic (Brown et al., 1990; see
Roch and McCarter, 1984a). In fish, MTs have been found in gills, intestine, kidney, and
liver with the latter receiving the bulk of attention.
MTs exist generally in a metal-saturated form (Hodson, 1988). Thus, in order for them
to detoxify additional metal, there must be synthesis of additional MTs (or other metal­
binding proteins) or displacement of one metal by another. All three of these mechanisms
come into play at times.
It is well established that a variety of metals induce the synthesis of additional MTs
(Hamer, 1986). It has also been found that acclimation to low levels of zinc (Hobson and
Birge, 1989), copper, (Buckley et al., 1982; Dixon and Sprague, 1981), or cadmium
(Klavercamp and Duncan, 1987) can result in elevated LC50 values (reduced toxicity) for
these metals. This increased tolerance has been associated with elevated levels of MTs
in one or more tissues. Elevated levels of MTs in gills or intestine may reduce the rate
of metal uptake into the blood (Petering et al., 1990), although the major mechanism of
increased tolerance is presumed to be the enhanced ability to sequester the accumulated
metal. Since a primary target organ from acute exposure of several metals is gill, the MTs
there may aid the increased tolerance. As Lauren and McDonald (1987) have pointed out,
for acute toxicity, there would be little benefit in protecting the liver with increased MTs
as there is no evidence that it is harmed (by copper at least) at these doses.
The ability of fish to acclimate to a metal varies with the particular metal. Chapman
(1985) reviewed the literature and ranked them in decreasing order: Zn > Cu > Cd > Cr.
This is the reverse of their binding affinities to MTs which is: Hg > Cd > Cu > Zn (Eaton,
1985), so the relationship of MTs to acclimation may not be as obvious as it appears. Roch
and McCarter (1984b) found elevated levels of MTs in field-exposed fish that did not
translate into higher metal tolerance. Part of the uncertainty regarding the relationship of
MTs to metal acclimation may be due to the fact that there are several other metal-binding
proteins in cells that may act in a manner similar to MTs. Hodson (1988) has emphasized
that the role of MTs will not be understood until we better understand these other proteins.
Mercury has an especially strong affinity for MTs, so much so that it displaces copper
and zinc from the molecule. According to Brown and Parsons (1978), this has the effect
of decreasing the normal tissue concentrations of these two essential elements when
salmon are chronically exposed to mercuric chloride.
In contrast to inorganic mercury, methylmercury apparently does not bind to MTs
(Weis, 1984), although it may bind to other cellular proteins that could provide some
capacity for sequestering it. Thus the form of the mercury to which a fish is exposed could
have a considerable bearing on the metabolism of other trace elements.
The kinetics of MT formation has been investigated by McCarter and Roch (1984).
With continuous copper exposure, hepatic MT rose steadily and then stabilized at 4 weeks
at a level dependent on the copper concentration in the water. Thus, the rate of MT
synthesis was controlled by the rate of copper uptake. Transfer of the acclimated fish to
water not contaminated with copper resulted in no significant loss in MTs after 4 weeks,
so once it is formed, MTs appear rather stable.
There is a widely held assumption that metals, in general, induce MT synthesis.
Evidently, however, not all metals are capable of doing this. Lead has been shown to not
 101

induce MTs when fed orally for a week to croakers, even though glutathione was induced
in the liver (Juedes and Thomas, 1984).
There has been considerable interest in using MT induction as a biomarker of metal
stress. The reasoning is that induction would indicate actual metal bioavailability, al­
though measurements of the concentration of individual metals in the tissues might do
essentially the same thing with less analytical effort. Also, in the field numerous stresses
that are not metal related can cause alterations in MTs. Thus, for fish it is generally
considered premature to utilize MT induction as a biomarker for metal contamination
until their normal physiological functions and controls are better understood (Stegeman
et al., 1992).

VIII. BIOCONCENTRATION OF ORGANIC POLLUTANTS

Very broadly speaking, the organic pollutants of concern for fish physiology fall into the
following groups: PAHs (mostly associated with petroleum), polychlorinated dibenzo-p-
dioxins, dibenzofurans, polyhalogenated biphenyls, and various insecticides and herbi­
cides.
As was mentioned at the beginning of this chapter, the route of uptake of a specific
chemical is going to depend on whether it is primarily in the food, dissolved in the water,
or both. Many organic pollutants that happen to be in the food (e.g., DDT, PCBs) are
taken up in direct proportion to the dietary level, which will depend on the quantity of
food consumed and the concentration in the food (Spigarelli et al., 1983; Hilton et al.,
1983). Factors that influence the metabolic rate of the fish, such as temperature or
simultaneous exposure to other pollutants (see Chapter 8), will alter the food consumption
and thereby the rate of accumulation of an organic contaminant.
During the 1970s, models were developed to approximate the bioconcentration factor
of organic chemicals that are dissolved in the water (Ernst, 1977; Krzeminski et al., 1977;
Vieth et al., 1979). These are based on the assumption that uptake and elimination rates
can be estimated by first-order kinetic expressions (i.e., they are in some manner propor­
tional to the concentration of the chemical in the environment and that in the fish). To
quote Veith et al. (1979, p. 1041): “Measurements of BCF can be made by exposing
organisms for a period sufficiently long that the steady state residue is observed, or
relying on the accuracy of a given kinetic model to measure appropriate rate constants and
calculate the BCF assuming the chemical behaved according to the model.” They go on
to make the point that the first method requires that the fish be small and the BCF must
not be too large, otherwise the time to reach equilibrium in a laboratory regime becomes
excessive. The kinetic model, however, can yield misleading results as uptake and/or
depuration rates may change during the exposure. As will be seen later in this section,
biotransformation processes are inducible so it is to be expected that depuration will
indeed change over time of exposure.
The bioconcentration factor for 30 common organic chemicals was determined in
fathead minnows by exposing them for 32 days in the laboratory (Vieth et al., 1979). The
BCF ranged from 2.7 to 194,000. Part of the aim of this study was to determine if the BCF
was related to the A?-octanol/water partition coefficient (P), which is a measure of the lipid
solubility of a molecule. It is well known that permeability of cell membranes by organic
molecules is highly dependent on their lipid solubility. With some exceptions, the BCF
was related to the partition coefficient.
More recently, however, Nichols et al. (1991; p. 388) “...have attempted with only
limited success to relate chemical log Kow to chemical accumulation in blood and tissues.”
They go on to remark that other information such as triglyceride partitioning or the
physical and chemical properties of molar volume, dipolarity/polarizability, and hydro­
gen bonding might be better for predicting partitioning in various tissues.
102

Laboratory derived estimates of residue accumulation from water neglect uptake via
the food, so they may greatly underestimate what would be found in fish taken from the
natural environment. They should also be looked upon as relative, rather than absolute,
as a host of things influence the accumulation of organics. These include size of fish, lipid
stores, temperature, age of the fish, and the processes of biotransformation and excretion.
Limited data (Vieth et al., 1979) suggest that within a species, age and body size has
little effect on BCF. However, an increase in temperature increases the BCF approxi­
mately twofold for each 10°C difference, and an oscillating temperature causes more
uptake than one which is constant. Spigarelli et al. (1983) exposed brown trout (Salmo
truttd) to PCBs from natural food and water under three thermal regimes: (1) a daily
temperature cycle ranging from 7-18 with a mean of 12.5°C; (2) a constant temperature
of 12.9°C; and (3) natural fluctuations of ambient inshore temperatures (4 °-ll‘^C) with a
mean of 7.7°C. Direct uptake from the water accounted for only about 10% of the total
PCB load, so most came from the food. The greatest PCB accumulation occurred in fish
from thermal regime number 1, and the fish exposed to the cycling temperatures fed and
grew at the rate they would have if acclimated to the maximum temperature experienced.
The authors conclude the temperature effect on PCB accumulation was because the
temperature controlled food consumption, growth, and lipid content of the fish.
In field studies, Gossett et al. (1983) analyzed for 27 organic compounds in livers of
fish taken in the vicinity of the Los Angeles, CA, wastewater treatment plant. They found
that the tissue concentrations of the compounds were correlated with the log P of the
compound (r = 0.63-0.75), but were not correlated with the environmental concentration.
While the overall correlation with lipid solubility was good, there were some notable
exceptions which the authors attribute to metabolism of the parent compound.
The molecular weight of a pesticide has been shown to affect the BCF (Kanazawa,
1982). When log BCF (Y) is plotted against the log molecular weight (X) of pesticides
of all sorts ranging in weight from 187^12, there is a correlation coefficient of 0.846.
There is a linear relationship between the lipid solubility of these same pesticides and
their BCFs, so the cause and effect here is not clear, but it is interesting that the larger the
pesticide molecule (at least up to a molecular weight of 412), the more it accumulates in
fish tissues. A similar relationship between molecular chain length and accumulation has
been found for linear alkylbenzene sulfonate (LAS), an anionic surfactant used in deter­
gents (Comotto et al., 1979).
Thus far in this discussion of accumulation of organics, the fish has been considered
as a single compartment, when it is, of course, made up of different types of tissues. In
comparison to the amount of information available on total body burden, the relative
accumulation of organic xenobiotics by different tissues is lacking. Pentachlorophenol
will now be used as a type of contaminant as there is considerable information about it,
thus a few generalizations begin to become evident.
Examination of Figure 11 illustrates how different tissues may accumulate markedly
different levels of an organic contaminant, and even compounds superficially similar may
accumulate in rather dissimilar ways. In this study, trout were exposed to either pen­
tachlorophenol (PCP) or pentachloroanisole (PCA) at a concentration similar to that later
found by others in contaminated natural waters (Delfino, 1979), but well below the lethal
concentration (Niimi and McFadden, 1982). PCP accumulated most in the liver whereas
PCA was accumulated far more in the fat. Neither compound showed much affinity for
muscle.
Glickman and Melancon (1982) reported BCF values after 48-h exposures for radio-
labeled PCP and 2.4.5-trichlorophenol (TCP) of approximately 50x for muscle, lOOx for
blood, lOOOx for liver, and 100,000x for bile. The test concentration in the water was
fairly high (0.05 mg/L), but the partitioning into different organs is especially striking.
 103

hours

Figure 11 Uptake of ^^C-labeled PCP and PCA in tissues of rainbow trout exposed to 0.025
jig/L in the water. Concentrations are calculated as |ig/g wet wt. Each point represents the mean
± the SE from at least six fish. (From Glickman, A. H., Statham, C. N. and Lech, J. J., Toxicol.
Appl. Pharmacol., 41, 649, 1977. With permission.)

The elimination of PCP may be relatively fast in that the half-life for fat is 24 h while
blood, liver, muscle, gills, and heart range from 6.2-10.3 h (Glickman et al., 1977). Of
course, the highest concentration is in the liver so the greatest amount was depurated from
there. This is to be expected since the liver is the primary organ for metabolism of PCP
(Lech et al., 1978). One final note on depuration of PCP: whereas the first half-life is quite
short, the data on this goes only out to 24 h (Glickman et al., 1977) and there may be a
low residue that remains for some unknown period of time. Indeed, Niimi and Cho (1983)
report a half-life for depuration of PCP in whole trout as 7 days, which is probably due
to longer retention time in some tissues than others.
The accumulation and elimination of PCP is greatly affected by the ambient salinity.
Freshwater acclimated killifish exhibited a BCF of 1680 (whole body) while seawater
acclimated killifish had a BCF of only 370 (Tachikawa et al., 1991). This is somewhat
surprising as seawater fish are about lOx more permeable to salt ions and water than are
those in freshwater (see Chapter 7). Furthermore, they drink a significant amount of
seawater to replace water lost by osmosis, but according to Tachikawa et al., (1991) the
amount of PCP taken in by the drinking of seawater-acclimated fish contributed only
“slightly” to the uptake. They attribute the reduced accumulation rate in seawater to a
slower rate of uptake and especially to a more rapid metabolism and excretion. The
mechanisms by which these processes would change with salinity acclimation are cur­
rently obscure.
104

The BCF for specific organs of organochlorine pesticide residues has been shown to
vary with the dose. Under acutely toxic doses, the liver, spleen, and fatty tissues have
much higher concentrations than either muscle or gill, but when the exposure is chronic,
concentrations in the liver decline to those found in muscle or gill while spleen and fatty
tissue have much higher levels (Holden, 1966). The lower levels in the liver with chronic
exposure are probably due to metabolism of the pesticide by that organ.
Fish captured in the field that are contaminated with organochlorine pesticides may
exhibit a seasonal difference in partitioning into different tissues. Trout and whitefish
captured from freshwaters in Russia showed highest concentrations of these pesticides in
the brain during the spring and summer; the liver accumulated them during late summer
and fall (Chernyaev, 1991).
Organophosphorus compounds accumulate very rapidly from the water reaching a
steady state in small fish such as guppies within 12-48 h. A biphasic relationship was seen
between accumulation rate and log Kow (De Bruijn and Hermens, 1991). Elimination rates
generally correlate with log Kow with some exceptions. These exceptions seem to be related
to different rates of biotransformation of parent compounds. This can also cause lower
bioconcentration factors than would be predicted. De Bruijn and Hermens (1991) review
several studies that show the impact that biotransformation can have on the accumulation
of various compounds. Welling and De Vries (1992) found that the organophosphorus
insecticide chlorpyrifos is eliminated rapidly from guppies but nearly all of the parent
compound is biotransformed before elimination. They go on to note that prolonged expo­
sures to the pesticide caused the elimination rate to actually decrease, which is the reverse
of what might be expected. Biotransformation enzymes are inducible (see Section IX) so
prolonged exposure would be expected to enhance excretory processes.
Fish accumulate aromatic petroleum hydrocarbons rapidly, so concentrations may
reach a maximum within a few hours of exposure (Anderson, 1979). As the number of
aromatic rings in the molecule increases, the accumulation and retention also increases.
Different petroleum hydrocarbons are accumulated to different equilibrium levels so the
concentrations in the fish will not necessarily represent the relative concentrations of the
different components in the environment. Longer exposures often produce lower tissue
concentrations of the parent compounds because of induction of biotransformation en­
zymes in the liver (Anderson, 1979).
Petroleum hydrocarbons are not partitioned into the tissues uniformly. In Table 1 data
are derived from work on dolly varden char (a salmonid) that had received a stomach dose
of either radioactive-labled toluene or naphthalene and then the tissues sampled 24 h later.
Thomas and Rice (1981) provided the percentage of administered label that appeared in
each organ and the average organ weights. In order to get a measure of relative concen­
tration in the different organs, I divided the average percentage of the total label in an
organ by the organ weight. It should be kept in mind here that these data reflect both the
parent compound and the metabolites. The relative percentage of these two forms varies
with the tissue. Several things then become evident in the data presented in Table 2:
1. The naphthalene has a greater potential for accumulation than does toluene, which
might be predicted as it is a two-ring compound whereas toluene is a one-ring molecule
(later we will see that toluene is excreted more rapidly than naphthalene).
2. Seawater acclimation caused naphthalene to be accumulated more than when the fish
were in freshwater, but this environmental variable had no affect on toluene accumu­
lation. The authors present other evidence to the effect that naphthalene is metabolized
more rapidly in freshwater-adapted fish, which provides a mechanism for the slower
accumulation of this chemical when the fish are in freshwater. Why the salinity would
affect this process seems obscure. It may be recalled that the opposite occurred with the
 105

metabolism of PCP in seawater and freshwater acclimated killifish. Seawater acclima­

tion stimulated metabolism and elimination of PCP (Tachikawa et al., 1991).
3. The concentrations in the various tissues differed by orders of magnitude. The bile
(called gallbladder by Thomas and Rice) had by far the largest concentrations, and gill
had remarkably high concentrations, even though the exposure was internal rather than
external. The high hydrocarbon levels in these two organs are probably due to their
central role in the excretion of both the parent compounds and their metabolites.
A problem often occurs when studying petroleum hydrocarbons in aquatic organs and
their environments in that many of these chemicals are “naturally occurring” in the
ambient waters, sediments, and fish tissues. Thus, there has been some interest in the

Table 1 Relative Concentration of

Radioisotope in Tissues 24 h After
Being Force-Fed Labeled Toluene or
Naphthalene Dose
Toluene
Tissue FW SW FW SW
Muscle 0.02 0.03 0.04 0.31
Gill 0.51 0.80 0.68 9.32
Bile 2.12 3.12 10.50 1.50
Liver 0.15 0.10 0.30 0.52
Brain 0.06 0.18 0.06 0.22

N o te : See text for method of calculation. FW =

Freshwater-acclimated fish. SW = Sea­
water-acclimated fish.
Data calculated from Thomas, R.E. and Rice,
S.D., 1981.

Table 2 Physiological Parameters Used

in a Physiological-Based Toxicoklnetic
Model for Adult Rainbow TrouP
Body weight (kg) 1.0
Ventilation volume (L H20/h 10.6
Effective respiratory vol. (L H20/h) 7.2
Oxygen consumption rate (mg 0 2 /h) 63.0
Cardiac output (L blood/h) 2.07
Arterial blood flow
To liver 0.06
To fat 0.176
Poorly perfused compartment 1.242
Richly perfused compartment 0.476
Kidney 0.116
Tissue group volumes (L)
Liver 0.012
Fat 0.098
Poorly perfused compartment 0.818
Richly perfused compartment 0.063
Kidney 0.009

From Nichols et al. (1990) using a trout at 12°C.

106

alkane squalane which is apparently correlated only with hydrocarbon sources such as
fossil fuels and other petroleum by-products (Matsumoto and Hanya, 1981). This material
has a high (about 40%) assimilation efficiency in trout (Cravedi and Tulliez, 1986). The
partitioning of this compound into various tissues is, however, rather different than other
hydrocarbons. In spite of its high lipophilicity, very little is accumulated in fat where
other hydrocarbons usually go (Cravedi and Tulliez, 1982). Instead, most accumulation
occurs in the liver, kidney, heart, and spleen. The authors (Cravedi and Tulliez) speculate
this may be due to uptake by the reticuloendothelial cells in these organs, although no
direct evidence is provided.
The detergents, LAS, and SLS, are accumulated rapidly in fish tissues (Comotto et al.,
1979; Tovell et al., 1975). Muscle and brain generally exhibit the lowest BCF and the
highest values are in gallbladder, liver, and gut. Apparently, metabolism of and excretion
of detergents are fairly rapid as a steady state is achieved at almost any dose after 3 days,
and depuration of over 85% of the accumulated material occurs within 4 days of being
placed in clean water.
In recent years there has developed an interest in creation of physiologically based
toxicokinetic models for fish exposed to waterborne toxicants (Nichols et al., 1990,
1991). The more traditional approach to modeling accumulation of a contaminant treats
the fish as a two or more compartment model and is based on measures of accumulation.
The physiologically based model uses a list of physiological variables to predict the time
course of contaminant accumulation. This theoretically then permits extrapolation to
other species and exposure conditions. Table 2 presents the physiological parameters used
in such a model. The actual values are from the literature. Other non-physiological
parameters used include concentrations of the test substance in the inspired water and
some characteristics of the chemical. One point of explanation is in order. In Table 2, the
arterial blood flow to a poorly perfused compartment may seem high as there is not much
blood flow there. However, in the fish this compartment is mostly white muscle which
makes up over 50% of the body, so even though the perfusion is limited, the total bulk
allows a considerable blood flow.
The physiologically based toxicokinetic model has been tested with pentachloroethane,
hexachloroethane, and tetrachloroethane (Nichols et al., 1991) using cannulated trout in
chambers similar to the one shown in Figure 3. The model proved reasonably good at
predicting the dynamics of accumulation of these chemicals. One of the outgrowths of
this type of study is how it illustrates the effects that chemical affinity and blood perfusion
have on accumulation dynamics. For example, fat tissue has a high affinity for the test
chemicals but is poorly perfused. As a result, uptake is slow into this compartment so that
its concentration may continue to rise long after an equilibrium concentration has been
reached in the blood.
While the physiologically based models have considerable potential, species differ­
ences will be an interesting challenge. For example, when the distribution of a single oral
dose of tetrachlorodibenzo-p-dioxin was compared in cod and in seawater-adapted rain­
bow trout, the highest concentrations in the cod were found in the liver and central
nervous systems. In the trout, on the other hand, the highest concentrations were in the
visceral and extravisceral fat depots and relatively little in the liver and central nervous
system (Hektoen et al., 1992). These differences are probably explained by differences
in physiology, which makes understanding comparative physiology a key issue for
different species.

IX. BIOTRANSFORMATION OF ORGANIC CONTAMINANTS

Biotransformation processes in fish have been reviewed extensively (e.g.. Lech et al.,
1982; Buhler and Williams, 1988; Goksoyr and Forlin, 1992; Andersson and Forlin,
 107

1992; Stegeman and Hahn, 1994). This is a very large and fast moving field so only a brief
presentation of some basic concepts will be attempted here.
The biotransformation reactions tend to make the relatively lipophilic parent com­
pound into one that is more hydrophilic (i.e., water soluble) and more polar. This
enhances the tendency for the substance to be excreted by the gills, bile, and kidney while
at the same time it reduces its affinity for plasma and tissue proteins and for adipose
tissue. Hydrophilic compounds are also less permeable to cell membranes so are less
likely to be accumulated in various tissues.
It is usually assumed that biotransformations are detoxification reactions. While that
may be true for a great majority of xenobiotics, there are examples in fish where the
transformation makes the substance more toxic or carcinogenic; the latter has serious
implications for human health but is clearly beyond the scope of this book. Increased
toxicity to the fish is seen with the classical case of the organophosphate insecticide
parathion. This insecticide must actually be converted to paraoxone by mixed function
oxidate (MFO) before it becomes an inhibitor of cholinesterase in the nervous system
(Ludke et al., 1972).
The biotransformation reactions are broadly divided into asynthetic (Phase I) and
synthetic (Phase II). The former group includes oxidation, reduction, and hydrolysis. The
synthetic reactions usually involve the conjugation of endogenous compounds such as
glutathione with the metabolites produced in the Phase I steps. However, in some cases
the conjugation is of the parent compound so it is not always necessary that a Phase I
reaction precede a Phase II reaction.
Most of the Phase I reactions are oxidations catalyzed by the cytochrome P450 system
referred to as the MFO, monooxygenase (MO), or CYPlAl system. For convenience, it
will be referred to here as the MFO system. It is a coupled electron transport system with
several accessory enzymes which acts to insert one atom of oxygen into the substrate and
reduces the second atom of oxygen to form water. The system is located predominantly
in the endoplasmic reticulum of the cells (microsomal fraction when separated in an
ultracentrifuge).
Phase I hydrolysis reactions are catalyzed by epoxide hydrolases which hydrolyze
various epoxides and diols. These enzymes are located both in the ribosomes and in the
cytosol of the cell (De Bruin, 1976).
Considerable species variability undoubtedly exists at the quantitative level in these
Phase I processes. The enzymes of the MFO system have very broad substrate specifici­
ties due to the presence of multiple isozymes (Andersson and Forlin, 1992). They have
also been found in the embryonic stages of several species so are functional at a very early
stage in the life cycle. Induction of MFO activity in larvae can occur via direct exposure
of the egg to crude oil (Goksoyr et al., 1991). Indeed, xenobiotics in the gametes of lake
trout have been shown to induce MFO activity in the livers of the offspring derived from
those gametes (Binder and Lech, 1984).
Increased activity of MFO enzymes is induced by exposure to xenobiotics. This has
been extensively studied in mammals and in fish. Classically, there were two major
classes of MFO-inducing chemicals referred to as the 3-methylcholanthrene (3-MC-like)
and phenobarbital-like. Now chemicals are classed by the specific family of P450 genes
that are activated. Over a dozen P450 forms have been purified from fish tissues; the most
extensively studied is from rainbow trout and is designated P4501A1 (Andersson and
Forlin, 1992). The inductive response is usually detected by measuring P450 catalytic
activity by assaying the ethoxyresorufin-O-deethylase (EROD) reaction which is low or
sometimes undetectable in non-contaminated fish.
The marine and freshwater environments are loaded with thousands of different
chemical effluents. It has been recognized since the mid-1970s that induction of MFO
might be a useful biomarker for environmental contamination as it would yield an
108

integrated signal for bioavailability of unknown xenobiotics and the defense responses by
the resident fish. Thus far, there has been considerable success in the utilization of this
biomarker, reviewed in Payne et al. (1987); Goksoyr and Forlin (1992); Collier et al.
(1992). Useful guidelines for the application of such procedures are found in Stegeman
et al. (1992).
The MFO system is not the “Holy Grail” of biomarkers, for it does have some
problems. To begin with, while a wide variety of xenobiotics induce increased MFO
activity in fish, the phénobarbital group of substances does not (Goksoyr and Forlin,
1992). The chlorinated hydrocarbon DDT falls in this category and fails to induce MFO
activity when fed to trout (Addison, 1977). Fortunately, most of the organic pollutants of
concern are inducers of this system.
The presence of cadmium (and possibly some other metals?) can lower MFO activity
(Forlin et al., 1986), so this might cancel out the effect of organic induction of MFO
activity. The sex of the fish affects basal levels; male trout have over twice the levels of
females (Williams et al., 1986), although sockeye salmon during the spawning migration
showed no sexual difference (Kennish et al., 1992). It appears that the sex hormones in
both sexes can have a considerable influence on MFO, but not in the same direction.
Andersson and Forlin (1992) review several studies which show that testosterone seems
to upregulate MFO whereas estradiol downregulates the same system. The situation in the
female during spawning season when both hormones rise is quite complicated and varies
with the species.
Another steroid interaction with the MFO system has recently been revealed using an
in vitro preparation of trout hepatocytes. Devaux et al. (1992) found that cortisol- or
dexamethasone-induced MFO activity, thus elevated cortisol levels from environmental
stress unrelated to pollutant contamination might produce high levels of MFO. This
deserves further study in intact fish.
Tributyltin is a common metal contaminant in harbors and has been shown to inhibit
MFO activity and the induction response (Fent and Stegeman, 1993). This could have
serious implications for fish exposed to mixed effluents which, of course, is the usual
situation in harbors and bays.
The process of induction of MFO enzymes can be looked on as a form of adaptation
to a contaminated environment. However, it also may have some adverse side effects.
This is because a function of the MFO system in non-contaminated fish is the metabolism
of steroid hormones. Lech et al. (1982) speculated that an enhanced rate of metabolism
of the sex hormones from pollutant-induced MFO activity could have harmful effects on
reproduction. Since then, several investigations have provided indirect confirmation that
elevated MFO can disrupt reproductive functions (e.g., Thomas, 1990), although direct
cause and effect mechanisms are not clear.
Because fish are poikilotherms, temperature might have a notable effect on MFO
function, however, current findings are in conflict on this matter. The elimination rate of
benzo[a]pyrene by bluegill (L. macrochirus) was increased threefold over a 10°C rise in
acclimation temperature and enzyme profiles indicated that biotransformation was simi­
larly affected (Jimenez et al., 1987). However, trout have been reported to exhibit ideal
temperature compensation for MFO activity (Blanck et al., 1989; Koivusaari, 1983) (i.e.,
the same rate is obtained at all acclimation temperatures if incubation temperatures for the
tests are run at the acclimation temperature). Members of the genus Lepomis generally
show “good” temperature compensation in their glycolytic and Kreb’s cycle enzyme
systems (Shaklee et al., 1977) so the lack of temperature compensation in the MFO
system of the bluegill deserves further investigation.
There are still a good many questions remaining concerning the MFO system in fishes.
Goksoyr and Forlin (1992) note that there is almost no information on the effect of
photoperiod, salinity, handling, etc., on this enzyme group. Of course, the extreme
 109

diversity of fish species presents a tremendous challenge to our further understanding of

this remarkable system.
The Phase II, or conjugation reactions involved in xenobiotic transformation, include
conjugations with glucuronide, sulfate, glutathione, or taurine. This has been referred to
as a protective synthesis (De Bruin, 1976) whereby both natural substances (e.g., steroids)
and foreign xenobiotics are coupled to endogenous substrates such as glutathione. There
are specific enzymes (e.g., glutathione transferases) which catalyze the transfer of the
conjugating agent from its bound coenzyme form to the foreign molecule. Obviously, this
is a greatly simplified summary of some rather complex biochemistry, much of which has
been learned from work on mammals. The interested reader is referred to the reviews of
Lech and Bend (1980), Smith et al. (1983), and George (1994).
Conjugated metabolites of a variety of organic chemicals have been found in fish
(Stehly and Hayton, 1989). The phenolics have probably received the most attention and
there are species differences in the conjugation of phenol and PCP. For example, the
goldfish and sheepshead conjugated a greater percentage of PCP with sulfate whereas
rainbow trout, fathead minnows, and firemouth utilized the glucuronide conjugation more
(Stehly and Hayton, 1989). The dose can also influence the conjugation mode. Nagel
(1983) found that when the phenol dose was increased the percent glucuronylated
increased while the percent sulfated decreased. Evidently, as has been seen in mammals,
the sulfate conjugation process is easily saturated, and when phenol is injected, as
opposed to putting it in the water, the material is rapidly transported to the gills where the
nonconjugated phenol is eliminated (Nagel and Ulrick, 1980).
Glutathione is seemingly a “molecule for all seasons”. Earlier in this chapter it was
mentioned that it plays a role, at present poorly understood, in the metabolic handling of
metals. In mammals, and probably fish, it serves a variety of functions in both natural
substrate and xenobiotic metabolism. The cell utilizes glutathione along with ascorbic
acid and vitamin E in the process of scavenging free radicals by superoxide dismutase and
catalase. The conjugation of glutathione to xenobiotics, or their Phase I products, is an
active field of study. The concentration of glutathione depends on the activity of several
enzymes, some of which catalyze its conjugation with other substrates, others catalyze its
synthesis.
Several investigators have reported elevated concentrations of glutathione in liver or
gill from exposures to various organic chemicals (Thomas and Wofford, 1984; Lindstrom-
Seppa and Oikari, 1991; Gallagher et al., 1992). Gallagher et al. (1992) showed that the
fungicide chlorothalonil induced an increased activity in hepatic gammaglutamylcysteine
synthetase which is a key enzyme in the synthesis of glutathione. Whether other chemi­
cals cause the rise in glutathione by this mechanism remains to be determined.
The importance of glutathione in counteracting toxicity was clearly shown by Gallagher
et al. (1992) when they depleted the liver and gill of glutathione with a drug. This caused
the toxicity of chlorothalonil to increase threefold.
Exposure of fish to either phenol or ammonia rapidly induced (i.e., within a day) a two-
to threefold increase in the activity of liver glutathione-5'-transferase activity. This en­
zyme catalyzes conjugation of glutathione with the xenobiotic. At the same time, there
was a transient rise in glutathione concentration (Chatterjee and Bhattacharya, 1984). The
concentration of glutathione will undoubtedly depend on the rate of synthesis minus the
rate of conjugation with xenobiotic.
Most of the work that is done on the transformation of xenobiotics by the liver has been
performed on fish sacrificed at particular times during an exposure regime, or following
an injection. In an attempt to get at some of the dynamics of the process, Forlin and
Andersson (1981) developed a system to use an in vitro perfused rainbow trout liver. The
isolated livers were kept functional for at least several hours which made it possible to
add a chemical to the “blood” going into the liver and measure the removal of the material
110

and the formation of metabolites as they appear in the blood and/or bile. This sounds
easier than it really is, and it is necessary to use large fish (>400 g) so work with small
species would be precluded.
It has been long assumed that biotransformation of xenobiotic organic chemicals
occurs mostly in the liver. It is now appreciated that other organs, most especially the
intestine and gills, also have considerable capacity as well (Van Veld, 1990; Miller et al.,
1989). Because the gills and intestine are the main modes of uptake for pollutants, having
the capacity for some metabolism of these compounds there has obvious advantages. Van
Veld et al. (1988) have developed a technique whereby the portal vein is catheterized so
the blood coming from the intestine can be sampled for metabolites produced by the
intestine. With this system, they found that most of the metabolites appearing in the blood
from the ingestion of benzo[a]pyrene were derived from the intestine instead of the liver.
There is probably considerable species variability in extrahepatic metabolism of xenobiotics
(Lindstrom-Seppa et al., 1981).
Intestinal microorganisms may transform organics present in the food (Addison,
1976). This can cause problems in determining the location where a metabolite was
formed, and for this reason, in laboratory studies the xenobiotic may be injected rather
than included in the food. (The injection mode of entry also precludes problems with
incomplete assimilation.)
The metabolic transformation of xenobiotic chemicals that occurs in fish has important
implications for monitoring programs. When doing residue analysis, if analysis for only
the suspected parent compound is done, misleading results may be obtained should part
or all of the chemical be transformed (Giesy et al., 1983). This can happen within a matter
of hours of exposure. In general, the bile will contain the conjugated products of the
transformations. The fact that these metabolites concentrate in the liver and bile argues
for analysis of these select tissues separate from muscle when doing residue analyses.
Biotransformations also make predictions of steady-state tissue concentrations based on
chemical characteristics quite suspect unless it is shown that the chemical is not signifi­
cantly metabolized during the period of exposure.

X. EXCRETION OF ORGANIC CONTAMINANTS

Depuration of some foreign chemical is usually measured by allowing the contaminated
fish to reside in non-contaminated water for some specified period of time and then
residue analysis is performed on the whole body or specific tissues. The latter sort of
analyses are far more informative as the rate of depuration varies greatly between
different tissues and this is related to their relative abilities for metabolic transformation
of the chemical. Depuration measurements give a general picture of the elimination of a
specific parent chemical which has practical application when there is concern for how
long a group of fish may remain contaminated following a chemical spill. Our interest
here is primarily with the physiological routes by which this is accomplished. (The
excretion of metals was reviewed earlier in this chapter.)
As with inorganic pollutants such as the metals, a fish has several possible routes for
excretion of an organic pollutant. These include the gills, skin, mucus, bile, feces, and
urine, which are the same as those “used” by metals, only the primary routes differ
considerably between different chemicals.
PCP is rapidly absorbed by fish from the water and it is rapidly conjugated with sulfate
and glucuronic acid. During exposure of goldfish to 0.1 ppm PCP, the conjugated PCP
began to appear in the bile within an hour and further accumulation there was linear for
up to at least 48 h, by which time the concentration factor was 12,000. Only the glucu-
ronide conjugate and a small amount of free PCP was detected in the bile (Kobayashi,
1978). In spite of the high concentrations of PCP conjugate in the bile, work in several
 111

species has shown that over 60% of the PCP is excreted mostly via the gills, but with some
through the urine (Stehly and Hayton, 1989; McKim et al., 1986).
Looking for metabolites in the surrounding water following exposure of fish to a
pollutant has caused some confusion in the literature as to what metabolites are actually
formed from phenol and PCP (Nagel, 1983). Perhaps the delay in releasing material from
the gallbladder may be a major problem here. This structure is in part a digestive organ
which contracts to empty its contents into the intestine when food is present. Because the
fish used in xenobiotic metabolism studies are generally postabsorptive, there may be a
tendency for material to stay longer in the gallbladder than would occur under “natural”
conditions where the fish is feeding.
A wide variety of organic materials and/or their metabolites have been found to
concentrate in fish bile, and this can occur very rapidly. Mention has already been made
of the accumulation of conjugated PCP there. Miyamato et al. (1979) reported consider­
able accumulation of fenitrothion (an insecticide) in the bile of trout after only 6 h of
exposure. Peterson and Guiney (1979) noted that PCB metabolites are excreted via the
bile of fish. Using nine very different isotopically labeled organic substances added to the
water for 24 h, Statham et al. (1976) obtained bioconcentration factors of 11-10,000 in
the bile of trout. In most cases, the material in the bile was actually the conjugated
metabolite. It is interesting that the lowest concentration factors in the bile are associated
with compounds with the highest lipid solubility. Recall that these have the high octanol/
water partition coefficients and therefore tend to be accumulated by fish to the greatest
degree. Evidently, they have a relatively low rate of metabolism or conjugation by the
liver, a characteristic that would contribute to their tendency to accumulate.
Finding a high concentration of some pollutant in the bile does not necessarily mean
this is the only, or even the dominant mode for excretion. In order to partition the
excretory modes it is necessary to use specially designed chambers similar to the one
shown in Figure 3. Using a chamber such as this, Thomas and Rice (1981, 1982) have
investigated the excretion of radiolabeled aromatic hydrocarbons associated with petro­
leum. The test chemical was inserted into the stomach in a gelatin capsule and the fish
allowed 24 h to absorb and excrete it. The gills turned out to be an especially important
organ for excretion of aromatic hydrocarbons. The rate of excretion of individual hydro­
carbons by the gills was shown to be inversely proportional to the size of the molecule
and the log Kow. More recent work by Le Bon et al. (1987) has shown that the isoprenoid
hydrocarbon, pristane, is metabolized first before being excreted via the gills.
The total isotope from naphthalene and toluene in the rear chamber, urine, and
gallbladder was less than 20% of that excreted via the gills (Thomas and Rice, 1981)
indicating that the latter organ is the primary mode of excretion of these two highly toxic
aromatic hydrocarbons. Even though high concentrations of these hydrocarbons were
found in the gallbladder and urine, the total volumes of fluid involved were so small that
they did not account for a very large amount of the overall hydrocarbon excretion
compared to that excreted via the gills. This points out again the fact that finding a high
concentration of some compound in the urine or bile does not necessarily establish it as
an important mode of excretion for that compound.
For larger organic molecules, the gills may not be particularly useful as an excretory
organ. Thomas and Rice (1982) concluded that excretion of anthracene and benzo[a]pyrene
via the gills was of little significance compared to the bile, although even that mode was
slow.
Phenol and cresol are rapidly excreted by the gills, bile, urine, and skin with the
greatest percentage coming out through the skin and bile (Thomas and Rice, 1981). The
skin and bile has also been shown to be an effective mode for excretion of naphthalene
metabolites in rainbow trout (Varanasi, 1979). In these fish, the bile contained little of the
hydrocarbon material over the first 24 h of exposure, but it became an important method
112

of excretion in the long run so it apparently takes some time for the excretory machinery
in the liver to get “geared up”.
Anthracene and benzo [a] pyrene are excreted very slowly compared to the aromatic
hydrocarbons (Thomas and Rice, 1982). In 24 h only somewhat less than 4% of the total
dose was excreted compared to 66% for phenol, 30% for toluene, and 10.8% for naph­
thalene. This delay is apparently due to the requirement that anthracene and benzo [a]pyrene
be metabolized before excretion. Anthracene depuration has been shown to be most rapid
at night (Lindstadt and Bergman, 1984). Such a periodic depuration could be character­
istic of a variety of compounds, but it seemingly has not been looked for in other studies.
It is well known that seawater-adapted fish excrete little urine volume compared to the
freshwater-adapted forms (even of the same species), and the osmotic flow of water
through the gills is inward in freshwater while outward in seawater. Thus, it is conceiv­
able that a greater amount of material might be excreted via the kidneys of freshwater fish.
In spite of these theoretical considerations, from very limited data it appears that the
salinity has little effect. The excretion of naphthalene and toluene via the urine was little
changed when the dolly varden used in the studies discussed above were adapted to
freshwater (Thomas and Rice, 1981). This represents only one species and two aromatic
hydrocarbons so the generalization should be treated as very tentative.
The detergent SLS is apparently excreted exclusively via the urine and bile. Using
radiolabled SLS Tovell et al. (1975) reported finding no isotope in the “gill” water of their
experimental goldfish. (Their fish had the rear half of the body enclosed in a plastic bag
which collected both urine and cloacal excretions.) Analysis of the bile showed very high
concentrations there of both the metabolized and non-metabolized form of the compound,
so it is evident that it is not necessary that SLS be metabolized before excretion.
Using catheterized channel catfish in a split chamber and LAS, another detergent,
Schmidt and Kimerle (1981) were able to partition the excretion more completely.
Ninety-six hours following injection of the isotope, 42% percent of the radioactivity had
been excreted. Of that, 60% appeared in the urine, 29% in the anal chamber (presumably
from bile), and 9.5% in the gill chamber.
Carboxymethyltartonate (CMT), a potential partial replacement for phosphates in
detergents was also tested. All of it was excreted in 96 h with 75% appearing in the urine,
16% in the anal chamber, and 8% was excreted via the gills. Thus, for this group of
organic compounds, little gill excretion occurs in freshwater fish, but instead they exit the
fish largely by way of the urine and to a lesser extent the bile.
The excretion of the lamprey poison Bayer 73 by teleosts occurs by both the urine
and bile in roughly equal amounts. Some may be lost across the gill tissue to the water
but this likely is relatively insignificant compared to the other two routes (Allen et al.,
1979).
Ethyl-m-aminobenzoate (commonly called MS 222) is widely used as a fish anesthetic
by adding it to the water. One of its virtues as an anesthetic is the rapid recovery of the
fish following their being placed in anesthetic-free water. This must be in part due to the
rapid clearance from the body of the fish. Stenger and Maren (1974) reported a 95%
clearance via the gills in 2 h. Renal excretion of the injected drug was less than 5% of the
total lost. An interesting sidelight to this study was the finding that IV injection failed to
produce anesthesia. Materials injected IV in fish must pass the gills before reaching the
brain, so evidently most of the drug was lost to the water upon one passage of the blood
through the gills (Hunn and Allen, 1974).
The commonly used carbamate pesticide, aldicarb, is rapidly distributed in the fish
following either oral administration or IP injection (Schlenk et al., 1992). It is also
excreted rapidly, mostly as the unmetabolized molecule via the gills. Its rapid exit through
the gills is probably due to it being a rather water-soluble molecule.
 113

The phenoxyacetic acid herbicides (e.g., 2,4-D), the primary metabolite of DDT
(DDA), and the metabolites of benzo[a]pyrene are all easily excreted by the teleost
kidney. Indeed, the kidney actively secretes them against a concentration gradient so the
urine contains a far higher concentration of the first two compounds than was present in
the blood (Pritchard and Bend, 1984). (The metabolites of BP were only slightly concen­
trated in the urine.) Pritchard and Bend (1984) note that a high lipid solubility tends to
reduce the ability of the kidney to excrete an organic xenobiotic. They also point out that
fish kidneys may be able to handle some compounds better than mammalian kidneys
because of the extensive renal portal system in the fish, which accentuates the role of
tubular transport, and the fact that the xenobiotics have a higher binding affinity to plasma
proteins in mammals.
It is difficult, if not impossible, to draw many generalizations regarding the excretion
of foreign chemicals in fish because these compounds are so diverse and the fish is
endowed with so many excretory modes. It does seem safe to conclude that the lipid-
soluble xenobiotics are usually metabolized into a water-soluble form before excretion,
and the products of that metabolism are then excreted largely by way of the bile and/or
urine. The gills can be an important mode of exit for small compounds that require little
if any biotransformation before excretion, and this is presumably a passive diffusion
process. Finally, the skin is especially important for small fishes.

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biotransformation of naphthalene by juvenile starry flounder {P la tic h th y s s te lla tu s ) and rock sole
( L e p id o p s e tta h ilin e a ta ). A rc h . E n v iro n . C o n ta m . T o x i c o l, 8, 673, 1979.
Veith, G. D., DeFoe, D. L. and Bergstedt, B. V., Measuring and estimating the bioconcentration factor
of chemicals in fish, J. F ish . R e s . B d . C a n ., 36, 1040, 1979.
Vetter, R. D., Carey, M. C. and Patton, J. S., Coassimilation of dietary fat and benzo[a]pyrene in the
small intestine: an absorption model using the killifish, /. L i p id R e s ., 26, 428, 1985.
Waldichuck, M., Some biological concerns in heavy metals pollution, in. P o llu tio n a n d P h y s io lo g y o f
M a r in e O r g a n is m s , Vemberg, F. J. and Vemberg, W. B., Eds., Academic Press, New York, 1974,
1.
Weis, P., Metallothionein and mercury tolerance in the killifish, F u n d u lu s h e te r o c litu s . M a r. E n v iro n .
R e s ., 14, 153, 1984.
Welling, W. and De Vries, J. W., Bioconcentration kinetics of the organophosphorus insecticide
chlorpyrifos in guppies { P o e c illia r e tic u la ta ), E c o t o x i c o l E n v iro n . S a f e ty , 23, 64, 1992.
Wicklund, A. and Runn, P., Calcium effects on cadmium uptake, redistribution, and elimination in
minnows, P h o x in u s p h o x in u s , acclimated to different calcium concentrations, A q u a t. T o x i c o l, 13,
109, 1988.
Williams, D. E., Regiospecific hydroxylation of lauric acid at the (Omega 1) position by hepatic and
kidney microsomal cytochrome-P-450 from rainbow trout. A r c h . B io c h e m ., 231, 503, 1984.
Williams, D. E., Masters, B., Lech, J. and Buhler, D., Sex differences in cytochrome P-450 isozyme
composition and activity in kidney microsomes of mature rainbow trout, B io c h e m . P h a r m ., 35,2017,
1986.
Winge, D. R., Premakumar, R. and Rajagopalan, K. V., Metal-induced formation of metallothionein
in rat liver. A rc h . B io c h e m . B io p h y s ., 170, 242, 1975.
Yarbrough, J. D. and Chambers, J. E., The disposition and biotransformation of organochlorine
insecticides in insecticide-resistant and susceptible mosquitofish, in. P e s tic id e a n d X e n o b io tic
M e ta b o lis m in A q u a tic O r g a n is m s , Khan, M. A. Q., Lech, J. J. and Menn, J. J., Eds., (ACS
Symposium Series 99), American Chemical Society, Washington, D.C., 1979, 145.
 Chapter
6
Liver
INTRODUCTION
The liver is of key importance when considering the action of toxic chemicals on fish. It
is the primary organ for biotransformations of organic xenobiotics, and probably also for
the excretion of harmful trace metals. Because many of these metals and organics tend
to accumulate to high concentrations in the liver (see Chapter 5), the cells there are
exposed to far higher (often several orders of magnitude) levels of harmful chemicals than
may be present in the environment, or in other organs of the fish. The liver serves a
number of functions related to other physiological activities, such as interconversions of
foodstuffs (Chapter 8) and metabolism of sex hormones (Chapter 13), so the potential
effects of xenobiotics on the liver are numerous, but often seen only indirectly.

II. STRUCTURE OF THE LIVER

The gross and histological organization of the fish liver has been described in consider­
able detail by Gingerich (1982) and Hinton et al. (1987). While they were able to derive
a number of generalizations, it must be emphasized that there is a fair amount of diversity
among the various fish groups and this variability could influence the toxicological and
physiological responses to pollutants.
The well-defined (histologically) lobular structure of the mammalian liver is appar­
ently not characteristic of fish. Instead, the hepatocytes are arranged as tubules of cells.
A tubule viewed in cross section is usually surrounded by five to seven hepatocytes with
their apices directed toward the central bile canaliculus and/or bile preductule. These
tubules are surrounded by the sinusoids which take the place of capillaries in the liver.
Blood enters the sinusoids from the hepatic portal system and hepatic artery and the
hepatocytes remove nutrients and xenobiotics from that blood. The bile is secreted into
the central bile canaliculus from which it ultimately flows into the gallbladder.
There seems to be some debate among histochemists as to the extent of specialization
(metabolic zonation) among hepatocytes in fish livers. Most seem to conclude that the
distribution of cellular specialization based on enzyme activity is relatively more uniform
in fish than in mammals (reviewed in Gingerich, 1982). However, Schar et al. (1985)

125
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conclude that rather extensive zonation is present in rainbow trout livers, although still
less than that found in mammals.
Parenchymal cells (hepatocytes) are the dominant type of cell present in liver. These
range in shape from oval to irregular polygons; their ultrastructure has been described by
Leland (1983). As would be expected from the functions of the liver, these cells are well
endowed with secretory and biosynthetic structures, such as Golgi bodies and rough
endoplasmic reticulum. Juvenile rainbow trout appear to have a near absence of these
cellular structures, which develop as the fish matures (Chapman, 1981; Leland, 1983).
This implies that biotransformation and excretion of xenobiotics may be severely limited
in younger fish. However, some induction of the biotransformation enzymes is possible
by xenobiotic exposure even in the embryos (Binder and Stegeman, 1983).
At any one time, some 12-15% of the liver mass of fish is blood (Stevens, 1968). This
is over twice the amount of most other organs. From a practical standpoint, it means that
when a crude homogenate of liver tissue is prepared for biochemical analysis, a signifi­
cant fraction of the content of that homogenate will actually be blood, rather than the
homogenized liver tissue.
In spite of there being a large amount of blood in the liver, the rate of blood flow
through the organ in fish is small in comparison with that in mammals. Thus, Gingerich
(1982) points out that clearance from the blood by the liver of endogenous and exogenous
chemicals is slower in fish than mammals, in large part due to this comparative lack of
blood perfusion.
Although the fish liver receives blood from the hepatic artery, it apparently lacks a
well-developed arterial blood supply in many species. This could imply a rather low level
of oxygen for liver cells, as they are perfused primarily by the portal vein which carries
venous blood from the intestine. Measurements of adenylate energy charge in this tissue
does indeed suggest a relatively low level of oxygen in these cells compared to those from
other organs (van den Thillart et al., 1980; Heath, 1984).

III. ALTERATIONS IN LIVER/SOMATIC INDEX

The ratio of liver to body weight differs greatly between and within various species of
fish. The liver/somatic index is usually expressed as the percentage of the wet weight of
the liver is to the whole body and is often also referred to as the hepatosomatic index
(HSI). The nutritional state of the organism at the time of capture has a considerable
influence on liver size. Indeed, the HSI is useful (among other measures) in assessing the
general condition of fish captured in the field (Goede and Barton, 1990). These transitory
changes in liver size are largely due to variations in glycogen and fat content. Because
fat is an important repository for many xenobiotics, the amount of such a chemical
deposited in the liver may depend to some extent on the nutritional state of the fish, and
variations between species are important; for example, flatfish (Pleuronectidae) and cods
(Gadidae) have especially large stores of lipid in comparison with most other teleost
groups (Gingerich, 1982).
Decreases in HSI are frequently seen in fish under stress (Lee et al., 1983; Ram and
Singh, 1988). This presumably occurs when a chronic stress imposes an energy drain on
the fish and is often correlated with loss of energy stores such as liver glycogen. The cause
of the decline in liver mass can actually be due to depressed feeding (Barton, 1988) and
is not necessarily a direct effect of some contaminant. As discussed in Chapter 8, fish that
are under stress often reduce or even cease feeding.
Increases in liver size are commonly seen in fish that have been exposed for long
periods of time to organic contaminants (especially petroleum hydrocarbons) in the
laboratory or field (Yarbrough et al., 1976; Poels et al., 1980; Fletcher et al., 1982; Oikari
and Nakari, 1982; Slooff et al, 1983; Vignier et al., 1992; Everaarts et al., 1993). The
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increase in HSI can be due to hyperplasia (increased cell number) and/or hypertrophy
(increased cell size). It may be associated with increased capacity to metabolize xenobiotics
so it could be considered an adaptation to the presence of pollution, rather than a
dysfunction. The hypothesis might be proposed that fish that are exposed to pollutant
stress may show decreases in HSI if food is limited or if the exposure is of short duration.
However, with chronic exposures where food is not limited and feeding rate is normal,
a rise in HSI may be the expected response.

IV. HISTOPATHOLOGICAL EFFECTS OF POLLUTANTS

There have been numerous reports of histopathological changes occurring in fish tissues
from exposure to pollutants and it is evident that a wide variety of chemicals cause lesions
in the livers of fish (Gingerich, 1982; Patton and Couch, 1984; Hinton et al., 1987,1992).
These chemicals include the metals arsenic, cadmium, copper, and mercury; assorted
industrial wastes such as ammonia, Aroclor, chlorine, and phenol, both organophosphate
and organochlorine pesticides such as endrin, dieldrin, Dursban®, and diazinon; the
carbamate insecticide carbofuran and petroleum hydrocarbons. It should be noted that
species differences can be considerable. For example, Bass et al. (1977) found histologi­
cal lesions in the livers of bluegill exposed intermittently to fairly high doses of chlorine,
whereas trout showed no changes in liver histology (at the level of light microscopy)
except for some loss of glycogen, even when the organ was examined from moribund
fish.
The histological changes observed in various studies on livers taken from fish exposed
to pollutants include increased vacuoles in the cytoplasm, enlarged lysosomes, changes in
nuclear shapes, focal necrosis (death of cells in a localized area), ischemia (blockage of
capillary circulation), hepatocellular shrinkage, regression of hepatocytic microvilli at the
bile canaliculi, fatty degeneration, and loss of glycogen. The latter two changes can be due
to depressed feeding and/or elevated levels of the stress hormones cortisol and adrenalin,
and therefore are not necessarily caused by the chemical acting directly on liver cells.
In general, the changes observed in liver tissue are rather non-specific so it is not
possible to identify the chemical causing the lesion (Gingerich, 1982; Patton and Couch,
1984). At the ultrastructural level, the smooth and rough endoplasmic reticulum (RER)
frequently become reorganized and proliferated in response to PCBs, DDT, or mixtures
of organic xenobiotics (Weis, 1974; Hacking et al., 1978; Kohler, 1989). It seems
reasonable to assume that this is associated with induction of xenobiotic biotransforma­
tion enzymes (see Chapter 5). Just such a correspondence between structural change and
function was found in mullet receiving injections of 3-methylcholanthrene (Schoor and
Couch, 1979). Thus, this could be interpreted as an adaptation rather than a pathological
change. Even so, it may come at a cost of resources to the fish.
Copper at 0.05 or 0.1 mg/L for 4-7 days caused a variety of alterations in cellular
structure revealed by transmission electron microscopy in livers of the snake-headed fish
(Channa punctatus; Khangarot, 1992). These included extensive proliferation of the
smooth endoplasmic reticulum, dilation of the RER, loss of mitochondrial structure,
proliferation of lysosomes, and accumulation of electron dense bodies. The most domi­
nant features were dilation of the RER, and reduction and shortening of mitochondria.
The RER changes are probably associated with detoxification while the mitochondrial
changes are clearly pathological.
Fairly acute exposures of tench to copper for 12 days caused accumulation of large
amounts of hémoglobinémie pigment in Kuppfer cells (Roncero et al., 1992). The
function of Kuppfer cells is removal of damaged erythrocytes so the accumulation of
hemoglobin in these cells from copper exposure suggests hemolysis of the blood cells. In
mammals, excess copper can cause hemolytic anemia by denaturing hemoglobin and
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oxidizing glutathione (Fairbanks, 1967), although anemia in fish from copper exposure
is not common (Chapter 4).
Exposing brown trout to a concentration of copper, which was below that which
inhibited spawning, produced some rather subtle changes in hepatocyte ultrastructure
(Leland, 1983). These included slight contraction of mitochondria and enlargement of
nuclei. At somewhat higher concentrations of copper, there was evidence of cell swelling,
and electron dense granules became prevalent. The latter may be areas of copper deposition,
but could also be points of cellular damage. Zinc caused similar changes so the lesions are
not specific for copper. At doses causing 50% mortality of juveniles in 42 days, Leland
(1983) notes (p. 353): “.. .loss in integrity of mitochondrial membranes, rupturing of plasma
and nuclear membranes, separation of granular and fibrillar nuclear components, fragmen­
tation of endoplasmic reticulum, and extensive autophagic vacuolization were significant
features of hepatocytes of surviving juvenile rainbow trout.” He further makes the cogent
point that aside from the possible sequestering of the metals, the ultrastructural changes
observed may be largely a non-specific response to stress.
Cytoplasmic inclusions seem to be a fairly common occurrence with fish that have
been exposed to either metals or organic xenobiotics (Patton and Couch, 1984; Hinton et
al., 1987). For example, trout reared on diets containing elevated amounts of copper had
electron-dense granules in the cytoplasm of their hepatocytes (Lanno et al., 1987). With
the aid of electron microprobe analysis, these were demonstrated to contain copper. The
authors suggest the granules may be involved in the process of excretion of copper into
the bile, or they may also be a mechanism for sequestering harmful chemicals.
Histopathological techniques are increasingly being used in field studies to assess the
impact of suspected contaminants (Kohler, 1990; Hinton et al., 1992; Meyers et al., 1992;
Young et al., 1994). Because the liver commonly accumulates xenobiotics to very high
concentrations, it exhibits exceptional sensitivity to these in the field. Good correlations
have been seen between the concentrations of various persistent chlorinated hydrocarbons
and metals and liver lesions (Kohler, 1990; Myers, 1992). Kohler (1989) examined the
question in flounder of how well the liver lesions regenerate following contaminant
exposure. She transferred contaminated flounder into a contaminant-free environment
and found partial or complete liver regeneration in 50% of the fish after 20 days and 70%
after 40 days. Interestingly, some of the flounder failed to show any regeneration at all
and these individuals retained their high levels of contaminants. While the field use of
histopathology certainly has promise, some biochemical changes have greater sensitivity
to pollutant stress and these will be discussed below.

V, MAJOR FUNCTIONS OF THE LIVER

A. INTERCONVERSION OF FOODSTUFFS
Because the liver is “down stream” from the intestine, foodstuffs absorbed from the diet
are often immediately acted upon by the liver. Conversions from one form to another
occur primarily in this organ (e.g., from protein to carbohydrate). Some of the key
enzymes involved in these conversions are sensitive to pollutants (see Chapter 9) and
these same enzyme systems are under control of several major hormones (Chapter 8,
Section X). One of the effects of this interconversion of foodstuffs is the production of
large amounts of ammonia from the deamination of amino acids (Wood, 1993).

B. STORAGE OF GLYCOGEN
The largest store of carbohydrate in the fish’s body is in the liver and this is the primary
source for blood glucose in the postabsorptive animal. Changes in liver glycogen concen­
tration are almost always seen in fish exposed to a variety of chemical and physical
stressors (Chapter 8).
 129

C. REMOVAL AND METABOLISM OF FOREIGN CHEMICALS IN

THE BLOOD
This is discussed at some length in Chapter 5. Metals and organic xenobiotics are often
sequestered there so cellular concentrations can become quite high.

D. FORMATION OF BILE
Bile has a number of things in it including the bile salts, which are necessary for normal
fat digestion; bile pigments, which are breakdown products of hemoglobin; and xenobiotics
or their metabolites which have been removed from the blood (Chapter 5). Harmful
metals are also excreted via this route.

E. SYNTHESIS OF MANY PLASMA PROTEINS

The complex mixture of proteins found in the blood largely originates from the liver. It
is broadly divided into the fibrinogens and albumins. (The immunoglobulin proteins
come from B lymphocytes so are not of liver origin.) The fibrinogens are required for
blood clotting, which incidentally, is quite rapid in fish compared to mammals. Fish under
stress exhibit an accelerated clotting, but this is associated with an increase in number of
thrombocytes rather than fibrinogen (Casillas and Smith, 1977). The albumins are impor­
tant in maintaining normal plasma osmotic pressure, blood pH buffering, as an amino acid
source, and as a transport molecule for hormones and exogenous chemicals such as metals
and organics.

F. SYNTHESIS OF CHOLESTEROL FOR USE IN STEROID HORMONES

AND CELL MEMBRANES
Cholesterol is the most abundant sterol in vertebrates. It functions to stabilize cell
membranes, plasma lipoproteins, and myelin in the nervous system. It also is a precursor
for bile acids and steroid hormones (Myant, 1981). Cholesterol is synthesized by the liver,
however, in mammals at least, the intestine and skin also synthesize significant amounts
of this compound.

G. EXOCRINE PANCREATIC SECRETION

The term “exocrine” means the portion of the pancreas devoted to the secretion of
digestive enzymes. This tissue is histologically observed surrounding the hepatic portal
veins inside the liver (Groman, 1982). A bony fish also has pancreatic tissue scattered
among the mesenteries so it is usually not a discrete organ as is typical of mammals. The
exocrine portion of the pancreas secretes a variety of digestive enzymes, but it is not clear
how important this pancreatic tissue within the liver is in digestion.

H. METABOLISM OF HORMONES
Many of the hormones of a vertebrate are metabolized by the liver. This avoids the
buildup in the blood of such things as estrogen, one of the key sex hormones, and cortisol.

VI. EFFECTS OF POLLUTANTS ON LIVER FUNCTION

Measurements of liver function per se are mostly based on clinical tests developed for
mammals (Gingerich and Weber, 1979). These are of three general types: (1) determining
the rate at which the liver clears some chemical (usually a dye) from the blood; (2)
monitoring the activity of plasma enzymes that are normally concentrated in liver cells,
but get into the blood when hepatocytes are damaged; and (3) determining changes in the
plasma concentrations of chemicals produced (e.g., albumin and cholesterol) or elimi­
nated (e.g., bilirubin) by the liver. From the brief listing above of the multitude of
activities associated with the liver, it is also safe to assume that a pollutant’s effect on liver
130

function may be reflected indirectly in such diverse physiological activities as sex drive
and digestion.

A. PLASMA CLEARANCE BY THE LIVER OF SPECIFIC DYES

An important function of the liver is the removal of hormones and potentially harmful
substances from the plasma and subsequent excretion in the bile. This process involves
several steps including uptake by the parenchymal cells, biotransformation of the sub­
stance by enzyme systems into a more polar product, and conjugation with another
molecule (e.g., gluconate) in preparation for excretion.
The primary interest of fish toxicologists has been the mechanisms for accumulation
and biotransformation of the xenobiotics which are discussed at some length in Chapter
5. The question has also been asked as to whether some substances may inhibit these
processes in the liver. In order to answer this, the dye clearance test developed for
mammalian systems has been applied to fish. Fortunately, the dye sulfobromophthalein
(BSP) is cleared from the blood in fish, at least in elasmobranchs (Boyer et al., 1976) and
trout (Gingerich et al., 1977), the same as it is in mammals.
The rate of clearance of BSP is measured by injecting the dye into a vein (usually the
caudal vein) and then sacrificing the fish for blood removal at intervals up to 2 h
postinjection (Gingerich et al., 1978). The concentrations of BSP in the plasma samples
is then determined colorimetrically. Considerable attention must be paid to establishing
an optimum dye dose and the nutritional state of the animal can influence the findings
rather greatly, thus the procedure is not as easy as it may appear. Because many fish alter
their feeding rate when in the presence of a pollutant, the application of dye clearance
tests to fish chronically exposed to some pollutant may not be justified.
Two chemicals, carbon tetrachloride and monochlorobenzene, are model hepatotoxic
agents in mammals. Intraperitoneal administration of carbon tetrachloride to rainbow
trout resulted in a dose-dependent plasma retention of BSP (Gingerich et al., 1978). In
other words, the clearance rate for BSP by the liver was reduced 24 h after receiving the
toxicant. The process of dye clearance from the blood involves several steps, any one of
which could be limiting. Thus this measure reveals an overall effect, but does not resolve
the mechanisms. Treatment with a chemical that specifically blocks formation of bile in
mammals also reduces the clearance of BSP in trout (Gingerich, 1982), but other toxicants
may act on different processes.

B. PLASMA ENZYMES OF LIVER ORIGIN

Clinical laboratories have long used the measurement of the activity of certain enzymes
in the blood plasma (sometimes called serum) as a diagnostic tool for assessing liver
damage in humans. Enzymes such as glutamic oxaloacetic transaminase (GOT), glutamic
pyruvic transaminase (GPT), and sorbitol dehydrogenase (SDH) are normally found in a
low concentration in blood, so if liver cells are damaged, they may leak these into the
plasma causing an increase in their catalytic activity there. Liver cells are particularly rich
in transaminases because it is the major organ for interconversions of foodstuffs. Al­
though GOT and GPT may become elevated from damage to other organs and are not
specific for liver, SDH apparently is quite specific.
When the hepatotoxic chemical carbon tetrachloride is administered to either fresh­
water or marine fish, increases in plasma GOT (PGOT) and plasma GPT (PGPT) are
usually observed (reviewed by Gingerich, 1982). Peak increases in enzyme activity are
seen 2-6 h after injection, which is even more rapid than the mammalian response to this
agent. However, the degree of change is generally not nearly as large as that seen in
mammals. A significant amount of PGOT can come from erythrocytes, so if hemolysis
occurs either in the fish, or in the process of blood collection, a false elevation may be
evident. Another source of these enzymes is the kidney, so increases may reflect damage
 131

to that organ, or even heart tissue. The most convincing evidence that CC14 causes release
of GOT and GPT from liver exclusively is found in the work of Inui. (1969). He showed
that eels with the liver removed exhibit no elevation in the enzyme activities when
injected with CC14, whereas sham operated controls do.
In an investigation by Casillas et al. (1983), histological lesions in the liver were
correlated with changes in plasma enzymes. Serum alanine aminotransferase (ALAT) and
asparate aminotransferase (ASAT) were elevated 40-60 times that of controls following
injection of CC14. These are much greater increases than GOT or GPT ever reach. Serum
albumin, total protein, bilirubin, and urea nitrogen did not change in relation to liver
lesions induced by CC14 injections, thus the plasma enzymes would seem to have better
diagnostic value for this type of liver damage than the other plasma constituents.
There has been some interest in using the changes in plasma enzyme activity as a
general measure of biochemical alterations induced by pollutants. McKim et al. (1970)
exposed brook trout chronically to 32-39 p.g/L copper and found a +60% increase in
plasma GOT during the first 21 days, but when measured at 11 months of exposure, the
PGOT was actually 30% below controls. The initial increase could have been due to heart
damage, but because the liver accumulates copper so much more readily than the heart,
that seems unlikely. The reduction observed at 11 months conceivably was due to a direct
inhibitory action of copper on the enzyme following copper accumulation in the liver.
Copper is a strong inhibitor of fish PGOT when the blood is exposed to the element in
vitro (Christensen, 1971).
Chronic exposure of a marine fish to mercury caused peak increases in PGOT and
PGPT within 4-8 days. The extent of change (>2x) was greater with PGOT than PGPT
(Hilmy et al., 1981). In the same study, it was reported that alkaline phosphatase exhibited
a 50% decrease in activity by 8 days of exposure. The authors think this latter effect may
be due to the mercury displacing the cofactor magnesium from the enzyme. Clearly there
is some direct inhibition of the enzyme in the plasma by mercury, but what the mechanism
is remains unclear. It may be worthwhile to note that Jackim et al. (1970) found that
alkaline phosphatase extracted from fish liver was weakly inhibited by mercury when the
exposure was in vitro, but the enzyme activity was actually stimulated when the exposure
was in vivo. In other words, mercury is inhibitory of alkaline phosphatase, but seemingly
induces synthesis of the molecule in the intact liver which more than compensates for any
inhibition.
When added in vitro, essentially all the transition metals cause inhibition of LDH and
GOT from white sucker plasma, although there is a fair amount of variation between
different metals as to the degree of inhibition (Christensen et al., 1977). The significance
of these in vitro findings is unclear because there are no data on the concentrations the
different metals may reach in the blood. Without that information, the finding of inhibi­
tion at some concentration in vitro means very little because the enzyme may never be
exposed to that concentration in the blood.
Exposure of pike for 8 days to phenol caused elevations in both GOT and GPT
(Kristoffersson et al., 1974), and Wieser and Hinterleitner (1980) found elevated levels
of GOT and GPT in plasma from rainbow trout taken near a sewage outfall (composition
apparently unknown).
Perhaps the best plasma enzyme for assessing liver damage in fish is SDH. Dixon et
al. (1987) has shown that this enzyme has activity in liver that is an order of magnitude
above nine other tissues examined in rainbow trout, thus its elevation in plasma is quite
specific for liver damage. They tested the effects of phenol, p-chlorophenol,
p-phenoxyphenol, carbon tetrachloride, cyanide, and copper on the level of this enzyme
in plasma. Both direct injections and additions to water were used as methods of
exposure. A dose-dependent marked rise was seen with all toxicants tested except
cyanide. The plasma activity peaked after around 48 h of exposure. When injections were
132

used as the mode of administration, there was a rise in SDH before histopathology was
detectable in the liver. Thus, the biochemical measurement in the plasma was even more
sensitive than histopathology.
As with the rainbow trout, SDH activity was found to be much higher in liver than in
six other tissues of mullet, a marine species (Ozretic and Ozretic, 1993). In the same
study, they found activity of glutamate dehydrogenase (a mitochondrial enzyme) is also
higher in liver than in other tissues, however, kidney has a level about one third that of
liver. Both enzymes exhibited marked increases in the plasma following injection of
either phenol or carbon tetrachloride.
Two other enzymes that rise in the plasma as a result of tissue damage are acid
phosphatase and A-acetyl-B-o-glucosaminidase (NAG). Both of these come from lyso­
somes that break down in the cells. Versteeg and Giesy (1986) found that chronic
exposure of bluegill to cadmium for up to 32 days produced increases in these two
enzymes in the plasma. Asparte and alanine transaminase enzymes were unaffected. The
changes in acid phosphatase and NAG were associated with a decrease in stability of liver
lysosome membranes (Versteeg and Giesy, 1985) which suggested a direct effect of the
metal on these organelles. It is interesting that no histological changes were seen in the
liver but decreases in growth rate of the fish were seen, so the biochemical alterations
were predictive of a higher order response to the pollutant.
Lysosomes accumulate a variety of metals and organic compounds (Moore, 1985). As
previously mentioned, Versteeg and Giesy (1985) showed that the membranes of these
organelles become less stable during chronic cadmium exposure. Kohler (1991) used this
measure to assess pollution in contaminated estuaries and found decreased lysosomal
stability in a gradient that correlated with the level of pollution. Her data suggest a two-step
response in that activation of the lysosomal system occurs first with increases in number and
size of lysosomes as they accumulate the contaminants. If this adaptive response is over­
come by the pollutant load, then the membrane stability of the lysosomes decreases
releasing their contained enzymes into cells and ultimately into the bloodstream.
In general, it appears that many toxicants will cause increases in at least some fish
plasma enzyme activities. A word of caution is in order here. We have found in this
laboratory that the enzyme activities in trout plasma are quite variable in control fish
when measured using an automated clinical analyzer. Part of this variability may be due
to the fact that these analyzers are designed for mammalian work and operate at 25°C or
higher. Trout enzymes are adapted to function at temperatures considerably below that
level, so one may be well beyond the temperature for optimum catalytic activity, espe­
cially for salmonids.

C. CHANGES IN PLASMA CONCENTRATION OF CHEMICALS

PRODUCED OR EXCRETED BY LIVER
The concentration of cholesterol in the blood is moderately sensitive to the presence of
pollutants, but the direction of movement seems to depend on the concentration of the
pollutant and on whether it affected osmoregulation of the fish. Gluth and Hanke (1985)
exposed carp to a variety of organic pesticide chemicals at sublethal concentrations for
72 h and noted decreases in cholesterol in all cases. Because plasma protein also
decreased at the same time and to approximately the same extent, they attribute the
changes to water uptake resulting from osmoregulatory failure (see Chapter 7). Decreases
in blood cholesterol were also seen with 96-h exposures of Ciarías to endrin or toluene
(Ghazaly, 1991). Because protein remained unchanged in this study, the decrease is
probably real and not due to water dilution. When the same species of fish was exposed
to mercury (both inorganic and organic) for periods up to 180 days, reductions in free and
esterified cholesterol were seen (Kirubagaran and Joy, 1992). This was also associated
with a reduction in several other lipids so it could reflect a general depletion of energy
 133

stores. The inorganic form of the mercury was more effective than the methylmercury in
lowering lipid levels, which is the reverse of what usually occurs with these pollutants.
Lead also caused a large decrease in cholesterol in blood, liver, ovary, and testes of
rainbow trout with chronic exposures of 30 or 60 days (Tewari et al., 1987). Decreases
in cholesterol may indicate impairment of the liver synthesis of this chemical, but there
is no direct evidence for this in the literature. Another possible cause would be increased
utilization for corticosteroid synthesis.
Marked increases in cholesterol have also been observed in some studies. In apparent
contrast to the above work on mercury, Dutta and Haghigh (1986) reported an increase
in plasma cholesterol over a 3-day period of exposure to methylmercuric chloride. Copper
has also been found to cause a steady increase in cholesterol with the levels reaching
almost twice that of controls after 30 days of continuous exposure (Singh and Reddy,
1990). Plasma protein and blood cell count actually went down so hemoconcentration
was certainly not the explanation for the rise in cholesterol.
DDT and endrin (another organochlorine insecticide), caused a 50% elevation in
serum cholesterol when eels and mullet were exposed to the insecticides for 96 h (Hilmy
et al., 1983). Interestingly, there was no dose effect over a 10-fold range in pesticide
concentration. Elevations in cholesterol could be explained by an enhanced production by
the liver (and other organs), or release of cholesterol from damaged cell membranes.
Regarding the first possibility, the hormone cortisol inhibits cholesterol synthesis and this
hormone becomes elevated in fish under stress (Chapter 8). Thus, a stimulation of
cholesterol synthesis does not seem likley in those fish under severe stress, however, fish
that are chronically exposed may not have this problem. Interestingly, it was found fairly
recently that chronic exposure to phenol (and perhaps some other substances as well?)
inhibits the conversion of cholesterol into steroid sex hormones (Mukherjee et al., 1991).
Phenol also caused increased cholesterol in the liver and blood, reflecting perhaps a
combination of “backing up” of cholesterol in the blood and stimulation of its synthesis
in the liver. It therefore appears that there are a number of ways that cholesterol metabo­
lism can be altered by toxic chemicals and the dose used may have a considerable impact
on the character of the effect.
It is well known that the liver is responsible for the elimination, via the bile, of the
products of heme breakdown, primarily bilirubin. If this process is impaired, a condition
of jaundice may develop and be reflected in a rise of bilirubin concentration in the plasma
(Mattsoff and Oikari, 1987). Oikari and Nakari (1982) found that exposure of trout for
11 days to kraft pulpmill effluent at a dose equivalent to 0.15 x 96 h LC50 produced a
marked elevation in plasma bilirubin. A concomitant inhibition of the enzyme UDP-
glucuronyltransferase in the liver tissue was also noted. Because this enzyme is required
for the normal conjugation reactions by which bilirubin is prepared for excretion into the
bile, its inhibition may be the primary cause of the jaundice. More recently, Rabergh et
al. (1992) found that the resin acids in pulpmill effluent also inhibit the uptake of bile acid
and bilirubin by isolated trout hepatocytes.
Liver dysfunction has been implicated in a study of high mortality of striped bass in
San Francisco Bay (Brown et al., 1987; Young et al., 1994). Moribund fish had elevated
levels of plasma bilirubin and uric acid was 80-fold in excess of the levels found in
reference fish. The plasma of the moribund fish was also yellow-orange in color. The
osmoregulatory picture was affected but only moderately so blood water percentage
changes were apparently minimal in these fish that were obviously under severe stress.
A mixture of agricultural, urban, and industrial contaminants were found in very high
concentrations in the livers of the moribund fish, but only low levels of these compounds
were found in reference fish from the Pacific Ocean (Cashman et al., 1992).
It is often said, but without much evidence presented, that nutrition can affect the
response of a fish to some stress. Dixon and Hilton (1981) have shown that a high
134

carbohydrate diet in trout reduces their tolerance to waterborne copper (i.e., lowers the
incipient LC50). What is perhaps more interesting is that Dixon and Hilton provided a
physiological mechanism: the high carbohydrate diet caused a greater accumulation of
copper by the liver. This is undoubtedly due to a reduced capacity for copper excretion
into the bile by the liver in fish with a high carbohydrate diet, although just why that
would be is obscure. A further aspect of nutrition will be presented next when discussing
ascorbic acid.

VII, ASCORBIC ACID AND POLLUTANT EXPOSURE

Ascorbic acid is better known as vitamin C. It plays a number of known biochemical roles
and undoubtedly several that are currently unknown. It is essential for the normal
synthesis of collagen. Other functions less well studied include involvement in the
metabolism of steroids by the liver, oxidation of tyrosine, and in conjunction with
superoxide dismutase and catalase, it helps prevent the buildup of free radicals in cells
(Lehninger, 1979). Ascorbic acid is discussed here primarily because most of the work
in fish has been on liver ascorbic acid, even though it is found in other tissues as well.
Species differences are striking; for example, Thomas and Neff (1984) found high levels
in the brain of mullet while Lopez-Torres et al. (1993) report that it was non-detectable
in the brain of trout.
It is not clear to what extent various teleosts have a dietary requirement for this
vitamin. Because a dietary requirement for ascorbic acid varies considerably among
different higher vertebrates, it would not be surprising to find that similar variability was
present among the teleosts. Salmonids, channel catfish, and mullet clearly do require it
in the food (Halver et al., 1975; Thomas et al., 1982), but the cypriniformes, which
include carp, possess the enzyme L-gluconolactone oxidase which is necessary for ascor­
bic acid synthesis (Yamamoto et al., 1978), and therefore they presumably do not require
this vitamin in the diet. Even in those species capable of synthesizing the vitamin, the
requirement may exceed that capability when stressed (Mayer et al., 1978).
In the tissues of fish, there appears to be two forms of ascorbate: L-ascorbic acid (Cl)
and L-ascorbic acid-2-sulfate (C2) (Benitez and Halver, 1982). The latter is generally used
as a dietary supplement as it is the more stable form (Halver et al., 1975). Benitez and
Halver (1982) suggested it may actually be the dominant form in which it is stored in the
tissues, but Thomas et al. (1982), working on mullet, found very little of C2 in gill, brain,
kidney, or liver, whereas there were considerable quantities of Cl in these tissues. The
enzyme C2 sulfatase catalyzes the removal of the sulfate from C2 yielding Cl. Benitez
and Halver (1982) have proposed that this enzyme serves as a sort of modulator of the
cellular levels of vitamin C in fish, in that as the Cl becomes utilized, the enzyme is
derepressed and increases in activity to promote replenishment of Cl. This mechanism
may work in those species with large amounts of C2, but if there is not much C2 available,
as in the mullet, such a function seems of little value.
In a rather interesting series of experiments, Mayer et al. (1978) exposed channel
catfish to various amounts of toxaphene (an organochlorine insecticide) in the diet for 150
days. They noted a dose-dependent depression of backbone collagen and spinal deformi­
ties, but when ascorbate was added to the diet at several different doses, the extent of
collagen depression and the incidence of deformities in the fish exposed to toxaphene was
decreased roughly in proportion to the ascorbate dose. Toxaphene was shown to cause a
depletion of vitamin C in the spine but not in the liver. Because vitamin C supplements
also reduced the whole-body residues of toxaphene, the authors hypothesized that this
molecule is used by the liver in detoxifying the toxaphene but at the expense of that in
the collagen.
 135

Exposure of mullet to a rather acute dose of pentachlorophenol (PCP) produced an

elevation in liver ascorbic acid in either 24 h (100 ppb PCP) or 48 h (200 ppb PCP). There
were corresponding increases in plasma cortisol which indicate interrenal activation
(Thomas et al., 1981). Because stress has been shown in trout to cause ascorbic acid
depletion in interrenal tissue (Wedemeyer, 1969), the possibility exists that ascorbate was
transported from the interrenal to the hepatic cells. Other hypotheses mentioned by
Thomas et al. include a reduction in ascorbic acid breakdown, or a stimulated synthesis,
caused by cortisol. The latter seems especially unlikely as mullet have virtually no ability
to synthesize ascorbic acid.
PCP is rapidly conjugated to glucuronid in goldfish (see Chapter 5) and this process
requires ascorbate in mammals, so the process of detoxification would probably cause
depletion of hepatic ascorbate. Indeed, in the mullet, at 120 h of exposure (when the
experiments were terminated), the liver ascorbate had returned to near “normal” (Thomas
et al., 1981). A more prolonged chronic exposure might eventually cause a lowering of
ascorbate as is seen in those fish exposed to waterborne metals (discussed next). How­
ever, it is interesting that perch taken from coastal waters polluted by kraft mill effluents
had elevated liver ascorbate levels (Anderson et al., 1988). These types of effluents have
several types of chlorinated phenolics including PCP (Oikari et al., 1985).
Mullet exposed to cadmium (10 mg/L) exhibited a brief 50% drop in liver ascorbate
at 6 h, but then by the third day the ascorbate became elevated. Then, continued exposure
produced a more-or-less steady depletion in hepatic ascorbate (Thomas et al., 1982).
Brain, kidney, and gill showed a somewhat similar pattern of change although the percent
depletion was not as great as that seen in the liver.
It is noteworthy that cadmium produced no initial (other than a very transient burst)
increase in liver ascorbate, as was seen in the PCP exposed animals. The hypothesis that
a rise in ascorbate is the result of increases in cortisol from the interrenal gland (as
proposed by Thomas et al., 1981) is indirectly supported by these cadmium data. This is
because work by Schreck and Lorz (1978) on salmon has shown that cadmium is rather
unique in that exposure to doses that are high enough to even cause death fail to activate
the interrenal gland, the way most other stressors do.
The mechanism responsible for the depletion of tissue ascorbate following cadmium
exposure is not known but must involve a stimulated rate of utilization of this vitamin.
Copper in the water has been shown to increase the dietary requirement for vitamin C in
trout (Yamamato et al., 1981), and supplementing the diet with vitamin C reduced the
accumulation of copper in the liver and gills. It could be this latter effect is due to an
enhanced excretion rate of copper produced by elevated vitamin C, but that is only
speculation. One cannot help but wonder if dietary supplementation with this vitamin
would also enhance the depuration of other metals and even organic compounds (Mayer
et al., 1978). Providing fish with high doses of vitamin C in the diet has been shown to
decrease the effect of waterborne thiotox and malathion on several enzymes in the gills,
liver and brain (Verma et al., 1982).
The depletion of tissue stores of ascorbate during chronic exposure to pollutants
(except chlorinated phenolics) may turn out to be common depending, of course, on the
ability of the species of fish in question to synthesize this vitamin. The extent of change
can be sizable. Six-month exposures to carbofuran caused a 44% decline in Indian
catfish (Ram and Singh, 1988), and a week of exposure to the watersoluble fraction of
No. 2 fuel oil caused a 45% loss of liver ascorbate in mullet (Thomas and Neff, 1984).
In the latter study it was also found that liver ascorbate was not affected much by
“natural” stressors such as changes in temperature, salinity, or handling, so measure­
ments of this trace nutrient in the liver may have potential for specifically indicating
chemical pollution.
136

From the standpoint of the fish, depletion of ascorbate can produce a variety of
pathological conditions including delayed wound healing, anemia, skin lesions, fin necro­
sis, scoliosis, etc. (Hilton et al., 1977; Thomas et al., 1982). Lipid peroxidation of cell
membrane lipids is a primary mechanism of cell injury by xenobiotics (Recknagel and
Glende, 1977) and has been seen in several studies of fish (see Thomas, 1990 for
references). Because one of the functions of ascorbic acid is prevention of peroxidation
in cells (Winston and Diguilo, 1991), a reduction in its concentration from various
chemical or physical stresses has obvious implications for possible cell injury.

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index of an Indian catfish, H e te r o p n e u s te s f o s s i lis , and its recovery, E c o to x ic o l. E n v iro n . S a fety , 20,
30, 1990.
Slooff, W., van Kreijl, C. and Baars, A., Relative liver weights and xenobiotic-metabolizing enzymes
of fish from polluted surface waters in The Netherlands, A q u a t. T o x i c o l, 4, 1, 1983.
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Delta of California, J. F ish B i o l , 44, 491, 1994.
 Osmotic and Ionic
Chapter
7
Regulation
I. OVERVIEW OF SOME GENERAL PRINCIPLES
Osmoregulation refers to the processes by which the osmotic pressure of the body fluids
and the water volume in an animal are held relatively constant. The osmotic pressure of
fish blood is mostly provided by inorganic salts and is approximately one third that of
seawater, with marine fish having slightly more salt in their blood than freshwater species.
Because gills are permeable to water as well as oxygen, CO2, etc., there is an osmotic flow
of water out of the marine fish because the salt concentration of its blood is less than that
of the ocean, and into the freshwater fish because of an opposite osmotic gradient. The
diffusion gradient for sodium chloride (the primary salt) is in the direction opposite to the
osmotic diffusion of water across the gill epithelium. Figure 1 illustrates these major ion
and water fluxes in typical teleost fish.
The water lost by diffusion from a marine teleost is replaced by the animal drinking
an equal amount of seawater. In order for the swallowed seawater to actually enter the
blood from the gut lumen, there must be active transport of NaCl from the lumen into the
intracellular space which sets up a local osmotic gradient for water movement into the
blood (Kirschner, 1991). Due to this uptake of salt from the gut and the passive diffusion
of salt into the animal, marine fish must constantly excrete a considerable amount of salt.
This is done through the gill and opercular epithelia by way of special “chloride cells”
(Payan and Girard, 1984). These cells are well endowed with mitochondria to generate
ATP and the enzyme Na,K-activated ATPase. The movement of sodium and chloride out
of marine fish by way of these cells is a complex process involving a combination of
electrical potential (inside positive) and an Na/K exchange. Sodium and chloride ions are
transported separately: sodium goes through the leaky paracellular junctions, chloride
directly through the chloride cells (Evans, 1993).
Freshwater fish have the opposite problem to their marine cousins in that excess water
gained from osmosis must be excreted. This is done by producing a relatively large
volume (5 ml/kg/h) of dilute urine. Because of a loss of salt from diffusion across the gills
and skin and a small loss via the urine, these fish must actively transport salt from the
water into the blood. This is presumably done by chloride cells in the gills (but not the
operculum), and possibly other gill epithelial cells as well, again utilizing energy from
ATP and catalyzed by Na,K-activated ATPase. The chloride cells in freshwater fish

141
142

Figure 1 Comparison of ion and water fluxes in a marine and a freshwater fish. Ion fluxes are
in mMkg/h. Thin arrows indicate passive processes, thick ones active transport. (Data from
Fletcher, C. R., J. Comp. Physiol., 124,157,1978; Eddy, F. B. and Bath, R. N., J. Exp. Biol., 83,
181, 1979).

transport sodium and chloride independently of each other. These electrolyte ions in the
water are exchanged for ammonia, hydrogen ions, and bicarbonate in the blood thus
aiding the excretion of these waste products (Payan and Girard, 1984).
Recently, it has been demonstrated that the adsorption of sodium to the gill surface
plays an important role in presenting sodium ions to the chloride cells (Handy and Eddy,
1991a). At low external sodium concentrations, as would be found in freshwater condi­
tions, calcium inhibits this adsorption process.
The ionic fluxes for marine fish are approximately an order of magnitude greater than
those for freshwater species (Figure 1). This reflects the greater permeability of the
marine fish to these ions. In order to achieve the high rate of salt excretion required by
such large fluxes, they have more chloride cells than do their freshwater relatives. It has,
however, been reported (Lauren and McDonald, 1985) that trout acclimated to soft water
have more chloride cells than those acclimated to hard (i.e., high calcium) water. There
are also marked species differences even among the freshwater fishes in ability to
transport electrolytes.
The differences in flux rates between marine and freshwater fish may actually be
greater for some species. Most comparisons are based on trout as the freshwater species,
whereas recently it was reported that gill permeabilities for electrolytes and water are
greater in trout than in other less active species such as yellow perch (Perea flavescens)
and smallmouth bass (Micropterus dolomieui) (McDonald et al., 1991a). These greater
fluxes in the trout are facilitated by a greater density of chloride cells and the individual
cells have a greater transport capacity when compared with a species such as the carp
(Cyprinus carpió) (Williams and Eddy, 1988a).
 143

Euryhaline fish, such as the well investigated Fundulus heteroclitus, have the ability
to reverse the active movement of sodium and chloride across the gill tissue, to change
the amount and composition of urine excreted, and to alter epithelial permeability, all
within a matter of hours. Anadromous and catadromous species, such as eels and salmon,
also do this reversal “act” but at a slower pace (i.e., days instead of hours).
The parr-smolt transformation in salmonids is a form of metamorphosis involving
physiological changes that produce an increased salinity tolerance (i.e., conversion into
a marine form). This is done in “anticipation of seaward migration” and is keyed to
photoperiod, environmental temperature, and perhaps an endogenous “clock” mechanism
(reviewed by Boeuf, 1993).
Acclimation temperature seemingly has little effect on blood electrolyte or osmolality
setpoints in either marine or freshwater fish (Burton, 1986) except for a tendency for the
setpoint to rise at temperatures near freezing in marine forms and fall in freshwater
species. This may, at least in some cases, represent a form of osmoregulatory failure at
temperatures near the extremes of the normal ranges. Rapid temperature changes, and
especially a sudden chill, can produce decreased plasma electrolyte levels in freshwater
species (see Eddy, 1981 for review).
Regulation of the various osmoregulatory processes is under hormonal control and is
only beginning to be understood (Bern and Madsen, 1992). Cortisol and prolactin are
probably the most important of the hormones controlling osmoregulation. In general,
cortisol promotes the gain of salt (and retention) in freshwater and its loss in seawater (see
Eddy, 1981). It achieves this by acting on NaVK*^ ATPase in the kidney, intestine, and
urinary bladder as well as the gills (Madsen, 1990). Prolactin acts to reduce membrane
permeability to ions and water. It also inhibits chloride secretion in marine species so it
is important in the adjustment to freshwater by anadromous fishes (Foskett et al., 1983).
In euryhaline teleosts, prolactin is the key hormone for adjusting to freshwater, whereas
cortisol is the main one involved in adjustment to seawater (Bern and Madsen, 1992).
Adrenalin has been shown to stimulate uptake of sodium from freshwater in an in vitro
preparation (Payan et al., 1975), but in seawater-adapted trout, it inhibits salt secretion
(Girard and Payan, 1980). Adrenalin also increases water permeability in the gills of
freshwater fish, presumably as a by-product of the increased oxygen permeability of the
gills associated with stress (Isia, 1984). Thus, adrenalin might not be considered as being
adaptive for osmotic and ionic regulation as it may actually exacerbate the problem of
living in freshwater.
A variety of other hormones have been shown to affect chloride cells. These include
somatostatin, urotinsins (from the caudal neurosecretory system), glucagon, and vasoac­
tive intestinal polypeptide. In seawater teleosts, the first three inhibit secretion of chloride
while vasoactive intestinal polypeptide and glucagon stimulate it (Foskett et al., 1983).
Thyroid hormones (T3 and T4) are instrumental in the physiological preparations of
salmonids for moving into seawater during the parr-smolt transformation (Grau et al.,
1981). Leatherland (1985) has also found that T4 injection reduces the elevation in
plasma sodium seen in rainbow trout abruptly transferred from freshwater to seawater.
Other hormones with effects superficially similar to T4 are the atrial natriuretic factor
(Arnold-Reed and Balment, 1991), which presumably comes from the heart, and a group
of hormones normally associated with regulation of growth: insulin-like growth factor-
1, growth hormone, and insulin (McCormick et al., 1991). Some of this latter group of
hormones may exert their osmoregulatory effects indirectly through stimulation of cor­
tisol secretion. Growth hormone is especially important in promoting seawater adaptation
in salmonids (Bern and Madsen, 1993; Boeuf, 1993).
Aldosterone, a major ion regulatory hormone in terrestrial vertebrates, may have
relatively little importance for teleost fish (Reinking, 1983). Interestingly, the renin-
angiotensin system, which in terrestrial animals is largly responsible for the regulation of
144

aldosterone levels, stimulates drinking in seawater teleosts and may thus be necessary for
normal adaptation to seawater (Carrick and Balment, 1983). It also appears to stimulate
urination in freshwater species, perhaps because it elevates blood pressure which would
cause increased filtration by the kidney (Arillo et al., 1981; Olson, 1992).
Along with the problem of maintaining internal osmotic pressure and monovalent
electrolyte cation ratios, a fish must also regulate divalent cations, most particularly
calcium. This element is critical in the maintenance of normal excitability in nerve and
muscle cells, stabilization of the branchial permeability, intracellular regulatory pro­
cesses, and it also plays a roll in reproduction, among other functions (Hunn, 1985). In
the ocean, fish are in a calcium-rich environment, while freshwater forms may or may not
be in a calcium-poor environment depending on the water hardness.
Higher vertebrates utilize bone as a calcium pool to help modulate plasma calcium
changes and this whole process is regulated by hormones from the parathyroid glands.
Cyclostomes have no calcified skeleton and Chondrichthyes have only calcified cartilage
which is in simple equilibrium with body fluids (Taylor, 1985), whereas the skeleton of
teleosts has considerable calcium in it. Taylor (1985) concludes that unlike higher animals,
it cannot undergo much remodeling and therefore serves only as a slight buffer of plasma
calcium and phosphate concentrations. The calcium pool that can be exchanged with the
environment represents only about 3% of the total body calcium content (Hobe et al., 1984).
The rate of calcium transport from the water into the blood of freshwater fish is only
about 3-10% of that of sodium and chloride (Hobe et al., 1984). Gills have been
considered to be by far the most important site, even for marine fish which drink large
amounts of calcium-rich water (Taylor, 1985). Sayer et al. (1991), however, have reported
that brown trout fry took up over 90% of their calcium from the intestine, perhaps because
they were in soft water and the calcium would have been largely dietary.
As in mammals, the blood calcium concentration is under hormonal regulation, but
there is a good bit of variation between different species of fish as to the normal
concentration of blood calcium (Hunn, 1985). Indeed, individual fish can tolerate rather
large variations in plasma calcium and it is not regulated as tightly as in tetrapods (Bern
and Madsen, 1992). The hormone calcitonin from the ultimobranchial bodies causes a
lowering of blood calcium by inhibition of Ca uptake in the gills. The Corpuscles of
Stannius, located on the surface of the posterior kidneys, produce the hormone stanniocalcin
which lowers the calcium concentration in the blood.
Whereas the above-mentioned hormones all cause a decrease in plasma calcium,
prolactin from the anterior pituitary stimulates uptake of calcium from the water by the
gills (Flick et al., 1984). However, in trout, prolactin is reputed to have little effect.
Instead, uptake of calcium from the environment in trout is stimulated by cortisol (Flick
and Perry, 1989). The mechanisms by which these hormones exert their effects are at
present unclear, but the gills and kidneys are most likely the target organs and bone
absorption or release of calcium seems rather unimportant (Taylor, 1985).
Obviously, the hormonal control mechanisms of electrolyte levels in fish are complex and
interrelated with other functions. For example, cortisol and adrenalin are important stress
hormones which affect a wide variety of functions in addition to osmotic and ionic homeo­
stasis. Thus, changes in hormonal levels under various environmental conditions may be in
response to altered blood ionic composition and/or to other homeostatic challenges. Little is
known about how the secretion rate of the various osmoregulatory hormones is regulated.

II. EFFECTS OF POLLUTANTS ON OSMOTIC AND

IONIC REGULATION
Changes in osmoregulation in fish exposed to some stressor have been generally eluci­
dated by measuring the blood plasma (or serum) electrolytes and/or total osmolality. The
 145

mechanisms of any changes in blood plasma composition are investigated by measuring

flux rates of electrolytes through gills or kidney using radioactive isotopes and by
assaying the rate of activity of the various cellular ATPases. Handling of a fish can,
however, cause changes in osmoregulatory parameters independent of environmental
pollutants, due to the phenomenon of handling diuresis (i.e., excess urination), and
changes in hormonal levels (Eddy, 1981; Ellsaesser and Clem, 1987). These changes may
last for several hours or even days. Other laboratory stresses and temperature changes also
affect osmoregulation in both marine and freshwater forms (Eddy, 1981,1982; Sleet and
Weber, 1983; Waring et al., 1992). Thus, there is a need for control fish that receive
exactly the same treatment as the experimentáis, except for exposure to the test pollutants.
Recent reviews on certain effects of pollutants on osmoregulation are Wendelaar
Bonga and Lock (1992) and Lauren (1991). Both correctly emphasize effects on gill
function including permeability and active ion uptake. The trends that have been observed
in osmoregulatory and ionoregulatory function when fish are exposed to a toxicant are
summarized in Table 1. What follows are some observations and elaboration of some of
the information in that table with an emphasis on the mechanisms involved.

A. COPPER
Copper causes a comparatively large upset in osmoregulation in freshwater fish; exposed
fish exhibit a rather rapid decrease in plasma electrolytes and/or osmolality. The cause of
death from acute exposure in freshwater fish appears to be a sequence of events which
start with the electrolyte loss. If this occurs rapidly enough (as it would with acute
exposures), a massive hemoconcentration follows which in turn causes a big increase
(ca. 2x) in arterial blood pressure. Finally, the heart apparently fails from having to deal
with the viscous blood and excessive pressure head (Wilson and Taylor, 1993a). This
sequence of physiological dysfunctions is very similar to that seen in fish dying from acid
exposure (Milligan and Wood, 1982, also see Chapter 10).
The mechanism of the copper effect on osmoregulation in freshwater appears to be an
inhibition of sodium and chloride uptake by the gills, although at fairly high copper
concentrations a stimulation of passive electrolyte efflux also occurs (McDonald et al.,
1988). The inhibition of electrolyte uptake is probably due to an inhibition by copper in
vivo of the enzyme Na,K ATPase in gill tissue (Lorz and McPherson, 1976). The
decreased plasma electrolytes in fish exposed to copper also causes a movement of water
from the blood into the tissues (Heath, 1984) which would facilitate the hemoconcentration
observed.
Decreasing alkalinity, pH, or hardness of the water enhance the toxicity of copper to
freshwater fish (Miller and Mackay, 1980). Lauren and McDonald (1986) showed that the
water alkalinity had a considerable effect on the osmoregulatory dysfunction induced by
copper whereas hardness had little effect in 24-h exposures. However, for chronic
exposures, hardness may become far more important because calcium has such a strong
influence on gill permeability, but it requires several days for a change in environmental
calcium to alter gill permeability (Lauren and McDonald, 1986).
Prolonged exposure to low copper concentrations may result in a decrease in blood
electrolyte levels initially, but then an adaptation to the toxicant occurs and the electrolyte
levels may recover (McKim et al., 1970; Christensen et al., 1972). Acclimation to
sublethal concentrations of copper makes the fish better able to tolerate subsequent higher
doses (Buckley et al., 1982). The acclimation process is fairly complex and time depen­
dent (Lauren and McDonald, 1987a). Initially, there appears to be a decrease in perme­
ability of the gills to sodium which reduces the efflux to below that of control fish in non-
contaminated water. Later, the rate of sodium influx shows a return toward normal due
to a synthesis of more Na,K ATPase in the gills, presumably to compensate for the copper
inhibition of this enzyme (Lauren and McDonald, 1987b).
146

Table 1 Osmoregulatory Effects of Water Pollutants

Substance and Type of
exposure water Effect observed Species Ref.
Copper
Acute FW Decreased plasma osmolality Golden shiner Lewis and Lewis, 1971
Decreased plasma osmolality Channel catfish Lewis and Lewis, 1971
Increased blood volume Striped bass Courtois and
Meyerhoff, 1975
Decreased Na, K, Cl uptake Rainbow trout Lauren and McDonald,
1985
Increased tissue water Bluegill Heath, 1984
Decreased plasma Na, Cl Rainbow trout Wilson and Taylor,
1993a
SW Increased K, Na, Mg, Cl Sheepshead Caudelhac et al., 1972
BR Increased plasma Na, K, Rainbow trout Wilson and Taylor,
Ca, Cl 1993b
SW No effect plasma ions Rainbow trout Wilson and Taylor,
1993
6,21 d FW Decrease Cl and osmolality Brook trout McKim et al, 1970
337 d FW No effect Brook trout McKim et al., 1970
7^2 d FW Decreased Na, Cl Flounder Stagg and
Shuttleworth, 1982
SW Increase Na, K, Mg, Cl Flounder Stagg and
Shuttleworth, 1982
14 d SW Decreased Ca upteike Flounder Dodoo et al., 1992
1-30 d FW Increased Na, K Indian catfish Singh and Reddy, 1990
30 d FW Decreased Cl Brown bullhead Christensen et al., 1972
1-172 d FW Reduced SW survival and Coho salmon Lorz and McPherson,
migration 1976
1-172 d FW Inhibition gill Na, K-ATPase Coho salmon Lorz and McPherson,
1976
Zinc
Acute FW No effect plasma electrolytes Rainbow trout Skidmore, 1970
FW Decreased Cl, Ca; no effect Na, K Rainbow trout Spry and Wood, 1985
FW Decreased plasma osmolality Channel catfish Lewis and Lewis, 1970
72 h FW Decreased Ca; increased Rainbow trout Spry and Wood, 1985
electrolyte efflux
30 d FW No effect osmolality or ions; Rainbow trout Watson and Beamish,
increased gill Na, K- and 1980
Mg-ATPase
Cadmium
1-178 d FW Electrolyte movement into Rainbow trout Giles, 1984
tissues; no effect kidney
function
25-50 d FW Decreased Na; increased Goldfish McCarty and Houston,
muscle Na 1976
2-8 wk FW Increased plasma Na Brook trout Christensen et al., 1977
4 days posthatch FW Decreased Na, K, Ca, water Atlantic salmon Rombough and
uptake Garside, 1982
20-180 h FW Time-dependent decline Rainbow trout Roch and Maly, 1979
plasma Ca
2-35 d FW Transient Ca decrease and Mg Tilapia Pratap et al., 1989
increase; no effect Na, K,
osmolality
Dietary FW Degeneration gill pavement Tilapia Pratap et al., 1989
cells and chloride cell activity
 147

Table 1 (continued) Osmoregulatory Effects of Water Pollutants

Substance and Type of
exposure water Effect observed Species Ref.
2-35 d FW Decreased osmolalitys; transient Tilapia Fu et al., 1989
decrease Na, Ca; increase
prolactin
Acute SW Increased Na, Cl Gunner Thurberg and Dawson,
1974
4-9 wk BR No effect NaCl; decreased K, Ca Flounder Larsson et al., 1981
Mercury
4h FW Decreased Cl influx; increased Lamprey Stinson, 1989
NaCl efflux
1 wk FW Decrease Na, Cl; slight increase K Rainbow trout Lock et al., 1981
2-8 wk FW Increase NaCl Brook trout Christensen et al., 1977
45-180 d FW Decreased cortisol Indian Catfish Kirubagaran and Joy,
1991
60 d SW Increase osmolality; decrease Ca Flounder Dawson, 1979
Injected SW No effect osmolality or ions Flounder Schmidt-Nielsen et al.,
1977
Chromium
Acute FW Decreased NaCl and osmolality Coho salmon Van der Putte et al., 1982
2 -A wk FW Reduced survival in SW Coho salmon Sugati, 1980
Lead
1 mo; 49 d BR Increase K; decrease Na; no Rainbow trout Haux and Larsson,
recovery effect Ca 1982
2-8 wk FW Increase NaCl Brook trout Christensen et al., 1977
Field FW Low Na, No effect K Whitefish Haux et al., 1985
Aluminum
Acute or chronic FW Marked ionregulatory Mostly See text
dysfunction in soft acid salmonids
water but not in hard water
At pH 7 ionoregulatory
dysfunction slight if at all
Tin
14 d BR No effect Na, K, Ca, Mg; Striped bass Pickney et al., 1989
increased gill Na, K- and Mg
ATPases
Iron
Acute FW Little effect Na Brook chair Gonzalez et al., 1990
Manganese
Acute FW Decrease Na Brook chair Gonzalez, 1990
Detergents
Acute FW Increase gill permeability to Rainbow trout Jackson and Fromm,
water 1977
FW Decrease plasma Na Rainbow trout McKeown and March,
1978
SW Increase plasma Na Rainbow trout McKeown and March,
1978
SW Increase Na,Cl, Ca, Mg; no Flounder Baklien et al., 1986
effect K
Phenol
Acute FW No effect plasma Cl Rainbow trout Swift, 1981
1 wk BR No effect Na, K, Mg, Ca,Cl Pike Kristoffersson et al., 1973
DDT
In v itr o Decrease intestinal water SW eel Janicki and Kinter,
uptake and Na, K-ATPase 1971
148

Table 1 (continued) Osmoregulatory Effects of Water Pollutants

Substance and Type of
exposure water Effect observed Species Ref.
Fed 18 d BR No effect Na, K, Ca, PO4 Flounder Haux and Larsson,
1979
Fed 2 wk Var. Increase osmolality in SW; no Rainbow trout Leadem et al., 1974
effect in FW
Acute SW Increase Na; decrease gut water Fundulus Miller and Kinter, 1977
uptake and Na, K-ATPase
4d SW(?) Increase Na and K Eels and mullet Hilmey et al., 1983
Lindane
Fed 30 d and FW Decrease plasma Na and Carp Demael et al., 1987
109 d muscle Na, K-ATPase
Fenvalerate
Acute FW Increase urine Na, K, and Rainbow trout Bradbury et al., 1987
osmolality
Endosulfan
24-240 h FW Decrease plasma Ca, Mg Tilapia Rangaswamy and
Naidu, 1989
Nitrite
Acute FW Decrease Na, K, Cl; rapid Rainbow trout Williams and Eddy,
recovery 1988b
FW Inhibition of Cl uptake by gills Rainbow trout Williams and Eddy,
and carp 1988a
1-12 d FW Decreased K; no change Na, Cl Atlantic salmon Bowser, 1989
Naphthalene
Acute Var. Transient increase osmolality Fundulus Levitan and Taylor,
in SW 1979
Increase ions SW; decrease ions
FW
Petroleum
Acute FW No effect plasma Na Rainbow trout McKeown and March,
1978
FW Transient decrease plasma Cl Rainbow trout Zbanyszek and Smith,
1984
FW Decrease NaCl and osmolality Rainbow trout Englehardt et al., 1981
SW Increase NaCl and osmolality Rainbow trout Englehardt et al., 1981
SW Decrease gill Na, K-ATPase, Sculpin Boese et al., 1982
no effect plasma osmolality
Ammonia
Acute FW Increase urine flow rate Rainbow trout Lloyd and Swift, 1976
FW Increase plasma renin Rainbow trout Arillo et al., 1981
FW No effect Cl Rainbow trout Smart, 1978
FW Slight increase osmolality Striped bass Oppenborn and
Goudie, 1993
FW Loss of Na, Cl, K Trout fry Paley et al., 1993
FW Increase plasma K; no change Na Rainbow trout Zeitoun et al., 1977
Chlorine
Acute BR No effect Na, K, or gill ATPase Morone Block, 1977
Ozone
Acute FW Decrease plasma NaCl Rainbow trout Wedemeyer et al, 1979
1-3 mo FW No effect plasma NaCl Rainbow trout Wedemeyer et al, 1979
Acid
Acute and chronic FW Large ionoregulatory effect Many species See Chapter 10
 149

Figure 2 Comparison of effect of waterborne copper on plasma chloride concentration in

seawater and freshwater-adapted flounder {Platichthys flesus). Copper concentrations were 170
|xg/L in seawater and 105 |ig/L in freshwater. Asterisks indicate means significantly different from
controls (p<0.05). (Data from Stagg, R. M. and Shuttleworth, T. J., J. Fish Biol., 20, 491,1982.)

There is one report of a rise in plasma sodium and potassium from exposure to copper
in a freshwater fish (Singh and Reddy, 1990). This was in an air-breathing catfish species
but that should not account for a rise in these electrolytes, which is opposite to what has
been found in all other published studies. Unfortunately, the authors do not attempt to
explain this, nor do they seem to recognize it as a contradiction.
In seawater, exposure to copper may increase the blood osmolality, but the extent of
deviation from controls is not nearly as great as that seen in freshwater fish (Stagg and
Shuttleworth, 1982). Figure 2 illustrates how it required 28 days of copper exposure of
seawater-acclimated flounders before a significant increase in plasma chloride was seen,
whereas only 7 days of exposure of the same species adapted to freshwater caused a
significant decrease. This was in spite of the copper concentration being lower during the
exposures in freshwater.
The mechanism by which seawater reduces the ionoregulatory dysfunction produced
by copper is partially revealed by the work of Wilson and Taylor (1993b). They exposed
rainbow trout which had been acclimated to either seawater or brackish water to the same
copper concentration (6.3 jiimol/L) and observed a rapid decline in plasma electrolytes in
those from brackish water but only minor changes occurred in the seawater-acclimated
fish. The authors propose the hypothesis that the basolateral location of the gill Na,K
ATPase protects it from copper in a seawater environment. Thus, the process of pumping
salt out of the fish would be unaffected by the metal. In dilute seawater, ionoregulatory
disturbances occur due to increased permeability caused by the copper displacing surface-
bound calcium. In full strength seawater, on the other hand, the higher concentration of
calcium (and Mg?) helps prevent the calcium displacement and thereby provides some
protection.
Exposure of sheepshead in seawater to a massive dose of copper (exceeding the 48-h
LC50) was followed by a large increase in all the plasma electrolytes. Cardeilhac et al.
(1979) suggest that the greatly elevated plasma potassium may be the cause of death from
exposure to copper in seawater because the levels they recorded would probably cause
heart stoppage. Because the plasma potassium concentration exceeded that in the seawater.
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the source must be leakage from tissue cells, although they failed to recognize that.
Internal hypoxia could cause this potassium leakage by an inhibition of the Na,K-pump
in cellular membranes, and the authors mention gill damage from these doses of copper
which certainly could induce internal hypoxia (see Chapter 3). They discount hypoxia as
being important, but do not offer evidence on which to reject this hypothesis, nor do they
suggest an alternative mechanism. Another one that could be mentioned is the possible
action of copper directly on cell membranes by inhibition of Na,K ATPase which
provides the power for the Na,K-pump. This would allow some of the potassium in the
cells to diffuse passively into the blood. Unfortunately, the high copper doses used are
probably not relevant to “real world” concentrations, and the cause of death could be a
combination of factors including high plasma potassium and low blood oxygen. It is
noteworthy that Stagg and Shuttleworth (1982) found no change in plasma potassium
with chronic exposure to copper in seawater-adapted flounders even though there were
slight increases in sodium and chloride. Thus, the threshold dose of waterborne copper
required to alter these blood ions must be lower than that required for changes in
concentration of potassium, at least in a marine environment.
Coho salmon {Oncorhynchus kisutch) migrate to sea during their second year of life.
At the time of this migration, they become more euryhaline. Chronic exposure of these
fish to copper while still in freshwater can cause a large reduction in survival when
transferred to seawater, a loss of ability to regulate plasma osmolality, and a concomitant
decrease in activity of the gill Na,K-activated ATPase (Lorz and McPherson, 1976). The
activity of this enzyme normally increases as the fish move into the seawater. A somewhat
similar effect of that seen with copper was observed for salmon exposed for 2 weeks to
sodium dichromate (Sugatt, 1980). In contrast to copper, however, dichromate in fresh­
water did nothing to the serum osmolality. Zinc produced different results than either of
the other two metals: chronic exposure at a comparable toxic concentration to that used
with copper did not affect either survival in seawater or ATPase activity in the coho
salmon (Lorz and McPherson, 1976).
While exposure to waterborne copper or dichromate reduces survival of salmonids
transferred to seawater, work on bluegill (Lepomis macrochrirus) suggests an opposite
effect on this strictly freshwater species (Heath, 1987). After being subjected to copper
at a sublethal dose for a week or more, the fish were able to then tolerate a hypertonic
(250 mM) sodium chloride solution considerably better than could controls. Analysis of
the plasma of those in the hypertonic solution revealed that those which had been exposed
to copper were regulating their plasma osmolality and chloride better than the controls.
It was hypothesized that chronic exposure to copper reduced the permeability of the gill
to sodium chloride thus slowing its passive influx. The acclimation studies mentioned
above (Lauren and McDonald, 1987b) lend some support for this although in their case,
the change was in passive efflux of NaCl.
One other aspect of copper toxicity that may deserve attention is the effect it has on
calcium metabolism. Copper apparently depresses the mechanism for uptake of calcium
from the water, even when the fish is in seawater (Dodoo et al., 1992), a phenomenon that
deserves further study.

B. ZINC
The results of studies on the effects of zinc exposure on osmoregulation may appear
somewhat contradictory (Table 1), but this is probably due to species differences and
different dose levels. Lewis and Lewis (1971) found a decrease in plasma osmolality of
channel catfish when the exposure was acute. However, it should be noted that it required
approximately five times the concentration of zinc to cause the same degree of blood
osmolality change as was produced by copper in the same fish species. Skidmore (1970)
observed no change in blood ions in trout exposed to a very high concentration of zinc
 151

(they died within 8 h). Skidmore’s fish were probably dying of internal hypoxia before
any changes in blood ions could take place. Within only a few hours, the blood oxygen
had dropped to a very low level.
The most definitive studies on zinc toxicity are those of Spry and Wood (1984, 1985)
on rainbow trout. They tested a high concentration (but not nearly as high as Skidmore)
and one approximating the 96-h LC50. At the high dose, the fish die of hypoxemia, but
at lower doses there was no hypoxemia. There was, however, a decreased plasma calcium
and stimulation of sodium efflux, which over a longer period of time would undoubtedly
cause osmoregulatory dysfunction. It is noteworthy that they observed some mortality
even though the plasma sodium chloride and blood oxygen was unchanged. They hypoth­
esize that the lethal mechanisms of the zinc at low environmental concentrations may be
at the cellular level and do not necessarily involve either osmoregulatory or respiratory
dysfunction.
Spry and Wood (1985) also found that after 48-60 h in water containing a high
sublethal concentration of zinc, there was a stimulation of sodium uptake by the gills. This
fits in with an earlier finding (Watson and Beamish, 1980) that chronic exposure to zinc
induces an increased ATPase activity in gill tissue of trout. It was attributed to a greater
passive flux of sodium out of the fish which then necessitated increased active uptake of
the electrolyte from the water. This would represent a form of physiological adaptation
by the fish to zinc in the water; a finding similar to what happens when fish are acclimated
to copper (Lauren and McDonald, 1987b).
It has been reported that in vitro exposure of the gill Na,K ATPase enzyme to zinc
caused an inhibition in activity, in apparent contradiction to the above finding of a
stimulation of activity when the exposure was in vivo (Watson and Beamish, 1980). These
observations point out the often conflicting results obtained when enzyme activities are
measured in tissues from fish exposed to some substance in the water, compared to adding
the same material to the incubation medium of an enzyme preparation (see Chapter 9).
The latter method yields results that have relatively little meaning, except perhaps as a
way of comparing the sensitivity of a given enzyme to more than one type of chemical.
There does not appear to be any information on the effect of zinc on whole-body
osmoregulation in fish adapted to seawater. There has, however, been some interesting
in vitro work. Using an isolated opercular epithelium preparation from seawater-adapted
F. heteroclitus, the killifish, Crespo and Karnaky (1983) found that copper or zinc at a
concentration of 4 x 10“^ M inhibited chloride transport when on the blood (serosal) side
of the preparation. Exposure of the other side had no effect, which suggests that the metals
are interacting with the ATPase molecules on the basal area of the chloride cells. This fits
with the hypothesis proposed by Wilson and Taylor (1993b) to explain why seawater
adapted fish are much less sensitive to metals like copper. Crespo and Karnaky (1983)
also present evidence that not all of the inhibition of chloride transport is caused by
inhibition of ATPase, but must also involve the NaCl carrier in the chloride cell.
Overall, zinc appears to cause only mild dysfunction in monovalent cation regulation
but the interesting effects on calcium regulation may warrant further study.

C. CADMIUM
This common pollutant appears to cause only moderate alterations in osmotic regulation,
but specific ionic regulation, especially that of divalent cations, is quite sensitive to
cadmium. First, we will examine what has been seen in studies of the effects of cadmium
on osmoregulation per se.
When tilapia were exposed to doses ranging up to 1000 p.g/L cadmium, they exhibited
a dose-dependent decrease in plasma sodium which became progressively more severe
over the test period of 2-35 days (Fu et al., 1989). Those in the highest dose all died in
8 days and had quite low plasma sodium levels, but when the exposure was at a more
152

realistic environmental concentration of 10 \igfL, the change in plasma sodium was only
slight and transient.
Goldfish exposed to cadmium experienced a modest decrease in plasma sodium and
a shift of sodium, potassium, and water into the muscles. McCarty and Houston (1976)
attribute these changes in part to an increased water uptake from the environment with
much of that water being localized in the extracellular fluid compartments of the body
thereby diluting the blood.
Giles (1984) was able to identify a threshold concentration for cadmium effects in
rainbow trout. Chronic exposures for up to 178 days to 3.6 pg/L caused no effect on blood
electrolytes while a dose of 6.4 pg/L resulted in a slight drop in plasma sodium,
potassium, calcium, and chloride but an elevated magnesium. Measurements of urine
flow and concentrations indicated that kidney impairment did not occur. Also, because
the urine flow rate was constant, this indicates that permeability did not change during
cadmium exposure. He explains his findings as being due to the redistribution of electro­
lytes that McCarty and Houston noted in the goldfish.
In apparent contrast to McCarty and Houston, and just about everybody else, Christensen
et al. (1977) reported a slight, but significant increase in plasma chloride in brook trout
exposed to very low levels of cadmium. They also reported elevations in plasma monova­
lent ions resulting from mercury and lead exposure. Two possible hypotheses for these
anomalous findings are presented later in this chapter in the section on mercury. (These
authors present no explanations, nor express surprise at the results.)
In seawater-adapted fish, a rather high concentration of cadmium is seemingly re­
quired before changes occur in plasma sodium and chloride. Thurberg and Dawson
(1974) used several concentrations in 96-h exposures, but only when they reached 48 ppm
cadmium, was there a change in plasma ions. This is a concentration an order of
magnitude higher than that which was required to alter osmoregulation in freshwater fish
(McCarty and Houston, 1976). In marine fish, oxygen consumption of gill tissue was
found by the same workers to be depressed at all cadmium concentrations above 3 ppm.
Because marine fish rely on gill function to excrete excess sodium chloride, it is interest­
ing that the in vitro metabolic rate of this tissue can be decreased by 50% without any
effect on the plasma electrolytes (Thurberg and Dawson, 1974). Of course, in vitro
measures of gill tissue metabolism may or may not have much similarity to the in vivo
metabolic rate of the same tissue (see Chapter 8). In any case, for marine fish, cadmium
seems to alter other functions, such as those of the kidney, more so than osmoregulatory
activity by the gills.
Larsson et al. (1981) studying the flounder, Platichthys flesus, found that exposure to
cadmium of fish adapted to brackish water (which was almost isoosmotic to the blood)
produced no changes in plasma sodium or chloride, but the concentrations of other ions
were changed. The brackish water environment of the fish had less potassium and calcium
but more magnesium than the plasma. Thus, the cadmium-induced changes in blood
electrolytes (elevated magnesium but decreased potassium and calcium) resulted from
enhanced net diffusion of magnesium in and calcium and potassium ions out. A fish
normally excretes excess magnesium via the kidney, so, since it rose in the plasma in
response to cadmium, this kidney function must have been inhibited. Histopathological
damage to the kidney from cadmium has been seen in euryhaline killifish (Gardner and
Yevich, 1970) and cunner (Tautogolabrus adspersus); (Newman and MacLean, 1974), so
this organ is certainly an important target organ for cadmium.
Cadmium appears to affect calcium metabolism in freshwater fish to a greater extent
than in seawater forms. The plasma calcium dropped by approximately 50% in a week
of exposure of rainbow trout to 0.3 ppm cadmium (Roch and Maly, 1979). Such a
concentration in the brackish water studies mentioned above caused only a slight drop in
 153

plasma calcium. This difference is expected, as fish in marine or brackish waters are in
a calcium-rich environment which provides less of a diffusion gradient for this element.
When tilapia were exposed to cadmium at 10 ftg/L, they exhibited a severe drop in
plasma calcium but no significant change in monovalent cations or osmolality (Pratap et
al., 1989). In the same laboratory, it was learned that upon continued exposure to this dose
of cadmium, there was an increased prolactin secretion rate and recovery of plasma
calcium (Fu et al., 1989). Interestingly, as time went on with the fish still in the water
contaminated with cadmium, prolactin activity returned to normal which suggests that
some secondary mechanism was induced. Pratap et al. (1989) also found that cadmium
in the food (but not in water) caused hypocalcemia in much the same way as the
waterborne metal, although the effect was somewhat delayed. They also concluded that
the effect of cadmium on this freshwater species was on the gills, not on the kidney, which
differs from that seen in marine species where the kidney is definitely a target organ.
Decreases in plasma calcium from cadmium exposure are apparently due to inhibition
of calcium influx alone, at least at modest exposure concentrations (Reid and McDonald,
1988; Verbost et al., 1987). Sauer and Watabe (1988) suggest the cadmium acts by
displacing calcium from protein carriers in the gill epithelium. Changes in calcium efflux
by either the gills or kidney are far less important, even though histological damage to
kidney occurs from cadmium (Gill et al., 1989). This damage, if produced by modest
concentrations of cadmium, seemingly does not result in altered calcium regulation by the
kidney in freshwater fish.
The alterations in plasma calcium may help explain the spasms and hyperexcitability
seen in some fish exposed to cadmium (Larsson et al., 1981). The altered calcium
metabolism does not affect the bone structure, probably because the bone is acellular in
many species (Larsson et al. 1981).
The early life stages of fishes are generally presumed to be the most sensitive to
cadmium (Benoit et al., 1976; Rombough and Garside, 1982). Thus, the investigation by
Rombough and Garside (1984) is particularly relevant. Atlantic salmon, which hatch and
undergo early development in freshwater, exhibit an almost linear increase in tissue
sodium, potassium, calcium, and water during the first 45 days posthatch (alevin period).
The ions increase 2.5- to 4-fold in concentration during this time. (The water content
increases by about 20%.) While much of the ion flux is from the yolk, approximately 65%
of the sodium, 45% of the potassium, and 75% of the calcium must be taken up from the
freshwater (Rombough and Garside, 1984). Cadmium concentrations ranging from 0.47
to 300 fig/L produced a dose-dependent decrease in the rate of uptake of the three ions
and water. The greatest effects were on potassium and calcium and the authors propose
that this may be a primary cause of mortality from cadmium poisoning in alevins. It is
further noteworthy that they detected reduced body potassium and calcium levels at
concentrations of cadmium far below that (300 |ig/L) required to produce histopathological
changes. The exact mechanisms of this reduction in uptake rate of ions and water from
the environment is not known. The inhibition of ion-activated ATPase enzymes, increases
in membrane permeability (due to low calcium), and loss of superficial mucus are
mentioned. Finally, the point is made by Rombough and Garside that the changes seen
in the alevins exposed to cadmium are not restricted to this metal alone. Many other
pollutants probably produce similar effects on larval fishes, but this has not been ex­
plored.

D. MERCURY
Mercury causes altered osmoregulation in both freshwater- and seawater-adapted fish,
but in common with other metals, the overall effect may not be as great in the latter
(Dawson, 1979). Inorganic mercury caused a slight elevation in plasma osmolality of
154

seawater-adapted flounders. It apparently was not due to an uptake of sodium, chloride,

or calcium as these plasma ions were unchanged or actually decreased in concentration.
Also, plasma protein concentration was unchanged as well. Increases in magnesium and/
or amino acid content of the blood are possibilities, but these were not measured.
Methylmercury injected into flounders on a daily basis for 13 days produced accumu­
lations of mercury in the gills up to 24 ppm (Schmidt-Nielsen et al., 1977). Still, this
treatment caused no effect on either intra- or extracellular electrolytes. There was also no
effect on the water content of tissues. The Na,K ATPase activity in gill and intestine
remained unchanged in the fish injected with mercury, however, the activity of this
enzyme in the bladder increased 2.5- and 1.5-fold in the kidney. Evidently, even though
mercury is an inhibitor of this ATPase in seawater flounder when measured in vitro
(Renfro et al., 1974), there must be a form of overcompensatory synthesis of the enzyme
in some tissues under more chronic exposures.
There was an increase in sodium and chloride when brook trout in freshwater were
exposed to methylmercury at very low concentrations (0.01-2.93 jig/L (Christensen, et
al., 1977). Such an unexpected increase in chloride was also found by the same authors
with cadmium exposure, as was cited earlier in this chapter. Although Christensen et al.
(1977) propose no explanations, there are at least two possible: (1) increased ATPase
activity in the gills or (2 ) increased rate of urination which would reduce the blood volume
and raise its electrolyte concentration. Currently there is no evidence regarding either of
these hypotheses other than to note that, under some circumstances, ATPase is stimulated
by methylmercury (Schmidt-Nielsen et al., 1977).
Much higher concentrations of mercury (5-200 \igfL) (Lock et al., 1981) decreased
plasma osmolality and sodium chloride in rainbow trout exposed for a week to either
inorganic mercury or methylmercuric chloride. The depression in osmolality is clearly
dose- and time-dependent. Sodium and chloride showed a similar pattern to the osmola­
lity changes except that the percent of alteration was greater for the ions.
It requires 10-20 times more mercuric chloride than methylmercury to produce the
same degree of lowering of osmolality and ionic concentrations (Lock et al., 1981). The
authors note that this difference is comparable to the differences between the 96-h LC50s
for the inorganic and organic forms of mercury. These they found to be 275 |xg Hg/L and
24 pg Hg/L respectively. Methylmercuric chloride is more lipophilic and accumulates
more rapidly in the tissues, so these results are not unexpected. The relative effectiveness
(potency) of the two forms of mercury is apparently due to their relative tendency for
accumulation, rather than to the specific action of the molecule. Lock et al. (1981) showed
that inorganic mercury is actually more potent than the organic form when tested in vitro
for effects on the uptake of water by the gills and ATPase activity, which again points out
the hazard of drawing conclusions from only in vitro work (which these authors are
careful to avoid).
One of the contributing causes of death from mercury poisoning in freshwater fish may
be osmoregulatory failure. Milligan and Wood (1982) have shown rather conclusively
that acid-stressed fish are killed by osmoregulatory failure when the plasma sodium
shows a greater than 30% drop, providing this decline is rapid. Trout exposed to the 96-h
LC50 of mercury for a week showed a +40% drop in sodium (Lock et al., 1981).
One of the most interesting conclusions to be drawn from the Lock et al. (1981) work
on mercury is that both inorganic and organic mercury cause osmoregulatory effects
primarily by producing an increase in the permeability of the gills to water. Using an
elegant gill perfusion arrangement, they were able to show that mercury compounds
stimulated water inflow at lower concentrations than those that resulted in inhibition of
gill Na,K ATPase. This enzyme was not affected except at rather acute concentrations of
mercury either with the gill perfusion work or with the in vivo exposures.
 155

Stinson and Mallatt (1989) have found that methylmercury increases permeability of
the gills of lampreys to sodium and chloride so there is a net increase in efflux of these
ions. They also, in agreement with Lock et al., 1981, found no inhibition of gill ATPase.
Thus, the relative lack of effect of mercury on gill ATPase has now been shown in both
seawater- and freshwater-adapted species.
The element selenium has been reported to reduce the toxicity of mercury to fish (Kim
et al., 1977). Heisinger and Scott (1985) subsequently showed that the osmoregulatory
dysfunction induced by injection of mercuric chloride into freshwater bullhead catfish
was completely blocked by simultaneous injection of a sodium selenite solution. A
protein-bound complex of mercury and selenium is apparently formed (Burk et al., 1974)
which reduces the toxic action of either element.
Finally, it should be mentioned that some of the ionoregulatory changes seen in fish
exposed to mercury may, at least in part, be induced by an effect of mercury on blood
cortisol levels. Recently, it has been learned that mercury inhibits the secretion rate of
cortisol even though the adrenocortical and pituitary ACTH cells exhibit hypertrophy
(Kirubagaran and Joy, 1991).

E. CHROMIUM
Sugatt (1980) exposed juvenile coho salmon in freshwater to chromium (as sodium
dichromate) for 1-4 weeks at a dose of 0.5 or 0.2 mg Cr/L. The exposed fish were then
challenged by transfer to seawater as might occur during the seaward migration. None of
the controls died from this challenge, but a high rate of mortality occurred among the
exposed fish over the following week. Serum osmolality was unchanged in either chro­
mate exposed or control fish while still in freshwater, however, upon transfer to the
seawater, those exposed showed a greater and more prolonged transient increase in
osmolality, so the physiological adjustments to seawater were delayed or even prevented
by the metal in the water.
The results for chromate are rather similar to those obtained for copper by Lorz and
McPherson (1976), which have already been discussed. They illustrate how the challenge
of another stressor (in this case salinity) on top of a chronic contaminant exposure can
cause physiological changes that might not have been observed with the chronic exposure
alone. It seems safe to predict that virtually any chemical pollutant that affects osmoregu­
lation would have an inhibitory effect on the migration of salmon (or other anadromous
and catadromous fish species), especially since the pollutants are frequently concentrated
in the lower reaches or mouths of rivers where the osmoregulatory adjustments must take
place.

F. LEAD
Rainbow trout kept in slightly hypotonic brackish water and exposed to lead (10, 75, or
300 jig Pb/L) for 30 days followed by a recovery of 49 days showed a dose-dependent
elevation in plasma potassium (Haux and Larsson, 1982). Given the low concentration of
potassium in the water, this element must have come from tissue cells. In vitro studies of
mammalian erythrocytes show that lead causes leakage of potassium out of the cells
(Hasan and Hernberg, 1966). Whether this holds for fish erythrocytes is unknown, but
even if it did, that would not limit the source to the blood cells, as high internal potassium
concentrations are typical of essentially all animal cells.
A more interesting question is the possible mechanism of this postulated flux of
potassium out of body cells. The Na,K ATPase of the cell membranes is responsible for
the differential concentration of potassium between body fluids and cytosol. The lead
may be inhibiting that enzyme in a wide variety of the body tissues, but if it did in the
study by Haux and Larsson (1982), there was not much effect on the regulation of sodium
156

by the gills. This may have been because the fish were in water nearly isotonic to the
blood so there was not much of a gradient for sodium either in or out of the fish.
In a study carried out by the same group on fish from lead-contaminated freshwater
lakes, the fish exhibited a decrease in plasma sodium but no change in plasma potassium
(Haux et al., 1985). The authors make no mention of this apparent contradiction, but one
possible explanation is that those fish in the lakes had experienced lead exposure for a
considerable period of time, maybe even for several generations, so some physiological
adaptations to the lead could have taken place.

G. ALUMINUM
Aluminum is the third most abundant element in the earth’s crust so it frequently gets
leached from soils experiencing “acid rain”, or runoff from land that has been stripmined
(Baker, 1982). A variety of studies and reviews have examined the interactions of acid
waters and dissolved aluminum on fish (e.g.. Baker and Schofield, 1982; Howells et al.,
1983; Neville, 1985; McDonald et al., 1991; Potts and McWilliams, 1989). The situation
is complex, to say the least, because aluminum affects different physiological functions
in the fish depending on the pH and calcium content of the water, and to a lesser extent,
the concentration of aluminum.
When exposed acutely to aluminum in soft water, rainbow and brook trout seemingly
die from progressive electrolyte loss if the pH is between 4 and 4.5. The initial cause of
electrolyte loss is enhanced passive efflux which is then followed in a few days by a reduced
NaCl uptake by the gills (Booth et al., 1988). The reduced NaCl uptake is probably caused
by inhibition by aluminum of Na, K ATPase in the gills (Staumes et al., 1984).
As the pH of the water is raised, the effect on blood electrolytes is reduced, but the fish
begins to experience an internal hypoxic condition (hypoxemia) which may prove lethal
(Neville, 1985; Wood et al., 1988; Malte and Weber, 1988). With trout, the threshold for
this transition appears to be around pH 4.5-5.0 so that at pH 4.8, a fish may suffer from
both loss of body ions and a decreased blood oxygen.
In acid water, raising the calcium reduces the ionoregulatory dysfunction induced by
aluminum (Wood et al., 1988). Finally, in hard acid (pH 5) water, aluminum causes no
ionoregulatory dysfunction at all but does cause an acute hypoxia (Malte, 1986). As
Wood et al. (1988) have noted, liming as an attempt to raise the pH and calcium levels
in lakes or streams has occasionally been associated with fish kills. They go on to point
out that such a change in water quality would merely promote respiratory failure while
reducing ionoregulatory dysfunction. It might be further mentioned that this is a good
example of how fish physiology research can have governmental policy overtones.
One other aspect of calcium needs to be mentioned. Verbost et al. (1992) have shown
a dose-dependent inhibition by aluminum of calcium uptake in carp. Calcium efflux was
less sensitive than the process of influx to the presence of aluminum. At the pH they were
using (5.2) there was little effect on the influx or efflux of sodium except at very high
aluminum concentrations. Thus, at higher pHs, aluminum may alter blood calcium levels
more than sodium, although this seemingly has not been examined. In any case, alter­
ations in calcium metabolism could have serious implications for reproduction (see
Chapter 13).
Trout are able to acclimate to the presence of aluminum and thereby raise the toxic
threshold concentration and reduce the physiological dysfunction. Some acclimation in
electrolyte balance occurs by four days’ exposure. The gills undergo a number of
physiological (McDonald et al., 1991b) and histological (Mueller et al., 1991) changes,
but perhaps one of the most important is a reduction in aluminum binding to the surface
of gill cells (McDonald et al., 1991b). Work by Reid et al. (1991) indicates that during
acclimation to aluminum there is an increased affinity of the gills for calcium associated
with a decreased binding of aluminum.
 157

Exley et al. (1991) have proposed a cellular mechanism for acute aluminum toxicity
involving the binding of aluminum both to the surface of lamellar epithelial cells and
subsequently to interior ligands. The sum effect is to increase permeability of the
epithelium, interfere in the second messenger system within the cells, and ultimately
accelerate cell death.

H. TIN
Tin compounds, generally in the organic form such as tributyltin, are used in antifouling
paints on ships, docks, etc. Striped bass, which often live in brackish water, were tested
with a variety of concentrations of tributyltin (Pinkney et al., 1989). They found a large
stimulation by the tin of Na,K ATPase and Mg ATPase activity in the gills. When tested
in vitro, however, these two enzymes were inhibited by tributyltin. A similar difference
between in vitro and in vivo effects on ATPase enzymes has also been seen with copper
(Stagg and Shuttleworth, 1982) and zinc (Watson and Beamish, 1980) exposures. It may
reflect a form of adaptation to the presence of the toxicant in that in vivo exposures
induced more enzyme synthesis. Indeed, Pinkney et al. (1989) found that the tin com­
pound caused no change in the plasma electrolytes so the adaptation was seemingly
successful, although it should be noted that the fish were in brackish water so diffusion
gradients between the environment and the fish blood were not large.

I. MANGANESE AND IRON

These two elements generally get into waterways as a result of strip mining. Manganese
has a very strong effect on sodium regulation (Gonzalez et al., 1990) when fish are
exposed to water with a concentration approaching their 96-h LC50. Body and plasma
sodium concentrations declined by 40-50% before death, which suggests this is a primary
mode of death in acute doses of manganese. On the other hand, iron at a concentration
even twice that of the LC50 caused very little change in plasma sodium so the mode of
toxicity of these two metals is markedly different.
Compared to the situation with metals, there is not a great deal known about the action
of organic pollutants on osmoregulation or control of electrolytes by fish. The large
number and extreme variety of anthropogenic organic chemicals that appear in water­
ways, however, make this a presumptively important lack of information.

J. DETERGENTS
With the aid of an isolated gill preparation, Jackson and Fromm (1977) demonstrated that
acute exposure to a detergent caused the gill to become more permeable to water. The
effect was dose dependent (5-100 ppm) and it reached a maximum in about 25 min of
exposure. That is probably too fast for much histological damage to take place except at
the highest concentrations tested (Abel and Skidmore, 1975). Concentrations below
5 mg/L caused no effect on the permeability of water over the 65-min exposure. This
altered permeability of gills to water could provide the mechanism for the decrease in
serum sodium which has been observed in freshwater fish exposed to an oil dispersant
(McKeown and March, 1978) that presumably acts much like a detergent. An increased
inflow of water would dilute the blood and/or result in a greater urine output, thereby
carrying some ions out of the body. A change in gill water permeability could also explain
the increased serum sodium in the seawater-adapted fish challenged with the same
dispersant (McKeown and March, 1978), because an increased permeability to water
produces a more rapid osmotic flow of water out of the fish.
Petroleum dispersants may also affect permeability of the gills to inorganic ions as
well as affect the ability to excrete an excess amount of these in marine fish. Baklien et al.
(1986) reported elevations in all blood ions except potassium when flounders were
exposed for four days to a fairly acute dose of dispersant, whereas the changes seen were
158

all statistically significant, they were not especially large. Because there was a fairly high
mortality among the exposed fish, the cause of death from this dispersant is probably
something other than osmoregulatory dysfunction.

K. PHENOL
Phenol seems to have little or no effect on plasma electrolytes even when exposures are
to a concentration one half the 48-h LC50 (Swift, 1981), or when exposed for a week to
a concentration approximating the incipient lethal level (Kristofferson et al., 1973). This
is in spite of the rather considerable damage that phenol does to gill tissue (Mitrovic et
al., 1968). It should be recalled that zinc is also quite damaging to gill tissue (Skidmore
and Tovell, 1972), yet does not necessarily alter blood sodium levels when exposure is
to acute levels (Skidmore, 1970). The usual type of histological damage to gills from
pollutants involves swelling and separation of the epithelium from the basement mem­
brane on the lamellum (see Chapter 3). Evidently then, the physical damage to the gill
lamellae does not necessarily result in an immediate failure to maintain osmoregulation,
although it certainly reduces respiratory gas exchange (Chapter 3).

L. DDT
DDT is a potent inhibitor of Na,K and Mg ATPase {in vitro) in fish tissues (Janicki and
Kinter, 1971). Hilmy et al. (1983) reported increases in plasma sodium, potassium, and
calcium in mullet and eels exposed to DDT or the other organochlorine insecticide endrin.
It is not clear in their paper whether the fish were adapted to freshwater or seawater.
Because there were increases in all measured electrolytes as a result of the insecticide
exposures, I have assumed they were in seawater. The authors noted a dose-dependent
increase in potassium which might indicate an inhibition by DDT of Na,K ATPase in the
cell membranes of muscle and other tissues (Demael, 1987). This would permit the
intracellular potassium to leak out down its natural concentration gradient. The authors
attribute the ionoregulatory dysfunction to liver damage, but the liver serves essentially
no such function in fish.
Janicki and Kinter (1971) have found that uptake of water from the gut in seawater-adapted
eels is inhibited by DDT, because the process is coupled to active sodium uptake which is
dependent on the Na,K ATPase enzyme. Miller and Kinter (1977) subsequently showed that
this inhibition of Na transport also impairs the uptake of amino acids from the gut. This finding
suggests that exposure to substances which inhibit ATPase, such as copper, in marine fish may
cause problems with the absorption of nutrients, which could have more practical implications
than a moderate osmoregulatory dysfunction. Freshwater fish, on the other hand, because they
drink very little, may not experience as great a problem from this.
DDT in the diet seemingly does not have much osmoregulatory effect on freshwater
fish compared to their seawater counterparts (Haux and Larsson, 1979; Leadem et al.,
1974). Leadem et al. (1974) fed DDT for 2 weeks to rainbow trout acclimated to
freshwater, one third seawater, or 100% seawater. The DDT treated fish in one third
seawater and 100 % seawater exhibited an elevated serum osmolality and sodium, but the
DDT caused no effect on these two parameters when the trout were acclimated to
freshwater. The enzyme Na,K ATPase in the gill tissue was inhibited in all the fish fed
DDT with the greatest percent of inhibition occurring in those adapted to seawater. This
was probably due to the enzyme being located basolaterally in the chloride cells in the
seawater-adapted forms. It is noteworthy that even though there was some inhibition of
this enzyme in the freshwater trout, there was no alteration in osmoregulation.

M. FENVALERATE
The pyrethroid insecticide fenvalerate is a potent inhibitor of a variety of ATPases (Clark,
1982; Desaiah et al., 1975). When trout were exposed to a highly acute dose, they showed
 159

a marked increase in concentration of various cations and osmolality of the urine (Bradbury
et al., 1987). The overall excretion rate of these cations also increased suggesting the
pesticide was inhibiting the reabsorption function in the kidney so the filtrate flowed
straight out. The net effect of this could be a loss of ions from the blood, although they
did not measure that parameter.

N, PETROLEUM HYDROCARBONS
It would appear that petroleum and the hydrocarbons that compose it have little effect on
osmoregulation. For example, Stickle et al. (1982) found that neither toluene or naphtha­
lene caused much effect on serum osmolality in coho salmon adapted to seawater or
freshwater unless the concentrations of the hydrocarbons exceeded the 48-h LC50.
Crude oil emulsions can apparently cause altered osmoregulation, but the concentra­
tions required may be rather high. Using a dose that produced considerable mortality in
7 days (in seawater), Englehardt et al. (1981) observed either depressions in serum
monovalent ions in freshwater or elevations of these ions in rainbow trout acclimated to
seawater. They also found considerable gill damage as a result of the crude oil emulsions.
Rainbow trout acclimated to freshwater and exposed to massive doses of a synthetic
oil mixture (similar to Prudhoe Bay crude oil) died in 1-4 h and the plasma chloride was
depressed (Zbanyszek and Smith, 1984). It probably was not, however, the direct cause
of death as the extent of chloride depression was not of sufficient severity (Milligan and
Wood, 1982). When trout were dosed with an oil mix concentration an order of magnitude
less, they experienced a slightly lowered chloride level at 24 h, but by 48 h of continuous
exposure it had returned to normal (Zbanyszek and Smith, 1984).
Using the wastewater from a petroleum refinery, Boese et al. (1982) found a 30%
inhibition of the gill ATPase, but only at a rather high concentration of waste (20%). In
spite of this enzyme inhibition, there was no change in the plasma osmolality. From this
observation and those mentioned earlier, it appears that it is possible to have a consider­
able inhibition of ATPase in the gills without a concomitant alteration in plasma electro­
lytes. Perhaps, the inhibition is compensated for by other hormonally mediated changes
(see summary at end of this chapter).
Salinity may affect the LC50 of various substances, and this is true of naphthalene and
other hydrocarbons derived from petroleum. In contrast to most metals, petroleum
components are more toxic at higher salinities (Stickle et al., 1982). Levitan and Taylor
(1979) attempted to investigate the physiological basis of this differential toxicity by
measuring serum osmolality of Fundulus exposed to naphthalene. At a concentration
which produced 85% mortality in 30 h, there was a transient increase in osmolality, but
it returned to normal within 12 h. While the authors think this change in serum osmolality
explains the increased toxicity in seawater, the evidence certainly does not support that
hypothesis. A more probable cause of the differential mortality is the increased intake of
naphthalene produced by increased drinking of seawater. They showed that, indeed,
naphthalene is taken up more rapidly when the fish are in hyperosmotic water. Once taken
up from the water, it will then concentrate in various tissues and other studies have found
that the distribution between tissues differs when the fish are in seawater as compared to
when in freshwater (see Chapter 5).
The available evidence seems to suggest that petroleum hydrocarbons have little effect
on osmoregulatory processes in either fresh- or seawater fishes. Exceptionally high doses,
which could certainly occur in areas of a petroleum spill, may produce severe osmoregu­
latory dysfunction, but chronic exposures probably affect other physiological processes
to a greater extent than those involved with osmoregulation.
Having said that, it should be pointed out that the use of oil dispersants to “clean up”
oil spills may cause a marked effect on fish electrolyte/osmoregulation (Baklien et al.,
1986), whereas either the oil or dispersant alone has relatively little effect.
160

O. AMMONIA
Elevated levels of unionized ammonia in the water caused trout to increase their urine
production in a concentration-dependent fashion (Lloyd and Swift, 1976). The stimulated
urine flow was explained by the authors as being due to increased inflow of water which
was then excreted. An alternative explanation later proposed by Arillo et al. (1981)
involves the circulatory system. They found that renin activity in the blood was elevated
by exposure to ammonia at concentrations and times of exposure that are comparable to
those used in the foregoing study.
Renin is released by the kidney and catalyzes the conversion of angiotensinogen to
angiotensin in the plasma. This latter protein is a powerful vasoconstrictor which causes
an increase in blood pressure (Olson, 1992). Smart (1978) noted a marked increase in
blood pressure in trout exposed to ammonia, although he used a very high concentration.
A rise in blood pressure would increase the filtration rate by the kidney and thus an
increase in urination.
With the theoretical and observational considerations noted above in mind, it appears
that ammonia causes little if any effect on the concentration of plasma electrolytes in adult
teleosts (Oppenborn and Goudie, 1993). This is so even when the concentration is lethal
and the fish are “overturning” (Smart, 1978; Swift, 1981). So, perhaps the compensatory
mechanisms are sufficient to prevent the electrolyte loss that would otherwise occur via
the increased urine flow.
The Oppenborn and Goudie (1993) study revealed two interesting aspects of ammonia
toxicity.
1. Striped bass in freshwater exposed to 0.5 mg/L unionized ammonia for 96 h exhibited
a slight rise in plasma osmolality. This suggests the gill regulation of electrolyes was
not compromised by ammonia. Instead, the stimulation of urine excretion rate probably
lowered the water content of the blood producing hemoconcentration.
2. Striped bass, Morone saxatilis, were less sensitive to ammonia than were hybrids
(M. saxatilis X M. chrysops). The mechanism of this differential sensitivity appears to
be that striped bass do not accumulate ammonia in their plasma as much as the hybrids
do when the external ammonia level is elevated. Whether this involves superior
excretion or less permeability of the gills to ammonia in striped bass is not known. It
is a somewhat surprising observation in that the hybrids are considered to be more
tolerant of changing environmental conditions.
Although ionic regulation in adult fish may not be especially sensitive to ammonia,
larval trout (and other species?) certainly are. Paley et al. (1993) have reported a marked
loss of sodium, chloride, and potassium from rainbow trout yolk sac fry when exposed
to ammonia for 24 h. The concentrations of unionized ammonia ranged from 7.2-36.2
p.mol/L and the effect on electrolytes was greatest for potassium. The loss of sodium was
probably due primarily to competitive inhibition of sodium uptake by ammonia. The
mechanism of potassium loss is less clear but may be caused by ammonia entry into the
cells and subsequent deprotonation which would cause a buildup of ions there (Paley
et al., 1993).

P. NITRITE
Nitrite is formed from nitrate by bacterial reduction. It appears to have relatively modest
effects on electrolyte regulation in fish (Williams and Eddy, 1988a,b; Bowser et al.,
1989). The effects on hemoglobin function (see Chapter 4) and other cellular activities
are probably more important in its toxicity. Nitrite is important in water of low ionic
content when nitrite/chloride ratio is high and fish take up nitrite instead of chloride
leading to formation of methemoglobin (Eddy and Williams, 1987).
 161

Q. CHLORINE AND OZONE

These are strong oxidizing agents which are quite toxic to fish (Hall et al., 1981). When
rainbow trout were subjected to a massive dose of chlorine (causing death within a few
hours) an elevation in plasma potassium was noted (Zeitoun et al., 1977). This would have
to have come from tissue cells, undoubedly as a result of the acute tissue hypoxia induced
by the gill damage (Bass et al., 1977; Bass and Heath, 1977). Unfortunately, the dose in
the Zeitoun et al. (1977) study was so high as to have little relevance to a real-world
situation. Using a much lower dose and a more prolonged exposure period. Block (1977)
found no effect of chlorine on blood electrolytes or gill ATPase on two species of brackish
water fishes. The lack of change in electrolytes may have been due to the fact they were
in water nearly isoosmotic to their blood, but the lack of an effect on the Na,K ATPase
enzyme may be indicative that, at least at low doses, chlorine may not have the potential
for much of an effect on osmoregulation.
Ozone is used as a disinfectant and as a substitute for chlorine in the antifouling of
steam-generating plants. Wedemeyer et al. (1979) found that acute exposure caused a
severe drop in sodium chloride, but chronic exposure caused no osmoregulatory effects,
even at concentrations resulting in limited histopathological damage to the gills. The
authors attribute death from high doses of ozone to massive gill damage followed by
osmoregulatory failure. An equally likely cause of death, perhaps in combination with the
osmoregulatory collapse, is internal hypoxia produced by the gill damage, as occurs in
acute exposure to chlorine (Bass and Heath, 1977) or zinc (Skidmore, 1970).

III. MUCUS
The importance of mucus in osmoregulation is beginning to be revealed (Shephard, 1982;
Handy et al., 1989). Handy and Eddy (1991b) have shown that mucus is usually absent
from the gill surfaces of rainbow trout that are free from stress. Thus, while it may aid
ionoregulation under some circumstances, it clearly is not required for “normal” function
in this species. Whether that is so for other species is not known.
It has been known since at least 1927 that exposure of fish to a variety of metals in
the water causes acute production of mucus by the gills, buccal cavity, and skin (Carpen­
ter, 1927). Over the years this observation has been repeated numerous times with a wide
variety of chemicals as well as acidic conditions. There now appears to be good evidence
both indirect and direct that the mucus helps maintain ionoregulation under these circum­
stances. When trout were exposed to either methylmercury or mercuric chloride at the
same ambient concentration, far more mucus was secreted by those in water with
mercuric chloride. (Lock et al., 1981; Lock and Overbeeke, 1981). It was further found
that it requires 10 -2 0 times more inorganic mercury in the water to cause the same degree
of osmotic dysfunction as that produced by a given concentration of organic mercury.
Thus, the mucus may be helping to counteract the increased permeability to water of the
gill poisoned by mercury.
Handy and Eddy (1989) have found that mucus of a freshwater fish has a considerably
higher concentration of Na and Cl than does the water. This reduces the gradient between
the blood and water, thus an increased mucus layer in fish experiencing pollutant stress
would reduce the passive diffusive loss of electrolytes. It also has a low permeation
coefficient for metals and thus may help protect the tissue from its action on the sensitive
gill epithelium (Handy et al., 1989).
Although increased mucus secretion in fish which are experiencing pollutant stress
appears to be adaptive regarding electrolyte regulation, it may be maladaptive from a
respiratory standpoint. This is because it adds an unstirred layer next to the lamellar
surface and therefore increases the diffusion distance for oxygen (see Chapter 3).
162

IV, CHLORIDE CELL PROLIFERATION

Zinc (Matthiessen and Brafield, 1973), copper (Baker, 1969), cadmium (Oronsaye and
Brafield, 1984), and nitrite (Gaino et al., 1984) have been found to stimulate an increase
in chloride cell number on the gills of fish. With zinc and cadmium, the authors felt that
this is a mechanism to excrete the accumulated metals, although osmoregulatory involve­
ment was not entirely discounted. There seems to be no direct evidence in support or
opposition of the hypothesis that these cells are excreting the metals.
Interestingly, as was mentioned above, nitrite is actively taken up from the water by
chloride cells (Williams and Eddy, 1988) so their proliferation under nitrite exposure
might appear to be maladaptive. Bath and Eddy (1980) have also found that nitrite ions
compete with chloride ions in the active uptake of the latter by the chloride cells (Bath
and Eddy, 1980). Thus the proliferation of more chloride cells caused by this substance
appears to be a compensatory mechanism to maintain normal uptake of chloride from the
water. It appears to be successful as nitrite exposure causes little effect on plasma chloride
content.

V. SOME SUMMARY COMMENTS REGARDING OSMOREGULATORY

AND ELECTROLYTE ALTERATIONS
In seawater-adapted fish, exposure to a wide variety of different pollutants induces an
osmoregulatory dysfunction which is reflected in a rise in plasma NaCl and osmolality.
In freshwater-adapted fish, the opposite will occur in that there is a loss of NaCl mostly
via the gills. Evidently, a large majority of pollutants affect osmoregulation and/or
ionoregulation, but there are considerable differences in their potency. Among the metals,
copper, aluminum, and methylmercury probably cause the largest amount of dysfunction.
Zinc and iron cause little or no effect on osmoregulatory variables, even though zinc, at
least, causes marked histopathological damage to the gill epithelium. Thus, physical
damage to the gills does not necessarily lead to ionoregulatory dysfunction. Indeed, the
hypersecretion of mucus that usually accompanies such histological changes may reduce
the ionoregulatory problems.
It appears that the percentage change in blood constituents produced by comparable
toxic concentrations of a given chemical may be greater for the freshwater-adapted fish
as opposed to marine, but this needs more systematic investigation. The mechanisms for
this difference are not obvious, since marine fish have flux rates for water and salts about
an order of magnitude greater than their freshwater cousins.
Osmoregulatory disruption by metals and most other chemicals, with the exception of
acids, usually involves inhibition of the Na,K ATPase enzymes in gill and perhaps also
in the gut. The latter would be of importance primarily in marine habitats where the fish
drink large volumes of water and this inhibition may cause a nutritional problem. This is
because the uptake of sodium in the gut is coupled to amino acid absorption so inhibition
of sodium uptake would inhibit the latter. The ATPase enzymes in the kidney may also
be a point of action, particularly in freshwater fish where a good deal of glomerular
filtration and reabsorption takes place, but this has not received much attention. Increases
in permeability of the gills may or may not take place.
While it has been assumed that altered osmoregulatory function is generally due to
effects of some toxicant on peripheral processes, the involvement of the brain and
endocrine tissues should not be ignored. For example, it has been reported that the
catecholamine content of the hypothalamus changes during acclimation to different
 163

salinities (Hegab and Hanke, 1981). Because this area of the brain is affected by a variety
of toxicants (Smith, 1984), there may be indirect effects on osmoregulation.
An altered osmotic and ionic makeup of the blood induced by some pollutant should
bring about hormonal changes as a compensatory response to restore homeostasis. It is
common to observe elevated cortisol levels with exposure to a variety of environmental
stressors (Pickering, 1993), and this hormone facilitates the active transport of ions by
both marine and freshwater teleosts (Madsen, 1991). Whether the cortisol elevation is part
of a generalized stress response or one caused by the electrolyte changes of the blood is
not clear. Indeed, it could easily be a bit of both. In any case, if the extent of ion alteration
is not too great, recovery of homeostasis may occur even with continued pollutant
exposure, and cortisol is probably a key mechanism here as it stimulates increased Na,K
ATPase activity in the gills (Madsen, 1991).
A stress severe enough to cause elevated adrenalin/noradrenalin levels may actually
accentuate disruption of hydromineral homeostasis because these catecholamines cause
a net loss of sodium and chloride (Vermette and Perry, 1987). Thus, from an osmoregu­
latory standpoint, these hormones appear to be maladaptive.
The hormone prolactin, from the anterior pituitary, has been found to increase mark­
edly during acid stress in tilapia (Oreochromis mossambicus); (Wendelaar Bonga et al.,
1984). This was associated with a subsequent reduction in water permeability of the gills
and restoration of plasma ion levels. A comparable hormonal response may occur with
other pollutants as well.
With a wide variety of substances causing osmoregulatory dysfunction, the question arises
as to whether this might add to or subtract from the energetic demands of the fish. It is a
difficult matter to approach because estimates of the energetic “cost” of osmoregulation vary
from negligible to 50% of the resting metabolic rate in freshwater (for review see Febry and
Lutz, 1987). Even closely related species may show huge differences. For example, Iwama
and Morgan (1990) report a value of 20% and 1% for the rainbow and steelhead trouts,
respectively. One thing that they most seem to agree on is that osmoregulation in freshwater
requires more energy than it does in seawater. This is intuitively rather suprising as the total
fluxes are much greater in the seawater forms. When there is an inhibition of the processes
catalyzed by ATPases, as occurs with a variety of pollutants, the demand for ATP might
actually go down. Thus, it is conceivable that pollutant exposure might thereby lower the
metabolic cost of osmoregulation, but that is pure conjecture at this time.
The calcium concentration in the ambient water critically influences permeability of
the gills to both sodium and water. Increasing calcium concentrations decreases the
branchial water permeability and rate of sodium loss caused by toxic chemicals.
As was mentioned at the beginning of this chapter, the usual method that has been used
to detect effects of pollutants on osmotic and ionic regulation has been to measure the
concentrations of Na, K, Ca, and Cl in the plasma after exposures. Recently, Wood (1992)
has proposed a major change in approach. He maintains that changes in flux rates are what
is actually of interest and these can be assessed by measuring the concentration of these
ions in the water in which the fish is residing (providing volumes are small). Such an
approach has several advantages such as the fact that since it is non-invasive, changes in
a single fish over time can be assessed. The technique is more sensitive to alterations in
osmoregulatory processes and can detect changes from toxic chemicals long before they
are reflected in internal concentration changes.
Finally, the single pollutant that probably has the greatest effect on electrolyte regu­
lation in fish is acid. Acidic conditions cause an exceptionally large dysfunction in
osmotic and ionic regulation which is discussed in some detail in Chapter 10.
164

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{S a lm o g a ir d n e r i) exposed to acutely lethal concentrations, J. F ish B i o l , 12, 93, 1978.
Smith, J. R., Fish neurotoxicology, in. A q u a tic T o x ic o lo g y , Vol. 2, Weber, L. J., Ed., Raven Press, New
York, 1984, 107.
Spry, D. J. and Wood, C. M., Acid-base, plasma ion and blood gas changes in rainbow trout during short
term toxic zinc exposure, J. C o m p . P h y s i o l B., 154, 149, 1984.
Spry, D. J. and Wood, C. M., Ion flux rates, acid-base status, and blood gases in rainbow trout.
S a lm o g a ir d n e r i, exposed to toxic zinc in natural soft water. C a n . J. F ish . A q u a t. S c l , 42, 1332,
1985.
Stagg, R. M. and Shuttleworth, T. J., The accumulation of copper in P la tic h th y s f le s u s L. and its effects
on plasma electrolyte concentrations, J. F ish B i o l , 20, 491, 1982.
Staurnes, M., Sigholt, T. and Reite, O. B., Reduced carbonic anhydrase and Na-K-ATPase activity in
gills of salmonids exposed to aluminum-containing acid water, E x p e r ie n tia , 40, 226, 1984.
Stickle, W. B., Sabourin, T. D. and Rice, S. D., Sensitivity and osmoregulation of coho salmon
O n c o rh y n c h u s k isu tch , exposed to toluene and naphthalene at different salinities, in. P h y s io lo g ic a l
M e c h a n is m s o f P o llu ta n t T o x icity , Vernberg, W. B., Calabrese, A., Thurberg, F. P. and Vernberg,
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Stinson, C. and Mallatt, J., Branchial ion fluxes and toxicant extraction efficiency in lamprey (P e tr o m y z o n
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1977.
 Chapter
8
Physiological Energetics
I. INTRODUCTION
This chapter is devoted to certain aspects of the subject of bioenergetics, an
immense subject encompassing levels of complexity from the cellular to the eco­
system. Physiological energetics, which is the main thrust of this chapter, includes
the study of rates of energy expenditure by individual fish, losses and gains of
energy in the body, and transformations and mobilizations of energy “pools” within
the fish. Growth, which is inextricably linked to energetics, is also included in this
chapter. Indeed, many fish bioenergetics studies are basically studies of growth
rate.
After a review of some basic concepts of energetics and methodologies applicable
to fish, the effects of pollutants on energy expenditure by individual fish will be
considered with implications for growth included where appropriate. The effects of
pollutants on growth rate per se of larval and juvenile fish follows. Swimming
performance will then be discussed at some length because the presence of toxic
chemicals may restrict this highly energy-dependent activity. Finally, a review of the
influence of pollutants on body energy stores of carbohydrate, lipid, and protein
closes the chapter.

II. GENERAL CONCEPTS

In biological studies, the basic unit of energy measurement has traditionally been the
calorie. With the transition to the SI system (Systeme International), the joule (1 cal
= 4.1868 J) is becoming the basic measure of energy expenditure, although some
continue to use the calorie (or Calorie = kcal). For a good discussion of the SI system
and how it relates to animal energetics, the review by Bartholomew (1982) is recom­
mended.
The following simple equation summarizes the principles of energy input and output
for any animal, the actual numbers involved become the energy budget.

171
172

I = G+M+E+F

where:

I = gross energy intake

G = tissue production (i.e., growth)
M = respiratory energy expenditure (also called metabolic cost or
metabolic rate)
E = energy lost via nitrogenous waste (NH3)
F = fecal loss (i.e., energy not absorbed by the digestive tract) ( 1)

The metabolic rate component can be further partitioned into standard (maintenance),
active, and postprandial metabolic rates; and the growth component can be divided into
somatic and gonadal (Wieser and Medgyesy, 1991). Bayne et al. (1979) have rearranged
Equation 1 slightly:

G = A - (M + E) ( 2)

where A refers to assimilation. They call “G” the “scope for growth” which emphasizes
how growth and energy metabolism are intimately related. Thus far, the scope for growth
concept has mostly been applied to pollutant effects on mollusks, but there is little reason
it could not also be used in fish studies.
Because fish are dynamic systems involving inputs and outputs which are constantly
changing, none of the symbols in Equation 1 are constants. A useful way of visualizing
what is happening to energy exchanges in a fish is to examine Figure 1.
In this hypothetical fish we start with an intake of 100 cal, 20 of which are lost as feces
and 7 as nitrogenous waste. (In fish, incidentally, most of the nitrogenous waste is
ammonia which is excreted via the gills, in contrast to terrestrial animals which excrete
these wastes via the urine.) The cost of digestion and assimilation of the food is 14 cal

100

27

(0)
44

(Mf + Ma) (Ms)

Figure 1 Average partitioning of dietary energy for a carnivorous fish. Numbers refer to
calories, letters to the symbols in Equation 1. See text for further explanation. (From Brett, D. J.
and Groves, T. D. D., Fish Physiology, Vol. 8, Hoar, W. S. and Randall, D. J., Eds., Academic
Press, New York, 1979, chap. 6. With permission.)
 173

(Specific Dynamic Action or postprandial) leaving a net energy to be used by the

organism of 59 cal. This net energy is partitioned between maintenance (i.e., basal
metabolic rate), swimming (active) metabolism, and growth. Clearly then, energy me­
tabolism (which is the same thing as energy expenditure) and growth are competing for
the net energy. Thus, if metabolism is elevated because of environmental stressors,
growth will be limited unless the intake of food is increased.
The above generalized energy budget applies to a typical carnivorous fish with
abundant food. Although 95% of the fish species are carnivorous, some important
commercial and aquaculture groups (e.g., cichlids, cyprinids) are herbivorous or omnivo­
rous (Pandian, 1987). Thus, it is interesting to compare generalized budgets for the two
different feeding styles (Brett and Groves, 1979):

Carnivores: 100 C = 29G + 44M + 7 E + 20F

Herbivores: 100 C = 20 G -h 37 M + 2 E -h 41 F

The assimilation efficiency of the herbivores is considerably less than that of carni­
vores, so a sizable amount of energy is lost via the feces in herbivorous species. Note how
the amount of energy lost via ammonia excretion (E) is relatively small compared to other
parts of the energy budget.
The percent of food assimilated varies with the diet composition. As a general rule,
carbohydrates are poorly assimilated in fish; about 60% is lost in the feces of carnivorous
fish. Omnivorous species such as carp do a bit better. Even so, for most fish, protein is
considered to be the major source of energy (Pandian, 1987). Having said that, however,
it should be further noted that lipids can be utilized by many species and these may reduce
the protein requirement. Furthermore, Brown et al. (1990) have shown that in channel
catfish {Ictalurus punctatus), at feeding levels of maintenance or below, lipids and
carbohydrates are utilized more efficiently than protein. As the daily ration is increased
above maintenance, protein and lipid are utilized more efficiently.
Protein, in addition to being a major intake energy source, is also a major form in
which energy is stored. Diana (1982, p. 395) has noted that: “Proximate composition of
fish under various feeding regimes indicated that energy gain or depletion from the body
was due to changes in amount of whole body tissue or body protein, rather than specific
utilization or storage of lipid.”
Tissues that synthesize a lot of protein may use a sizable proportion of their total
energy expenditure for protein synthesis. Recently, Pannevis and Houlihan (1992) have
found that isolated fish liver cells utilized an average of nearly 80% of their total oxygen
consumption for this purpose. This contrasts with a value of 2.8% for the Na,K ATPase
activity which maintains the transmembrane electrolyte balance. Thus, changes in stan­
dard oxygen consumption of whole animals may at times reflect changes in protein
synthesis rates, a point rarely considered in the literature.
The generalizations about the importance of protein as an energy source in fish applies
to whole-animal studies. Individual tissues may utilize other substrates preferentially. For
example, fish gill (which is a mixture of several cell types) prefers glucose and lactate
over amino acids for oxidation (Mommesen, 1984). Even though protein is a major source
of energy in fish, the stress of exercise (Driedzic and Hochachka, 1979) or severe
environmental hypoxia (Heath and Pritchard, 1965) causes rapid depletion of stores of
carbohydrate, primarily liver and muscle glycogen. A considerable amount of work has
shown that certain pollutants may do the same thing (discussed later in this chapter).
Since the early 1970s, there have been a number of attempts to compile energy budgets
for several species of fish in the laboratory and in the field (see Brafield, 1985; Soofiani
and Hawkins, 1985; Adams and Breck, 1990 for review of methodologies used). Hewett
174

and Johnson (1992) have also created a generalized bioenergetics model for fish growth
for microcomputers. It enables an experimenter to alter various factors (e.g., oxygen
consumption rate or temperature) and follow changes in aspects of the energy budget over
time. It is set up to operate with 20 different species including lampreys, several salmo-
nids, centrarchids, and percids.
Most physiological studies of the effects of pollutants on energetics in fish have
involved measures of energy expenditure and that will be the emphasis in this chapter.
Bartholomew (1982, p. 947) has pointed out that: “The rate of energy metabolism
probably integrates more aspects of animal performance than any other single physiologi­
cal parameter.” Because all physiological activities require energy, measurements of the
rate of its expenditure by a fish tells us a lot about how the fish is functioning in its
environment, and when the variations over time are observed, the relationship of temporal
changes in the animal to those in its environment can also be investigated.

III. METHODS OF MEASURING ENERGY EXPENDITURE IN FISH

The most common method of measuring the energy expended by a fish is to measure the
rate of oxygen consumption (sometimes called respiration). This is not to be confused
with breathing movements which some authors designate by the same name. The fre­
quency of breathing movements is not a good indicator of metabolic rate because gill
damage which causes a reduced flow of oxygen from the water to the blood, and thus a
decreased consumption of oxygen also causes increased breathing (see Chapter 3).
Attempts have been made to measure heat production (i.e., direct calorimetry) in fish
(Brafield, 1985), however, difficult technical problems associated with this approach
have prevented its wide application. One can calculate the joules released from oxygen
consumption data (19.38 J/ml oxygen consumed). This assumes, however, no anaerobic
energy expenditure. If the fish is being forced to swim at burst speed or is under severe
hypoxic or pollutant stress, then there will be an anaerobic component. In order to
measure this, whole-body lactic acid levels can be determined and then the energy
released by anaerobic metabolism computed (0.27 mg lactic acid/4.186 J).
It is also possible with some effort to measure oxygen consumption, and carbon
dioxide and ammonia excretion rates all simultaneously (Brafield, 1985). This yields
information on substrate utilization but has not been applied much at this writing.
A method to estimate maintenance (i.e., basal metabolism) energy needs is to feed
groups of fish at different rates and determine the daily percent energy change in the
whole body by bomb calorimetry. When feed rate is plotted against percent energy
change per day, a straight line is obtained which passes through the zero energy change
(i.e., maintenance level) at some feed rate that can then be used to estimate maintenance
feed consumption (Brown et al., 1990).
Oxygen consumption rate is by far the most common way to measure energy expen­
diture in any animal and fish are no exception. The consumption of oxygen by a fish is
usually measured by confining the animal in a chamber through which water slowly flows
(Cech, 1990). The difference in concentration of dissolved oxygen between the inflowing
and outflowing water multiplied by the flow rate yields the oxygen consumed. Various
intermittent flow arrangements have also been devised to avoid problems of lag. In nearly
all the literature, oxygen consumption data on fish are expressed as either mg 02 /kg body
wt/h, or as ml 02 /h. There has also been a not too successful effort to get investigators
to express such data in p.mol O2 (31.251 p-mol = 1 mg) (Gnaiger, 1983).
Data on consumption of oxygen (metabolic rate) by fish are classified into three types.
1. Standard metabolic rate. This approximates the basal metabolic rate commonly used
in human energetics but it is difficult to define with precision in fish. Thus, the fish is
 175

allowed a period of acclimatization to the chamber (e.g., 12-24 h), is free from any
external disturbances, has been acclimated to the test temperature for approximately a
week, and is in a postabsorptive state. Most importantly for the determination of
standard metabolic rate is the complete elimination of body movement. Because this is
clearly impossible to do without subjecting the organism to stress, and anesthetics cause
undesirable side effects, some quantitative measure of movement is often obtained and
the data relating oxygen consumption to body movement are extrapolated to zero body
movement (Beamish and Mookherjii, 1964; Cech, 1990). The standard rate is a measure
of the cost of homeostatic regulation in the animal. Thus, all the various requirements
of energy for pumping blood, regulating ionic composition, nervous conduction, tissue
repair, etc., are included in this.
2. Routine metabolic rate. This rate of oxygen consumption is similar to the standard rate,
however, it is more variable because random body movement is not eliminated as a factor.
For this reason, the values will be higher. If the fish is a relatively inactive type and the
respirometer chamber is small, it will come close to yielding the standard metabolic rate.
By far the majority of “resting” metabolic rate data on fish are of this type and some would
argue that it has more environmental realism than does the standard rate.
3. Active metabolic rate. In order to obtain this, the fish must be forced to swim at a
maximum sustained speed while the consumption of oxygen is determined. Various water
tunnels and other devices have been devised for this purpose (Cech, 1990; Fry, 1971).
The difference between the standard and active rate is termed the metabolic scope
(Bartholomew, 1982). It is a measure of the maximum rate of energy that a fish can
expend above that required to keep the homeostatic machinery functioning. It would
probably be more precise to refer to this as aerobic scope, for as fish approach a maximum
swimming speed, anaerobic metabolic processes begin to play a part in the total expen­
diture of energy (Driedzic and Hochachka, 1978). Precisely where the line separating
aerobic and anaerobic expenditure of energy lies is not known but undoubtedly varies
greatly between species of fish and their swimming habits (e.g., sculpins vs. tunas).
When measuring energy metabolism in fish, it is important to realize there are many
variables that may have large effects on metabolic rate. These must be recognized and
kept as constant as possible when measuring the effect of some pollutant on this rate,
unless one wishes to investigate potentially complicating interactions.
Water temperature probably has the greatest effect of any natural environmental
variable on metabolic rate. Because fish (except for some species such as tuna) are
ectothermic animals, their bodies are for all intents and purposes at the same temperature
as the water. Routine metabolic rate of a species of fish from the temperate zones averages
an increase of 2.3-fold for a rise of 10°C (i.e., the QIO = 2.3) in the midpoint of the
thermal range for that species (Brett and Groves, 1979). While acclimation for a period
of days to a new temperature may reduce the extent of this increase, it will not generally
eliminate it, and as we shall see, chronic exposure to a pollutant (e.g., copper) may delay
the process of temperature acclimation.
The feeding of fish has a large effect on their rate of energy metabolism. Feeding
causes an increase in the rate which is roughly proportional to the size of the ration up
to a saturation level. In seven species investigated by Jobling (1981), there was an average
doubling of the routine metabolic rate when fed at saturation level. This postprandial
elevation in metabolism persists for a few hours to several days. In the plaice, at least, the
size of the ration has little influence on the duration of the postprandial elevation. Most
workers have made their measurements either 24 or 48 h postfeeding, which seems to be
a reasonable compromise.
Because long-term exposures to pollutants may reduce appetite, when doing chronic
studies it is probably best to give controls the same ration size as experimental animals
176

receive, even though this may be less than they were getting before. However, excessive
food deprivation can cause an abnormally low rate of metabolism (Beamish, 1964).
Bouts of severe exercise can cause an oxygen debt so the oxygen consumption rate
after the exercise may remain elevated for 3-12 h, depending on species and extent of the
exercise (Heath and Pritchard, 1962; Goolish, 1989). The same phenomenon occurs
following severe hypoxia or anoxia (Heath and Pritchard, 1965; Van den Thillart and
Verbeek, 1991).
The greater the body size of an animal, the slower its rate of metabolism per gram of
tissue. This rule holds for most poikilothermic and homeothermic animals. Therefore, it
is best to either group fish by size or use a limited size range thereby eliminating this
factor as a variable.
These precautions may seem obvious to the experienced investigator, but unfortu­
nately, in many studies of metabolism adequate attention has not been paid to them. It is
hoped this brief review will raise the consciousness of those who plan to pursue these
kinds of studies in the future. Still, even with careful elimination of extraneous variables,
there is an element of truth in the comment by Wood and McDonald (1982, p. 199): “In
our experience, one only has to think evil of a fish to double its rate of oxygen consump­
tion.”
Fry (1971) has devised a system of classification for environmental factors based on
how they affect the metabolic rate of a fish. A factor that reduces the active oxygen
consumption is called a “limiting factor”. An example of this would be the concentration
of dissolved oxygen. At progressively lower levels of oxygen in the water the active
oxygen consumption decreases in proportion to the severity of the hypoxia (cf. Chap­
ter 2). A factor that alters the standard metabolic rate by changes in the flow rates of
molecular components through the cellular metabolic pathways is referred to by Fry as
a “controlling” factor. Temperature is an example of this.
If an environmental factor influences the metabolic cost of maintenance, this has
been designated as “masking” (Fry, 1971). An example is the requirement for energy
associated with osmotic and ionic regulation. As the salinity (i.e., the masking factor)
changes these costs increase or decrease but not necessarily in proportion to the change
in osmotic or ionic gradient (Rao, 1968). The possible interaction of these three
categories of factors should not be overlooked. For example, it is well known that low
dissolved oxygen has a more limiting effect at high rather than low temperatures (Fry,
1971). On the other hand, acclimation temperature apparently does not affect the
metabolic cost of osmoregulation (Rao, 1968). Pollutants may act as one or more of
these environmental factors.

IV. EFFECTS OF METALS ON METABOLIC RATE

A. COPPER
We begin this section with a review of the work involving copper as the “pollutant”
because there is probably more known about the effects of this on energy metabolism in
fish than any other chemical. The observations should not, however, be interpreted as a
“typical” response to a metal, as will be seen when the other metals are discussed.
When routine oxygen consumption of bluegill (Lepomis macrochirus) was measured
while they were exposed to copper for 7 days, there was an initial stimulation followed
by inhibition (O’Hara, 1971). Both the stimulation and inhibition were concentration
dependent.
The initial stimulatory effect is most likely due to simple irritation of the mucous
membranes of the oral cavity which causes fish to move around more. With subsequent
adaptation of the receptors, this effect could abate in a day or two. More interesting is the
depression of metabolism with prolonged exposure to copper. Felts and Heath (1984)
 177

more recently observed that bluegills exposed to 0.21 mg/L copper showed no inhibition
in oxygen consumption until sometime between 9 and 30 days of continuous exposure.
This concentration of copper is below any of those tested by O’Hara (1971) and because
the inhibition required so long to occur, it suggests the extent of inhibition might be
dependent on the amount of copper accumulated from the water. If this were the case,
individual tissue respiration rate could be affected. To test this hypothesis, the in vitro
oxygen consumption of liver, brain, and gill tissue was measured (Felts and Heath, 1984).
At 30 days of exposure (20°C), only the respiration rate of the liver was different from
that of controls, and it was slightly elevated rather than inhibited; thus the hypothesis is
rejected. The stimulation of liver energy consumption may reflect an increase in the
detoxification activity and associated protein synthesis in the liver. Also, copper has been
found to stimulate respiration when added in vitro to fish liver mitochondria (Zaba and
Harris, 1978).
The explanation for the whole-body metabolic depression produced by copper must
then lie with a reduction in muscle metabolism. This could be due to: (1) inhibition of
energy metabolism enzymes by copper, (2) decreased muscle tone, or (3) decreased
spontaneous activity. The first possibility is probably not important, at least at this
concentration of copper, because such an inhibition should have caused reduced respira­
tion in the other tissues measured and that was not observed in the Felts and Heath study.
A reduction in spontaneous activity of bluegill exposed to sublethal concentrations of
copper has been quantified (Ellgaard and Guillot, 1988). Ellgaard and Guillot suggest this
might be a direct effect of the copper on the enzymatic reactions of respiration. However,
the in vitro tissue respiration work of Felts and Heath (1984) does not support that
hypothesis.
In work by Waiwood and Beamish (1978) the effect of body movement was kept
constant by measuring the oxygen consumption of fish swimming at controlled speeds
following their exposure to copper for 5 days (Figure 2). The concentration of copper used
was approximately that which would cause 20% mortality in 240 h. Even at this low
concentration, there was a significant effect on metabolism. Copper caused the oxygen
consumption to become elevated at all swimming speeds suggesting an increased cost of
maintenance (masking effect), contrary to the results discussed above. The metabolic
scope was also reduced (limiting effect), which is a separate issue to be considered later.
Unfortunately, Waiwood and Beamish (1978) were using trout while the other work
mentioned was done on bluegill; therefore there may be a species-specific difference. A
more likely explanation is that the copper caused an increased maintenance cost in certain
tissues (e.g., liver) while at the same time it reduced spontaneous movement in fish not
forced to swim. Recall the discussion at the beginning of this chapter regarding the high
cost of protein synthesis in liver.
Support for the hypothesis of increased maintenance cost of copper-exposed fish is
found in work on perch growth and food consumption. Collvin (1985) found that growth
was suppressed at very low levels of copper even though food consumption remained
unchanged. Copper presumably does not cause a reduction in assimilation efficiency in
fish (Lett et al., 1976), so the only logical explanation for the reduced growth is a greater
utilization of energy by the fish leaving less to be used for growth.
In summary, it appears that exposure of fish to sublethal levels of copper causes
metabolism in some non-muscular tissues (e.g., liver and gill) to increase while depress­
ing spontaneous muscular activity. This latter effect may involve some effects of the
metal on the central nervous system. The net effect on whole-animal oxygen consumption
will depend on whether one measures it in a swimming fish, where muscle metabolism
is kept the same in experimental and control animals, and therefore any differences are
due to changes in other tissues, or in “resting fish”, in which case small variations in
muscular activity are probably the dominant influence on metabolic rate.
178

Figure 2 Oxygen consumption rate of rainbow trout (Salmo gairdneri) at different swimming
speeds measured on fish which were first exposed to waterborne copper for 5 days. The
concentrations of copper are in iig/L. (From Waiwood, K. G. and Beamish, F. W. H., Water Res.,
12, 611, 1978. With permission.)

With the close coupling between metabolic rate and growth, changes in the latter are
to be expected when metabolism is altered. So, it is not surprising that chronic exposure
to copper depresses the rate of growth in young trout (Lett et al., 1976). However, over
a period of 10-40 days of continued exposure there appears to be an acclimation to the
copper and return to normal growth. The speed of return to normal is inversely propor­
tional to the concentration of copper in the water.
Lett et al. (1976) have also noted a virtual cessation of eating upon initial exposure to
copper. Then over a period of 5-15 days, the fishes’ appetite returns to normal. As with
growth, the rate of recovery depends on the concentration of copper. Because the appetite
returns to normal more rapidly than the growth rate, it appears that a lack of eating is not
the sole cause of the slowed rate of growth, although it must contribute to it.
Collvin (1985) also found that perch acclimated to the copper so that after 20 or more
days (depending on copper concentration in the water), the effects on growth were
reduced or disappeared. In his study, however, food consumption was constant so it raises
the possibility that once the detoxification mechanisms (metallothionein?) are fully
induced, the energetic demands are reduced so they are not competing as much with
growth for the energy intake.
Suppression of appetite is commonly observed in fish when they are exposed to metals
(Lett et al., 1976 and personal observation). The mechanism for this has not been
determined, but probably is in part caused by hormonal changes. Fish under almost any
type of stress show elevated concentrations of plasma corticosteroid (Donaldson, 1981)
and adrenaline (Mazeaud and Mazeaud, 1981). These hormones could cause a direct
 179

inhibition of eating or they might even do it indirectly by producing changes in the blood
glucose level. Colgan (1973) has presented evidence that appetite in fish is suppressed by
high levels of glucose in the blood. The higher the blood glucose, the less the food intake.
The above-mentioned hormonal changes cause mobilization of liver glycogen into blood
glucose, so in essence the system may be fooled into “accepting” the caloric intake as
being more than adequate. The acclimation phenomenon whereby the appetite returns to
normal could reflect a return to “normal” levels of the corticosteroid hormones, although
no direct evidence is available on this. A literature review on the control of appetite in fish
(Fletcher, 1984) emphasizes the multifactorial nature of this process.
At present, changes in the maintenance cost as a result of copper or any other metal
is not well explained. Excretion of copper and repair of damaged tissue could contribute
to this as could alterations in osmoregulation (see Chapter 7). Waiwood and Beamish
(1978) make the interesting suggestion, based on their results and others that the masking
effect could be due to copper causing some blockage of nervous transmitters or other
neuronal malfunctions of the central nervous system. If this speculation is correct, the net
result would be a reduction in coordination and, thereby, swimming efficiency. It is
hypothesis that does not appear to have been further tested.
One final interaction of copper with energetics has to do with the question of whether
exposure to the metal affects the ability of fish to acclimate to a temperature change.
Figure 3 shows how raising the water temperature 10°C while bluegill (L. macrochirus)
were exposed to a sublethal level of copper caused essentially the same amount of
increase in metabolism in controls and exposed fish, but upon continued exposure to the
copper and elevated temperature, the controls showed a classical type III (Precht, 1958)
partial compensation while those in copper exhibited a greatly delayed acclimation to the
new temperature. While increased temperatures generally enhance growth rate (Brett and
Groves, 1979), it is evident from these data that copper can also cause the demand for
energy to be “excessively elevated” following a temperature rise, and this could compete
with growth for the available energy intake.

Figure 3 The effect of copper exposure (0.21 mg/L) on metabolic acclimation to a temperature
change in bluegill (L macrochirus). Between days 3 and 4 of exposure, the temperature was
increased from 20-30°C (represented by the vertical line) and held at 30°C for the remainder of
the exposure period. Bars represent 2 SEM above and below the mean. Open circles = controls.
Closed circles = copper exposed. (From Felts, P. and Heath, A., J. Fish Biol., 25,445,1984. With
permission.)
180

B. MISCELLANEOUS METALS
Mercury is well known for causing neurological toxicity in a variety of animals. From the
standpoint of energetics of fish, three studies are noteworthy. MacLeod and Pessah (1973)
measured active metabolism in rainbow trout following exposure to inorganic mercury
(as HgCL) for 96 h. The mercury caused a depression of active metabolism and the effect
was increased at higher temperatures. At first glance, these results may seem different
from those of Waiwood and Beamish (1978) for copper, but recall, however, that copper,
while it caused an increased maintenance demand, also resulted in a decrease in the
maximum oxygen consumption (i.e., a limiting effect). Active metabolism, as measured
by MacLeod and Pessah (1973), is displayed at maximum sustained swimming speed, so
the two sets of data are actually similar. Both results may be due to altered nervous
function, although other mechanisms such as inhibition of cellular enzyme activities
could also be occurring (Brown and Parsons, 1978). As has been emphasized in several
places, observed effects may be attributable to more than one cause.
Mercury may inhibit routine as well as active metabolism in some species. Panigrahi
and Misra (1978) reported an 84% decrease in “resting” oxygen consumption in Anabas
exposed for 10 days to 3 mg/L mercuric nitrate. After continued exposure for 45 days
these fish were still alive even though the metabolism remained down. Survival at a
resting metabolic rate of only 16% of “normal” for this length of time is remarkable.
These fish are commonly called the “climbing perch”, so they are quite capable of
breathing air. Unfortunately, the authors do not adequately describe their methods, but
one is left with the feeling the mercury either reduced spontaneous activity, which was
probably quite high in their controls (0.19 mg/g/h) and/or caused the fish to utilize more
breathing of air, which presumably was not measured. Such a high dose of mercury would
probably cause considerable gill damage (Olson et al., 1973) and thus the reduced oxygen
consumption from water would reflect this.
The oxygen consumption of larval fish rises dramatically with hatching and as the yolk
sac is absorbed. There is then a rapid drop in the oxygen consumption when active feeding
begins. Storozhuk and Smirnov (1982) found that these metabolic changes were almost
eliminated and the overall rate of metabolism was depressed by exposure of pink salmon
larvae to mercury at either 25 or 1 pg Hg/L (Figure. 4). The extent of metabolic inhibition
was not dependent on the concentration of mercury in the medium. This is somewhat
surprising in that the concentration of mercury in the tissues was 13 times higher when they
were kept in the 25 pg Hg/L than when in 1 pg Hg/L. The authors cite earlier work in their
laboratory which showed that exposure of salmon larvae to mercury causes a decrease in
concentrations of copper, zinc, and iron in the tissues. Consequently, they attribute the
metabolic inhibition to a replacement of these elements by mercury in metal-containing
enzymes.

Figure 4 Oxygen consumption by pink

salmon (O. gorguscha) larvae immedi­
ately after hatching. Circles = controls.
Squares = exposed to 25 pg Hg/L. Ar­
rows indicate changeover to active feed­
ing. (From Storozhuk, N. G. and Smirnov,
B. F., J. IchthyoL, 22, 168, 1982. With
permission.)
 181

Aluminum has a variety of physiological effects and apparently, damage to the gills
is one of them. Make (1986) found a marked drop in arterial P02 when trout were exposed
to 2 mg/L for up to 72 h. The drop in arterial P02 was preceded by an elevation in oxygen
consumption and this was maintained even in the face of the severely dropping Po2.The
increased rate of oxygen consumption was probably due to the big increase in energy
demand of the ventilatory muscles (ventilation frequency increased almost threefold) in
response to the falling arterial P02.
Chromium, zinc, and lead at sublethal concentrations have all been found to increase
oxygen consumption, (Brafield and Mathiesse, 1976; Somero et al., 1977; Pamila et al.,
1991). For chromium and zinc, at least, the cause of the increased energy expenditure
appears to be increased spontaneous activity. Irritation of skin or mucous membranes
clearly causes greater spontaneous locomotor activity in a fish, as it would almost any
other animal. Ellgaard et al. (1978) have devised a clever method of quantifying this
spontaneous activity in bluegill, which could presumably be used for other species, as
well. A 10-gal aquarium is divided into two equal compartments by a transparent partition
containing four holes 4.5 cm in diameter. A group of fingerlings is introduced into one
side and the number found on the opposite side is counted at time intervals. The data are
then treated mathematically as an opposed first-order chemical reaction. With this simple
system they were able to show a dose-dependent increase in spontaneous locomotor
activity for fish exposed to zinc, cadmium, and chromium (Table 1). These changes
reached a plateau after 3 days’ exposure and remained at that level for the remainder of
the 2-week studies.
The extent of the increase in locomotor activity correlates well with the relative
toxicities of the metals tested. For example, cadmium caused greater changes than the
other two and it has the lowest LC50. (The decline in locomotor activity in 0.5 ppm
cadmium is an artifact due to the fact that considerable mortality was occurring and some
of the fish were dying and therefore not moving much.) Because locomotor activity is
very much a “driver” of the rate of metabolism, these observations suggest that these
metals cause an elevation in routine metabolism, at least in part through this mechanism.
In later studies, Ellgaard and colleagues have shown that crude oil (1979) and assorted
acids (1984) cause reductions in spontaneous activity of bluegill. Thus, their method
holds promise for quantifying both increases and decreases in activity.
The effects of cadmium on metabolic rate has recently been used to model the effects
of this metal on growth in bluegill. Sandheinrich and Atchison (1993) successfully

Table 1 Spontaneous Activity of Bluegill

Exposed to a Metal in the Water for 3 Days
Metal added Concentration % of controls
None — 100
Cadmium 0.1 mg/L 148
0.25 778
0.5 60
Chromium 0.05 118
2.4 364
24.0 650
Zinc 0.1 130
5.0 380

N o te : See text for description of method for quantifying

activity.

Data from Ellgaard, E. G., Tusa, J. and Malizia, A., J. F ish

1, 19, 1978.
B io l,
182

predicted 28-day growth rates from intitial measurements of oxygen consumption. It is

to be hoped that more of this approach will be used in the future.
The effect of lead may depend on whether the fish is freshwater or marine. Ellgaard
and Rudner (1982) observed no effect of lead on spontaneous activity of bluegill, even
when tested at lethal concentrations. Somero et al. (1977), however, exposed Gillichthys
mirabilis (a marine goby) to lead in the water for up to 87 days and found a greatly
elevated oxygen consumption throughout this period. There was no gill damage and there
was no effect of the lead on in vitro respiration of gill tissue even though the gills
accumulated high levels of it. They did note (subjectively) a greatly elevated amount of
physical activity in the tanks along with the heightened rate of oxygen consumption; so
the effect of lead could have been either neurogenic or irritating. Somero et al. (1977)
concluded it was the former.
In summary, metals generally cause changes in metabolic rate but the direction of the
effect varies with the metal — some stimulate, others inhibit. These changes in routine
metabolic rate are generally mediated through effects on motor activity of the fish. Metals
can also alter the enzyme systems within cells of liver, and presumably other tissues (see
Chapter 9), but what effect this has on whole-animal metabolism cannot be predicted
from those findings as changes in the metabolic rate of some tissue such as liver may be
masked by changes in motor activity brought about by the metal acting via the nervous
system, either by inhibiting or stimulating it, or by irritating sensitive epithelial tissues.

V. GILL TISSUE METABOLISM: EFFECTS OF METALS AND

POSSIBLE RELATION OF GILL METABOLISM TO WHOLE-BODY
METABOLIC RATE
The rate of oxygen consumption by gill tissue has been of interest in the study of pollution
by metals because this tissue is relatively easy to remove and work with from a variety
of animals, both finfish and shellfish, and also because gill tissue is one of the first
damaged by metal exposure (see Chapter 3). It has also been thought that such a
physiological measure might be useful in determining the extent of damage from metal
exposure to a sample of organisms drawn from a population in the field suspected of
suffering from some effluent. In other words, a biomarker, although all this work was
done before the use of the term biomarker came into vogue. The idea may still have merit,
however, few measurements of this type have been made in recent years.
It should be first pointed out that measurements of the respiration rate of gill tissue
from fish exposed in the laboratory to very high doses means little as the respiration
obviously will decrease as the cells are rapidly killed within a few hours by the metal.
Also, changes in gill respiration should not be interpreted as representing similar alter­
ations in whole-body respiration rate. The two measurements may or may not be related.
Exposure of cunner (Tautogolabrus adspersus) and winter flounder {Pseudopleuronectes
americanus) to mercury (5-10 ppb) for 30-60 days caused elevated oxygen consumption
of gill tissue. This is in contrast to what was found with striped bass {Morone saxatilis)
by the same authors, in which the gill tissue respiration was depressed by a comparable
exposure to mercury (Calabrese et al., 1977). They further found that the bass is actually
more tolerant of metal exposure than the two other species, so the gill respiration findings
are not indicative of overall metal tolerance by the species.
In apparent contrast to mercury, chronic exposure to cadmium resulted in a 30%
depression of gill tissue respiration in cunner, but it is noteworthy that the amount of
depression was approximately the same at all concentrations tested (3-48 ppm), so the
effect was not concentration dependent (Thurberg and Dawson, 1974). The respiration of
gill tissue from striped bass and flounder was also slowed by cadmium exposure (Calabrese
et al., 1977).
 183

Tort et al. (1984) exposed dogfish sharks (Scliorhinus canicula) to 50 ppm cadmium
for 6 days. The 24-h LC50 for this species is 200 ppm so this concentration is sublethal.
They observed a severe depression in in vitro gill tissue respiration at 2 days’ exposure,
but by 6 days of continued exposure, the respiration had returned almost to the control
level. There was a marked increase in lactate at 2 days and drop in ATP which suggests
a reduction in oxygen availability to the gill tissue, probably due to histological changes
(see Chapter 3). These variables also returned to control levels by day six of exposure.
Thus, the dogfish exhibited a rather good ability to adapt to the presence of cadmium in
the water.
These findings might be taken to indicate that cadmium is a general respiratory poison
for gill tissue, but such a generalization would have to be limited to teleost fish. For
example, exposure of oysters caused an increase in oxygen consumption of the gill tissue.
Moreover, the increase tends to be proportional to the amount of cadmium accumulated
in the tissue (Engle and Fowler, 1979). Copper causes a somewhat similar result in the
oyster. Engle and Fowler attribute the increased rate of respiration to an increase in
permeability of cellular and/or mitochondrial membranes. Such a hypothesis was sup­
ported by their electron microscopic observations of the tissues which showed damage to
these membranes. Zaba et al. (1978) found that copper causes an increase in permeability
of fish liver mitochondria to potassium and an increase in respiration at low doses, but
an inhibition at high doses.
Studies by Tort et al. (1982) on dogfish exposed to zinc for 4-21 days showed
inhibition of gill tissue respiration. The concentrations required to cause inhibition (10
ppm minimum) seem high so the relevance for “real world” situations is questionable.
However, since these fish were able to tolerate such seemingly high doses for at least 21
days, perhaps acute gill damage from zinc does not occur in elasmobranchs as it does in
teleosts (see Chapter 3).
In bluegill exposed to copper for up to a month, oxygen consumption by isolated gill
tissue was unaffected except in one case. It was stimulated by the copper exposure in fish
that had also experienced a rise in temperature from 20-30°C on the third day of exposure
and held there for 9 days while still receiving copper (Felts and Heath, 1984). The
physiological significance of this is that whole-animal oxygen consumption was also
elevated above that of the non-exposed fish (see Figure 3), thus copper caused the process
of temperature acclimation to be delayed in both whole fish and gill tissues.
The changes observed in gill respiration that have been reviewed here are relevant to
research on the percent of the whole-body oxygen requirement consumed by gill tissue.
Two studies suggest it is very high. Daxboeck et al. (1982) measured the difference
between the amount of oxygen removed from the water and the amount actually appear­
ing in the blood. This was done using spontaneously ventilating, artificially perfused
preparations of trout and the results were used to estimate the in vivo consumption of
oxygen by gill tissue. They concluded that a median of 21% of total whole-body oxygen
uptake is utilized by this tissue alone. That seems a remarkably large figure when it is
realized that gill tissue represents only 3.9% of the total body weight. Johansen and
Pettersson (1981) using perfused head and isolated gill arch preparations, estimated that
in cod the contribution of gill to whole-body consumption of oxygen is 6.6%, which is
considerably less than that found by Daxboeck et al. (1982) but is still large compared to
the percent of body mass.
It may be relevant to note that the Daxboeck et al. (1982) paper is not cited in more
recent work (Perry and Walsh, 1989) and reviews on gill metabolism (Mommesen, 1984).
Perhaps it is now felt the values are unrealistically high.
Measuring the rate of respiration in isolated tissues requires their removal from the
fish, mincing or slicing, and then the measurements of oxygen utilization must be made
quickly in a microrespirometer of some sort (LeFavre et al., 1970; Oikawa and Itazawa,
184

Table 2 Oxygen Consumption of Gill Tissue Measured In V itro or In V iv o

Species Temp. Gill Whole-animal Ref.

In Vitro
Bluegill 20 21.56 2.66 Felts and Heath (1984)
Bluegill 20 31.25 11.87 O’Hara (1971)
Pumpkinseed 20 15.02 2.68 Roberts (1967)
Rainbow trout 16 21.25 6.88 Evans et al. (1962)
Carp 20 8.04 Itazawa and Oikawa (1983)
Gunner 20 6.70 Thurberg and Dawson(1974)
Flounder 20 5.35 Calabrese et al. (1975)
Tilapia 25 4.82 Perry and Walsh (1989)
Toadfish 25 13.82 Perry and Walsh (1989)

In Vivo
Rainbow trout 7 11.16 Daxboeck et al. (1982)
Cod 15 4.24 Johansen and Pettersson (1981)

N o te : All values are expressed as iimol/g wet wt/h. Comparisons with whole-body oxygen
consumption are included where these were done by the same author(s).

1983). Unavoidably, the procedures cause breakage of many cells so the question is
frequently raised as to how realistic are the rates obtained in vitro. Table 2 compares in
vitro and in vivo data for freshwater and marine species.
Several points are presented by this table. Both the in vitro and in vivo values for the
freshwater fish are generally higher than those from marine fish, which is somewhat
surprising given the much greater ionic flux in the marine forms (Chapter 7). At first
glance the in vivo values for gill respiration seem low compared to those in vitro.
However, the former were measured at lower temperatures. If a Q10 of 2 is assumed, the
two rates are remarkably similar. Finally, in all cases, the metabolic rate per gram of tissue
weight of gill tissue was several-fold greater than that of the whole animal.
In a more recent study. Perry and Walsh (1989) showed by cellular isolation experi­
ments that the chloride cells have a considerably higher rate of energy metabolism than
the other cells of gill tissue. They further noted that the number of chloride cells in the
gills is greater in a marine-adapted species, which does not fit the data shown in Table 2
where the marine species have the slower rate of respiration. Still, everybody seems to
be in agreement that gill tissue has a very high energy requirement.
It is tempting to speculate that changes in whole-body metabolism of a resting fish are
due mostly to changes in the metabolism of tissues other than muscle. Even though
muscle makes up some 50-60% of the body mass, it appears to contribute relatively little
to the total metabolism except when the fish is swimming. Thus changes seen in fish
exposed to pollutants may reflect predominantly effects on non-muscular tissues when
measurements are made on resting fish (i.e., standard metabolic rate), but when the fish
is forced to swim or when spontaneous activity is altered by the chemical, differences in
metabolism between controls and exposed specimens probably reflect effects on muscle
metabolism or on the process of muscular coordination and the central nervous system.
This latter point is especially noteworthy in fish exposed to pesticides, which will now
be discussed.

VI. EFFECTS OF PESTICIDES ON WHOLE-BODY AND

INDIVIDUAL TISSUE RESPIRATION
In probably the first study of this sort on fish exposed to an insecticide, Ferguson et
al. (1966) observed that when mosquitofish (Gambusia ajfinis) were exposed to
 185

endrin (a chlorinated hydrocarbon) at a concentration of 20 ppb, there was no effect on

metabolism for a period of days. Then suddenly hyperactivity and spasms would occur
which caused the oxygen consumption to rise dramatically just before death. Similar
results have been obtained on white suckers exposed to the organochlorine insecticide,
methoxychlor (Waiwood and Johansen, 1974). The response appeared to be an all-or-
nothing phenomenon in that fish which survived the exposure exhibited no change in
oxygen consumption, while in those that did not survive, a big increase in metabolism
took place shortly before death.
Bansal et al. (1979) found that chlordane also stimulates oxygen consumption but in
their fish it was only in a transient way. When they exposed carp to this insecticide at
concentrations ranging from the LC50 to 1/12 the LC50, they found that at concentrations
below the LC50, there was an initial phase, which they called the sensitization phase,
lasting 6 h irregardless of the concentration. Then there followed a period of hyperactivity
and elevated oxygen consumption (two- to fourfold increase). By the end of 24 h of
continued exposure, the level of activity and oxygen consumption of the fish had returned
to normal, as if the receptors that were stimulating the activity had adapted to the
insecticide. (Those fish exposed to the LC50 showed a steady decline in metabolic rate
until death at 10 h of exposure.)
Using a system to quantify spontaneous swimming activity (described earlier in this
chapter) Ellgaard et al. (1977) found a concentration-dependent increase in activity of
bluegill exposed to sublethal concentrations of DDT. The maximum hyperactivty was
generally achieved in about 8 days and continued for at least 16 days of continuous
exposure. It is important to further note that the effects of DDT on activity persisted for
at least 2 weeks in fish transferred to non-contaminated water.
In general then, it appears that organochlorine insecticides cause hyperactivity in fish.
The character of the response depends on the species of fish. Hyperactivity and spasms
are probably, at least in part, due to increases in the sensitivity of peripheral receptors to
stimuli that would not normally be especially excitatory (Bahr and Ball, 1971). Of course
there are probably also direct effects of the pesticide on the central nervous system.
The herbicide triclopyr seems to act on fish in a manner similar to organochlorine
insecticides. Johansen and Green (1990) quantified both muscular activity and oxygen
consumption in coho salmon exposed for 96 h to trichlopyr. At a concentration <0.10
mg/L it caused hypersensitivity to external stimuli (such as lights being turned on) and
elevated activity and oxygen consumption. At somewhat higher concentrations the fish
became lethargic and oxygen consumption declined.
Organophosphate insecticides appear to inhibit metabolic rate both in whole-body
measurements and in tissues removed from exposed fish, although a transient stimulation
in oxygen consumption may occur (Bansal et al., 1979; Rao et al., 1985).
Rath and Misra (1979) found a concentration-dependent inhibition that varied with the
size of the fish. The amount of respiratory inhibition was size dependent in that small ones
showed a greater inhibition after 15 days of exposure, but, they recovered more rapidly
when returned to uncontaminated water. These observations would seem to suggest a
faster uptake and rate of detoxification of the insecticide in smaller fish which fits with
their overall faster rate of metabolism.
In a follow-up study. Rath and Misra (1980) measured in vitro respiration in brain, gill,
and muscle tissues from fish exposed to diclorovros, as was done in their whole-animal
investigation. Again there was an inverse relationship between body size and metabolic
rate, and the percent inhibition was greater in tissues from smaller fish. Periods of
exposure were for up to 28 days and, throughout, gill tissue exhibited the greatest percent
of inhibition which might relate to its proximity to impact from the dissolved pesticide.
Methyl parathion (an organophosphate insecticide) also produces a marked decrease
in metabolic rate of both whole-animal and specific tissue respiration (Murty et al., 1984;
186

Figure 5 Energy cost of stress for rainbow trout due to stress of permethrin detoxification and
tissue repair in relation to exposure time in 5 and 10% of the 96-h LC50. In calculating the energy
cost, 3.25 cal/mg of oxygen consumed was used. (From Kunaraguru, A. K. and Beamish, W.,
Comp. Biochem. Physiol., 75A, 247, 1983. With permission.)

Rao et al., 1985; Bala and Mohideen, 1992). The pyrethroid insecticide fenvalerate also
produced a severe decrease in tissue and whole-animal respiration in 48-h exposures
(Reddy and Philip, 1992). One possible problem with the latter two studies cited is that
the investigators apparently measured control respiratory rate only before the pesticide
exposures were commenced. With continued confinement in a respirometer, a fish may
show a decline in respiration due to acclimation to the chamber and to starvation (Fry,
1971), thus a decrease could be normal rather than due to pesticide exposure.
Pyrethroid insecticides appear to stimulate respiration. In an excellent study on rain­
bow trout by Kumaraguru and Beamish (1983), permethrin caused the standard metabolic
rate to rise over 7 days of exposure, but then under further exposure it gradually declined
toward the level of the controls at a rate depending on concentration (Figure 5). Standard
metabolism was measured in this study with swimming tunnels which permitted measure­
ments of oxygen consumption at several rates of swimming. Then, by extrapolation to
zero activity, the calculated standard rate is obtained (they refer to it as “basal”). Thus,
the stimulation in metabolic activity caused by the pyrethroid was due at least in part to
factors other than increased spontaneous activity of the fish (which was not measured).
Kumaraguru and Beamish (1983) attribute the increased metabolic cost to detoxication
and tissue repair. Figure 5 illustrates how they used the data obtained to obtain an actual
energy cost of this increased maintenance which could be incorporated into an energy
budget. The figure also illustrates the phenomenon of physiological acclimation to the
pesticide as the curves return toward zero with time.
The implications of an elevated maintenance cost from pesticide presence in the water
was also shown in work on carbaryl, which acts similarly to organophosphorus insecti­
cides. Arunachalam et al. (1980) found that over a 27-day exposure to carbaryl, there was
a decreased growth rate and conversion efficiency. Conversion efficiency refers to the
percentage of food converted into growth so if this goes down, it implies either a
reduction in assimilation or a greater cost of maintenance. Because assimilation was
 187

unchanged, the maintenance cost must have been higher even though respiration rate was
not measured in this study.
Rotenone is another widely used insecticide. It is also used as a fish poison. Its
mechanism of action is to block the flow of electrons from NAD to the cytochrome
system and thus causes an inhibition of cellular respiration (Skadsen et al., 1980).
Rainbow trout were forced to swim in a water tunnel at various speeds while being
exposed to rotenone for only a few hours. Exposure to a concentration equivalent to the
96-h LC50 caused no effect on the standard metabolic rate (i.e., zero relative perfor­
mance). It did, however, reduce the maximum metabolic rate so at that concentration it
was acting as a limiting factor (Fry, 1971). At one half of the LC50, the standard
metabolic rate was elevated (a masking response) but the maximum rate was not signifi­
cantly reduced. Then at a still lower concentration of rotenone, an even further elevation
in the standard rate of metabolism was found. The authors do not attempt to explain the
basis of this inverse concentration response. Evidently, the stimulatory effect is only
manifested at very low concentrations of rotenone. Perhaps, higher concentrations impair
gas exchange at the gill surface to such an extent that even though the energy demand is
up, the consumption of oxygen becomes inhibited. It would be interesting to see what
chronic exposures to these low concentrations would do to the swimming capacity and
resting rates of metabolism.
It has been repeatedly shown herein how difficult it is to determine the relative
importance of spontaneous activity and intrinsic cellular respiration in explaining in­
creases or decreases in whole-body metabolism during exposure to some chemical. Thus,
the study by Peer et al. (1983) is of interest because they measured oxygen consumption
and spontaneous activity simultaneously in mullet (Rhinomugil corsula) exposed to
pentachlorophenol (PCP). By extrapolating to zero activity one can get a good estimate
of the standard rate of metabolism. They observed that PCP is acting as a masking factor
by increasing the maintenance cost. Because the slopes of activity vs. metabolism were
not significantly different, the effect of the chemical is on cellular metabolism instead of
on spontaneous activity.
Pentachlorophenol is classed as a metabolic stimulant which acts by uncoupling
oxidative phosphorylation by the electron transport system in the mitochondria (Webb
and Brett, 1973). Thus, oxygen is consumed but without the concomitant formation of
ATP. Webb and Brett (1973) observed that growth of sockeye salmon was depressed in
a concentration-dependent fashion by PCP. All fish received the same ration and assimi­
lation was unaffected by the PCP. Therefore, the depressed growth is caused by the
maintenance costs competing for the available energy.
The relationship between energy taken in, growth, and maintenance costs was empha­
sized in the equation and diagram at the beginning of this chapter. Using these concepts,
one can prepare an energy budget for fish exposed to some pollutant. This was done for
the cichlid, Cichlasoma bimaculatum, exposed to 0.2 ppm pentachlorophenate (Krueger
et al., 1968). The heat of combustion of a sample of fish was determined on day one and
again on the tenth day of exposure. In addition, the heat of combustion of the food eaten
was obtained. Basal metabolic rate, nitrogen loss, etc., were obtained from other studies
in that lab. It appears that PCP stimulated food intake but depressed growth in the cichlids.
Webb and Brett (1973) found no effect of PCP on the appetite of salmon.
The cichlids exposed to PCP clearly had a higher “cost of living”, or to quote the
authors: “The pentachlorophenol did not destroy or damage the growth machinery; it
merely made it more costly to operate.” (Kruger et al., 1968, p. 124). When body
composition was determined, it was found that the exposed fish stored less fat than
controls. Unfortunately, the calories attributed to carbohydrate and protein were com­
bined in their report, so it is not possible to determine changes in these individual stores
188

of energy. Because fish exposed to PCP tend to mobilize liver glycogen into blood sugar
(Thomas et al., 1981; Hanke et al., 1983), there were probably some large decreases in
carbohydrate relative to protein.
Samis et al. (1993) report that bluegill exposed to PCP at a concentration of 173 pg/L
for 22 days exhibited a depression in both growth and feeding during the final 10 days
of exposure. However, when the PCP exposure concentration was at 48 pg/L, growth
declined but food consumption was unaffected, thus appetite can be affected by PCP but
the concentration must be fairly high.
The effluent from kraft pulpmills contains several potentially harmful substances
including some chlorinated organics that resemble PCP in molecular structure (Davis,
1976). Exposure of salmon to a concentration of the effluent equivalent to one third of
the 96 h LC50 causes an elevated oxygen consumption even though the arterial oxygen
content is reduced (Davis, 1973). Thus, the increased energy demand is, at least in part,
due to hyperventilation brought on by the internal hypoxia.
Based on an increased utilization of energy for maintenance, one would predict an
inhibition of growth rate in fish receiving pulpmill effluent. Surprisingly, this does not
necessarily occur. Indeed, several workers have noted stimulation of growth, providing
the fish are fed an excess ration (see McLeay and Brown, 1979 for review). So the
exposed fish apparently ate more food, if it was available, and this more than compen­
sated for the increased energy demand.
Cyanide is considered a metabolic depressant as it binds in place of oxygen to the
heme of cytochrome oxidase, the terminal enzyme of the electron transport system in
cells. Because cyanide blocks the utilization of oxygen by the cells, oxygen consumption
rate must decrease. This seems to have not been systematically investigated in fish,
although a study by Dixon and Leduc (1981) on rainbow trout reveals an interesting
phenomenon. Trout were exposed to two different concentrations of cyanide for 18 days
after which time they were placed in respirometers with no cyanide present. The oxygen
consumption was then determined over a period of 144 h. The cyanide-exposed fish
initially respired at a lower rate than the controls and this was dose dependent, so the
cyanide was acting as a depressant. Then, those that had previously been exposed to
cyanide exhibited a greatly elevated rate which reached a peak at 72-96 h. Respiration
then returned to a lower rate which was still above the controls. So, several days after the
initial exposure, cyanide acted as a metabolic stimulant even though it was no longer
present, and this was prolonged for at least a week.
An elevated rate of oxygen consumption after some sort of stress might be due to
oxygen debt. However, the oxygen debt acquired after exhaustive exercise in trout is
“paid back” in about 6 h (Scarbabello et al., 1991) so that cannot be the explanation for
the prolonged high rate of respiration. Leduc (1984) notes that mammals chronically
exposed to cyanide develop goiter due to failure of the thyroid to take up iodine. This
causes an elevated amount of thyroid-stimulating hormone from the anterior pituitary. He
further speculates that if the same thing occurs in fish, there would probably be an excess
amount of thyroxine produced from the enlarged thyroid gland after the cyanide was no
longer present. This might stimulate oxygen consumption and growth rate, both of which
take place following exposure to low levels of cyanide.
The mudskipper (Boleophthalmus boddaerti) is a goby that inhabits burrows on
mudflats that are frequently anoxic. Because it has high resistance to lack of oxygen, it
was thought that it might also show good tolerance of cyanide. Chew and Ip (1992) found
that indeed it is quite tolerant of cyanide but not because of its high anerobic capacity, nor
because it slows metabolism down. When exposed to cyanide at concentrations that
would normally kill other fish, this species shows no slowing of oxygen consumption
even though cytochrome oxidase is inhibited by 50%. Evidently, it has a considerable
 189

excess of this enzyme present, although there is no obvious reason why such an adaptation
evolved.

VII. EFFECTS OF PETROLEUM HYDROCARBONS ON

ENERGY METABOLISM
Petroleum hydrocarbons may stimulate or inhibit oxygen uptake of juvenile or adult fish
depending on the exposure concentrations. At concentrations approaching those that are
lethal, the effect is apparently to inhibit oxygen uptake due to damage to the gill
epithelium (Hawkes, 1977; Prasad, 1987). However, there may be an initial stimulatory
effect for a few hours due to the increased breathing of the fish which has a considerable
energy demand from the respiratory muscles (Thomas and Rice, 1979), but this is
followed by a rapidly falling oxygen consumption.
Most studies on the effects of oil on fish have been done on embryos, larvae, or juveniles.
At these stages, petroleum at chronic levels of exposure probably increases oxygen con­
sumption, however, the evidence is mostly indirect. Vignier et al. (1992) exposed Atlantic
salmon parr for up to 40 days. They observed a reduction in growth rate of oiled fish, even
in those that were feeding normally. Higher levels of oil can inhibit feeding rate and thus
growth rate (Fletcher et al., 1981; Kiceniuk and Khan, 1987). The results of Vignier et al.
suggest that there is a reduction in food conversion efficiency which may be due to
increased metabolic demand. It could also be due to direct effects of oil on the process of
food absorption by the intestine, but no evidence is available on this.
Hose and Puffer (1984) observed an increased oxygen consumption by grunion
embryos exposed to a very low concentration of benzo[a]pyrene. However, at higher
concentrations, there was an inhibition of oxygen consumption. This can be contrasted
with Ostrander et al. (1989) who were unable to detect any metabolic changes in coho
salmon eggs or larvae exposed to benzo[a]pyrene. They did, however, note serious effects
on behavior of the larvae as they attempted to emerge from the gravel after hatching so
the petroleum hydrocarbon was not without effect.
Cod eggs exposed to North sea crude oil exhibited no changes in oxygen consumption,
but the activity of the larvae was suppressed (Serigstad and Adoff, 1985). This is in
contrast to the findings of Eldridge et al. (1977) in which Pacific herring eggs were more
sensitive to benzene than were the larvae, although their data on the egg oxygen consump­
tion is difficult to interpret. The higher dose caused increased respiration while the lower
dose caused decreased respiration compared to controls. Clearly, the information cur­
rently available does not permit generalizations regarding the effect of petroleum on
energetics. Indeed, there have even been examples of where it seemed to stimulate growth
(Eldridge et al., 1977; Laughlin et al., 1981), possibly due to a stimulation of feeding.

VIII. METHODS APPLICABLE TO MEASUREMENT OF ENERGY

EXPENDITURE IN THE FIELD
As the various studies of the effects of pollutants on oxygen consumption show, changes
in bodily activity are frequently the reason for at least part of the changes in metabolic
demand. In all these studies, however, there is the problem that the fish are confined in
a chamber where “normal” movement is greatly restricted. A fish living in the wild will
expend energy for feeding, escaping predation, and exploratory behavior. None of this is
recorded in the present methods of measuring oxygen consumption, and yet if one is to
prepare a complete energy budget, such data are required. Two techniques that have been
used successfully with terrestrial animals to obtain measurements of energy expenditure
in the wild are the telemetry of heart rate and the double-labeled water method.
190

Because blood flow is proportional to oxygen consumption, it follows that as the latter
changes, so will the cardiac output. With the aid of biotelemetry, the heart rate can be
recorded in a free-ranging animal on a continuous basis. This method has been used with
considerable success in both birds and mammals (Gessamen, 1980). In fish, however, the
relationship between heart rate and cardiac output is not always consistent because large
changes in stroke volume may occur with little frequency change. With considerable
effort, Priede and Tytler (1977) were able to calibrate individual fish and obtain some
estimates of metabolism in free-ranging trout and cod by telemetry of the heart rate. More
recently, Sureau and Lagardere (1991) were able to relate distance moved (and presum­
ably energy expended) with telemetered heart rate in soles {Solea solea), but the tech­
nique did not work with sea bass (Dicentrarchus).
Rogers and Weatherly (1983) were able to detect the electromyogram from the
opercular muscles and transmit this to a recorder. The voltages produced by the action
potentials of the muscles were integrated and compared with the oxygen consumption of
fish either swimming spontaneously in a chamber, or swimming at fixed speeds in a water
tunnel. They obtained a good correlation between the average integrated voltage and
oxygen consumption, although the curves describing these values were different if the
fish was swimming spontaneously as opposed to being forced to swim in the tunnel. This
was explained as being at least in part due to the use of different muscles for the two
different types of swimming. In any case, this method offers a range of possibilities for
continuous recording of expenditure of energy by fish in ponds, rivers, lakes, estuaries,
and in laboratory settings as well. Kasello et al. (1992) have further refined this technique
for measuring activity in free-ranging fish for months at a time.
The double-labeled water method is an ingenious way to measure energy expenditure
over a period of time in free-ranging terrestrial animals. However, it depends upon several
critical assumptions, and one of these precludes its use with aquatic animals. Namely,
there is the requirement that little or no unlabeled water enter the body by way of the
respiratory or skin surfaces (Nagy, 1980). With fish, there is a considerable flux of water
across these surfaces so this method is not applicable to them.
The oxygen consumption rate of an animal is in reality a measure of the end result of
many enzyme reactions in the cells. Biochemical research has revealed that certain
enzymes are rate limiting in that they catalyze non-equilibrium reactions in certain
metabolic pathways and their substrate is at concentrations approaching or exceeding
saturation. In essence then, these key reactions set the pace for the other reactions in the
metabolic chain (Hochachka and Somero, 1984; Newsholme and Crabtree, 1986). The
activity of these enzymes can be assayed using fairly standard procedures. Selection of
appropriate enzymes is based on two criteria: (1) enzyme has been shown to reflect
whole-animal oxygen consumption rate or growth and (2) is stable when stored frozen for
days or weeks. Enzymes that meet these criteria for fish include cytochrome oxidase and
citrate synthase.
The relationship between oxygen consumption or growth and enzyme activity has
been investigated to only a limited extent, but in all cases a good correlation exists
(Goolish and Adelman, 1987; Kaupp and Somero, 1988; Koch et al., 1992). Others
(Simon and Robin, 1971; Smith and Chong, 1982) have found the rate of enzyme activity
changes in response to body size and/or starvation and season in exactly the same manner
as oxygen consumption rate, although they did not measure the latter. In some of these
studies, skeletal muscle was the test tissue; in others, it was liver or heart muscle. The
various tissues gave similar trends. A possible weakness in the potential use of enzyme
activities for assessing pollution effects is that they may not respond to increased
swimming activity (Johnston and Moon, 1980; Goolish and Adelman, 1987). Further­
more, cytochrome oxidase in some species may be present in considerable excess, so it
would not be a rate-limiting step (Chew and Ip, 1992).
 191

Cytochrome oxidase has the largest database, but Somero (personal communication)
reports that in marine organisms it is sometimes rather unstable when frozen compared
to citrate synthase. Goolish and Adelman (1987), however, found good stability for this
enzyme in frozen largemouth bass (a freshwater species).
Some environmental conditions including the presence of pollutants may stimulate
increased amounts of anaerobic metabolism. A good enzyme for assessing changes in this
type of metabolism is lactate dehydrogenase (Kaupp and Somero, 1988).
Assessing metabolic activity in free-ranging fish by biotelemetry of muscle electrical
activity or by the measuring of tissue enzyme activities involves two different time frames.
In the first method, the dynamics are important whereas the latter method will presumably
give a sort of average of recent metabolic activity. Clearly, they are not mutually exclusive
procedures, but instead they may offer opportunities to pursue different questions in the
overall effort to understand how pollutants affect the physiological energetics of fish.

IX. EFFECTS OF POLLUTANTS ON LARVAL AND

JUVENILE GROWTH
Growth is essentially the laying down of new flesh through the process of protein
synthesis, but this is the endproduct where there are several physiological processes that
precede and parallel it. These include feeding behavior, availability of food, type of food
eaten, digestion, absorption, assimilation, excretion, energy expenditure for maintenance
and movement, and changes in certain hormonal levels. All of these physiological
phenomena are directly or indirectly affected by pollutants, some of which have been
discussed at some length in this chapter. Thus, any change in growth produced by some
exogenous chemical will usually have multiple causes. Therefore, growth is a sort of
integrator of a variety of physiological and environmental factors (Weatherly, 1990).
Some stages of the life cycle are often more sensitive to chemical insult than is growth
and thus lend themselves better to determinations of maximum acceptable toxicant
concentrations (MATC) based on early life-stage studies (McKim, 1977; Woltering,
1984). However, growth is obviously a critical factor governing recruitment into the
population and therefore deserves considerable attention.
It will come as no surprise for many people to learn that sublethal levels of a very wide
variety of substances have been found to slow the growth of larvae or juveniles (reviewed
in Woltering, 1984). In most cases, the mechanisms involved have not been investigated.
In his comprehensive review of the influence of environmental factors (excluding pollu­
tion) on growth of fish, Brett (1978) emphasizes the interdependence of energy and
growth. Metabolic expenditure by fish for maintenance and muscular contraction “steals”
energy that could, at least in theory, be used for making more fish. This relationship
between energy intake, energy expenditure, and growth is diagramed in Figure 1. In a
sense, growth gets the energy that is left over after all the other needs are met. A pollutant
may cause an increase in maintenance cost which then requires a greater food intake if
the energy balance is to be maintained. This is nicely illustrated in a study wherein perch
were exposed to copper at five concentrations (1-89 |ig/L) for 30 days (Collvin, 1985).
At concentrations above 22 |Lig/L there was a slowing of growth although no effect on
food consumption. Instead, the slowing in growth was attributed to an increased meta­
bolic maintenance cost due to detoxication. It might be recalled that Waiwood and
Beamish (1978) have shown increased standard metabolic rate in rainbow trout exposed
to copper at a concentration of 40 pg/L for 5 days. Prolonged exposures (40 days) of
salmon parr to crude oil have been shown to depress food conversion and therefore
growth, again by apparently increasing the metabolic cost of maintenance (Vignier et al.,
1992). This has also been observed in fish exposed to cadmium and 2,4,dichlorophenol,
two very different types of water pollutants (Borgmann and Ralph, 1986).
192

Feeding causes a surge in oxygen consumption (as mentioned earlier in this chapter).
Feeding also causes a concomitant rise in protein synthesis but the time course of the
change in protein turnover varies with different tissues (Houlihan, 1991). The reason for
the stimulation in protein synthesis with feeding is unclear as it increases in liver before
the blood levels of absorbed amino acids rise following assimilation of protein, so amino
acids must not be the stimulus.
In a variety of animals it appears that the primary stimulus for muscle growth is dietary
protein rather than energy (Millward, 1989). Of course, fish utilize protein for energy
more than many other types of animals so the stimulus may be difficult to differentiate
in them.
If food intake or assimilation is reduced then there will be less energy and protein
available for growth so the rate will decline. For example, Farmanfarmian and Socci
(1984) reported that the absorption of leucine (an essential amino acid) by the intestine
was inhibited 20-80% by mercury concentrations that might be expected in the gut of
Fundulus inhabiting waters contaminated with mercury. Methylmercury was somewhat
less potent in this respect.
It is also common to observe that fish exposed to sublethal concentrations of some
chemical may exhibit a reduced appetite for reasons that are unknown. Wilson et al.
(1994) exposed rainbow trout to aluminum in acid water at a concentration of 162 |Lig/L.
Even though the fish were fed to satiation, for the first 10 days of exposure they decreased
their feeding rate which caused reduced growth that was only partially recovered during
the subsequent 40-day exposures. The authors noted that the trout in the tanks contami­
nated with aluminum displayed reduced spontaneous activity which would lower the
routine metabolic rate. This actually caused a rise in gross food conversion efficiency
(38% compared with 31% for controls), but the combination of a higher standard
metabolic rate and depressed appetite caused slowed growth. Acid water (pH 5.2) without
added dissolved aluminum caused no effect on growth.
Growth hormone from the pituitary enhances appetite and also improves gross conver­
sion efficiency [(Growth/Ration) x 100]. Donaldson et al. (1979) discuss some possible
mechanisms by which it might do this. These include: (1) stimulation of fat mobilization
and oxidation which would free amino acids for synthesis rather than utilization for
energy; (2) increased protein synthesis; and (3) stimulation of insulin synthesis and
release. Recent studies reviewed by Houlihan (1991) confirm increased protein synthesis
occurring from growth hormone stimulation in fish.
In mammals (and possibly also fish) muscle growth is accomplished with nuclear
replication which is considered to be under control of growth hormone. Cytoplasmic
growth is under control of insulin, among other things. This latter hormone is influenced
considerably by pollutant exposure, but not always in the same direction, as is discussed
in the section in this chapter on carbohydrate changes. For example, cadmium causes
damage to the insulin-secreting cells in the pancreas so the level of this hormone may fall,
whereas many other pollutants stimulate insulin production indirectly by bringing about
a hyperglycemia due to rises in “stress hormones”. The high blood glucose then stimu­
lates the beta cells to secrete more insulin. While this might be interpreted as stimulating
growth, one of the primary stress hormones, cortisol, suppresses protein synthesis and
stimulates protein breakdown (Van der Boon et al., 1991).
In general a physiological response to a stressor is amplified when that stressor is
applied suddenly, as opposed to slowly. This was exemplified in a growth study by Seim
et al. (1984). They measured growth and survival in steelhead trout exposed to several
copper concentrations during development from 11-85 days post-fertilization. The unique
part of the study was the comparison of intermittent with continuous exposure. The
intermittent exposures were for 4.5 h daily and the concentration in the water followed
a sort of square wave (concentration/time). The area under the concentration curve was
 193

used to calculate the average concentration as if it were given continuously. Growth was
inhibited more with the intermittent exposures and they accumulated more copper in their
tissues. If this phenomenon holds for other toxicants, it could have a marked impact on
the estimates of MATC, for the natural environment, which we attempt to simulate in the
laboratory, is rarely constant.
There have been reports of growth being stimulated in fish by exposure to a toxic
pollutant. McLeay and Brown (1974) and Mason and Davis (1976) both reported elevated
growth of alevins and juvenile salmon (Oncorhynchus kisutch) when exposed to kraft
pulpmill effluent. It has also been seen more recently with European perch {Perea
fluviatilis; Sandstrom et al., 1988), although decreases in growth were seen in lake trout
(Salmo trutta) exposed to the same effluent (Oikari et al., 1988). In the last study cited,
there was the presence of a fair amount of chlorophenolic compounds which would have
caused an elevated maintenance metabolism.
A variety of hypotheses have been proposed to explain a stimulated growth rate. These
include hormone analogs in the wood extractives that simulated growth hormone, stimu­
lation of appetite, and inhibition of territorial behavior by the dark color. No direct
evidence is available regarding any of these. In some cases, it may be a form of hormesis.
The term “hormesis” refers to an overcompensation to some inhibitory challenge. It was
seen in crab zoeae which grew faster after being exposed for 5 days to petroleum hydrocar­
bons (Laughlin et al., 1981). Note that the exposure was for only 5 days, which simulates
an oil spill. Compensatory growth has also been seen in larval or juvenile fish subjected to
starvation or reduced ration and then given food ad lib (Weatherley and Gill, 1981;
Dabrowski et al., 1986; Miglavs and Jobling, 1989). In one study (Miglavs and Jobling,
1989), compensatory growth was associated with improved food conversion efficiency.
Growth was also stimulated in minnows (Phoxinus phoxinus) that were fed a diet
contaminated with PCBs (Bengtsson, 1979). Other work (Mayer et al., 1977) has shown
that these chemicals stimulate thyroid activity in fish which provides a possible mecha­
nism for the growth enhancement, although the influence of thyroid on growth in fish is
very dependent on day length and/or season (Donaldson et al., 1979).
Finally, growth stimulation has been observed in larval striped bass exposed to the
herbicide molinate (Heath et al., 1993). In this study, larvae were exposed to molinate for
4 days at a concentration of molinate less than one half the 96-h LC50. Then they were
reared for 10 days in non-contaminated water. The exposed fish exhibited both an
increased dry weight and RNA/DNA ratio.
Extensive studies utilizing isotopically labeled amino acids have begun to reveal the
complex interactions between growth, protein synthesis, and protein degradation (i.e.,
protein turnover). The concepts emerging have been extensively reviewed by Houlihan
(1991). Protein synthesis is obviously required for growth to occur, but degradation of
proteins is also constantly occurring. It is now known that protein synthesis rates and
degradation are intimately linked, but differ considerably in different tissues. For ex­
ample, they are exceptionally high in gill tissue, but efficiency of protein retention is low
there (around 4%) compared with white muscle where it is approximately 75%. In other
words, there is a lot of protein turnover in gill tissue, perhaps because so much of it is
epithelial tissue with a high degree of sloughing off.
There is a linear relationship between protein synthesis and growth rates as there is
between growth and degradation rates. However, as growth rate increases, the rate of
protein degradation does not increase as fast as synthesis (Houlihan, 1991). In other
words, the slopes are different. Thirty to forty percent of the protein synthesis in a fish
occurs in white skeletal muscle (Fauconneau, 1985), and this tissue is most responsive to
the nutritional state of the fish (Lied et al., 1985). Houlihan (1991) argues that the
fractional protein synthesis rate of white muscle can be used as a good indication of
general growth rate of the whole organism.
194

Most growth studies of fish require the maintenance of the animals for weeks or
months in the laboratory under well-defined conditions. Such long durations increase the
likelihood of mechanical failures (e.g., floods) or disease ruining an experiment. Bulow
(1970) was probably the first to report that the ratio RNA/DNA concentrations in the
tissues of fish correlated with feeding intensity and, more importantly from our standpoint
here, the recent growth rate of the animal. This ratio also changes with the season in wild
fish and decreases when the fish are subjected to the stress of low dissolved oxygen
(Bulow et al., 1981; Peterson and Peterson, 1992). Buckley (1984) further refined the
technique for the assessment of growth of larval fish in the sea.
Barron and Adelman (1984) measured these nucleic acids and protein content in larval
fathead minnows exposed for 96 h to hexavalent chromium, cyanide, /7-cresol, ethyl
acetate, or benzophenone. By using several doses and also measuring the growth for 28
days, they were able to relate the changes in biochemistry with the actual growth of the
fish. Figure 6 shows the sort of data obtained in this study. In contrast to Bulow’s work
(which they do not cite), the absolute concentrations of the nucleic acids were more
sensitive to the toxicant effect and more indicative of growth changes than were the ratios.
Generally, the RNA content gave the best correlation with growth. In that regard, it has
been reported (Venugopalan, 1967) that when growth hormone is injected into intact fish,
there occurs a marked stimulation of liver RNA content, but a very variable effect on that
of DNA. One cannot necessarily conclude from this that the pollutants were causing an
inhibition of growth hormone because numerous other factors could also play a role, but
it is an interesting relationship.
Evidently, tissue RNA can change quite rapidly when the organism is subjected to a
toxicant. Barron and Adelman (1985) have found that within 24 h of exposure to a

Figure 6 Effect of hexavalent chromium on growth,

nucleic acid content, and protein of larval fathead
minnows (P. promelas). Data are normalized as per­
centage of the mean control. All points are the means
±S.E. (From Barron, M. G. and Adelman, I. R., Can. HEXAVALENT CHROMIUM
J. Fish. Aquat. Sei., 41,141,1984. With permission.) (mg/L)
 195

sublethal concentration of cyanide, there is a significant difference in total body RNA

between exposed and control fathead minnows.
The original procedure used by Bulow (1970) for measuring RNA and DNA was
extremely tedious. Now several newer techniques (Bulow, 1987) and even more recent
modifications make it possible to do over 50 samples per day (Heath et al., 1993).
Another biochemical methodology for assessing growth in larval and juvenile fish is
the measurement of the enzymes citrate synthase, cytochrome oxidase, and lactate
dehydrogenase. The activity of these enzymes, two aerobic and one glycolytic, correlated
well with recent growth rate in the saithe, a marine teleost (Mathers, 1992). As was
discussed earlier in this chapter, these enzymes also correlate well with overall metabolic
rate so they, along with nucleic acid analyses, may offer considerable potential for
assessing the impact of chronic exposures to various pollutants in the field and laboratory.

X. SWIMMING PERFORMANCE
In the foregoing discussions of the effect of various pollutants on energy metabolism, it
was noted that some may reduce the maximum (active) rate of oxygen consumption.
More accurately stated, aerobic scope was reduced. In this section, the effects of pollu­
tants on swimming performance will be dealt with, realizing that aerobic scope and
swimming performance are inextricably related.
Beamish (1978) provides an extensive review of the methodologies used in studying
swimming capacity in fish. He classifies swimming performance into three major catego­
ries: sustained, prolonged, and burst. “Sustained swimming” includes all levels of activity
from spontaneous movements to cruising for periods in excess of 200 min. In essence,
sustained swimming can be maintained indefinitely. “Prolonged swimming speed” is of
shorter duration and results in fatigue. The term “critical swimming speed” (under the
category of prolonged swimming) is frequently used to designate a speed that is a
maximum that can be maintained for a measured period of time. To determine this, a fish
is placed in some sort of water tunnel and the speed is then increased stepwise with
intervals of 30 min to 2 h at each step until the fish fails to maintain position for the full
interval at a given step. Brett (1964) has devised a precise method of calculating the
critical swimming speed which includes consideration of the duration of time a fish was
able to breast the maximum speed. Most of the studies up to now involving pollutants
have measured critical swimming speed (Ucrit).
The highest speed that a fish can attain is called the “burst speed”, one which can be
maintained for probably less than a minute, and in some species is probably reached for
only a few seconds. It is powered primarily by anaerobic metabolic processes (Driedzic
and Hochachka, 1979; Beamish, 1978). For all three swimming types, the time frames
mentioned here are based largely on work using salmonids. Somewhat different times
may be more appropriate for other fish groups and some species are incapable of
maintaining a sustained speed at all.
Randall and Brauner (1991) recently reviewed the effects of temperature, environmen­
tal pH, and salinity on swimming performance of fish. They make the point that aerobic
swimming speed is affected by muscle contractility and/or the rate of gas transfer by the
gills and the circulatory system. An environmental factor such as temperature may affect
swimming by altering muscle contractility whereas a change in pH affects the oxygen­
carrying capacity of the blood. Although they do not discuss pollutants, it might be further
pointed out that the nervous system can be an important target for many chemical
contaminants and this could affect swimming ability.
This review is limited to studies done in the laboratory. While there have been attempts
to measure swimming performance in the field, the problem of control and replicability
196

make the results obtained rather anecdotal (see Beamish, 1978). Spontaneous locomotor
activity and the avoidance of pollutants by fishes is taken up in Chapter 12.
In order to measure swimming performance in a controlled manner, the fish must be
induced to swim at some specified speed. For this purpose a variety of swimming
chambers have been developed ranging from a simple trough with a paddlewheel to move
the water, to complex water tunnels, some quite large, where water is pumped through a
swimming chamber in which the fish are confined (Fry, 1971; Beamish, 1978; Graham
et al., 1990). In most investigations of the effects of pollutants on swimming performance,
the fish were exposed to the test substance for some interval of time before being placed
in the swimming chamber.
Blocking the transfer of oxygen across the gill should result in a reduction of aerobic
swimming. This was shown with coniferous fibers from pulpwood which tend to clog the
gill lamellae and thus interfere with normal water flow through them, and this probably
reduces oxygen uptake. Exposure to these fibers in the water caused a dose-dependent
reduction in endurance of fathead minnows forced to swim at a slow speed. Support for
the hypothesis that oxygen transfer was impaired was the observation that lowering the
oxygen in the water, or raising the temperature, accentuated the effect of the fiber on
swimming (McLeod and Smith, 1966).
In the above-mentioned study, stamina at a fixed slow speed was measured. The critical
swimming speed (Ucrit) of fish is also expected to be especially sensitive to impairment of
oxygen transfer in gills because this is one of the primary determinants of aerobic scope.
Kraft pulpmill effluent has been shown to impair the gas exchange process resulting in
depressed levels of arterial oxygen (Davis, 1973). In a rather thorough study, Howard
(1975) measured critical swimming speeds in coho salmon (O. kisutch) following exposure
of the fish to pulpmill effluent at several different concentrations and lengths of time. There
was a significant reduction in critical swimming speed at all concentrations above 1/10 the
96 h LC50 and the effect was concentration dependent. A plateau in the extent of swimming
inhibition occurred after some 18^8 h of exposure. This may suggest that damage to the
gills is not cumulative. Support for this hypothesis was found when coho were exposed to
these same concentrations for 90 days, there was no impairment of critical swimming speed,
so physiological compensation, or repair of the damaged tissue must have taken place.
McLeay and Brown (1979; p. 1056) make the cogent point that “...these compensations
should not be considered as acclimation responses unless it can be demonstrated that they
occur without an increased energy cost or without a debilitation of other physiological zones
of tolerance required for survival in the natural environment.”
The ability of brook trout fry to swim in the presence of aluminum (300 Fg/L) was
found to be very dependent upon the pH of the water (Cleveland et al., 1986). The
aluminum had no effect on swimming of the fry at pH 7.2 or 4.5. However, at the
intermediate pH of 5.5, they were essentially unable to swim, but after the fish reached
37-67 days of age, the aluminum had no effect on Ucrit at any of the pHs. Aluminum in
low pH waters causes problems for both ionoregulatory processes and oxygen transfer at
the gills of adult trout (Wood, 1989). The effect, especially on oxygen transfer, seems to
be greatest at a pH of around 5-6 which would fit with the larvae data in the Cleveland
et al. (1986) work. Still, why there was no effect on Ucrit of juveniles is peculiar and no
attempt is made by the authors to explain it.
Acclimation to aluminum has been explored using rainbow trout juveniles (Wilson
and Wood, 1992). The fish were exposed for a period of 22 days to water of pH 5.2 in
the presence of only 30 pg/L aluminum and other fish were kept without the metal.
Figure 7 shows how the aluminum caused a rapid reduction in Ucrit and then a gradual
restoration over time as the fish acclimated to the aluminum. Although complete Ucrit
recovery did not occur in 22 days, the authors reported that complete restoration of
ionoregulatory function occurred at that time.
 197

Figure 7 Critical swimming velocities (Ucht) of rainbow trout expressed as body lengths per
second, for fish exposed for 22 days to pH 5.2 (open circles) or pH 5.2 plus 30 |xg Al/L (filled
circles). The dashed line represents the combined mean value for control fish (maintained and
swam at pH 6.5, zero aluminum) on days 0 and 24 (Ucht = 4.82 ± 0.11 BL/s, n = 30). Asterisks
indicate means different (p <0.05) from the 6.5/0 control group, and daggers indicate means
significantly different from the 5.2/0 group (From Wilson, R. W. and Wood, C. W., Fish Physiol.
Biochem., 10, 149, 1992. With permission.)

The reduction in swim speed from aluminum exposure is associated with histopathological
changes in the gill epithelium (Mueller et al., 1991). These changes increase the diffusion
distance for oxygen across the gill epithelium and thus are probably responsible for reducing
the aerobic scope. Niki and Parrel (1993) quantified both the changes in lamellar structure
and Ucrit in juvenile salmonids exposed to a wood preservative at several concentrations
and observed a strong relationship between reduction in Ucrit and extent of gill damage
(Figure 4, Chapter 2).
Ucrit can also be reduced by pollutants that do not necessarily affect the transfer of
oxygen across the gill. After only 5 h of exposure of trout to copper at 0.02 ppm (in soft
water), there was a drop in critical swimming speed to about 55% of controls, but after
10 h of continuous exposure, swimming speed had returned to approximately 73% of
normal where it remained for up to 30 days of exposure (Figure 8). The interaction with
water hardness is noteworthy in this study. As the water gets harder, the effect on
swimming is less. Waiwood and Beamish (1978) conclude that critical swimming perfor­
mance showed impairment at copper concentrations as low as 0.25 of the 96-h LC50

Figure 8 Effect of copper, water

hardness, and the duration of expo­
sure on the critical swimming speed
of rainbow trout (pH 6, 12°C). Har­
ness (mg/L CaCOg) is shown by the
larger numbers. (The general pattern
at pH 7.5 was similar except the criti­
cal swimming speeds were higher.)
(From Waiwood, K. G. and Beamish,
W., Water Res., 12, 611, 1978. With
permission.)
198

which are comparable to those thresholds affecting reproduction in this species. However,
under conditions of high water hardness and high pH, swimming performance is not
nearly as sensitive to copper.
The physiological basis for the impairment of swimming speed by copper is complex
and not clear. Oxygen transfer by the gills at these concentrations is probably not a
problem as Sellers et al. (1975) exposed trout to copper concentrations considerably
higher than these and observed no change in arterial oxygen in resting fish. The increased
energy demand induced by the copper that was discussed earlier in this chapter would
reduce the aerobic scope, and this was indeed observed in the Waiwood and Beamish
(1978) work. As was mentioned earlier, effects on the nervous system which might cause
some loss in muscular coordination would contribute to less efficient swinuning resulting
in an increased energy demand at any swimming speed, and thus a decrease in the critical
swimming speed.
Lead seems to have relatively little effect on swimming speed. Freadman et al. (1985)
tested striped bass at several concentrations of lead for 60 days and observed inhibition
only at a concentration of 200 pg/L which is fairly high.
It was mentioned earlier in this chapter that petroleum hydrocarbons often stimulate
resting metabolism in fish. It would therefore be predicted that a reduction in aerobic
scope would follow. This seems to have been confirmed in work on the maximum
swimming speed of coho salmon exposed for 48 h to a range of concentrations (25-75%
of the LC50) of crude oil (Thomas and Rice, 1987). The effect on swimming performance
was not, however, very large and a concentration at 75% of the LC50 was required to
produce an effect on swimming performance. Rather large increases in plasma cortisol
were seen so the fish were under significant stress from the crude oil.
Respiratory poisons exert their effects at the cellular level through more than one
mechanism. For example, cyanide is known to be a blocker of the enzyme cytochrome
oxidase so the cell is unable to utilize oxygen as a terminal electron acceptor. In a sense,
then, it simulates hypoxia. Pentachlorophenol, on the other hand, blocks oxidative phos­
phorylation so that oxygen is used, but the generation of ATP aerobically is reduced.
Cyanide has been found to be a potent inhibitor of swimming speed (reviewed in Leduc,
1984). Various workers have used exposures of up to 36 days and seen inhibitions in
swimming speeds of 50% or more. Pentachlorophenol at chronic levels has been shown
by two independent studies to have little effect on this measure of performance in trout
or a cichlid (Webb and Brett, 1973; Peer et al., 1983). Thus, it cannot be predicted that
metabolic poisons will necessarily alter the Ucrit.
Fish taken from polluted waterways have also been tested for swimming performance.
Striped bass from the Hudson River exhibited reduced Ucrit when compared with the
same species from other East Coast rivers or hatcheries (Freadman et al., 1985; Buckley
et al., 1985). Interestingly, the two studies cited here were conducted approximately at the
same time but presumably independent of each other. The explanation for the reduced
performance capacity is probably complex as is the mix of pollutants in the Hudson.
Freadman et al. (1985) measured PCBs in the parts per million range in the Hudson River
fish. Sustained swimming (which they measured) is dependent almost exclusively on red
muscle. PCBs are sequestered in the lipids, and, coincidentally the red muscles utilize
lipids predominantly for energy so the authors suggest that these organochlorine com­
pounds may alter lipid mobilization during swimming.
Buckley et al. (1985) found that their Hudson River striped bass had heavy parasitic
infections in the muscles. Because several studies (reviewed in Beamish, 1978) have
shown that parasitic infections can adversely affect swimming performance, the sugges­
tion was made that this may be at least one mechanism for the poor performance of the
contaminated fish.
 199

Substances that act on the nervous system can have a marked effect on not only
spontaneous activity, as was discussed above in the section on whole-body energy
metabolism, but also on the critical swimming speed. Brook trout exposed to the orga-
nophosphate insecticide fenitrothion showed a dose-dependent inhibition of critical speed
(Peterson, 1974). These insecticides are well-known acetylcholinesterase inhibitors. Post
(1969) noted a 50% reduction in coho salmon “stamina” following exposure to malathion,
another organophosphate insecticide, and this corresponded to an 86% reduction in
acetylcholinesterase activity in the nervous system. When he tested rainbow trout there
was a somewhat greater reduction in stamina but less inhibition in the enzyme activity.
With the use of several concentrations of two organophosphates, Cripe et al. (1984)
observed a marked dose-dependent inhibition of acetylcholinesterase but a relatively high
concentration was required before critical swimming speed in sheepshead minnows was
reduced. Whether there is an actual relationship between cholinesterase and swimming
performance in fish is not clear from these studies. When one starts altering the functions
of the nervous system, all sorts of indirect effects may take place, such as “motivational”
disturbances.
Larval fish have varying abilities to swim, depending on species and stage of devel­
opment. Heath et al. (1993) tested swimming performance of newly hatched striped bass
larvae exposed for four days to the insecticides methyl parathion, carbofuran, or an
herbicide, molinate. Two concentrations were tested, one at one half the 96-h LC50 and
one more than an order of magnitude lower. Swimming performance was assessed in a
simple fashion using a petri dish with a smaller dish in the center forming a circular “race
track”. Radial lines were marked under the petri dish. Single larvae were chased with a
glass rod around the track for 1 min and the number of lines that were crossed were
counted. This arrangement probably measures a combination of burst and prolonged
swimming. Few of the larvae became exhausted during the 1-min duration. Significant
decreases were seen in swimming performance of larvae in the methyl parathion at both
concentrations and in the molinate at the one half LC50 concentration. Interestingly, in
both the methyl parathion and molinate groups at the higher concentration, there was
depressed swimming ability even after 10 days recovery in non-contaminated water.
Acetylcholinesterase was inhibited in the exposed fish but this could not be unequivocally
associated with the impaired swimming performance as this enzyme was also inhibited
in the carbofuran-exposed fish as well, but no effect of the pesticide on their swimming
was detected.
While several authors have noted an inhibition of swimming capacity by pesticides.
Little et al. (1990) found no effect of methyl parathion, chlordane, or PCP on this capacity
in small (0.5-1.0 g) rainbow trout. They used exposures of 96 h and concentrations as
high as one half the 96-h LC50. They also tested the herbicides DBF (an organophos­
phate) and 2,4-DMA (a phenoxy compound) and found an enhanced swimming ability
following exposure to low concentrations but an inhibition at high (but still sublethal)
concentrations. The stimulation of swimming ability may be an example of hormesis
wherein a modest stress causes enhanced ability to grow or perform other functions
(Stebbing, 1987). Of course, there may be other explanations as well.
It should be emphasized here that swimming performance tests of Ucrit probably have
validity only with those species that are migratory or normally swim for long periods
(e.g., many salmonids). For, as Beitinger and McCauley (1990) have emphasized, swim­
ming stamina is probably not important in many inactive fish species who rely more on
burst swimming for the capture of food and evasion of predation. We currently know little
about how pollutants affect this type of swimming, or if there is any effect at all. It seems
most likely that those chemicals that affect the nervous system will have the most impact
on burst swimming, since aerobic capacity is unimportant for that style of movement.
200

It is evident that a variety of mechanisms may be responsible for reductions in

sustained swimming capacity. Under acute exposure to different substances, respiratory
gas exchange is often inhibited due to gill damage, and this will reduce the aerobic scope
and thereby the critical swimming speed. However, this is probably not as important
during chronic exposure where gill damage is less, if there is any damage at all. Then, if
swimming effects are seen, they may be due primarily to a greater metabolic demand for
detoxification and repair which would reduce the aerobic scope.
Chronic exposure can also produce alterations in nervous function which reduces
neuromuscular coordination and/or motivation. One of the most potent inhibitors of
swimming performance is cyanide which reduces the ability of muscles to utilize oxygen.
A final factor that may be important in limiting swimming stamina is the loss of energy
stores (largely carbohydrate) that frequently occurs when fish are exposed to pollutants
(see next section). While these are not particularly important for the resting fish, a fish
swimming at maximum speed utilizes them rapidly and exhaustion takes place when they
are expended (Driedzic and Hochachka, 1979).

XI. CHANGES IN CARBOHYDRATE, LIPID AND

PROTEIN ENERGY STORES
A. INTRODUCTION
It was mentioned early in this chapter that fish utilize protein and lipids for energy to a
greater extent than do mammals. However, when under acute stress from abiotic or biotic
factors, fish are somewhat similar to other vertebrates in that they mobilize and use
carbohydrates. Changes in the tissue concentration of carbohydrates have received a good
deal of attention among students of the effects of pollution because of their relationship
to the “traditional” stress responses of other vertebrate animals. First, some basic concepts
will be reviewed so the changes induced by xenobiotics can be better understood. Figure 9
shows the major energy stores. The diagram emphasizes carbohydrates and their hor­
monal controls because these change rapidly during stress, but it should be emphasized
that there is actually far more energy available to the fish in protein from muscle and fat
in other parts of the body.
The greatest amount of carbohydrate is stored in the liver as glycogen. Skeletal muscle
glycogen is also an important store, but the concentrations found in fish are generally at
least an order of magnitude less than those in the liver (Black et al., 1962). Blood glucose
is the form in which carbohydrate is transported and the concentration in the blood is
controlled by cortisol, adrenaline, and insulin. In general, the homeostasis of blood
glucose in fish is poor. The classical picture of insulin maintaining a tight control of blood
glucose, as is typical of mammals, does not hold for fish. This hormone has relatively
little effect on blood glucose; it instead is mostly involved in stimulating amino acid
incorporation into protein and reducing the conversion of amino acids into glycogen
(glyconeogenesis; Ablett et al., 1981). Thus, in fish insulin secretion rate by the beta cells
in the pancreas is controlled more by amino acids than by glucose (Cowey et al., 1977).
The dynamics of intermediary metabolism are greatly influenced by any sort of
stressor and these mechanisms are outlined in Figure 9. A stressor is detected as some sort
of change that alters the homeostasis of the animal (including psychological, Schreck,
1981). Operating via the hypothalamus, the first effect is to activate the chromaffin cells
which, in teleosts, are located in the walls of the cardinal veins and in some cases the head
kidney (Mazeaud and Mazeaud, 1981). There is no well-defined adrenal medulla, as is
found in mammals. Chromaffin cells release adrenaline and a small amount of noradrena­
line which stimulate the conversion of liver glycogen into blood glucose and the utiliza­
tion of glucose by muscle. These adrenergic effects may result in increases of blood sugar
within minutes of the onset of the stress. Elevations in the concentration of blood lactic
 201

EXTERNAL AND INTERNAL C = CORTISOL

STIMULI (STRESSORS) a = ADRENALIN
I = INSULIN

CATECHOLAMINES

INSULIN

BLOOD GLUCOSE AMINO ACIDS ^ BLOOD GLUCOSE BLOOD GLUCOSE

AMINO ACIDS
FATTY ACIDS C- FATTY ACIDS c-
1 +
c+
PROTEIN
(GLY COGENESIS) (GLYCOGENOLYSIS) FAT
-GLYCOGEN PROTEIN
I I (GLUCONEOGENES'lS) FAT
(GLYCOLYSIS)
GLYCOGEN-^ t
LACTIC ACID

LIVER SKELETAL MUSCLE Mise. TISSUE

Figure 9 Generalize(J diagram of energy flows and their hormonal controls emphasizing
carbohydrates and the generalized stress response. The letters designating specific hormones
with a plus or minus beside them indicate the process is stimulated or inhibited by the hormone.
(See text for further explanation.)

acid frequently occur coincident with the stress, but it is not clear whether this is due to
adrenergic stimulation of glycolysis, or a direct effect of the stress (e.g., hypoxia) on the
muscle tissues. It could, of course, involve both mechanisms.
If the stress persists, other mechanisms may also be involved in mobilization of energy
stores. Classically, cortisol from the adrenal cortex (in mammals) acts to reduce glucose
utilization by muscle and other tissues and helps form more glycogen in the liver. The net
effect is to maintain an elevated blood glucose level. It has long been assumed that similar
mechanisms occurred in fish since exogenous administration of cortisol (by injection)
generally causes an elevated blood glucose (for review, see Van der Boon et aL, 1991).
However, this picture may need some revision, at least in trout. Andersen et al. (1991),
in a rather sophisticated series of experiments, have cast doubt on the role of cortisol as
a glucocorticoid. They implanted osmotic pumps into rainbow trout which provided an
elevated level of cortisol without the usual attendant handling associated with injections.
The fish were also fitted with dorsal aortic cannulae so serial blood samples could be
taken, thus daily glucose, amino acid, and cortisol measurements were possible from the
same fish. With this arrangement, they found upon elevation of cortisol that blood amino
acids increased in concentration, but there was no elevation of blood glucose and no
change in liver enzyme activities associated with glyconeogenesis. These findings sug­
gest that prolonged blood glucose elevations in fish, as seen with chronic stress, must be
maintained by mechanisms other than cortisol (adrenaline?). There may also be large
species differences so generalizations from rainbow trout may not always be valid (Van
der Boon et al., 1991).
While cortisol per se may or may not act directly to raise blood glucose levels, recent
studies have revealed that it can alter the responsiveness of hepatocytes to adrenaline.
Reid et al. (1992) have shown that chronically raising the blood cortisol level causes a big
202

increase (fourfold) in number of adrenoreceptors on the hepatocyte membranes and this

enhances glucose production in response to adrenaline. Thus, cortisol could contribute to
hyperglycemia indirectly by this mechanism.
The dynamics of the stress response in mammals (and to a certain extent in fish)
generally is characterized by an alarm phase during which the level of adrenaline rises
rapidly causing a mobilization of liver glycogen into blood glucose. This is followed by
a resistance phase in which the glucose levels remain elevated and liver glycogen may or
may not be low, depending on the extent of hyperglycemia, diet, etc. Should the stressor
continue, then either adaptation or exhaustion occurs.
Adaptation implies changes in several related physiological processes which permit
the return of homeostasis. When blood sugar is involved, this means a return to near
normal levels. Exhaustion may occur if the extent and duration of stress is sufficient and
is characterized by a depletion of liver glycogen, decreasing levels of cortisol, depressed
immune response, and a host of other changes that make the organism less able to survive,
especially if other types of stressors are present (Selye, 1973; Pickering, 1981). There is
also accumulating evidence that chronic high levels of cortisol in fish cause utilization of
protein for energy so growth is inhibited (Van der Boon et al., 1991).

B. EFFECT OF PULPMILL EFFLUENT

Kraft pulpmill effluent has received considerable attention from fish physiologists, and
investigation of the changes in energy stores is no exception. Exposure of coho salmon
to a concentration of effluent equivalent to 0.8 of the 96-h LC50 produced an immediate
hyperglycemia (80% increase in blood glucose). After 48 h of continuous exposure, blood
glucose increased even further reaching levels almost four times those of controls. By this
time, liver glycogen had decreased to nearly zero (McLeay and Brown, 1975). The
authors feel these effects are primarily due to elevated adrenergic activity instead of
cortisol because the latter stimulates synthesis of liver glycogen, and they observed a loss
of glycogen. This interpretation may be correct, however, it seems just as likely that the
extent of liver glycogen breakdown merely exceeded the somewhat slower process of
glycogen formation.
The changes in concentrations of carbohydrate in response to pulpmill effluent were
explained by McLeay and Brown (1975) as an indirect response to internal hypoxia.
Work by Davis (1973) has shown that this effluent causes damage to the gills producing
a lowering of arterial oxygen. Because environmental hypoxia (which also lowers arterial
oxygen) has been shown to cause hyperglycemia and mobilization of liver glycogen
(Heath and Pritchard, 1965), the hypothesis has support.
In a later study, McLeay and Brown (1979) exposed cohos to levels of pulpmill
effluent ranging from 0.1-0.5 of the LC50 for periods of up to 200 days. They observed
hyperglycemia and a lowering of liver glycogen, but the extent of the changes was not
as great as with the acute exposure, and there were indications of recovery by 90 days in
this chronic study. The authors mention a condition of “exhaustion” at 200 days, but the
data obtained at that time were not much different than those obtained at 90 days. When
Oikari and Nakari (1982) subjected trout to components of pulpmill effluent for 11 days,
they did observe a clear-cut exhaustion in that the liver glycogen became almost com­
pletely depleted and the blood glucose decreased by 35%. They also noted elevated
concentrations of ammonia in the blood. The ammonia apparently came from lateral
muscle as its protein concentration decreased by 13%. These latter changes would be
expected as cortisol stimulates the conversion of protein into free amino acids which are
then deaminated by the liver for gluconeogenesis.
The dynamics of protein and lipid changes during exposure to pulpmill effluents are
complex. Oikari et al. (1985) report elevated liver protein and muscle lipid after 30 days’
exposure to a very low level of effluent. Muscle protein was depressed. They conclude
 203

this indicates a shift from protein to lipid metabolism. The high levels of liver protein at
this time may be due to the induction of very large amounts of the biotransformation
enzyme ethoxyresorufin-O-deethylase (EROD; Larsson et al., 1988). This also results
usually in liver enlargement due to the fat and protein synthesis. In general, there are
minimal changes in carbohydrates from these effluents if the exposures are realistically
low (Andersson et al. 1987; Larsson et al., 1988).

C. METALS
As with pulpmill effluent, zinc at acute levels causes gill damage and consequently an
internal hypoxia in fish exposed at acute concentrations (see Chapter 3). Such exposure
results in elevated lactic acid and utilization of much of the muscle glycogen (Hodson,
1976). Such changes are in part, at least, a direct effect of hypoxia on the muscle tissue.
Zinc also produces a hyperglycemia (presumably from adrenergic stimulation), which
may be further potentiated by a depression of insulin secretion from damage to pancreatic
beta cells (Wagner and McKeown, 1982) in much the same way that cadmium works (see
below).
The dynamics of the response to some chemical insult can be as interesting as the final
outcome. For example, Schreck and Lorz (1978) measured cortisol in coho salmon at
several time intervals during exposure to different concentrations of copper. There was
a dose-dependent rise in blood cortisol within 2 h of the start of exposure, but then this
returned to normal at 24 h, only to again become elevated later. These changes in cortisol
could be a physiological response to correct an altered blood electrolyte concentration
(see Chapter 7), especially the delayed rise in hormone.
Copper causes hyperglycemia in fish, whether the exposure is for a short time or even
up to a month. With a very high dose (5 mg/L), carp exhibited a marked hyperglycemia
that, however, returned to control level at 6 days. With a much lower concentration (0.25
mg/L), the Indian catfish (Heteropneustes fossilis) exhibited a steadily rising blood
glucose over a period of 30 days (Asztalos et al., 1990). Therefore, it seems probable that
the return to control levels seen in the 1-week exposure of carp was due to exhaustion of
liver glycogen, rather than any sort of adaptation to the presence of the metal.
A limited amount of work has shown that fish exposed to a stressor (e.g., a metal) for
days or weeks will respond more when challenged by a subsequent but different stressor
than do controls that have not had the previous exposure (reviewed in Schreck, 1990).
Heath (1991) found that bluegill exposed to copper for a week exhibited more of a
hyperglycemic response to environmental hypoxia than did controls. The copper also
prolonged the time required for recovery from a hypoxic stress.
Elasmobranchs may respond to copper in a way considerably different than teleosts.
At least, that seems to be evident in the study by Tort et al. (1987) in which they exposed
dogfish sharks to five different concentrations of copper for 48 h. The highest concentra­
tion (16 mg/L) approximated the 24-h LC50 for this species of fish, which is very high
compared to teleosts. There was a rather severe and dose-dependent decrease in blood
glucose, which is the opposite of what would be expected in a teleost. They also found
a marked increase in liver glycogen, but no changes in liver lipid or protein. They attribute
the hypoglycemia to hemodilution (a decrease in hematocrit was seen) which is possible
since most elasmobranchs are slightly hyperosmotic to seawater and copper has notable
effects on osmoregulation (see Chapter 7). The rise in glycogen could be due to an
inhibition by copper of glycogen conversion into blood sugar. An interesting hypothesis
to test would be whether the enzymes responsible for mobilization of glycogen to blood
sugar were being inhibited by the accumulation of copper in the liver.
The concentration of copper in the water has recently been shown to have a large
qualitative effect on lipid metabolism. When exposed to a lethal concentration for three
days, Sivaramakrishna et al. (1992) found that Labeo lost total lipids from nearly all
204

tissues and there was an increase in free fatty acids and glycerol. On the other hand,
exposures to sublethal concentrations for up to 30 days produced increases in total lipids
and transitory increases in free fatty acids. The largest changes were seen in liver with
lesser effects of the copper in gill, muscle, and brain, in that order. It seems probable that
the rise in lipids could be due to conversion from protein although no direct evidence is
available.
While metals, in general, may cause conversion of liver glycogen into blood glucose
at acute and perhaps even at chronic concentrations, the mechanism by which this is done
is certainly different for cadmium, depending on the fish species. In the Schreck and Lorz
(1978) work on copper mentioned above, they also measured cortisol in cohos exposed
to cadmium at a variety of concentrations including some that were clearly lethal. Much
to their surprise, there were no significant changes in cortisol in the fish exposed to
cadmium, even in those that were moribund, but a cadmium-induced hyperglycemia has
been noted (Gill and Pant, 1983; Larsson and Haux, 1981; Das and Banerjee, 1980;
Ghazaly, 1992; Pratap and Bonga, 1990) as well as hypoglycemia (Gill and Pant, 1983;
Das and Banerjee, 1980), depending on the dose, and liver glycogen may or may not
become depressed, depending on the species. For example, when rainbow trout and
flounder (P.flesus) were exposed to cadmium at two different concentrations for 80 days,
there was a marked reduction in liver glycogen in the trout, but an 80% elevation in the
founder. Both exhibited a hyperglycemia, but the effect was greater in the trout (Larsson
and Haux, 1981). The differences reflect the rather large difference in metabolic activity
of these species and undoubtedly, there are some differences in their hormonal responses
as well.
The cortisol response to cadmium may be quite species specific too. While Schreck
and Lorz (1978) observed no change in coho salmon exposed to very high doses of
cadmium, tilapia, on the other hand, showed a large increase in cortisol which persisted
for 14 days’ exposure. By 35 days of continuous cadmium exposure, the tilapia had
returned to the control level suggesting an adaptation to the presence of the metal (Pratap
and Bonga, 1990). Interestingly, blood glucose also went up, but returned to control level
in only four days so the elevated cortisol did not correlate with the hyperglycemia. This
perhaps fits in with the discussion at the beginning of this section relative to the possible
lack of a glucocorticoid activity of cortisol in fish.
The effects of cadmium on carbohydrate metabolism are complicated by the fact that
this element causes damage to the insulin-producing cells in the pancreas (Havu, 1969).
In mammals cadmium has been shown to decrease the levels of circulating insulin
(Ghafghazi and Mennear, 1973). A reduction in insulin secretion rate causes hyperglycemia,
such as occurs during insulin diabetes. Thus, elevations seen in blood glucose during
chronic exposure to cadmium might be in part due to this “diabetic” condition. There is
probably also some adrenergic stimulation, especially if the doses are high, which could
cause losses in liver glycogen, as occurred in the trout mentioned above. If chronic
exposure results in hypoglycemia, it is probably due to a combination of liver glycogen
depletion, reduced feeding, and even excretion of glucose by the kidney, as occurs in
mammals poisoned by cadmium (Ghafghazi and Mennear, 1973).
In sharks, as was discussed above, copper produced a lowered blood sugar. In the same
laboratory, it was found that cadmium caused just the opposite; a marked hyperglycemia
and loss of liver glycogen and protein occurred during 96-h exposures (Hemandez-
Pascual and Tort, 1989). In other words, cadmium caused a more “traditional” stress
response in this elasmobranch.
A lowering of blood sugar was observed in trout exposed to lead for 16 weeks (Haux
et al., 1981). The authors suggest this was due to damage of kidney tubules which they
say occurs in lead-exposed mammals. Another possibility, which they did not mention,
is the fact that lead depresses gluconeogenesis in the livers of rats (Cornell, 1974) and
 205

may do the same thing in fish. The situation regarding lead in fish is somewhat confusing
in that the same laboratory observed an elevated blood glucose (60% increase) in fish
from a lead-contaminated lake (Haux et al., 1985). Of course, in this field study, other
factors besides lead could have been present to cause the elevated glucose levels.
Mercury (as HgCl) has also been shown to cause a lowering of blood glucose and liver
glycogen in the freshwater snakehead (Channa punctatus) (Sastry and Rao, 1981). Sastry
and Rao (1982) subsequently found that this element caused inhibition of the enzyme
glucose-6-phosphatase in the liver of these fish. This is a key enzyme in the conversion
of glycogen to glucose, so these findings provide a mechanism for the observed changes
in the carbohydrates. These workers further found an elevated rate of activity for the
enzymes glutamate dehydrogenase, amino acid oxidase, and xanthine oxidase which
suggests a shift to more amino acid catabolism in the fish exposed to mercury. Such a
change in metabolism could be viewed as a cellular-level compensation for the reduced
energy available from carbohydrates.
We see from the foregoing review that the responses of fish to some metals follows
a classical general adaptation syndrome of hyperglycemia, lowered liver glycogen, and
elevated cortisol. Other metals such as mercury produce stress, but the response does not
necessarily follow the classical adaptation syndrome.

D. PETROLEUM HYDROCARBONS AND SURFACTANTS

Petroleum and its individual constituents may be examples of substances that cause
“stress”, but the classical response of hyperglycemia is absent or minimal. McKeown and
March (1978) used a 96-h exposure of rainbow trout to Bunker C oil and Zbanzszek and
Smith (1984) a 1- to 72-h exposure of the same species to a model mixture of aromatic
hydrocarbons. In the first study, a slight hypoglycemia was observed even though gill
damage and some mortality took place. In the second investigation cited, depending on
the concentration, significant changes occurred in hematological components but blood
glucose remained the same, even in moribund fish. McKeown and March (1978) specu­
late that petroleum affects the kidney, causing loss of glucose through the urine.
Benzene, an important component of petroleum, was found to cause irritation of
sensitive membranes such as gill epithelium and a considerable accumulation of benzene
in tissues of the fish (MacFarlane and Benville, 1986). However, in spite of evident stress,
striped bass adults exposed to this material for up to 21 days exhibited only transient
elevations in cortisol, blood glucose, and lactate with apparent adaptation to the stress
within 48 h.
Detergents have been shown to cause severe gill damage (see Chapter 3). Fingerling
carp exposed to a sublethal concentration of linear alkyl benzene sulfonate for up to 96 h
exhibited progressive loss of glycogen from gill, liver, and kidney but a gain in protein
in these organs (Misra et al., 1991). Lactic acid increased considerably in all tissues so
internal hypoxia must have occurred and this would explain the loss of glycogen due to
enhanced anaerobic metabolism. The increased protein synthesis is difficult to explain but
might conceivably reflect general repair of damaged tissue.

E. PESTICIDES
Pesticides seemingly cause a variety of changes in carbohydrate metabolism in fish,
however, care must be taken in interpreting results. For example, high doses can cause
an immediate hyperglycemia followed by hypoglycemia before death (Hanke et al.,
1983). If the blood glucose were measured at only one time interval, one could conclude
very different things regarding responses.
Exposure of eels (Anguilla anguilla) to 0.1 ppm of PCP for 8 days causes a marked
hyperglycemia, but peculiarly, no changes in liver glycogen (Holmberg et al., 1972).
High glucose levels persist even after 55 days of recovery in non-treated water. Holmberg
206

and associates do not attempt to explain these findings other than to conclude the PCP
caused a “hypermetabolic state”. A high glucose level without a concomitant loss of some
glycogen and the persistence of the hyperglycemia argues for some sort of alteration in
the ability of tissues to metabolize glucose for energy, so it would tend to accumulate in
the blood. Because PCP enhances tissue metabolism (see section on whole-animal
metabolism), there would seem to be the possibility that the PCP caused damage to the
insulin-producing beta cells in the pancreas. This mechanism has already been hypoth­
esized to explain the effects of exposure to cadmium, but is only a tentative speculation
at this time for PCP.
Organophosphate insecticides may suppress aerobic metabolism of tissues (see dis­
cussion in earlier part of this chapter); the action would therefore appear to be different
from that of PCP. Hyperglycemia associated with liver glycogen mobilization has been
observed in fish exposed to a variety of sublethal concentrations of organophosphate
compounds (Koundinya and Ramamurthi, 1979; Sastry et al., 1982; Awasthi et al., 1984;
Rani et al., 1990; Gill et al., 1990). Marked elevations in lactic acid, even after a month
of exposure, suggest that the cause of the carbohydrate mobilization is probably due in
part to adrenaline. A reduction in aerobic metabolism due to suppression of the aerobic
metabolic machinery by the insecticides would also cause a stimulation of anaerobiosis
and utilization of carbohydrate for energy.
Exposure of C. punctatus to quinalphos (another organophosphate insecticide) for 15
and 30 days produced slight but significant increases in liver glycogen and muscle
glycogen and a mild hypoglycemia (Sastry et al., 1982). This is the opposite of the
findings reviewed in the preceding paragraph; the authors feel their observations may in
part be explained by elevations in insulin. As support for that hypothesis, they note that
Eller (1971) found hyperplasia of pancreatic beta cells following chronic exposure to
endrin (a chlorinated hydrocarbon insecticide). The mechanism of the hyperplasia in­
duced by the pesticide is unknown, and we do not know whether an organophosphate
insecticide can cause such an effect, nor is it known if the hyperplasia actually causes
elevations in insulin, or is merely a compensatory response to inhibition of insulin
synthesis or secretion by the pesticide.
A more definitive study of an organophosphate compound (dimethoate ) may shed
some light on the contradictory findings discussed above. Pant and Singh (1983) found
that acute exposure produced a hyperglycemia and glycogenolysis, but when they ex­
posed the fish to concentrations equivalent to 1/11 or 1/7 of the 96-h LC50 for 15 and 30
days they observed hypoglycemia even though liver glycogen was only partially depleted.
The acute responses can easily be explained as a classical stress response mediated by
adrenergic and possibly cortisol stimulations. They propose that hypoglycemia following
chronic exposure may be due to the hyperplasia of beta cells already mentioned. Again,
however, it is not known if there actually is an elevation in insulin.
It appears that chlorinated insecticides cause mobilization of liver glycogen into blood
glucose in much the same fashion as seen with organophosphate compounds (Gill et al.,
1991). For example, Verma et al. (1983) exposed three different species of freshwater fish
to a chlorinated hydrocarbon and an organophosphate insecticide for 30 days at concen­
trations equivalent to 1/4, 1/8, and 1/16 of the 96-h LC50. With both compounds there
was a dose-dependent hyperglycemia ranging up to almost a 100% increase and a dose-
dependent loss in liver and muscle glycogen (up to 70%). Organochlorine compounds
also cause big increases in lactic acid and the effect increases with duration of exposure
(Gill et al., 1991).
Gill et al. (1990; 1991) noted a marked drop in brain glycogen from organophosphorus
or organochlorine exposure. Because brain tissue is dependent upon carbohydrate for its
metabolism (Hawkins, 1985), this depletion of that energy store could have serious
implications and could provide a mechanism for neurological changes.
 207

More recent studies have revealed changes in lipids and protein associated with
pesticides. The effect depends on the tissue examined. The organochlorine endosulfan
caused total lipids, free fatty acids, and proteins to increase in liver, whereas the total
lipids decreased in skeletal muscles and ovary (Gill et al., 1991). A similar pattern of
change was seen with an organophosphate insecticide (Gill et al., 1990).
Changes in lipids may have an impact on reproduction. Singh (1992) examined
changes in female Heteropneustesfossilis (catfish) exposed to malathion (an organophos­
phate) for 4 weeks during various phases of the annual reproductive cycle. During the
preparatory phase malathion caused a reduction in free fatty acids, monoglycerides,
triglycerides, phospholipids, and free cholesterol in liver and gonad. Liver esterified
cholesterol was increased. As spawning approached, the liver free fatty acids rose while
all other lipids (except esterified cholesterol) remained low. At spawning, malathion
caused the ovarian levels of triglycerides and esterified cholesterol to be elevated while
other lipids were still low. Evidently, cholesterol biosynthesis is unaffected by the
pesticide but the hydrolysis of esterified cholesterol to free cholesterol is inhibited. This
could affect sex steroid biosynthesis. Also the mobilization of various hepatic lipids to the
ovary may be inhibited by the malathion.
When carp were exposed to the synthetic pyrethroid cypermethrin for up to 96 h, they
exhibited a steady decline in glycogen and lipid in liver, gill, and brain. Muscle, on the
other hand, showed a steady increase in these energy stores over the same time (Piska et
al., 1992). Interestingly, protein concentrations changed in exactly the opposite direction
with increases in liver, gill, and brain, possibly due to synthesis of detoxification en­
zymes, while muscle protein exhibited a steady decline in concentration.
Lipid levels may have promise as one of several bioindicators of water contamination
in field studies. Adams et al. (1992) measured serum and total body triglycerides in sunfish
{Lepomis) taken from four sites along a stream receiving mixed contaminants. Declines in
these measures were seen in the more contaminated sites with the serum triglycerides being
the most sensitive of the two biochemical measures. The lower energy reserves were also
reflected in lower growth rates and fecundity in the same populations of fishes.

F. MISCELLANEOUS POLLUTANTS
Ozone is a strong oxidizing agent which is an example of a toxicant that causes massive
gill damage and subsequent internal hypoxia. Ozone is sometimes used as a substitute for
chlorine in sewage disposal and in steam electric generating plants. Chronic exposure to
5 pg/L resulted in only mild gill damage and no changes in osmoregulation or blood
glucose (Wedemeyer et al., 1979). However, only one day’s exposure to 7 pg/L caused
acute hyperglycemia, presumably mediated by adrenaline. This rather sharp threshold
between a “no effect” level and a strong response has also been noted in simple toxicity
tests on chlorine (Heath, 1977).
Lowering the the pH of the water to 5.2 caused a mild elevation of plasma glucose in
rainbow trout (Brown et al., 1984). A further lowering to 4.7 resulted in a large hyperglycemic
response which got steadily greater for up to 20 days of continuous exposure. Plasma
cortisol was unaffected by lowering the pH to 5.2 but greatly elevated at pH 4.7, so the
hyperglycemia may reflect some effect of cortisol as well as adrenergic action.

G. CONCLUDING COMMENT
From the foregoing discussion it is clear that levels of energy stores are sensitive to
pollution stress. Thus, they may lend themselves to utilization as a biomarker in field
monitoring programs, but if we are to understand the mechanisms involved in the changes
observed, future studies should incorporate measures of hormones, especially catechola­
mines, insulin, and cortisol. Glucagon and growth hormone may also be important as
well.
208

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 Alterations in Cellular
Chapter
9
Enzyme Activity,
Antioxidants, Adenylates,
and Stress Proteins
L INTRODUCTION
Essentially all chemical reactions in cells are catalyzed by enzymes, thus the action of a
foreign chemical in the cell almost always involves disturbances in enzyme function. The
subject of biochemical toxicology is immense and much is beyond the scope of this
treatise as it involves subjects such as induction of neoplasms, etc. This chapter follows
naturally from the one on whole-body energetics (Chapter 8), because in attempting to
understand the mechanisms by which pollutants produce alterations in energetics, we are
often led to the cellular level of biological complexity in order to gain a fuller understand­
ing of the underlying mechanisms. Because many of the cellular enzyme systems in cells
are associated with the generation of ATP, adenylates will be considered here. Antioxi­
dants and stress proteins are included in this chapter, somewhat arbitrarily, because they
are cellular constituents.
A recent book on aquatic animal biochemical toxicology (Malins and Ostrander, 1994)
deals with xenobiotic transformation processes, cellular histological biomarkers, DNA
adducts, neoplasia, etc. In the presentation here, coverage will be limited to the action of
particular chemicals on intermediary metabolism enzymes, adenylates, and stress pro­
teins and antioxidants. Enzymatic transformations of xenobiotic materials and changes in
tissue glutathione are covered in Chapter 5. Alterations in tissue ascorbic acid are
discussed in Chapter 6 and of acetylcholinesterase in Chapter 12.

II. SOME COMMENTS ABOUT ENZYME METHODOLOGY

It is often assumed that xenobiotic “A” affects only enzyme “B” (or perhaps C, D, and E);
such specificity of action rarely occurs. Rather, there is usually a broader spectrum of
effects in that many chemicals affect the same enzyme, and many enzymes are affected
by the same chemical. Nevertheless, the direction of the change (i.e., increase or decrease
in activity) and the relative extent of the effect make these sort of studies fruitful.

217
218

providing one realizes that reports of an altered activity of some enzyme does not mean
that it is the only one affected, and is therefore the “mode of action” of that contaminant.
Other enzymes were probably also affected but were not assayed. Because cells have
many hundreds of types of enzymes, the possibilities are considerable.
The measurement of an enzyme’s activity is usually done using only partially purified
systems. The test tissue (e.g., liver) is homogenized in order to break up the cells.
Differential centrifugation may or may not then be used to separate constituents such as
mitochondria from other components. The activity of the enzyme in question is then
quantified by measuring the production of a product, or the change in a coenzyme (e.g.,
NAD) while the reaction mixture is provided with an excess of substrate specific for the
enzyme in question. Assuming the pH and temperature are appropriate, the activity of the
enzyme is then limited by the amount of enzyme present. The activity is expressed as
micrograms of enzyme in the extract, as units of activity, or as the amount of some
product formed per unit time. When foreign chemicals are present, there may be inhibi­
tion or acceleration of the catalyzed reaction rate. The mechanism of these effects can
involve changes in enzyme quantity, or they may be due to a direct effect of the chemical
on the enzyme molecules affecting affinity for substrate, etc.
Obviously, one measures enzyme actvity in vitro. There have been numerous studies
where the test substance (e.g., a trace metal such as cadmium) was added directly to the
reaction mixture and the activity of some enzyme is then measured. It is quite obvious to
anyone who has studied biology that cells are far more than a mere bag of enzymes. Cells
are incredibly complex in both structure and function. Enzymes are packaged, for the
most part, in organelles, and the ultramicroscopic structure of these is altered by common
water pollutants (e.g., Somasundaram et al., 1984). The penetration of foreign chemicals
into these structures may be considerably different than into the cell interior as a whole.
As was indicated in Chapter 5, cells often have mechanisms for sequestering and/or
excreting potentially harmful substances (Fowler, 1987).
From all these considerations it is obvious that the effects on enzymes may be quite
different if the material is added in vitro as opposed to exposing the animal to the test
substance for a period of time and then measuring the enzyme activity in tissues removed
from the exposed animals (i.e., an in vivo test). For example, Jackim et al. (1970) and
Hilmy et al. (1985) found little correlation between the in vitro and in vivo effects of
several trace metals on a variety of liver enzymes in fish. Gill et al. (1990) examined this
question in several organs of the rosy barb following mercury exposure and found little
correspondence between the effects seen. Changes produced by a chemical can be in
opposite directions when comparing in vitro and in vivo exposures. This is strikingly seen
in the work of Bostrom and Johansson (1972) on eels exposed to pentachlorophenol
(PCP). Of the seven liver enzymes they measured, cytochrome oxidase was the most
sensitive to in vitro inhibition by PCP. However, in vivo exposure of the eels to the same
pesticide resulted in a considerable stimulation of cytochrome oxidase activity. Thus,
there may be enzyme induction in the intact tissue but enzyme inhibition when exposed
to the chemical in vitro.
Another problem with in vitro studies, and this is an especially important one, has to
do with the metabolism of organic molecules, such as pesticides. Many, if not most,
xenobiotic organic chemicals are rapidly converted to other metabolites in the liver (see
Chapter 5). Therefore, the lesions produced in enzymatic activity observed from exposure
of a fish to some organic chemical may be markedly different both quantitatively and
qualitatively than that seen if the chemical is simply included in the homogenate reaction
mixture. In DeBruin’s (1976; p. 690) massive review of the largely mammalian literature,
he concludes at one point: “Examples of additional inhibitory effects of poisons on
enzyme systems may be multiplied almost indefinitely. However, many of the studies in
 219

this field apply to in vitro experiments and offer, in general, little value for elucidating
basic mechanisms of toxic action in vivo''
In the review here, we will limit our consideration to those studies where the exposure
was to intact fish, and the extensive mammalian literature will, for the most part, be
omitted.
For the discussion that follows, a point that should be kept in mind is that enzymes of
energy metabolism are sensitive to nutritional stress so that some of them decrease during
starvation. These include but are probably not limited to several Kreb’s cycle enzymes
(Smith and Chong, 1982; Goolish and Adelman, 1987; Houlihan et al., 1993). It is
common for fish to greatly decrease their food consumption when under pollutant stress,
so some apparent enzyme inhibitions from long-term exposures may be due to this
mechanism rather than some direct effect of the pollutant on the enzyme.

III. ALTERATIONS IN CELLULAR ENZYME ACTIVITY RESULTING

FROM METAL EXPOSURE
Table 1 summarizes many of the studies of the action of metals on enzyme activity. For
all of the work listed, exposures were to the waterborne element and tissues were then
removed from the fish following the interval of time indicated. In one of the first studies
on this topic, Jackim et al. (1970) exposed killifish {Fundulus heteroclitus) held in
seawater to a variety of metals individually at a concentration equal to their 96-h LC50.
Only surviving fish were used. The enzymes chosen were all metal-containing enzymes,
so the presumption was that traces of other cations might displace or affect the metal
moiety. From examination of Table 1 it is obvious that there is considerable qualitative
variety in the effect of the five different metals on these enzymes. For example, lead was
a stimulator of alkaline phosphatase but a strong inhibitor of both acid phosphatase and
xanthine oxidase. Copper was the lone stimulator of xanthine oxidase, although it was a
weak inhibitor at all concentrations when tested in vitro. This would seem to indicate a
compensatory biosynthesis of the enzyme in order to “adapt” to the presence of copper,
a phenomenon noted by Gould (1977) in flounders exposed to cadmium. Jackim et al.
(1970) found cadmium was the weakest inhibitor of catalase and xanthine oxidase, but
the strongest inhibitor of RNAase.
Cadmium, along with mercury, has received considerable attention because of its high
toxicity and the fact that there is no known function in cells for these elements. Not
surprisingly, high concentrations of cadmium can result in enzyme inhibition, (Gould and
Karolus 1974). However, chronic exposures, which are more relevant to the real world,
cause some far more interesting biochemical effects. When the the same species (the
marine cunner) was exposed to quite low levels (50-100 ppb) of cadmium for 30 days,
liver aspartate aminotransferase was inhibited by about 20%. In the same fish, the enzyme
glucose-6-phosphate dehydrogenase increased in activity in a dose-dependent manner
(Macinnes et al., 1977). The authors attribute this to increased pentose shunt activity in
order to provide metabolites for increased biosynthesis (for repair of cadmium damaged
tissues?). These enzyme changes occurred even though there were no detectable increases
of cadmium in the liver itself.
There was a clear-cut increase in carbonic anhydrase activity in flounders emersed in
water with cadmium (Gould, 1977). This is probably the enzyme associated with eryth­
rocytes, rather than the kidney tubules, as there is little renal carbonic anhydrase present
in marine teleosts (Gould, 1977). Christensen and Tucker (1976) found a strong inhibition
by heavy metals, including cadmium, of carbonic anhydrase from fish erythrocytes when
measured in vitro. Gould (1977) further notes that this enzyme contains zinc, and
cadmium is known to displace metal ions from enzymes and thereby reduce their activity.
Table 1 Changes in Enzyme Activity Following Exposure of Fish to Test Metal
Duration
Chemical Enzyme@) Effect Species of exposure Concentration Organ@) Ref.
Pb, Hg Alkaline phosphatase Fundulus heteroclitus 96 h LC50 Liver
Cu, Cd Alkaline phosphatase F. heteroclitus 96 h LC50 Liver
Ag Alkaline phosphatase F. heteroclitus 96 h LC50 Liver
Pb, Cd, Cu, Hg Acid phosphatase F. heteroclitus 96 h LC50 Liver
Pb, Hg, Ag, Cd Xanthine oxidase F. heteroclitus 96 h LC50 Liver
Cu Xanthine oxidase F. heteroclitus 96 h LC50 Liver
Ag, Pb, Cu, Hg Catalase F. heteroclitus 96 h LC50 Liver
Cd Alkaline phosphatase Barbus conchionius 96 h LC50 Liver, gills
Cd Alkaline phosphatase B. conchionius 96 h LC50 Kidney, ovary
Cd Alkaline phosphatase B. conchionius 96 h LC50 Gut
Cd Acid phosphatase B. conchionius 96 h LC50 Gut, ovary
Cd Glutamate and pyruvate transaminase B. conchionius 96 h LC50 Liver, muscle
Cd Lactic dehydrogenase B. conchionius 96 h LC50 Heart
Cd Cu-Zn-Superoxide dismut. Scorpaena guttata 113 96 h LC50 Intestine
Cd Aspartate aminotransferase Tautogolabrus 3 and 24 ppm Liver
Cd Aspartate aminotransferase Tautogolabrus 50 and 100 ppb Liver
Cd Glucose-6-PO, dehydrogenase Tautogolabrus 50 and 100 ppb Liver
Cd Leucine aminopeptidase Pseudopleuronectes 5-10 ppb Kidney
Cd Carbonic anhydrase Pseudopleuronectes 5-10 ppb Kidney
Cd Aspartate aminotransferase Mugil cephalus 96 h LC50 Heart, gill
Cd Alanine aminotransferase M. cephalus 96 h LC50 Heart, gill
Cd Alanine aminotransferase M. cephalus 96 h LC50 Liver
Cd Acid phosphatase M. cephalus 96 h LC50 Heart, gill, liver
Cd Lactic dehydrogenase M. cephalus 96 h LC50 Heart, gill, liver
Cd Several enzymes Herring eggs 10 P P ~
Hg Glycolytic enzymes Carassius auratus 0.25 ppb Muscle
Hg Alkaline phosphatase C. auratus 0.25 ppb Liver
Hg Protein synthetase enzymes C. auratus 0.25 ppb Liver, muscle
Hg Acid phosphatase Puntius conchonius 96 h LC50 Liver, gills, kidney
Hg Alkaline phosphatase P. conchonius 96 h LC50 Liver, gills, kidney
Hg Two amino acid transferase P. conchonius 96 h LC50 Liver, gills, kidney
Hg Glycolysis < G a m b u s ia h o lh ro o k i 28 h 0.86 ppm Muscle 10
Hg Kreb’s cycle activity > G. h o lb r o o k i 28 h 0.86 ppm Muscle 10
Hg Na,K, Mg ATPases < N o to p te r u s n o to p te r u s 30 d 17-88 ppb Brain, gill, kidney 17
Hg Na,K, ATPase < C h a n n a p u n c ta tu s 30 d 0.3 ppm Brain 18
Hg Acid phosphatase < C. p u n c ta tu s 30 d 0.3 ppm Brain 18
Hg Alkaline phosphatase > C. p u n c ta tu s 30 d 0.3 ppm Brain 18
Hg Glucose-6-phosphatase < C. p u n c ta tu s 30 d 0.3 ppm Brain 18
Lactic and pyruvate dehydrase
Hg, Pb Succinic, malic, lactic dehydrogenases < 3 Freshwater teleosts 48 h “Sublethal” Liver, muscle, brain 11
Pb, Hg Delta-aminolevulinic A. dehydrase < F u n d u lu s h e te r o c litu s 4-14 d 0.8-10 ppm Liver, kidney 19
Ag, Zn Delta-aminolevulinic A. dehydrase > F. h e te r o c litu s 4-14 d 0.8-10 ppm Liver 19
Pb Alkaline, acid and glucose phosphatase < H e te r o p n e u s te s 4-30 d 3.8 ppm kidney, ovary 23
Cu Alkaline phosphatase < C. p u n c ta tu s 24 h 7.5 ppm Liver 20
Cu Acid and alkaline phosphatase > P u n tiu s c o n c h o n iu s 48 h 96 h LC50 Gut, kidney 12
Cu Acid phosphatase < P. c o n c h o n iu s 48 h 96 h LC50 Liver 12
Cu Alanine aminotransferase < P. c o n c h o n iu s 48 h 96 h LC50 Gill, kidney, muscle 12
Cu Lactate dehyrogenase < P. c o n c h o n iu s 48 h 96 h LC50 Liver, gill 12
Cu Alkaline phosphatase > C. p u n c ta tu s 24 h 7.5 ppm Kidney 20
Cu Acid phosphatase > C. p u n c ta tu s 24 h 7.5 ppm Liver, kidney 20
Cu Glycogen phosphorylase, glucose-6- > L a b e o r o h ita 1-30 d 0.2 ppm Liver, muscle 21
phosphorylase, lactate dehydrogenase
Cu Succinic dehydrogenase < L a b e o r o h ita 1-30 d 0.2 ppm Liver, muscle 21
Cu Succinic dehydrogenase < T ila p ia m o s s a m b ic a 1-14d 1.5 ppm Muscle, liver 22
Cu Succinic dehydrogenase > T. m o s s a m b ic a 1-14d 1.5 ppm Liver, muscle
Cu, Zn Succinic dehydrogenase < O r e o c h r o m is 96 h 96 h LC50 Liver, brain, muscle 13
Cu, Zn Glyceraldehyde dehydrogenase > O r e o c h r o m is 96 h 96 h LC50 Liver, brain, muscle 13
Cr Three oxidases ns S a lm o g a ir d n e r i 48 h 2.5 ppm Several 14
Cr Na,K ATPase < S. g a ir d n e r i 48 h 2.5 ppm Kidney, intestine 15
Cr Na,K ATPase ns S. g a ir d n e r i 48 h 2.5 ppm Gill, liver 15

N o te : Where more than one chemical or tissue is listed in sequence, arranged in order of decreasing effect; ns = no significant change. References are as follows: 1. Jackim
et al., 1970; 2. Gill et al., 1991; 3. Bay et al., 1990; 4. Gould and Karolus, 1974; 5. Macinnes et al., 1977; 6. Gould, 1977; 7. Hilmy et al, 1985; 8. Nicholls et al., 1989;
9. Gill et al., 1990; 10. Kramer et al., 1992; 11. Shaffi, 1993; 12. Gill et al., 1992; 13. James et al., 1992; 14. Buhler et al., 1977; 15. Kuhnert et al., 1976; 16. Mounid IS)
et al., 1975; 17. Verma et al., 1983; 18. Sastry and Sharma, 1980; 19. Jackim, 1973; 20. Srivastava and Pandley, 1982; 21. Radhakrishnaiah et al., 1992; 22. lO
Balavenkatasubbaiah et al., 1984. 23. Sastry and Agrawal, 1979.
222

Therefore, the induction of more enzyme in chronically exposed fish is interpreted as an

adaptation at the molecular level to the partial enzyme inhibition caused by the poison.
This is a nice example of the greater value of in vivo studies compared to those done in
vitro.
The enzyme glucose-6-phosphate dehydrogenase is a key control point in the pentose
phosphate shunt. It is not a metalloenzyme but its activity is normally modified by the
concentration of magnesium. Cadmium caused a loss of sensitivity to this magnesium
activiation (Gould, 1977). Metabolic flexibility is necessary for adapting to rapidly
changing temperatures (Hochachka and Somero, 1984) so this could have subtle impli­
cations for the flounders in their estuarine habitat where temperatures are notoriously
variable.
Enzymes from different tissues may be affected very differently by cadmium. An
example of this is seen in the study by Hilmy et al. (1985) in which juvenile mullet were
exposed to cadmium at a fairly high concentration. Enzyme activities were measured
daily for 4 days. In contrast to the inhibition of aspartate aminotransferase in the liver of
cunner, which was mentioned above, they observed a strong stimulation of this enzyme
in the heart and gill by cadmium, and this increased over the 4-day period. Enzyme from
liver exhibited an initial stimulation followed by inhibition, which suggests that a low
tissue concentration of cadmium stimulated activity, but as the concentration built up over
time, inhibition of enzyme activity occurred. Another transferase, alanine aminotrans­
ferase, was also induced by cadmium in the two non-hepatic tissues of the mullet, but
inhibited in the liver.
Tissue-specific effects of cadmium were also observed in the freshwater rosy barb.
Alkaline phosphatase was unaffected in liver and gills, stimulated in kidney and ovary,
and inhibited in the gut (Gill et al., 1991). It is difficult to understand stimulation of
alkaline phosphatase in kidney as cadmium tends to accumulate there (Chapter 5). Acid
phosphatase may be stimulated or inhibited depending on species of fish and tissue
examined (Gill et al., 1991).
From the studies discussed here, it seems that cadmium can cause either induction or
inhibition of a variety of cellular enzymes during acute exposures. However, more
chronic exposures may yield a different picture. When rainbow and brown trout were
exposed to high but still sublethal concentrations of cadmium for either 2 weeks or 3
months, no changes were noted in the activities of some 10 different enzymes in a variety
of tissues (Roberts et al., 1979). This is in spite of the fact that significant amounts of
cadmium accumulated in the liver, kidney, and gills of the test fish. It is noteworthy that
two of the enzymes (glucose-6-phosphate dehydrogenase; aspartate aminotransferase)
examined in the trout are the same ones that showed changes in marine fish exposed
acutely to cadmium. Perhaps, with more prolonged exposures, adaptation at the molecular
level takes place. Time-course studies are needed to confirm this.
In developing herring eggs, the enzyme phosphoenol-pyruvate carboxykinase in­
creases in activity by about two orders of magnitude during the time between the early
blastodisk and just before hatching. When herring eggs were exposed to 10 ppm cadmium
during this time, a decreased activity was observed (Mounid et al., 1975). Somewhat less
extensive decreases in enzyme activity were also noted for the NAD- and NADP-malic
enzymes and propionyl-CoA-carboxylase. Rosenthal and Alderdice (1976; p. 2055) point
out that: “Since the enzymes studied are involved in biosynthetic processes, it is specu­
lated that the depressive effect of cadmium on activity of the enzymes explains why
cadmium exposure results in smaller, inactive larvae at hatching”.
Long-term exposures of scorpionfish (a marine species) to cadmium resulted in no
changes in alkaline phosphatase, succinic dehydrogenase, or glyceraldehyde phosphate
dehydrogenase in several tissues (Bay et al., 1990). In other words, they obtained results
similar to those of Roberts et al. (1979) on trout. However, they did detect a severe
 223

(eightfold) inhibition of Cu-Zn-superoxide dismutase in intestine with a trend (although

not significant) toward less of this enzyme in the other tissues. It might be speculated that
the high sensitivty of the intestine might be due to the fact that this is a marine species
which must drink water to correct for osmotic loss. Freshwater fish would not have that
mode of cadmium entry into the body.
The dismutase enzyme helps protect cells from superoxide anion radicals produced
during oxidative metabolism so lipid peroxidation of membranes might follow cadmium
exposure. Bay et al. (1990) further present evidence that cadmium displaced copper and
zinc from the enzyme pool of the cells and thereby disrupted copper and zinc homeostasis.
It also should be noted that the same laboratory (Brown et al., 1989) has found that the
subcellular distribution of cadmium differs greatly depending on whether exposures are
acute or chronic.
While cadmium seemingly has a relatively small effect on oxidative enzymes, mer­
cury has a considerable effect on these enzymes. Lactate, pyruvate, and succinic dehydro­
genase enzymes are all key control points in the flow of carbons through glycolysis and
the citric acid cycle. Mercury is apparently a strong inhibitor of these enzymes as
exposure of the freshwater teleost, commonly called the murrel, for 60 days to 3 ppb of
mercury caused a greater than 85% inhibition of lactate and pyruvate dehydrogenases in
muscle and of succinic dehydrogenase in gill (Sastry and Rao, 1981). Such a level of
inhibition would certainly slow the ability to produce ATP, especially when the fish is
under some sort of stress or when forced to swim (see MacLeod and Pessah, 1973).
In a follow-up study, Sastry and Rao (1982) measured the activities of several
enzymes in six different tissues of the murrell following 15, 30, and 60 days of exposure
to 3 ppb of mercury. Most of the changes seen were in the 60-day samples and are
summarized in Table 2 . The percent change from control fish varies between different
tissues; brain and muscle seem to be the least affected, while kidney and liver show the
greatest changes. This is most likely due to differential rates of uptake and retention of
mercury in these tissues (see Chapter 5). This illustrates how it would be useful in future
investigations of this sort to have simultaneous measures of the tissue concentrations of
xenobiotic chemicals.
Inhibition of glucose-6-phosphatase and hexokinase (Table 2) would cause a reduction
in both glycogenolysis and gluconeogenesis (i.e., the breakdown of glycogen and the
synthesis of glucose from non-carbohydrate molecules, respectively). Thus, we have a
possible mechanism for the decreases in blood glucose of fish exposed to mercury (Sastry
and Rao, 1981), instead of the hyperglycemia which is generally observed in fish under
stress (see section on carbohydrate metabolism in Chapter 8). Such a reduction in the
availability of carbohydrate for energy was partially compensated for by increases in the
activity of glutamate dehydrogenase and L-amino acid oxidase, both of which are central
to the utilization of amino acids for energy. In a different study on the same species.

Table 2 Percent Change in Enzyme Activity Following 60 Days Exposure of

the Freshwater Murrel, C h a n n a p u n c ta tu s , to 3 ppb of Mercuric Chloride
Brain Gill Intestine Kidney Liver Muscle
Glucose-6-phosphatase ns 39 48 40 37
Hexokinase ns ns ns 52 49 ns
Malate dehydrogenase 27 29 18 27 34 33
Glutamate dehydrogenase ns ns 41 30 51 ns
L - Amino acid oxidase 23 43 ns 36 33 ns
Xanthine oxidase ns ns 52 ns 38 ns

N o te : ns indicates no significant change.

224

Sastry and Sharma (1980) observed inhibition (10-43%) of several key enzymes involved
in energy metabolism in the brain.
Recently, Shaffi (1993) reported on activities of lactate dehydrogenase, succinic
dehydrogenase, and malate dehydrogenase in three freshwater species following 24- or
48-h exposures to mercury. For all three enzymes the greatest inhibition occurred in liver
with progressively less inhibition in muscle, brain, kidney, and gill, in that order. Unfor­
tunately, the actual concentration of mercury used is not specified. Because there was a
marked inhibition of enzyme activity in 24 h, it must have been fairly high, even though
the author refers to it as “sublethal”.
According to the findings of Verma et al. (1983), Na,K-ATPase is inhibited by
mercury more in brain tissue than gill, liver, or kidney. Thus, even though the brain does
not accumulate much mercury compared to other tissues (Massaro, 1974), at least one of
the enzymes there is quite sensitive to the metal. In spite of this seeming anomaly, there
is indirect evidence that accumulation of mercury may be an important variable in several
of the other tissues. In nearly all the above-mentioned studies, in addition to the long-term
exposures, observations were also made at shorter time intervals, such as 7 or 15 days.
In most cases, there were non-significant changes seen in enzyme activity at those shorter
exposure periods which could reflect the time required for the metal to accumulate in the
tissues (see Chapter 5).
It is well known that mercury in natural waterways is frequently methylated by
microbes located in sediments (Bryan, 1976). More relevant to the laboratory studies
discussed in this review is the observation that the liver of fish may also methylate the
element, although déméthylation can also occur (Bryan, 1976). The reason for raising this
point is that there is some evidence that methylmercury and mercury may not always act
in the same manner in cells. For example. Fox et al. (1974) found inhibition of Kreb’s
cycle activity in brain slices of guinea pig when subjected to methylmercury but not when
exposed to the inorganic form of mercury at the same concentration. Thus there is a
possibility that the inhibition of Kreb’s cycle enzymes found in these fish studies may be
due to methylmercury rather than inorganic mercury. Furthermore, Southward et al.
(1974) claim that inorganic mercury did not affect ATPase activity in their in vitro
mammalian cell extract studies, so the inhibition of ATPase could also be due to
methylmercury. However, the two studies mentioned were done in vitro using mamma­
lian tissues, so their relevance to fish in vivo activity is uncertain.
The enzyme delta-aminolevulinic acid dehydrase catalyzes the first step in heme
synthesis and the ultimate formation of hemoglobin. A depression in the activity of this
enzyme is used in human medicine as a diagnostic indicator of chronic or acute lead
poisoning (DeBruin, 1976). Not surprisingly, when fish are exposed to lead, this enzyme
is inhibited (Jackim, 1973; Johansson-Sjobeck and Larsson, 1979). For the most part, this
effect is limited to lead although mercury may also cause a slight inhibition (Jackim, 1973).
Silver and zinc actually stimulated the liver to produce more enzyme (Jackim, 1973) so
delta-aminolevulinate dehydrase inhibition seems to be relatively specific for lead poi­
soning in both fish and humans. This is one of the only examples of a specific action of
a contaminant. Johansson-Sjobeck and Larsson (1979) observed that 30-day exposures of
trout to 75 ppb lead caused a 74% lowering of the activity of this enzyme but the hemoglobin,
hematocrit, and red blood cell count remained unchanged. So delta-aminolevulinate dehydrase
must have a large “safety factor” in its activity and/or the rate of turnover of erythrocytes in
fish may be relatively slow. Because they are nucleated, their lifespan is probably greater than
those of mammals who have an average lifespan of 3 months.
Moving on to copper (Table 1), a 24-h exposure of the green snakehead (Ophiocephalus
punctatus) to this element at a concentration approximating 1/10 the 48-h LC50 concen­
tration produced a 24 and 37% inhibition of acid phosphatase in the liver and kidney,
respectively (Srivastava and Pandley, 1982). The effect on alkaline phosphatase was, on
 225

the other hand, rather different. There was a 10% inhibition in the liver, but a 20%
stimulation in the kidney. Gill et al. (1992) also observed a stimulation of alkaline
phosphatase in kidney (and intestine). Srivastava and Pandley (1982) speculate that since
this enzyme is involved in glucose reabsorption in the kidney tubules, there may be an
enhanced activity of it because of the high glucose levels that occur in the blood during
exposure to copper. This kind of response is, however, not limited to the kidney or gut,
or to this element. Recall that Sastry and Sharma (1980) observed a stimulation of this
same enzyme by mercury in the brain.
An increase in acid phosphatase was seen after copper exposure in some tissues
(Srivastava and Pandley, 1982; Gill et al., 1992). This enzyme is associated with lysoso­
mal activity and Gill et al. (1992) speculate that its elevation reflects proliferation of
lysosomes in an attempt to sequester the toxic copper ions. They note that Weiss et al.
(1986) have found large secondary and tertiary lysosomes in fish following exposure to
copper and hepatic copper tends to be sedimented in this fraction of homogenate.
Copper exposure causes mobilization of liver glycogen into blood glucose (Chapter 8).
At the enzyme level, this is seen in the stimulation of glycogen phosphorylase
(Radhakrishnaiah et al., 1992). Copper also seems to inhibit oxidative metabolism at both
the whole animal level (Chapter 8) and at the enzyme level and this is compensated for
by a stimulation of glycolysis which is reflected in an enhanced lactate dehydrogenase
and glyceraldehyde dehydrogenase activity (James et al., 1992; Radhakrishnaiah et al.,
1992; Balavenkatasubbaiah et al., 1984).
Limited evidence suggests that chromium may have little effect on enzymes of energy
metabolism (Buhler et al., 1977). This element did inhibit the enzyme Na,K ATPase in
kidney and intestine, but not in gill or liver. Na,K ATPase is critical in the function of the
so-called “sodium pump” located in cellular membranes. From these results, one could
predict that chromium would cause loss of sodium in the urine of freshwater fish and a
reduction in the uptake of sodium from the water, thereby producing an altered electrolyte
homeostasis (Chapter 7).

IV, ENZYME EFFECTS FROM ORGANIC CHEMICALS

Most of the information on the action of organic chemicals is, as is to be expected, on
pesticides (Table 3). Before considering them, however, mention should be made of the
petroleum hydrocarbons. There has been a great deal of physiological work done on their
effects in fish and invertebrates (see Anderson, 1979). Enzymatic studies have mostly
been directed at understanding the biotransformations and detoxification reactions (see
Chapter 5). Little attention has been paid to their possible impact on enzymes associated
with energy metabolism. Heitz et al. (1974) exposed mullet to a crude oil for 4 days and
then assayed 13 enzymes in several tissues. They found essentially no significant changes.
With crude oil it is difficult to determine what the LC50 is, but the authors note that the
concentration they used is well below the lethal level. Perhaps it was below the threshold
for any enzyme effects, or as they concluded, crude oil has little effect on the enzymes
of energy metabolism in fish.
Such a conclusion may depend on the species of fish and the doses of petroleum used
in experiments. Elevation (Davison, 1992) and suppression (Prasad, 1987) of whole-
animal metabolic rates have been reported upon exposure to sublethal concentrations of
petroleum components. Prasad (1987) also reported a severe dose-dependent suppression
of in vitro tissue respiration rate gill, liver, kidney, and muscle excised from oil-exposed
carp. Thus, there is a high probability that petroleum hydrocarbons would suppress
enzyme activity.
Pentachlorophenol is a powerful uncoupler of oxidative phosphorylation in mitochon­
dria (see Rao, 1978 for review). When eels were exposed for 4 days to this pesticide.
 IO
IO
o>

Table 3 Changes in Enzyme Activity Following Exposure of Fish to Test Chemical

Chemical Enzyme(s) Effect Species Exposure Concentration Organ Ref.
Crude oil 13 enzymes ns M u g il c e p h a lu s 4d ca. 4 ppm Muscle, brain, liver and gill 1
Pentachlorophenol Pyruvate kinase and lactic dehydrogenase < A n g u illa a n g u illa 4d 0.1 ppm Liver 2
Pentachlorophenol Hexokinase, G-6-P04, dehydrogenase, > A . a n g u illa 4d 0.1 ppm Liver 2
fumerase cyto. oxidase, succinic and
pyruvate dehydrogenase
Pentachlorophenol Succinic and pyruvate dehydrogenase < N o tr o p te r u s 30 d 2.8-8.3 ppb Brain, liver, gill 3
Pentachlorophenol Lactic dehydrogenase > N o tr o p te r u s 30 d 2.8-8.3 ppb Brain, liver, gill 3
Parathion Aldolase and phosphorylase “a” > T ila p ia m o s s a m b ic a 48 h 1/3 LC50 Gill, liver, brain 4
Parathion Phosphorylase “a” < T. m o s s a m b ic a 48 h 1/3 LC50 Muscle 4
Parathion Phosphorylase “b” < T. m o s s a m b ic a 48 h 1/3 LC50 Gill, liver, brain 4
Metasystox Succinic dehydrogase < C h a n n a s tr ia tu s 30 d 1/3 LC50 Gill, brain, muscle, liver, 5
kidney
Metasystox Lactic dehydrogenase > C. s tr ia tu s 30 d 1/3 LC50 Muscle, brain, gill, liver, 5
kidney
Quinalphos Hexokinase, lactate, pyruvate and succinic < C. p u n c ta tu s 30 d 0.025 ppm Liver, gill, brain 6
dehydrogenases
Malathion Acid and alkaline phosphatase < B r a n c h y d a n io 7d 0.5-1.1 ppm Liver 8
Phosphamidon Alkaline phosphatase > P u n tiu s c o n c h o n iu s 48 h 96 h LC50 Gill 7
Phosphamidon Acid phosphatase < P . c o n c h o n iu s 48 h 96 h LC50 Liver 7
Phosphamidon Two transaminases > P . c o n c h o n iu s 48 h 96 h LC50 Heart, muscle 7
Phosphamidon Lactic dehydrogenase < P . c o n c h o n iu s 48 h 96 h LC50 Heart, liver, muscle, gill 7
Aldicarb Alkaline phosphatase > P. c o n c h o n iu s 48 h 96 h LC50 Kidney, gill 7
Aldicarb Two transaminases > P . c o n c h o n iu s 48 h 96 h LC50 Gill, heart 7
Aldicarb Lactic dehydrogenase <> P. c o n c h o n iu s 48 h 96 h LC50 >Gill, <heart 7
Carbofuran Acid and alk. phosphatase > C h a n n a p u n c ta tu s 6m 4.5 ppm Liver 9
Endosulfan Acid and alk. phosphatase > P . c o n c h o n iu s 48 h 96 h LC50 Kidney, liver, ovary 7
Endosulfan Lactic dehydrogenase < P . c o n c h o n iu s 48 h 96 h LC50 Heart, muscle, liver 7
Endosulfan Lactic and malic dehydrogenase < C la r ia s b a tr a c h u s 7d 0.001 ppb Liver, muscle 10
Endosulfan Acid and alk. phosphatase < C hanna gachua 15-30 d 2.25-5.6 ppb Liver, kidney, muscle 11
Dieldrin Glutamate dehydrogenase < S a lm o g a ir d n e r i 240 d Dietary Brain 12
Dieldrin Glutamate dehydrogenase > S. g a ir d n e r i 240 d Dietary Liver 12
Glutamine synthetase
Dieldrin Glutamate synthetase > S. g a ir d n e r i 240 d Dietary Brain
Paraquat Glycogen phosphorylase > S. g a ir d n e r i 1-7 d 0.5-10 ppm Liver 13
G-6-Phosphatase

N o te : Reference numbers are as follows: 1. Heitz et al, 1974; 2. Bostrom and Johansson, 1972; 3. Verma et al., 1982; 4. Rao and Rao, 1983; 5. Natarajam, 1984; 6. Sastry
et al, 1982; 7. Gill et al., 1990; 8. Kumar and Ansari, 1986; 9. Ram and Singh, 1988; 10. Shukla and Tripathi, 1991; 11. Sharma, 1990; 12. Mehrle and Bloomfield,
1974; 13. Simon et al., 1983.

lo
ro
228

Bostrom and Johansson (1972) observed increases in several enzymes of the citric acid
cycle, the respiratory chain, and the pentose shunt but decreases in the enzymes of glyco­
lysis (Table 3). This is to be expected as there would be a need for increased respiration if
the efficiency of ATP synthesis was reduced because of the uncoupling mentioned above.
The opposite results were obtained by Verma et al. (1982) on Notopterus in which it
was found that glycolysis was stimulated and aerobic respiratory enzymes were inhibited
by PCP. They make no attempt at reconciling these differences with previous work (they
are not even mentioned). Rather their findings are interpreted as indicating that the PCP
induced a hypoxic condition in the fish by damaging the gill tissue. An unpublished Ph.D.
dissertation is cited in support of this hypothesis. The work on eels mentioned above
involved even a more acute type of exposure so one would think that such a treatment
would have caused hypoxia there too. Krueger et al. (1966) obtained results on a cichlid
that support the Bostrom and Johansson (1972) work. Other than a species difference,
there does not appear to be an obvious explanation for the contradictory results.
Organophosphorus (OP) insecticides are well-known inhibitors of acetylcholine­
sterase in a variety of organisms. This aspect of organophosphorus action is discussed
in Chapter 12. There is accumulating evidence that they also cause some interesting
effects on cellular enzymes, especially those involved in energy metabolism in fish
(Table 3).
Parathion has been found to cause an increase in active phosphorylase “a” in a variety
of tissues except muscle, where the enzyme activity went down after parathion exposure
(Rao and Rao, 1983). The inactive phosphorylase “b” enzyme decreased in the different
tissues, but again muscle was the “non-conformist” in that it remained unchanged there.
These phosphorylase changes are required for the rapid mobilization of glycogen and this
phenomenon was observed in the same study. It seems paradoxical that muscle enzymes
did not respond enzymatically in the same way as the other tissues, since there was a 50%
loss in muscle glycogen (Rao and Rao, 1983). Parathion also caused an increase in
aldolase, and since this is a key enzyme in glycolysis, there must have been an increased
utilization of glucose via that metabolic pathway in the liver.
Other work (Natarajan, 1984), also on an OP insecticide (metasystox), has shown an
inhibition of succinic dehydrogenase, an enzyme of Kreb’s cycle, but a stimulation of the
glycolytic enzyme lactate dehydrogenase. Under these circumstances, an increase in
lactate should occur, however, this metabolite was not measured. Natarajan (1984) cites
work done in India which showed that Sevin® (a carbamate insecticide) also decreased
the activities of Kreb’s cycle enzymes. And finally, the OP pesticide sumithion has been
shown to suppress these oxidative enzymes while stimulating lactic dehydrogenase
(Koundinya and Ramamurthi, 1979).
Increased glycolysis and reduced oxidative metabolism can be interpreted in at least
two different ways. Natarajan (1984) attributes these alterations to gill damage by the
insecticide resulting in “histotoxic anoxia”. No data on gill histology are presented, but
an increased red blood cell count and hemoglobin content of the blood was noted,
responses frequently observed in hypoxic fish (see Chapter 2). However, when one
measures enzyme activity, the maximum rate of activity is actually measured and there
seems little reason for oxidative enzyme activity to decrease as a result of a hypoxic
condition. More to the point, in vitro studies of OP insecticides suggest a direct inhibition
of the pesticide on the enzymes involved in aerobic respiration (Hiltibran, 1974), and
Channa striatus has the ability to breath air so gill damage should not be as much of a
problem for it as it might be for some other teleost.
Sastry et al. (1982) reported inhibition of both aerobic and glycolytic enzymes in most
of the tissues assayed from snakehead fish which had been exposed chronically to
quinalphos. Unfortunately, they make no attempt at reconciling their seemingly contra­
dictory findings. It is noteworthy that they also found decreases in blood glucose, lactic
 229

acid, and hemoglobin along with increases in glycogen. Thus, the response they report to
this insecticide is almost exactly opposite to that seen with other organophosphates!
A large dose-dependent inhibition of alkaline and acid phosphatase was found in livers
of zebra danios exposed to malathion (Kumar and Ansari, 1986). The investigators
attribute the decrease in acid phosphatase to leakage of lysosomal enzyme into the
plasma, an interesting hypothesis supported by other work where elevations were seen in
this enzyme in the plasma of chickens exposed to malathion. They go on to point out that
alkaline phosphatase inhibitions have several possible explanations including the fact that
they have serine residues at active sites. Organophosphorus insecticides inhibit enzymes,
such as acetylcholinesterase, which possess this characteristic.
The OP insecticide phosphamidon also caused a decrease in acid phosphatase activity,
but an increase in alkaline phosphatase (Gill et al., 1990). They attribute the stimulation
of the latter enzyme to glucocorticoid influence but the evidence is tenuous at best. The
transaminase enzyme stimulation could be associated with gluconeogenesis which is
stimulated by glucocorticoids so it is surprising that liver transaminase activity was not
affected by phosphamidon (Gill et al., 1990), for that organ is where traditionally most
gluconeogenesis occurs.
Phosphamidon caused a strong inhibition of lactate dehydrogenase from all tissues
examined by Gill et al. (1990). Such a response is probably due to leakage from the cells
into the plasma. It is perhaps noteworthy that the same authors found a marked stimula­
tion of this same enzyme from heart when the pesticide was added directly in vitro.
The carbamate insecticides seem to cause a consistent stimulation of phosphatase
activity in a variety of tissues (Ram and Singh, 1988; Gill et al., 1990). Aldicarb also
stimulated transaminase activity in several tissues but inhibited lactate dehydrogenase in
the same tissues (Gill et al., 1990).
The organochlorine (OC) insecticides are no longer used much in industrialized
countries because of their persistence in the environment, but they still get heavy usage
in many so-called “third world” countries. Nearly all the studies of enzyme effects in fish
have been conducted in vitro, with all the weaknesses associated with that methodology.
The general conclusion from that work is that these insecticides reduce metabolic energy
production by cells (see Hiltibran, 1982 for review).
With in vivo exposure to the OC insecticide endosulfan. Gill et al. (1990) found
marked inhibition of lactate dehydrogenase in several tissues. This was confirmed in a
different species by Schukla and Tripathi (1991) who also found inhibition of malate
dehydrogenase, a Kreb’s cycle enzyme. The latter workers found that the fish synthesized
new enzyme over a period of 28 days if transferred to non-contaminated water, thus the
loss of activity was not permanent. They point out that low doses of OC pesticides are
antithyroid so the effect on the enzymes of cellular energy metabolism may in part be
mediated by decreases in these thyroid hormones. Support for this hypothesis was
obtained when they treated their fish with the thyroid hormones T3 and T4 and got normal
enzyme activity even after endosulfan exposures.
The evolution of insecticide resistance by insect populations has been a continuing
problem that even gets into the popular press. Less well known is the resistance which
is developed by some mosquitofish {Gambusia) populations exposed for many genera­
tions in nature to a particular pesticide. Ferguson (1967) concluded this was genetically
based. A biochemical mechanism for it was proposed by Yarbrough and Wells (1971)
who were able to show that the membrane of mitochondria in livers and brains from
resistant mosquitofish apparently excluded the OC endrin from contact with the dehydro­
genase enzymes therein. When they disrupted the mitochondrial membranes by freezing
and thawing, they got inhibition of succinic dehydrogenase by endrin in mitochondria
from both susceptible and resistant fish, but when the mitochondria were intact, there was
no inhibition of this enzyme if the mitochondria were obtained from endrin-resistant fish.
230

Overall, the limited evidence suggests that OC compounds inhibit various enzymes
associated with the release of energy in cells. The action on alkaline and acid phosphatase
enzymes appears to depend on duration of exposure. Short term, the effect was to
stimulate activity (Gill et al., 1990) whereas exposures of 15 or 30 days (Sharma, 1990)
caused a consistent strong inhibition that was dose-dependent (as much as 66%). For OC
compounds which have very long persistence in the environment, such long-term expo­
sures are probably more realistic than those short term. However, for OP insecticides with
their limited persistence, exposures for only a few days may be quite realistic.
Dieldrin given in the diet at several doses to rainbow trout caused a decreased brain
glutamate dehydrogenase activity, however, the activity of this enzyme increased in the
liver (Mehrle and Bloomfield, 1974). The higher doses caused the concentration of
ammonia in the brain to rise, which is to be expected when the ammonia detoxifying
enzyme is suppressed. The authors suggest that part of the mechanism of dieldrin
intoxication is due to ammonia acting on the brain. Ammonia is certainly toxic to the
brain as it seems to inhibit ATP production of nerve cells (see Tomasso et al., 1980 for
review).
A variety of ATPases have been found to be sensitive to OC insecticides when tested
in vitro (Hiltibran, 1982). In some cases even stimulation is observed (Hiltibran, 1982).
However, Davis et al. (1972) made the important observation that in vivo exposure of
trout to oral doses of DDT that produced death in 36 h caused no significant changes in
Na,K ATPase from gills, brain, and kidney. Thus, the in vitro effects were not confirmed
in the intact animal.
Paraquat is a widely used herbicide. When carp were exposed to doses ranging from
0.5-10 ppm, there was a doubling of the liver phosphorylase activity in fish from the
higher concentrations. Glucose-6-phosphatase activity in the liver also was elevated some
40-50%. Using a dose of 0.5 ppm for several days produced a gradual rise in these
enzyme activities reaching a fourfold rise at 7 days for the phosphorylase (Simon et al.,
1983). No information was given on the LC50 for this chemical, but the fish would appear
to have been under considerable stress as these enzyme activations would mobilize
carbohydrate. The authors also cite unpublished work that showed that paraquat caused
increased lactate dehydrogenase activity. All these changes mean a mobilization of
energy from glycogen and an enhanced rate of glycolysis which were probably mediated
by increased epinephrine and cortisol.

V. CONCLUDING COMMENTS ON ENZYME EFFECTS

From the above discussion, we see that a variety of enzymes can be affected by a given
substance, and many substances affect the same enzyme. Stimulation of an enzyme may
occur in one tissue while inhibition is seen in another, within the same species of fish.
Thus, with a few exceptions, the idea of there being a single mode of action at the enzyme
level is generally not a viable concept for a wide variety of pollutants. Of course cells have
a multitude of different enzymes so further work may yield some examples of specific
metabolic lesions induced by certain chemicals such as the inhibition by lead of delta-
aminolevulinic acid dehydrase. In order to make meaningful interpretations, there is a
need for measures of the concentration of xenobiotic chemicals in the same tissues in
which enzyme activity is assayed. Furthermore, partitioning of the chemical into specific
organelles should not be overlooked.
The effects seen in the in vivo studies reviewed herein probably involve a combination
of endocrine influences (especially the stress hormones cortisol and epinephrine), direct
action of the chemical on enzyme molecules, and/or enzyme induction by inhibition of
negative feedback. While in vitro methods may indicate relative sensitivities of different
 231

enzymes to the direct action of a particular chemical, they probably have little relevance
to the “real world” because they may or may not simulate the concentration that the
enzyme is exposed to in vivo, and of course, the other two potential mechanisms
mentioned above are eliminated in an in vitro preparation.
Finally, at the risk of sounding unintentionally provincial, it should be noted that the
majority of the in vivo work has been done in India, using local species of fish, many of
which are air breathers. Whether similar findings will be obtained on other freshwater and
marine teleosts remains to be seen.

VL ANTIOXIDANTS
Oxygen free radicals are one, two, or three electron reduction products of molecular
oxygen. They include the superoxide anion radical (02~), hydrogen peroxide, and the
hydroxyl radical ( OH). They are produced as by-products of many cellular reactions
including both cytoplasmic (e.g., xanthine oxidase, mixed function oxidase) and mito­
chondrial (e.g., electron transport chain) enzymes. These oxyradicals can cause consid­
erable cellular damage including oxidations of membrane lipids, proteins, and nucleic
acids. The polyunsaturated fatty acids of membranes are especially susceptible to
peroxidation and the techniques for the detection and measurement of this have been
reviewed by Gutteridge and Halliwell (1990). The oxidative lesions in cells are also
related to induction of tumors in fish (Malins and Haimanot, 1991).
Normally, the accumulation of oxyradicals is controlled by antioxidant enzymes and
low molecular weight molecules such as glutathione and ascorbate. Metallothionein,
which is recognized as a metal detoxification agent, has also been suggested as a possible
antioxidant (Shi, 1990).
A wide variety of organic and inorganic toxic contaminants cause an increased
production of oxyradicals in cells (Stegeman et al., 1992). This in turn serves to induce
production of more of the antioxidant enzymes superoxide dismutases, catalases, peroxi­
dases, glutathione reductase, and glutathione. Ascorbate is also involved in antioxidant
activities and is discussed in this book in Chapter 6 while glutathione is taken up in
Chapter 5. Here we limit our discussion primarily to the antioxidant enzymes.
Basal antioxidant enzyme activities tend to be higher in tissues which exhibit high
oxidative metabolism. These include red muscle, the gas gland in the swim bladder, heart,
and blood (Filho et al., 1993). Enzyme activity levels also differ between fish species;
those that are more physically active tend to have the higher activities (Filho et al., 1993).
When trout were exposed to cadmium for 180 days at concentrations 13 and 25% of
the LC50, elevations in liver superoxide dismutase were seen. This induction of the
antioxidant enzyme could be interpreted as a response to an increase in oxyradicals. On
the other hand, glutathione reductase went down and catalase was unchanged (Palace et
al., 1993). Ascorbate also showed a marked decline with cadmium exposure presumably
due to its utilization as an antioxidant. In the same study, they observed that a diet
deficient in ascorbate caused elevations in superoxide dismutase in the absence of
cadmium, which the authors suggest may be due to a compensatory antioxidant enzyme
induction.
When dab (a flatfish) were exposed to sediments contaminated with polynuclear
aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs) for 25 to 140 days, the
activity of all three antioxidant enzymes declined in both contaminated and non-contami-
nated (reference) fish. However, superoxide dismutase, catalase, and lipid peroxidation
were higher in contaminated fish compared to those from the reference sediments after
80 days, but not at 140 days. Glutathione peroxidase did not change (Livingston et al.,
1993). The authors conclude that the PAHs and PCBs caused transient oxidative stress.
232

Fish sampled from areas along the coast of Spain contaminated with iron, copper, and
several aromatic hydrocarbons exhibited elevations in antioxidant enzymes when com­
pared with a reference site (Rodriquez-Ariza et al., 1993). Enzymes which increased
included superoxide dismutase, glutathione peroxidase, catalase, and glutathione reduc­
tase. The largest increase was in glutathione peroxidase, especially the Se-dependent
isoenzyme. This enzyme is particularly important in protection against lipid peroxidation
and seems to have been successful in these fish as little lipid peroxidation was detected.
These observations exemplify an important concept in that toxic chemicals may induce
various types of biochemical changes that are an adaptation to the chemical stressors.
These include detoxification enzymes such as those associated with cytochrome P450,
metallothionein, and the antioxidant enzymes described here. It is useful, as was done in
the study just mentioned, to determine whether the adaptive changes are successful in
reducing or eliminating toxicity.

VII. ADENYLATES
The release of energy from organic foodstuffs in cells by the various metabolic processes
occurs in a stepwise fashion. The energy so obtained is “stored” temporarily in high
energy phosphate bonds. The term adenylates refers to the nucleotides adenosine tri­
phosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).
Creatine phosphate, while being a high-energy phosphate molecule, is not a nucleotide,
although it is often measured along with the adenylates. Clearly, ATP has the most energy
stored in it so the relative concentrations of these adenylates reflects the energy imme­
diately available to the cell for muscular contraction, maintenance, ionic pumping,
biosynthesis etc. They also act as modulators of enzyme activity in glycolysis and the
Kreb’s cycle (Atkinson, 1977). The adenylate energy charge (AEC) is the ratio of ATP +
0.5 (ADP) to total adenylate concentration in the tissue. This value can vary from 0-1.
In invertebrates and microorganisms, unstressed individuals show an AEC above about
0.8, and decreases in the AEC have been useful in assessing environmental stressors in
these forms (Atkinson, 1977; Ivanovici, 1980; Livingston, 1985).
ATP is the most variable component of the adenylate pool. In fish it decreases during
muscular exercise (Schulte and Hochachka, 1990) and hypoxia (see Chapter 2), and
different temperatures can result in significant changes (Walesby and Johnston, 1980;
Kindle and Whitmore, 1986). For the most part, ADP and AMP change comparatively
little with these kinds of alterations in activity or environment. With decreases in ATP,
one might expect a rise in AMP, but this does not necessarily occur because it can be
converted to inosine monophosphate (IMP).
Seasonal changes in adenylates can be quite large (Dehn, 1992). The redear sunfish
(a centrarchid) showed the lowest muscle AEC (0.5-0.75) in the winter but liver dropped
to below 0.5 in April during the spawning season. Dehn also noted that liver exhibited a
lower AEC than muscle at all times of the year.
MacFarlane (1981) tested the effect of water pH on brain, muscle, gill, and liver tissue
adenylate concentrations of gulf killifish (F. grandis). The test organisms were exposed
for periods of up to 96 h at four different pHs ranging downward from the control level
of 7.8. The greatest decreases in ATP, total adenylates, and AEC were observed in brain
and gill. There were relatively small changes in total adenylate and energy charge
compared to ATP which decreased as much as 72%. The effect of hydrogen ions in the
water on brain ATP appeared to be dose dependent and this depression increased with
time of exposure, as well.
McFarlane (1981) explained the declines in ATP and total adenylates in the brain as
being due to decreased sodium levels in the blood induced by the acid. He cites a study
on fish electric organs to the effect that neural activity causes release of ATP into the
 233

extracellular fluid which is then degraded extracellularly to adenosine before being taken
back into the cells. When the sodium concentration is reduced, this uptake of adenosine
is inhibited. Another factor (discussed by MacFarlane) which probably contributes to
lowered ATP in tissues other than brain is an elevated energy demand in the gills from
pumping hydrogen ions out of the fish and in the muscles from the considerable muscular
activity caused by the acid.
Copper at high sublethal levels has been shown to cause a lowering of ATP in liver
and muscle (Heath, 1984). Bluegill (Lepomis macrochirus) were exposed to copper at a
concentration equivalent to the LC20 and tissues sampled at intervals of up to 96 h of
exposure. Significant decreases in ATP occurred first at 48 h with the greatest changes
in liver. Brain exhibited no change in ATP. Measurements of tissue lactic acid established
that tissue hypoxia did not occur, therefore other hypotheses to explain the lowered level
of ATP must be proposed. Heath (1984) noted increases in tissue water content concomi­
tant with the drop in ATP. It was calculated that this dilution of the cell contents
accounted for 25-30% of the decrease in nucleotide concentration. The remainder of the
decrease is attributable to one or more of the following causes: (1) bluegill exposed to
sublethal copper show elevated liver tissue energy demand (Felts and Heath, 1984),
presumably due to detoxification processes and increased demand for energy by cells
generally pulls ATP down (Walesby and Johnston, 1980); (2) when fish liver mitochon­
dria are exposed to copper {in vitro), there is a large increase in permeability and oxygen
consumption (Zaba and Harris, 1978), so a direct effect of copper on mitochondrial
respiration is probable; (3) inhibition of some enzyme systems probably occurred (Table
1); and (4) copper may alter cellular concentrations of glutathione. Stacey and Klaassen
(1981) noted a lowering of glutathione in rat hepatocytes exposed to copper {in vitro).
Schole (1982) has proposed a central role for glutathione in the regulation of energy
metabolism in cells so changes in the concentration of this compound could have large
effects on these chemical reactions. This latter mechanism is, of course, highly specula­
tive.
There seems to have been little work on the effects of pesticides on adenylates. The
carbamate insecticide carbofuran and the synthetic pyrethroid fenvalerate had only slight
effects on these parameters during 10-day exposures to doses a bit less than one half the
96-h LC50 (Hohreiter et al., 1991). This relatively small effect may in part be due to the
failure of these substances to be taken up by the fish during short-term exposures. Because
some insecticides certainly affect energy metabolism at the molecular level (Table 2) and
whole-animal metabolism (Chapter 8), it seems likely that adenylates would also be
affected as well. The kinetics of the response may be as important to reveal as the overall
effect.
There has been some interest in the use of adenylates as biomarkers in field and
laboratory studies (partially reviewed in Mayer et al., 1992). Thus far, mollusks seem to
yield useful results better than fish. In part, this is probably because of the effect of
struggling during capture on muscle adenylates. This can be considerable in fish and
occurs within seconds so accurate baseline values are extremely difficult to obtain
(Schulte and Hochachka, 1990). Another problem is the large effect of season on these
parameters which was discussed earlier. In general, it seems that substances that accumu­
late in the liver, such as copper, or some of the lipophilic organics, might have by far the
greatest effect on adenylates there. Also, liver adenylates are not affected by handling
stress so variability should be less (assuming spawning season is avoided).

VIII. STRESS PROTEINS

The stress proteins are a group of cellular proteins whose synthesis increases when the
cell is subjected to any sort of stress. They were first discovered in Drosophila that had
234

experienced a fairly rapid increase in temperature and were therefore called “heat shock
proteins” (hsp; Welch, 1993). It is now known that the induction of these molecules is
much broader than just from heat so the name “stress proteins” is becoming more
accepted. There are four major groups of stress proteins based on their size and these are
designated hsp90, 70, 60, and 16-24. The newer terminology is “stress 90”, “stress 70”,
and “chaperonin”, for the 60-kDa family and low molecular weight (LMW) for the 16-
to 24-kDa group (Sanders, 1993).
Stress proteins are not limited to stressed cells. They are found in nearly all cells and
their function is to regulate folding, assembly, and aggregation of other proteins. They are
often collectively called chaperones for this reason. A primary mechanism of toxicity
involves protein dénaturation which results in molecular aggregation and misfolding
(Hightower, 1991). This causes binding of a protein called heat shock factor (HSF) to
controlling genes (promoter) which then activate transcription of the stress protein genes.
Synthesis of stress proteins then occurs to facilitate the repair of the denatured proteins.
As the proteins are repaired, stimulation of the HSF declines slowing the induction
process so the feedback loop is completed.
The specific stress proteins induced vary with different tissues, probably in part due
to different tissue sensitivities to particular toxicants. Within a tissue, the stress protein
induction process appears to be non-specific (i.e., all stressors cause essentially the same
response although the kinetics may differ). Also, work with a number of groups other than
fish indicates that stress 70, chaperonin, and the LMW stress proteins have different
isoforms and their induction may differ with the specific stressor. Future studies may
reveal more stressor-specific proteins involved in other types of cellular repair as well
(Sanders, 1993).
Part of the process of acclimation to a toxicant may involve stress proteins. In a variety
of animal cells, it has been found that previous treatment with a heat shock or toxicant
stress makes the cells more tolerant of a subsequent stress (Welch, 1993). This is,
incidentally, separate from the induction of detoxifying enzymes such as those associated
with cytochrome P450 or metallothionein which are discussed in Chapter 5.
Thus far, research on stress protein induction relative to water pollution has largely
been limited to work on mollusks and grass shrimp (reviewed in Sanders, 1993). The
situation will undoubtedly change rapidly in the future, although the techniques for
measuring induction are currently difficult for those not familiar with molecular biology
methodologies. The potential for future utilization of the stress protein response is great
because it should integrate damage due to multiple stressors while at the same time
possibly yielding diagnostic information as to the cause of the damage (Sanders, 1993).

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Shi, C., Metallothionein as a scavenger of free radicals. T r a c e E lem . M e d ., 1 , 48, 1990.
Shukla, S. and Tripathi, G., Endosulfan toxicity on metabolic enzymes and protective role of
triiodothyroine in the catfish C la r i a s b a tr a c h u s , P e s tic . S c i., 32, 363, 1991.
Simon, L. M., Nemcsok, J. and Boross, L., Studies on the effect of paraquat on glycogen mobilization
in liver of common carp {C y p r in u s c a r p io L), C o m p . B io c h e m . P h y s io l, 75C, 167, 1983.
Smith, J. C. and Chong, C. K., Body weight, activities of cytochrome oxidase and electron transport
system in the liver of the American plaice H ip p o g lo s s o id e s p la te s s o id e s . Can these activties serve
as indicators of metabolism?. M a r. E c o l P r o g . S e r., 9, 171, 1982.
Somasundaram, B., King, P. E. and Shackley, S., The effects of zinc on the ultrastructure of the brain
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Southward, J., Nitisewojo, P. and Green, D. E., Mercurial toxicity and the perturbation of the mitochon­
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Stacey, N. H. and Klaassen, C. D., Copper toxicity in isolated rat hepatocytes, T o x ic o l A p p l P h a r m a c o l,
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 Chapter
10
Acid Pollution
I. INTRODUCTION
Acid precipitation has become a widespread and increasing problem in several countries
in Europe and North America. Acid falls as rain and snow and even dry gases and small
particles. Some people may be surprised to learn that the latter two account for approxi­
mately two thirds of the total acid precipitation in the U.K. (Mason, 1989). The major
acids of concern are sulfuric and nitric which are formed in the atmosphere from
emissions of coal-fired power plants and automobiles.
The pH of uncontaminated rainwater is not neutral, instead due to equilibrium with
carbon dioxide, it is around 5.6. Rain and snow even in remote areas are almost always
more acidic than this and in areas where air pollution is acute, the pH can easily fall below
5. The effect on streams and lakes depends greatly on the hydrogeology (Henriksen, 1982;
Norton, 1982). Those bodies of water surrounded with soils or rock rich in base minerals
(e.g., Ca/Mg carbonates and bicarbonates) are relatively immune from effects of acid
precipitation. In large areas of North America and Scandinavia, the buffering capacity of
the fresh surface waters is extremely limited so over the past decades there has occurred
a rapid (in the historic sense) drop in their pH.
Atmospheric pollutants are deposited episodically such that there is usually an autumn
and a spring decline in pH. The autumn decline coincides with the spawning and early
embryonic development of species like lake trout (Salvelinus namaycush), lake white fish
(Coregonus clupeaformis), and lake herring (C. artedii) (Peterson et al., 1982). Very rapid
spring reductions of pH can occur due to snow melt and this may coincide with the late
embryonic and/or larval development of fall-spawned eggs. As will be seen later, the
larval (alevin in salmonids) stages are often the most sensitive developmental stage to
acute acid stress. Furthermore, the spring spawning run of many species such as the
rainbow trout may occur at a time of very low ambient pH.
The reductions in pH of surface waters mobilize metals, especially aluminum (Baker,
1982). The increases in aluminum from acid precipitation are generally from natural input
due to the ubiquitous distribution of this element. Because fish are more often than not
receiving the impact of both acid water and aluminum, in recent years there has been a

239
240

tendency for laboratory studies to combine these. This chapter will, however, emphasize
pure acid toxicity with some reference to action of aluminum where appropriate. Alumi­
num is also discussed in some detail in Chapter 7.
The sensitivity of different fish species to low pH differs, even between different
salmonids. For example, cutthroat trout {Oncorhynchus clarki), and golden trout
(O. aguabonita) are probably the most sensitive of the family while rainbow trout are
intermediate followed by brook {S. fontinalis) and brown trout (Salmo truta) (Delonay et
al., 1993). As a general rule, warmwater species are more tolerant of acid conditions than
are salmonids.
The literature on acid rain is immense and certain aspects of the physiological effects
of acid toxicity to fish have been reviewed by Fromm (1980), Peterson et al. (1982),
Wood and McDonald (1982), Howells (1984), and Wood (1989). Throughout this book,
individual pollutants have been discussed within the context of how they affect particular
organs or physiological processes. Here we devote a whole chapter to the one pollutant
because of the extensive information available. In addition, it gives an opportunity to
attempt to integrate several physiological processes in the context of one pollutant. The
organization of this chapter will therefore be different from others in this book. Instead
of the usual normal physiology followed by a consideration of the effects of pollutants
on that physiology, we will instead use a life-cycle approach. We will start with spawning
and then consider the effects of acid (and often aluminum) on the different life stages. For
those relatively unfamiliar with basic fish physiology, reference to the early parts of the
chapters on reproduction, respiration, and osmoregulation may be helpful.

II. SPAWNING
Failure to lay eggs when the mature adults are subjected to chronic low pH has been
noted in many studies involving both salmonids and non-salmonid species (e.g., Weiner
et al., 1986; Peterson et al., 1982; Tam and Pay son, 1986). Some reduction in numbers
of eggs laid has been seen at nearly any pH below control levels, thus there seems to
be no overall threshold pH that can be applied to all fish species, or even to all
salmonids. Moreover, the effects of a lowered pH on egg production is also very
dependent on environmental calcium levels; the lower the calcium, the greater the
effect (Parker and McKeown, 1987). It has been suggested that the primary physiologi­
cal mechanism for decreased numbers of eggs is an inhibition in the production of yolk
proteins (Peterson et al., 1982).
Many female fish exhibit elevations in plasma calcium during ovarian maturation.
This elevation may be important in the process of vitellogenesis. Vitellogenin, a phos-
phoprotein synthesized in the liver, is transported to the ovaries as a calcium-vitellogenin
complex for use in yolk synthesis. Female white suckers from acidified lakes were
reported to have abnormally low blood calcium levels (Beamish et al., 1975). This often
cited reference has been used to explain failure of reproduction in fish residing in
acidified waters. However, Munkittrick (1991) has pointed out that the calcium levels in
the Beamish et al. (1975) female fish did not differ significantly from controls. Instead,
Beamish et al. based their conclusion on the ratio of female to male calcium level, and
it turns out that the four male control fish had abnormally low concentrations of calcium
in their blood. Indeed, Weiner et al. (1986) found no effect on plasma calcium of low pH
per se in the laboratory on rainbow trout females, although it did cause a reduction in
calcium in the males. This finding can be contrasted with that of Parker and McKeown,
(1987) in which a low pH caused a failure of female rainbow trout to exhibit the normal
surge in plasma calcium during oogenesis. They also noted that low calcium levels in the
water reduced the plasma concentration of vitellogenin and lowered the gonadosomatic
 241

index (an indication of reduced egg production). It was concluded that the depressed
calcium availability inhibited vitellogenin formation by the liver. More recently, Roy et
al. (1990) utilized a homologous radioimmunoassay to measure vitellogenin in rainbow
trout at three pHs (7.6,5.6,4.5) during spawning. There was a surge in vitellogenin during
the 20-day exposure period in the pH 7.6 and 5.6 groups, but the fish in pH 4.5 failed to
exhibit the surge. Thus, low pH can suppress vitellogenin formation, but whether calcium
is involved may depend on the species, hardness of water, and pH.
Assuming there is a reduction in blood calcium associated with low pH water, the
question may be asked as to what is the mechanism of this reduction? Limited data
suggest that sublethal low pH alone does not affect the activity of Ca^+ ATPase in the gills
so calcium uptake from the environment may not be a critical problem, rather it appears
that increased calcium loss from the body may be more important (Parker et al., 1985;
Parker and McKeown, 1987). A further mechanism that could be involved is the effect
of acid on sex hormone levels in the female fish. Estradiol induces hypercalcemia in
maturing female fish (Whitehead et al., 1978), but Weiner et al. (1986) reported no effect
of pH 4.5, 5.0, or 5.5 on female sex hormones during the final 6 weeks of reproductive
maturation in rainbow trout. Interestingly, in contrast to just about everybody else, they
also saw no effect of pH on plasma calcium levels in female fish.
The above discussion suggests that egg production per se is quite sensitive to reduced
pH. However, a recent study casts some doubt on that hypothesis. Mount (1988) worked
with brook and rainbow trout in soft acid water and observed reduced fecundity, but this
was due to decreased growth in the female. Egg production per unit body weight of the
female was actually normal in those exposed to acidified water, but those fish experienc­
ing stress, as reflected in loss of plasma sodium and decreased feeding, showed a severely
reduced body size. Mount also made the important observation that progeny from brook
trout that had been in very low calcium were more sensitive to acid than were those
spawned in water with higher calcium levels. He concludes that recruitment failure in soft
acid waters is not due to failure of egg production but is instead due to the progeny being
excessively sensitive to subsequent acid.
This hypothesis gets some support from the work of Weiner et al. (1986) who found
that exposure of rainbow trout females or males to acid waters (pH 4.5-5.5) during
reproductive maturation causes the subsequent eggs and larvae to be more sensitive to low
pH . This occurs even if only one of the parents was exposed to acidity (Weiner et al.,
1986). It is tempting to speculate that this increased sensitivity of progeny might be due
to reduced vitellogenin which could result in a decreased yolk deposition. However, since
exposure of males only also produced increased sensitivity (although not as severe), other
mechanisms are additionally possible.
Androgen levels in male salmon have been shown to be sensitive to low pH. Normally,
these hormones undergo a considerable surge just before spawning. Examination of fish
collected from two rivers (pH 5.6 and 4.7) revealed a reduction in this surge in those from
the low pH river (Freeman and Sangalang, 1985). The cause of this could be due to a
generalized stress response and reduced feeding in the acid-exposed fish, although direct
effects on either gonadotropin release or sex hormone synthesis are not excluded (Free­
man and Sangalang, 1985; Wendelaar Bonga and Balm, 1989).
Sperm motility and fertilization has also been shown to be sensitive to low pH. Daye
and Glebe (1984) reported that duration of movement by Atlantic salmon sperm declined
in a linear fashion with pH until about pH 4.5. Below this point, motility rapidly declined
to zero. Fertilization success was unaffected by a pH above 5 but at pH of 4 it was zero.
Even a very short exposure duration can have a marked effect on subsequent develop­
ment. An acute exposure to pH 5 at the time of fertilization caused high mortality in early
development in both salmonids and cyprinids (Gillet and Rombard, 1986).
242

III. EMBRYONIC DEVELOPMENT AND HATCHING

As embryonic development of fish eggs proceeds, sensitivity to acid changes. The most
sensitive stage is early cleavage before organ development occurs (Peterson et al., 1982).
At this time, the zona radiata (chorion) acts as a reservoir for hydrogen ions rather than
a barrier allowing diffusion of these ions into the perivitelline fluid. Apparently, this thick
membrane decreases in permeability to hydrogen ions later so as the embryo develops,
resistance to acid waters increases by about 0.5 pH units (Daye and Garside, 1979).
However, sublethal morphological effects on the developing embryos of Atlantic salmon
occur at a pH as high as 5. These effects include abnormal development of various organs
such as gills, brain, and heart (Peterson et al., 1982). At hatching there is a big increase
in sensitivity to acidity which emphasizes the protective effect of the chorion (Siddens et
al., 1984).
Newly shed salmon eggs take up water until they become turgid (water hardening).
This process is inhibited when the pH is 4.5 or lower (Peterson and Martin-Robichaud,
1982). The developing embryo is surrounded by perivitelline fluid which is encased in the
chorion. This fluid accounts for about 7% of the egg and contains macromolecules which
facilitate water hardening. It also has some buffering capacity so it, along with the
chorion, help protect the developing embryo from hydrogen ions (Eddy and Talbot,
1985).
When the embryo reaches the eyed stage it begins to accumulate sodium from the
environment. This process is severely inhibited at a pH of 4 (higher pHs were not tested).
Eddy and Talbot (1985) showed that uptake only is affected and there is no effect of the
acid on sodium efflux from the egg. The perivitelline fluid acts as an “ion trap” binding
sodium and other ions thus reducing efflux (Eddy and Talbot, 1985).
Studies of the direct effect of low pH on hatching are made more difficult by the fact
that pH adversely affects development of the embryo so a delay or failure in hatching
could be due to that. A key step in the hatching process is the softening of the egg capsule
which is catalyzed by a hatching enzyme. This enzyme is apparently a protease and like
all enzymes exhibits an optimum pH. Several studies (cited in Peterson et al., 1982) have
shown this optimum to be above pH 7, yet the pH of the perivitelline fluid (in spite of its
buffering capacity) is generally only 0.5 pH units above that of the ambient water
(Peterson et al., 1980). Inhibition of chorionase activity at pH 4.5 delayed or prevented
hatching (Waiwood and Haya, 1983). Problems with hatching at low pH may, in addition
to chorionase enzyme inhibition, be due to reduced embryo activity because of the effects
on development mentioned above (Peterson et al., 1980).

IV. LARVAE FROM HATCHING THROUGH SWIM-UP

The physiological process most affected in larvae by acid (or aluminum) is probably
ionoregulation. During larval development in a wide variety of species, there normally
occurs a marked uptake of sodium, potassium, and calcium from the water while at the
same time body magnesium declines (Peterson et al., 1982; Wood et al., 1990a,b). The
ionic uptake apparently occurs via chloride cells in the yolk-sac epithelium, gills, and skin
(Hwang and Hirano, 1985). Wood et al. (1990a) demonstrated that brook trout fry
exposed continuously from fertilization to swim-up to either acid water (pH 4-5.2) or acid
water plus aluminum failed to take up ions at the normal rate. However, perhaps a more
important finding of this study was that the environmental calcium concentration influ­
enced the net electrolyte accumulation much more than either pH or aluminum. De­
creased uptake of electrolytes could have been due to altered ion exchange rates or to
reduced rate of development of the larvae, or both. Of course, as Wood et al. (1990a) point
out, the rate of development might be limited by the net electrolyte accumulation. They
 243

conclude that both development and ion exchange rates are important with the latter
dominating in the yolk-sac stage.
The importance of environmental calcium for electrolyte uptake in larvae is striking,
and at pH 4.8, a calcium level of 8 mg/L provides almost complete protection from the
acid (Wood et al., 1990a). This level of calcium is at the upper end of what is usually
considered the soft water range which, unfortunately, is not generally found in areas
where acid stress occurs.
Spawning brook trout have been shown to actively avoid low pH by selecting areas
of alkaline upwellings from groundwater in which to build their redds (Johnson and
Webster, 1977). Thus, the young hatchlings avoid the most severe acid conditions until
emerging from the gravel. Because this emergence may occur at the time of snow melt,
there has been some interest in the effect of acid and/or aluminum exposures at that time.
When brook trout fry at the yolk-sac stage were exposed for 21 days to several different
combinations of acid, aluminum, and calcium. Wood et al. (1990b) found that water pH
was the primary determinant of whole-body ion status, rather than calcium as in the
above-mentioned study. In part this is due to the fact that in the previous study, eggs and
early larvae did not survive the lowest pHs so no electrolyte data were obtained on them.
Nevertheless, to quote Wood et al. (1990b, p. 1612): “Fry surviving a continuous 91-day
exposure from fertilization will maintain an ionic status most dependent upon the water
Ca during exposure, while alevins surviving a 21-day challenge in the yolk-sac or swim-
up phase will exhibit a status most dependent upon the pH of the challenge.” They go on
to emphasize that the latter is probably the more environmentally realistic as calcium is
usually low in waters that are impacted by acid. Because growth was not influenced by
pH, aluminum, or calcium in the yolk-sack and swim stages, the authors conclude that the
mechanism of ionoregulatory failure is depressed active uptake and changes in perme­
ability, rather than a reduced development.
A surprising finding of the Wood et al. (1990a,b) studies was the protective effect of
aluminum at intermediate concentrations. Ionoregulatory failure was reduced at alumi­
num concentrations of 37 and 111 |ig/L. Indeed, in the exposures at the later larval stages,
it seemed to stimulate electrolyte uptake so that aluminum exposed fish had higher ion
concentrations than controls. At aluminum levels higher and lower than these, the metal
had a harmful effect on ionoregulation. The same laboratory had earlier shown a protec­
tive effect of aluminum in adults from acute acid exposure. The physiological basis of this
phenomenon is unclear; long-term exposures of adult salmonids to aluminum causes
proliferation of chloride cells in the gill epithelium (Tietge et al., 1988). If this occurred
in the larvae, it would facilitate uptake of electrolytes, but it is not known if a similar
adaptation to aluminum occurs in larvae, and the bimodal nature of this response to
aluminum is perplexing.
Exposures to acid or aluminum do not necessarily need to be prolonged to result in
significant effects on subsequent development in some species. When lake trout embryos
were exposed for 5 days to aluminum (100, 200 |ig/L) in water of pH 5, there were no
mortalities, but alevins examined after 21 or 32 days in water lacking aluminum were
smaller, had less calcified skeletons, and whole-body calcium and potassium concentra­
tions were down by 18-22% (Gunn and Noakes, 1987). Evidently, the short exposures
caused permanent damage to ionoregulatory mechanisms as well as to feeding ability and
yolk utilization.
A number of studies (see De Lonay et al, 1993 for references) have documented acid
effects on various aspects of larval behavior. The most consistent finding in both salmo­
nids and centrarchids is a reduction of spontaneous swimming activity. This would
probably contribute to reduced feeding and therefore growth. Furthermore, the ability to
maintain position in a current was reduced in brook trout exposed to acid waters (Cleve­
land et al., 1986).
244

V. JUVENILE AND ADULT: BLOOD ACID-BASE BALANCE AND

ELECTROLYTE CHANGES FROM ACUTE EXPOSURES
The arterial pH can be affected by ambient water pH, but the calcium concentration in the
water very much controls this effect. At a low calcium level, lowering the water pH
around rainbow trout to 4.3 (equal to the 7-day LC50 for this species) has no effect on
arterial pH. On the other hand, a considerable acidosis occurs at higher calcium levels (see
Figure 1). Even with a decrease in blood pH as occurs in high calcium acid water, there
is little effect on intracellular pH of brain, heart, or skeletal muscle. Thus, the mechanism
by which acid causes death at realistic pHs in trout is not acidosis (Wood, 1989).
Acid may act on carp {Cyprinus carpio) a bit differently than it does on trout. At a
pH of 4 and an intermediate level of calcium in the water carp experienced a drop in
arterial pH concomitant with electrolyte alterations. Ultsch et al. (1980) attributed this
blood pH drop largely to a loss of base through the gills. It appears then that the impact
of acidified water on blood pH differs with the species and may be more severe in
warm water species.
Numerous studies (beginning with Packer and Dunson, 1970) of fish exposed acutely
to acid have shown a considerable drop in blood or whole-body electrolytes (see reviews
by Wood, 1989; McDonald et al., 1989). Both influxes and effluxes of sodium and
chloride through the gills are affected; but of the two, however, influx may be the more
sensitive process. Influx is inhibited and efflux stimulated so the net effect is a massive
loss of ions from the body. This becomes progressively worse at lower pHs so that at a
pH of approximately 4 in trout influx is completely stopped and efflux is increased almost
threefold over that of controls at pH 7. Wood (1989) concludes that Na+ influx is inhibited
by acid due to the competition of H"^ with Na+ for transporter at the gill. The inhibition
of chloride uptake is more obscure because it is not clear to what extent, if any, this
process is linked to sodium transport. Normally, chloride in the water is exchanged for
bicarbonate and hydroxyl ions in the blood so there may be a depletion of these in the gill
cells of fish in acid conditions, or the chloride carrier may be damaged by the acidified
water (Wood, 1989).
The large increases in outward electrolyte diffusion are believed due to a massive
increase in ion permeability produced by the leaching of calcium away from the paracellular
channels in the gill epithelium (McDonald et al., 1989). The tight junctions between
pavement cells and chloride cells in the gills of rainbow trout are very sensitive to changes
in pH (Freda et al., 1991). These changes can occur very rapidly (i.e., within minutes of
exposure) and may involve only minor changes in permeability of the gills to water
(Jackson and Fromm, 1980).
As with other physiological process affected by acid, water hardness (expressed as
CaC03 ) is an extremely important variable. Figure 2 illustrates the effect that water
hardness has on body sodium of white suckers {Catostomus commersoni) exposed to acid.
Note that the exposure duration for the fish in pH 4 soft water was for only 1.5 days
compared to 6-19 days for the fish in hard water. This was because in soft water at pH
4, there was 100% mortality within 48 h, but only 16% in hard water at the same pH after
19 days. The pumpkinseed {Lepomis gibbosus) is a more acid-tolerant species than is the
white sucker. When it was exposed to pH 4 water, (Fraser and Harvey, 1984) it eventually
lost the same amount of sodium as did the sucker, but the latter lost sodium at an 11-fold
faster rate. These observations support the suggestion (McDonald, 1983) that lethality
from acid water is determined more by the rate of sodium loss than the total amount.
Milligan and Wood (1982) have proposed that the etiology of death in acid water can
be traced to the ionoregulatory failure discussed above, although the final cause is
actually circulatory, rather than ionic. In this proposed sequence of events, (Figure 3), if
the initial branchial ion loss causes a rapid drop in plasma Na'^ and/or Cl" by more than
 245

♦ 0.2
ApM .
0 %

a - 0.2
UJ
E
-0.4 -

A h ^hi
0

♦4 0

♦8 0

0
A'
10
j APIasma (Na^]
a
UJ
E -20
- " f '
f j
-30

0
APlasma [ c r ]
-10
O’ ir '
UJ
E -20

-30

0 1 2 3
Watar (ca“'^] (mEq L)

Figure 1 Relationship between concentration of calcium in the water and the extent of acid-
base and ionic disturbances in the arterial blood of adult rainbow trout exposed for 3 days to pH
4.3. (From Wood, C., in, Acid Toxicity and Aquatic Animals, Morris, R., Taylor, E., Brown, D. and
Brown, J., Eds., Cambridge University Press, New York, 1989, 125. With permission.)

30%, it triggers a reduction in plasma volume due to water loss to the environment and
tissues. The increased hematocrit and plasma protein produce a rise in blood viscosity
which in turn causes arterial blood pressure to rise. Hematocrits as high as 70% and
massive increases in blood viscosity have been observed in fish exposed to acid (Wood
and McDonald, 1982). Wood (1989) has suggested that catecholamines may facilitate
these various changes, a point supported by the findings of Ye et al. (1991) in which they
measured increases in catecholamines during acid exposure in rainbow trout. Wood
(1989) further points out that the blood acidosis seen when fish in hard water are exposed
to a low pH is probably secondary to the electrolyte loss as a cause of death. This
mechanism seems to apply to all fish species and even amphibians (Freda and McDonald,
1988).
246

Figure 2 Whole-body sodium concentration in white sucker exposed to acidified soft water
(Ca = 0.207 mE/L) and hard water (Ca = 2.11 mE/L. Exposures were 6-9 days except for pH
4 in soft water which was for 36 h. Points are means of 15-19 fish except for pH 4 in soft water
where N = 4. (Redrawn from Fraser, G. A. and Harvey, H. H., Can. J. Zoo!., 62, 249, 1984.)

Tolerance to low pH varies between species and at least some of the mechanisms
involved have been revealed that facilitate this tolerance. In trout and shiners (Notropis
cornutus), both rather sensitive to acid, calcium definitely moderates the rate of sodium
loss, but Freda and McDonald (1988) have found that it has little effect on the loss of
sodium from yellow perch {Perea flavescens), a rather acid-insensitive species. This and
other evidence suggests that calcium displacement from the gill by ions does not occur
in the perch as it does in other species which are less able to tolerate acid waters.
The sunfish {Enneacanthus obesus) is rather acid tolerant, even more so than other
members of the genus (Gonzalez and Dunson, 1989). Its tolerance rests in part with the
fact that it has gills which have a high affinity for calcium so it is not displaced as readily
by ions. In addition, however, this species compensates for sodium losses by shifting
this element from bone into the extracellular fluid. In addition, low ambient pH causes

Acid . Branchial Circulatory Failure ?

Exposure Ion Loss

Figure 3 Proposed sequence of events through which environmental acid exposure may
cause death of a fish. See text for discussion. (From Wood, C. M. and McDonald, D. G., Acid
Rain/Fisheries, Johnson, R. E., Ed., American Fisheries Society, Bethesda, MD, 1982,197. With
permission.)
 247

decreases in body water and potassium concentration which helps to reduce the net
movement of water from the extracellular fluid compartment into the intracellular fluid.
Electrolyte losses via the kidney are small in soft acid water, probably because of fluid
shifts into the muscle intracellular compartment and a subsequent rise in plasma oncotic
pressure which causes urine volume to fall (Wood, 1989). In hard water, however, it
should be recalled that a rapid influx of H+ through the gills occurs. Under these
circumstances, the kidney plays an important role in excreting the excess hydrogen ions.
Thus, the response of fish to acid in hard water is one of the few examples in which the
teleost kidney plays a significant roll in acid-base regulation (Heisler, 1989).
The above discussion regarding responses to acute exposures simulates conditions
such as a rapid snow melt or heavy precipitation of low pH rain. A more gradually
induced acidification may shift threshold pHs to a lower level. For example. Van Dijk et
al. (1993) exposed carp to pH 4 water over 4 h and then monitored them for 48 h. Contrary
to the findings of Ultsch et al. (1981) where severe losses of plasma ions and a decline
in plasma pH occurred in carp with a rapid drop in water pH, they saw no changes, nor
were there any changes in stress hormones. A somewhat similar finding was reported by
Balm and Pöttinger (1993) with rainbow trout which were gradually exposed to pH 4
waters. Thus, the kinetics of the acid exposure may have a big effect on the ultimate
response. This may be a nice example of the need to consider the “psychological” aspects
of a stress (Schreck, 1981). The sudden imposition of any sort of change in a fish’s
environment may induce a stress response out of proportion to the severity of the stressor.
This is a point of which we physiologists need to be more cognizant.

VI. JUVENILE AND ADULT: BLOOD CHANGES FROM

CHRONIC EXPOSURE
Many fish experience a chronic type of exposure that may last for many days or even
months and years. Basically, there are two types of response that might occur with this
prolonged exposure: ( 1 ) there may be a chronic depression of plasma electrolytes that
persists more or less indefinitely or (2 ) over time in the acidified water there may be a
partial or complete compensation indicating the fish were successful in adjusting to the
acid water. Both types of response have been seen, but the character of the response
depends on species and on the presence of other ions such as calcium or aluminum. In
addition, a third possibility must be considered in that populations may become geneti­
cally selected for better tolerance of acid waters.
Juvenile Atlantic salmon were kept in soft water of pH 4.5-5.2 for one or three months
and it was found that ionoregulation was impaired throughout the whole time (Lacroix et
al., 1985). Moreover, the fish exhibited reduced growth and branchial ATPase activity
and failed to undergo smoltification (Johnston et al., 1984). When adult rainbow trout
were exposed to pH 4.8 in soft water for three months there was an initial net loss of
sodium and chloride (Audet et al., 1988). By 30-50 days of continuous exposure to the
low pH, a new equilibrium was reached in which influx and efflux rates were both lower
than in controls and plasma levels of these ions were stabilized at an abnormally low, but
not lethal level. Plasma glucose rose steadily during the exposure reaching a maximum
of 20 mmol/L which is approximately seven times the control level. No acid-base
disturbance was seen but ammonia excretion rate increased with time, which suggests an
increased protein catabolism. The extreme hyperglycemia along with the increased
ammonia excretion rate suggests the fish were under a considerable degree of stress
(Audet et al., 1988). In a subsequent study using the same experimental regime (Audet
and Wood, 1993), an elevated cortisol level was found throughout the exposure time but
catecholamines remained unchanged. Prolonged sublethal acid stress apparently weakens
the fish for, if they are subsequently exposed to a lethal pH level, they lose electrolytes
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more rapidly and are less tolerant of the acid than those that had not received a pre­
exposure. Thus, rainbow trout do not acclimate to low pH alone (Audet and Wood, 1988).
Plasma potassium and calcium remained unchanged in the Audet et al. (1988) study.
The lack of an effect on calcium is interesting in light of the debate mentioned in the
beginning of this chapter concerning the relationship between a presumed decline in
blood calcium and reproductive failure in female salmonids from acid lakes.
There has been recently published an apparent contradiction to the conclusion that
rainbow trout cannot acclimate to acid. Balm and Pöttinger (1993) exposed trout to pH
4 for 14 days in soft water and found an increased turnover rate of choride cells, little
change in plasma electrolytes, no increased sensitivity to confinement stress, and no
elevation in cortisol. In other words, their fish seemed to adjust to the acid fairly well,
although they did not test their tolerance to a subsequent more acute acid stress. They
were using two European strains of rainbow trout whereas the Audet and Wood work was
done with Canadian fish. This may or may not explain the discrepancy in the results.
Another difference is that Balm and Pöttinger subjected their fish to a gradual lowering
of the pH whereas Audet and Wood’s fish experienced a more rapid decline.
The ability to acclimate to low pH is quite species dependent. African Tilapia showed
fairly good recovery of osmolarity when exposed to pH 3.5 by day 15 of exposure but
goldfish did not (Wendelaar Bonga et al., 1984). There are also populations within a trout
species that are more resistant to acid waters (McWilliams, 1982), but since acclimation to
acid by trout may not occur, this is probably due to genetic selection (Swaarts et al., 1978).
Adaptation to acid by salmonid fishes can certainly occur in the presence of hard water
(see Audet et al., 1988 for references), a condition which is not common in areas receiving
acid precipitation. Interestingly, aluminum is frequently associated with acid pollution
and it seems to actually aid adaptation by fish to these conditions. Wood et al. (1988a)
and Wilson and Wood (1992) have found that if sublethal concentrations of aluminum are
combined with a chronic acid stress, the fish show an initial ionoregulatory dysfunction
which may be greater than with acid alone, but then as time goes on, those with the
combination of aluminum and low pH show partial or complete restoration of normal
plasma sodium and chloride. Also, fish acclimated to acid + aluminum are able to better
tolerate a subsequent challenge with a lower pH and high concentrations of aluminum
(Wood et al., 1988b). Thus, trout in soft water can acclimate to aluminum but not acid
alone. Indeed, Wood et al. (1988b) propose that low levels of aluminum may be beneficial
to fish under chronic acid stress.
The mechanism of this beneficial effect of chronic aluminum exposure seems to rest
with a proliferation of chloride cells (McDonald and Milligan, 1988) and reduced perme­
ability of the branchial epithelium. This latter phenomenon may be due to epithelial
swelling (Tietge et al., 1988) which increases the blood to water diffusion distance. Such
a change would probably reduce the transfer capacity for oxygen which would limit
exercise ability, a point taken up later.
Yellow perch {P.flavescens) is a species with high acid tolerance, but even within this
species, there are populations that have diverged physiologically (Nelson and Mitchell,
1992). Environmental acidity may act synergistically with swimming to upset blood acid-
base. Thus, it is interesting that fish from populations in acid lakes were able to maintain
acid-base balance in their blood better than those from circumneutral lakes when exer­
cised in acid waters (Nelson and Mitchell, 1992).
Exhaustive exercise increases the toxicity of acid waters to trout (i.e., increases the
lethal pH) (Graham and Wood, 1981). This is not surprising as severe exercise depresses
blood pH, cellular pH, and causes losses in blood ions via the gills; it can even cause death
of fish in circumneutral pH waters (Wood et al., 1983). In addition, a fish actively
swimming in acid waters brings much more water in contact with its gills than when at
rest. When carp were exposed gradually to pH 4 water, they exhibited no alterations in
 249

blood electrolytes, even after 24 h. However, if they were forced to swim constantly at
two body lengths per second, while being gradually subjected to the acid water, they
exhibited a steady decline in plasma pH, Na+, and Cl~, so the increased gill water flow
exacerbated the effect of acid (Van Dijk et al., 1993).
Warmwater species of fish frequently experience a greater seasonal temperature
change than do salmonids. This can have a serious implication if they are also exposed
to low pH conditions. When young largemouth bass (Micropterus salmoides) were
exposed to acid waters along with a temperature simulating overwintering (3.8°C), they
lost blood osmolality more than did those exposed to the acid at a warmer temperature.
McCormick and Jensen (1992) propose that the first winter for juveniles may be the most
critical stage in the life cycle in northern populations which experience both severe winter
and acid conditions.
Before leaving this section, brief consideration should be given to what happens to fish
when exposed to alkaline conditions. The critical alkaline pH for salmonids appears to be
between 9 and 10. Acute exposure to these conditions causes an inhibition of ammonia
excretion by the gills and respiratory alkalosis (i.e., elevation of plasma pH). The
inhibition of ammonia excretion is “...probably due to a reduction of the diffusion
gradient for NH3 across the gills when the environmental pH approaches the negative
logarithm of the dissociation constant for ammonia” (Wilkie and Wood, 1991, p. 1070).
When rainbow trout were exposed to pH 9.5 for 72 h, they initially showed alkalosis but
blood pH was eventually stabilized around 8, in part by accumulation of lactate even
though blood oxygen was not limiting. Perhaps more interesting was the observation that
nitrogen excretion shifted toward formation of urea, but even so, ultimately, plasma
ammonia stabilized at a level about six times that of controls (Wilkie and Wood, 1991).
Changes in plasma sodium and chloride were comparatively minor with alkaline expo­
sure. Wilkie and Wood (1991) point out that while the rainbow trout seems to be able to
adapt to pH 9.5, it is not without cost so the fish may become more susceptible to death
from other causes.
There is a strain of cutthroat trout {O. clarki henshawi) that thrives in Pyramid Lake,
NV, at pH 9.4. Apparently these fish have a lower rate of ammonia excretion, a higher
proportion of nitrogen excretion via urea, a higher rate of renal excretion, a lack of
coupling between ammonia excretion and sodium influx, and a high plasma ammonia
level which facilitates diffusive excretion of ammonia across the gills (Wright et al.,
1993). When these fish were challenged with water of pH 10, they exhibited a doubling
of plasma ammonia concentration and urea excretion and a decreased level of plasma
sodium chloride. Survival was poor during 72-h exposures and those that did not survive
showed highly elevated plasma ammonia and severely depressed sodium and chloride
(Wilke et al., 1993). Thus, these fish which are living at pH 9.4 are existing at near their
limit which may be exceeded in the future as Pyramid Lake becomes more desiccated.
There is an African species of Tilapia living in a pH of around 10 and is able to do this
in part because it excretes its nitrogen primarily as urea (Randall and Wright, 1989).

VII. HORMONAL RESPONSES

Cortisol secretion rate increases rapidly with acid exposure (Wendelaar Bonga and Balm,
1989), as it does with most environmental stressors. In Tilapia, the cortisol level returns
to normal in a few days even though the fish is still in the acid water. This is not due to
a decrease in secretion rate of the interrenal cells, however. Instead, there is an elevation
in peripheral cortisol clearance rate which could be due to metabolism of the hormone in
the liver or excretion by the kidney. Such an enhanced rate of peripheral clearance of
cortisol was not found in brook trout exposed to high acidity. Instead, Wood et al. (1988b)
found high cortisol levels even after 10 weeks of continuous exposure in this species.
250

The elevated cortisol levels seen in acid-exposed fish are probably in response to the
ionoregulatory dysfunction since cortisol stimulates chloride cell proliferation, and Na,K
ATPase activity in the gills (Wendelaar Bonga and Balm, 1989). Furthermore, the cortisol
elevates blood glucose which reduces the osmoregulatory load produced by the loss of
sodium and chloride ions.
Two hormones from the anterior pituitary have been shown to be sensitive to acid
stress: prolactin and arginine vasotocin. Prolactin rises in fish experiencing acid exposure
and it can remain elevated for months (Wendelaar Bonga and Balm, 1989). This hormone
is important in freshwater fish where it reduces ion and water permeability of epidermis
and gill epithelium (Bern, 1983).
Arginine vasotocin may or may not be involved in osmoregulation (Evans, 1993). The
concentration of this hormone is generally measured in the pituitary rather than the blood
because of extremely low concentrations in blood. Hontela et al. (1991) showed that this
hormone rose in concentration in brook trout whenever acid exposure was sufficient to
lower plasma sodium levels. The same laboratory (Hontela et al., 1993) went on to test
this hormone as a biomarker for acid stress in feral fish. It appears to have some promise
but they had to develop ANCOVA models to adjust for body size and season, which have
a considerable effect on the pituitary levels of this hormone.
Hormones from the caudal neurosecretory system are also stimulated by acid waters
(Hontela et al., 1989). These hormones along with cortisol, prolactin, and possibly
vasotocin are all involved in regulating blood ion concentrations so their increase in fish
experiencing osmoregulatory dysfunction is probably an attempt to restore homeostasis.
Thyroid activity has also been shown to be sensitive to acid exposure. In general, a
decrease in the blood level of thyroid hormones is seen and they may or may not show
recovery when the fish is transferred to circumneutral pH waters (Brown et al., 1990). The
mechanism by which acid waters depress thyroid activity is currently unknown.
Mention has already been made at several points about catecholamines. Changes in the
level of these hormones are probably very much influenced by the degree of stress the fish
is psychologically perceiving. Insofar as the regulation of plasma electrolytes is con­
cerned, catecholamines may not necessarily be considered a homeostatic group of hor­
mones because they increase gill blood perfusion and increase the area of the gill lamellae
in direct contact with the water (Wendelaar Bonga, 1993). Thus, catecholamines could
actually do more harm than good for fish in acid waters, although this hypothesis needs
direct confirmation.

VIII. VENTILATION AND BLOOD GASES

Measuring ventilation in fish, especially salmonids, requires an assessment of both
breathing frequency and depth (i.e., stroke volume), for the latter may change while the
former does not. Better still is to measure the actual ventilation volume but this requires
a two- (or three-) compartment chamber in which the fish can be restrained with a
membrane around the head separating the mouth from the postopercular waters. For
example, Janssen and Randall (1975) found that rainbow trout in low pH increased their
ventilation volume by increases in ventilatory stroke volume with little change in
breathing frequency. The response to the low pH was not immediate, but instead
developed gradually which may have been due to a production of mucus on the gills
which reduced oxygen transfer. Neville (1979) found that ventilation increased with
mild acid exposure in this species only if there was a rise in external PCO2 and Janssen
and Randall (1975) found that this gas was a more powerful stimulant to ventilation
than was acid alone.
 251

The observation that CO2 is such a strong stimulant is important in that addition of H+
to water can cause the PCO2 to rise due to the reaction H+ + HC03" > H2CO3> CO2+ H2O.
Thus the increases in ventilation often observed with elevated acidity may in fact be due
to the elevated CO2 (Giles, 1984; Neville, 1979; Walker et al., 1988).
Arterial P02 seems to be relatively unaffected by low but sublethal pH levels in soft
water. Indeed, it may actually increase with acid exposure due to the carbon dioxide-
induced hyperventilation just discussed (Walker, 1988). If the water pH is low enough,
however, mucus formation on the gill surface may restrict oxygen diffusion and cause a
decrease in arterial P02 (see Chapter 3). The threshold for this in carp is between pH 3.5
and 4.0 (Ultsch et al., 1980). It is not clear where that threshold is for salmonids, but in
soft water it must be above pH 4.8 (Walker et al., 1988). In hard water, it is probably
higher than in soft water as alterations in gas exchange seem to be greater in hard water
at a given pH (Wood et al., 1988b). Also, the addition of aluminum at 300 p,g/L causes
a marked drop in arterial P02 of brook trout in pH 4.8 (Wood et al., 1988b).
Increases in arterial PCO2 cause a drop in HBO2 due to the well-known Bohr effect.
The pH of red blood cells does not necessarily fall as much as that of the plasma, however.
This is due to compensatory Na/H exchanges through the erythrocyte membrane which
are facilitated by catecholamines (Nikinmaa, 1983). In spite of this, in acid-exposed
rainbow trout, the Bohr effect is not completely eliminated (Ye et al., 1991). Thus, a drop
in actual concentration of oxygen in the blood occurs and this, rather than changes in P02
or pH, may be the major stimulus for increased ventilation in salmonid fishes during acid
exposure (Randall, 1982).

IX. OXYGEN CONSUMPTION, SWIMMING PERFORMANCE, AND

SWIM BLADDER INFLATION
The rate of standard oxygen consumption is generally not influenced much by changes
in pH of the water, except at very low levels (pH <4.0) where it begins to decline (Ultsch,
1978; Ultsch et al., 1980; Ye et al., 1991; Van Dijk et al., 1993). However, the critical
oxygen tension (i.e., that level of oxygen in the water below which oxygen consumption
becomes dependent on oxygen tension) is affected by water pH in both carp and trout.
Ultsch et al. (1980) found that at lower pHs, the critical oxygen tension increased, thus
the fish were less able to deal physiologically with environmental hypoxia if the pH was
also low.
Critical swimming speed of trout appears to not be influenced much by pH over a
range of 6 to 8 (Waiwood and Beamish, 1978). However, 7-day exposures of rainbow
trout to various levels of pH revealed a threshold of 4.4-4.6 below which critical
swimming speed declined linearly by about 4% per 0.1 pH unit (Graham and Wood,
1981). A somewhat similar finding was reported for arctic chair where the effect of low
pH was somewhat greater but the threshold was the same (Hunter and Scherer, 1988). The
above studies involved fish that had been exposed for a week so some adaptation to the
acid may have occurred. When trout were exposed for only 24 h to water of pH 4 and 5,
a 45 and 33% decline, respectively, in critical swimming velocity was observed (Ye and
Randall, 1991). It is interesting that in the same study, exposure to a pH of 10, produced
a 39% impairment in swimming capacity. The effects on swimming could be due to a
reduction in oxygen content of the blood caused by the plasma pH decline and resulting
Bohr shift, as swimming speed is very dependent on blood oxygen content (Jones, 1971).
This hypothesis sounds attractive, but arterial pH is not very sensitive to the water pHs
used in these studies. Also, as was mentioned above, catecholamines help maintain
intracellular pH of the erythrocytes, so altered oxygen content of the blood is probably
252

not the primary mechanism of decreased swimming capacity, except perhaps at a very
low pH.
While oxygen content of the blood may not change too much with minor changes in
pH, oxygen transport could. This is because the blood may become more viscous due to
hemoconcentration brought on by the acid and the blood circulation to the muscles could
then be impaired (Butler et al., 1992). A further mechanism for impaired swimming
capacity has been recently revealed by Butler and Day (1994). Using electromyography
and biochemical measurements of lactate and glycogen, they found that brown trout
exposed to pH 4 failed to recruit white muscle fibers as swimming speed increased.
Because most of the muscle bulk of a fish is white muscle, this could have a considerable
effect on maximum swimming speed.
Acid water can also affect recovery from exhaustion. Fish that had been fatigued were
able to restore blood acid-base balance within 2 h of rest if they were in neutral or alkaline
waters, but not if the water was acidic (Ye and Randall, 1991).
Swim bladder function in fish may be especially sensitive to depressions in pH,
however, only limited information is available on this. When fathead minnows (Pimephales
promelas) experienced a 4-day exposure to pH 5.3 water they had difficulty in adjusting
buoyancy with the swim bladder (Jansen and Gee, 1988). This dysfunction persisted for
at least 32 days and the authors suggest it could contribute to the elimination of fathead
minnows (and other species?) from acidified environments. Overall, this would appear to
be an interesting subject to pursue in other species, and if confirmed, to examine possible
mechanisms.

X. BEHAVIOR
Laboratory experiments using pH gradients have shown that a wide variety of fish species
can detect and then avoid water of a particular level of acidity (assuming that less acid
water is available). Peterson et al. (1989) report that the threshold for avoidance ranges
from pH 4.1 for yellow perch, through 4.7 for brook chair, to 5.9 for rainbow trout. The
threshold avoidances correspond fairly well to the limiting pH suggested by field surveys.
Of course, as the authors point out, the presence of aluminum in field situations may be
more important than the pH per se. Avoidance of alkaline conditions seems to be slight
although waters above pH 10 were not tested.
When fish experience acid stress, they are usually less active and tend to lose their
appetite (Jones et al., 1987). At an intermediate pH of 5, Arctic chair were found to look
normal as far as activity was concerned but were less responsive to an extract of food
(Jones et al., 1985). A pH of 4.5 caused a considerable effect on behavior (decreased) and
an increase in thigmotaxis (Jones et al., 1987). When returned to control conditions
following 16 days in the acid treatment, behavior returned to normal within 2 weeks.
Some may have recovered sooner, so effects were not permanent.
Chemoreception is an important process in feeding behavior for many fish species.
Lemly and Smith (1987) devised a behavior assay to test the effect of low ambient pH
on chemoreception by fathead minnows. They observed a sharp threshold of pH 6. At that
pH and lower, there was a complete cessation of response to a pulsed liquid food stimulus.
The olfactory sensory organs were examined with scanning electron microscopy but no
lesions were observed. It is interesting that this seems to be a rather high pH for blockage
of feeding response but it corresponds to the level where fathead minnows become
eliminated from natural waters.
Finally, mild acid exposure may even affect the ability of fish to learn a maze. Goldfish
were exposed to pH 5.2 for 11 days and then, using operant conditioning, taught a maze.
 253

Those exposed to the low pH were significantly slower to learn the maze than were
controls (Garg and Garg, 1992).

XL CONCLUDING COMMENT
The amount of information we now have on the effects of acid on fishes exceeds that for
any other single pollutant. Acid stress clearly affects a multitude of functions in fishes,
but it is interesting that many, if not most, of these dysfunctions can be traced to an initial
effect on ionoregulation at the gills wherein the fish loses sodium, chloride, and to a lesser
extent calcium more rapidly than it can be recovered. This same etiology appears to hold
for both acute as well as more chronic types of exposure and for different stages of
development. The major difference being that with chronic exposures, the fish may
achieve a different equilibrium level, while under a severe acute exposure they die of
circulatory failure.

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Hunter, L. A. and Scherer, E., Impaired swimming performance of acid-exposed Arctic chair, S a lv e lin u s
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Hwang, P. P. and Hirano, P., Effects of environmental salinity on intercellular organization and
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Jackson, W. F. and Fromm, P. O., Effect of acute acid stress on isolated perfused gills of rainbow trout.
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Janssen, R. G. and Randall, D. J., The effect of changes in pH and PCO2 in blood and water on breathing
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 255

Johnston, C., Saunders, R., Henderson, E., Harman, P. and Davidson, K., Chronic effects of low pH
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Lemly, A. D. and Smith, R. J. F., Effects of chronic exposure to acidified water on chemoreception of
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rainbow trout, S a lm o g a ir d n e r i. I. Branchial and renal net ion and H+ fluxes, J. E x p . B io l., 102,
123, 1983.
McDonald, D. G. and Milligan, C. L., Sodium transport in the brook trout, S a lv e lin u s fo n tin a lis : effects
of prolonged exposure to low pH in the presence and absence of aluminum. C a n . J. F ish . A q u a t. ScL,
45, 1606, 1988.
McDonald, D. G., Reader, J. P. and Dalziel, T., The combined effects of pH and trace metals on fish
ionoregulation, 'm ,A c id T o x ic ity a n d A q u a tic A n im a ls , Morris, R., Taylor, E., Brown, D. and Brown,
J. , Eds., Cambridge University Press, New York, 1989, 221.
McCormick, J. H. and Jensen, K., Osmoregulatory failure and death of first-year largemouth bass
{M ic r o p te r u s s a lm o id e s ) exposed to low pH and elevated aluminum, at low temperature in soft
water. C an . J. F ish . A q u a t. ScL, 49, 1189, 1992.
McWilliams, P. G., A comparison of physiological characteristics in normal and acid exposed popula­
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Milligan, C. L. and Wood, C. M., Disturbances in haematology, fluid volume distribution and circulatory
function associated with low environmental pH in the rainbow trout, S a lm o g a ir d n e r i, J. E xp. B i o l ,
99, 397, 1982.
Mount, D. R., Physiological and toxicological effects of long-term exposure to acid, aluminum, and low
calcium on adult brook trout (Salvelinus fontinalis) and rainbow trout {S a lm o g a ir d n e r i) , Ph.D.
dissertation. University of Wyoming, p. 184, 1988.
Munkittrick, K. R., Calcium-associated reproductive problems of fish in acidified environments: evolu­
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Nelson, J. and Mitchell, G., Blood chemistry response to acid exposure in yellow perch {P e r e a fla v e s c e n s ):
comparison of populations from naturally acidic and neutral environments. P h y s io l. Z o o l , 65, 493,
1992.
Neville, C. M., Ventilatory response of rainbow trout {S a lm o g a ir d n e r i) to increased H'^ ion concentration
in blood and water. C o m p . B io c h e m . P h y s i o l , 63A, 373, 1979.
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P h y s i o l , 152, 67, 1983.
Norton, S. A., The effects of acidification on the chemistry of ground and surface waters, in A c i d R a in /
F ish e r ie s, Johnson, R. E., Ed., American Fisheries Society, Bethesda, MD, 1982, 93.
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Parker, D. B. and McKeown, B. A., Effects of pH and/or calcium-enriched freshwater on plasma levels
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Peterson, R. H., Daye, P. G. and Metcalf, J. L., Inhibition of Atlantic salmon hatching at low pH, C an.
J. F ish . A q u a t. S c i , 37, 770, 1980.
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metal stress, in A c i d R a in /F is h e rie s , Johnson, R. E., Ed., American Fisheries Society, Bethesda, MD,
1982, 176.
Peterson, R. H. and Martin-Robichaud, D. J., Water uptake by Atlantic salmon ova as affected by low
pH, T ra n s. A m . F ish . S o c ., I l l , 772, 1982.
Peterson, R. H., Coombs, K., Power, J. and Paim, U., Responses of several fish species to pH gradients.
C an . J. Z o o l , 67, 1566, 1989.
Randall, D., The control of respiration and circulation in fish during exercise and hypoxia, J. E xp. B i o l ,
100, 275, 1982.
Randall, D. and Wright, P., The interaction between carbon dioxide and ammonia excretion and water
pH in fish. C an . J. Z o o l , 67, 2936, 1989.
Roy, R. L., Ruby, S., Idler, D. and So, Y., Plasma vitellogenin levels in pre-spawning rainbow trout,
O n c o rh y n c h u s m y k iss, during acid exposure. A rc h . E n v iro n . C o n ta m . T o x ic o l, 19, 803, 1990.
Schreck, C., Stress and compensation in teleostean fishes: response to social and physical factors, in.
S tr e s s a n d F ish , Pickering, A. D., Ed., Academic Press, New York, 1981, chap. 13.
Siddens, L. K., Siem, W. K., Curtis, L. R. and Chapman, G. A., Toxicity of environmental acidity on
various life stages of brook trout, P r o c . W est. P h a r m a c o l S o c ., 27, 265, 1984.
Swaarts, F. A., Dunson, W. A. and Wright, J., Genetic and environmental factors involved in increased
resistance of brook trout to sulfuric acid solutions and mine acid polluted waters, T ra n s. A m . F ish .
S o c ., 107, 651, 1978.
Tam, W. H. and Payson, P. D., Effects of chronic exposure to sublethal pH on growth, egg production,
and ovulation in brook trout, S a lv e lin u s f o n tin a lis . C an . J. F ish . A q u a t. S c i , 43, 275, 1986.
Tietge, J., Johnson, R. D. and Bergman, H., Morphometric changes in gill secondary lamellae of brook
trout {S a lv e lin u s f o n tin a lis ) after long-term exposure to acid and aluminum. C an . J. F ish . A q u a t. S c i ,
45, 1643, 1988.
Ultsch, G., Oxygen consumption as a function of pH in three species of freshwater fishes, C o p e ia , 1978,
272, 1978.
Ultsch, G., Ott, M. and Heisler, N., Acid-base and electrolyte status in carp {C y p r in u s c a r p io ) exposed
to low environmental pH, J. E xp. B i o l , 93, 65, 1980.
Ultsch, G., Ott, M. and Heisler, N., Standard metabolic rate, critical oxygen tension, and aerobic scope
for spontaneous activity of trout {S a lm o g a ir d n e r i) and carp {C y p r in u s c a r p io ) in acidified water.
C o m p . B io c h e m . P h y s i o l , 6 1 A , 329, 1981.
Van Dijk, P., van den Thillart, G., Balm, P. and Wendelaar Bonga, S., The influence of gradual water
acidification on the acid/base status and plasma hormone levels in carp, J. F ish B i o l , 42, 661, 1993.
Van Dijk, P., van den Thillart, G. and Wendelaar Bonga, S., Is there a synergistic effect between
steady-state exercise and water acidification in carp?, J. F ish B i o l , 42, 673, 1993.
Waiwood, B. A. and Haya, K., Levels of chorionase activity during embryonic development of S a lm o
s a la r u n d e r acid conditions. B u l l E n v iro n . C o n ta m . T o x i c o l, 30, 511, 1983.
Waiwood, K. and Beamish, F. W. H., Effects of copper, pH and hardness on the critical swimming
performance of rainbow trout {S a lm o g a ir d n e r i) . W a te r R es., 12, 285, 1978.
Walker, R. L., Wood, C. M. and Bergman, H. L., Effects of low pH and aluminum on ventilation in
the brook trout {S a lv e lin u s fo n tin a lis ). C an . J. F ish . A q u a t. S c i , 45, 1614, 1988.
Weiner, G. S., Schreck, C. B. and Li, H. W., Effects of low pH on reproduction of rainbow trout, T rans.
A m . F ish . S o c., 115, 75, 1986.
Wendelaar Bonga, S. E., Van der Meij, J. C. A., Van der Krabben, W. A. and Flik, G., The effect
of water acidification on prolactin cells and pars intermedia PAS positive cells in the teleost fish
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601, 1984.
Wendelaar Bonga, S. E. and Balm, P. L. M., Endocrine responses to acid stress in fish, in. A c i d T o x ic ity
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Wendelaar Bonga, S. E., Endocrinology, in T h e P h y s io lo g y o f F ish es, Evans, D. H., Ed., CRC Press,
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Whitehead, C., Bromage, N. and Forster, J., Seasonal changes in reproductive function of the rainbow
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Wilkie, M., Wright, P. A., Iwama, G. K. and Wood, C. M., The physiological responses of the Lahontan
cutthroat trout {O n c o r h y n c h u s c la r k i h e n s h a w i), a resident of highly alkaline Pyramid Lake (pH 9.4),
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1991.
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Wood, C. M., Turner, J. D. and Graham, M. S., Why do fish die after severe exercise?, J. F ish B i o l ,
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evidence of acclimation to acid/aluminum stress in adult brook trout {S a lv e lin u s fo n tin a lis ) . I. Blood
composition and net sodium fluxes. C an . J. F ish . A q u a t. S c l , 45, 1587, 1988a.
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C an . J. F ish . A q u a t. S c l , 45, 1597, 1988b.
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F ish e r ie s, Johnson, R. E., Ed., American Fisheries Society, Bethesda, MD, 1982, 197.
Wood, C. M., McDonald, D. G., Ingersoll, C. G., Mount, D. R., Johannsson, O. E., Landsberger, S.
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Wood, C. M., McDonald, D. G., Ingersoll, C. G., Mount, D. R., Johannsson, O. E., Landsberger, S.
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A q u a t. S c l , 47, 1604, 1990b.
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175, 153, 1993.
Ye, X. and Randall, D., The effect of water pH on swimming performance in rainbow trout {S a lm o
g a ir d n e r i, Richardson), F ish P h y s i o l B io c h e m ., 9, 15, 1991.
Ye, X., Randall, D. and Xiqin, H., The effect of acid water on oxygen consumption, circulating
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P h y s i o l B io c h e m ., 9, 23, 1991.
 Chapter
11
The Immune System
I. OVERVIEW OF FISH IMMUNOLOGY
In common with other vertebrates, the fish immune system is used to defend against
harmful bacteria, viruses, fungi, and parasites. The fish immune system has many
similarities with that of the mammalian system, but there are also some important
differences. For example, temperature and life style have a considerable influence on
immune function in fish. Species living a solitary existence in cold waters have rather
poorly developed immune systems compared to social species in warm waters where
pathogens are more easily passed between fish and the pathogens have shorter generation
times due to the higher temperatures (Post, 1983). Within a species, temperatures at the
low end of a species thermal range may reduce immunological competency. Zeeman
(1986) emphasizes how daily and seasonal cycles can greatly modify the immune
response in fish.
Our current understanding of fish immunology is far behind that of mammals even
though many of the techniques and concepts worked out on the latter are now being
applied to fish (Stolen, 1993, 1994). Reviews of fish immunology include those by Post
(1983), Satchell (1991), Weeks et al. (1992), and the book on the subject edited by
Manning and Tatner (1985).
The first line of defense in a fish includes the physical barriers of scales and skin and
the chemical barrier, mucus. The latter contains lysozymes which are bacteriolytic
enzymes (Lie et al., 1989). A wide variety of pollutants and low pH as well as the stress
from handling stimulate increased secretion by mucus glands in the gills and skin.
Whether the composition of the mucus changes as a result of such a hypersecretion is
apparently unknown, but it does serve to potentially trap harmful organisms and then
slough them off (Anderson, 1990). Another initial defense is the acidity of the stomach
which, at least in mammals and presumably also in fish, kills many bacteria.
If the initial barriers are penetrated by a pathogen, then the other components of the
immune system come into play. At first, certain immune cells are attracted to the site of
entry. These include tissue macrophages and monocytes in the blood which phagocytize
the microbes. Macrophages may actually be monocytes (a type of leukocyte) that have

259
260

entered the tissues and differentiated. Macrophages are considered to be of greater

immunological importance in fish than in the other vertebrates (Weeks et al., 1992), so
their activity is frequently assayed as a test for immune function.
Neutrophils (another leukocyte) are attracted to the site of invasion, where they release
enzymes, including lysozyme, that destroy harmful organisms. Neutrophils and throm­
bocytes (which are primarily involved in blood clotting) have also been reported to be
phagocytic (MacAithur and Fletcher, 1985). All the reactions thus far described are
collectively termed the “non-specific response” because they are essentially the same
irregardless of the pathogen involved. If these defense mechanisms are overcome, the
microorganisms may rapidly multiply and kill the fish unless the specific immune
response is induced in time.
The specific immune response must be induced in reaction to individual antigens. These
are specific molecules, usually protein, that are on the surface of foreign organisms such as
bacteria or viruses, or are toxins secreted by those organisms. Antigens are basic to the
recognition of non-self by the various components of the immune system. An antigen is
transported by the monocytes to melanomacrophage “centers” of the anterior kidney and
spleen where the antigen is presented to lymphocytes that are responsible for producing the
antibodies, and to T cells which are specific for that particular antigen. The transporting and
presenting of an antigen by monocytes is referred to as antigen processing.
The classic picture in mammals of B lymphocytes formed in the bone marrow and
residing in the lymph nodes and responsible for humoral immunity must be modified in fish.
Fish have no bone marrow or lymph nodes and the production of lymphocytes is apparently
in the head kidney, gut-associated tissue, and the spleen. Some B lymphocytes become
activated to divide (thus becoming plasma cells) by an antigen and start secreting antibodies
toward the specific antigen. Other B lymphocytes differentiate into memory cells that serve
to “remember” what the antigen was so that upon subsequent exposure to the same antigen,
the antibody response (i.e., the secondary response) is much more rapid and larger.
The antibodies are proteins which are referred to as immunoglobulins and, in mam­
mals, are grouped into five classes designated with the prefix “Ig”. In fish, however, only
the IgM class has been found (Jurd, 1985). Antibodies do not destroy antigen-bearing
invaders. They instead inactivate antigens and mark them for destruction by macrophages
and complement, another protein component of the immune armament. They also may
neutralize viruses or toxins and agglutinate them, which makes them more accessible for
phagocytosis by macrophages.
The other major arm of the specific immune response is designated “cell mediated”.
It depends on the function of T lymphocytes, which, in mammals, are formed in the very
young by the thymus from cells that originated in the bone marrow. The thymus then
largely degenerates as the animal matures. The presence of T lymphocytes in fish was for
some time controversial but now they are generally considered to be present (Anderson,
1990; Satchell, 1991). Mammals, and presumably fish, have subpopulations of these cells
which aid in antibody production and others that suppress it. The thymus in fish involutes
at sexual maturity in some species but retains its normal size in others (Satchell, 1991).
There are other parts of the immune system of fish that are not well understood. These
include natural killer (NK) cells, which destroy tumors; interferon, which interferes with
replication of viruses; transferrin in the blood, which deprives microorganisms of iron;
and an assortment of other molecules which agglutinate pathogens and aid phagocytosis
(Alexander, 1985; Satchell, 1991).

II. EFFECTS OF POLLUTANTS ON IMMUNE FUNCTION

The field of immunotoxicology, even in mammals, is relatively young (National Research
Council, 1992). It has been presumed for decades that environmental pollutants or other
 261

stressors (e.g., handling) can affect one or more of the immunological functions in a fish,
for it is almost common knowledge that fish frequently then become more susceptible to
various diseases (Snieskzko, 1974). The field of fish immunotoxicology (or
immunomodulation as it is sometimes called) has, however, only begun to really grow
since the mid-1980s. Earlier observations were usually peripheral to other aims of a
toxicological or immunological study and have been extensively reviewed by Zeeman
and Brindley (1981). In many cases, the major observation was the occurrence of
histological lesions in kidney or spleen following exposures to some pollutant at often
unrealistically high environmental concentrations.
There are a wide variety of tests that have been developed to assess how well a
particular component of the immune system is functioning. Most of these are modified
from similar ones used in mammalian clinical laboratories and vary considerably in how
difficult they are to perform. They fall into five broad groups: (1) susceptibility of fish
to infection or tumor formation; (2) changes in leukocyte numbers or differential count;
(3) alterations in phagocytic response or melanomacrophage centers; (4) measures of
antibody production; and (5) scale or fin graft rejection times (as a measure of cell-
mediated immune function). Anderson (1990), Stolen (1993, 1994), and Weeks et al.
(1992) provide useful guidance for those wishing to begin investigations in this rapidly
growing discipline.

A. METALS
1. Cadmium
This metal causes both immunological inhibition and stimulation in mammals, depending
on a variety of factors, especially the particular immune function measured (Koller,
1981). For example, T-lymphocyte activities are usually suppressed by cadmium whereas
the effects on B lymphocytes are more varied. The same sort of variability may also
prevail for fish. Thuvander (1989) tested rainbow trout after 12 weeks in very low
concentrations of cadmium (0.7 or 3.6 jiig Cd/L) and observed suppression of T-lympho-
cyte function whereas the B-antibody response to a bacterial challenge was enhanced in
the fish exposed to cadmium. The metal exposure caused no other clinical effects in the
fish. One cannot, however, conclude that cadmium stimulates antibody capability in fish.
Tests in the same species at comparable cadmium concentrations, but using a response
by the intact fish to the injection of human blood cells, found a modest inhibition of
antibody production (Viale and Calamari, 1984).
The data presented by Robohm (1986) points up how it is hazardous to conclude much
about effects of cadmium on immune responses in fish if it is based on only one species.
He exposed cunners and striped bass to cadmium for 96 h and tested the antibody
response of intact fish to a bacterium. Cadmium caused inhibition of serum antibody titers
in cunners, but it caused a sixfold stimulation in striped bass. Exposures were at the same
concentration in both fish species (10-12 |Lig/ml) a cadmium concentration that is about
half the LC50. There does not appear to be a clear explanation for this large species
difference.
Cadmium can have a marked effect on differential leukocyte counts in fish. Newman
and MacLean (1974) reported a dose-dependent increase (threefold) in neutrophils and a
dose-dependent decrease (also nearly threefold) in lymphocytes in the cunner, a marine
teleost. Three-week exposures of goldfish to cadmium yielded rather similar results
except the effect was not as large or was it dose dependent (Murad and Houston, 1988).
Murad and Houston speculate the lack of dose dependency may reflect differences in
cadmium sensitivity of lymphocyte subpopulations so that as a threshold is reached,
essentially all of those from one population are eliminated. Then, unless the threshold of
the other population(s) is achieved, there is no further change in lymphocyte number with
rising cadmium concentration. The primary reason for the decrease in lymphocytes is
262

evidently due to a reduction in their rate of production. Evidence for this is that the blast
cells, which are lymphoid progenitors, also decreased and other investigators (Stromberg
et al., 1983) report lesions in the hematopoietic areas following cadmium exposure.
Stimulation of the numbers of neutrophils does not currently have an explanation.
Because it has now been observed following exposure to cadmium in two very different
sorts of fish, it may be a fairly widespread phenomenon. While the depression of
lymphocytes by cadmium probably compromises immune function, it is not clear what
effect stimulation of neutrophils may have. It might conceivably compensate for the lack
of lymphocytes. A suggestion of this is seen in the work of MacFarlane et al. (1986) who
exposed juvenile striped bass to cadmium at 12 or 30 pg/L for 5 days and then challenged
them with the bacterium which causes columnaris disease. The members exposed to
cadmium were slightly less susceptible to the disease than were controls. It would be
premature, however, to jump to any broader conclusions from this single study.
A variable that could have a considerable effect on the modulation of immune
response in fish exposed to cadmium is their lack of a cortisol response to this metal. Most
fish exhibit an elevated plasma cortisol level in response to nearly any stressor, however,
cadmium appears to be an exception to this generalization (Schreck and Lorz, 1978;
Thomas and Neff, 1985). As will be discussed later in this chapter, elevated cortisol may
be a primary mechanism for immune system suppression in fish exposed to a variety of
pollutants. It is not clear why cadmium alone among the metals fails to induce this
hormonal change, but it may help explain the sometimes unexpected results when fish are
exposed to this metal.

2. Copper
Copper sulfate is frequently used to control external columnaris infections of pond fishes.
Thus, it is not surprising that exposure of juvenile striped bass for 5 days to this metal
improved their resistance to this disease bacterium (MacFarlane et al., 1986). However,
this metal caused immunosuppression of antibody-producing cells in rainbow trout when
tested in vitro (Anderson et al., 1989), and air-breathing catfish (Saccohranchus fossilis)
exposed to copper at several very low concentrations for 28 days exhibited a dose-
dependent depression of antibody production, phagocytic activity of spleen and kidney
macrophages, and a prolongation of eye allograft rejection time (Khangarot and Tripathi,
1991). The latter indicates a suppression of T-cell activity.
Hetrick et al. (1982) exposed striped bass to copper at 7 and 10 pgL~‘ for 96 h prior
to challenge with two naturally occurring bacterial species {Vibrio anguillarum and
Pasteurella piscicidd). Copper exposure increased susceptibility of the fish to the disease
organisms. So, while copper may be protective for some external diseases, defense
against internal infections can be compromised by prolonged exposure to the metal.
When zebrafish were exposed to copper or zinc at several different concentrations for
7 days, a dose-dependent suppression of kidney lymphocyte number and natural cytotoxic
cells was observed (Rougier et al., 1994). Copper was more potent in this regard than was
zinc. The effect on macrophage activity was, however, more complex. Copper caused a
marked decrease in macrophage activity both in vitro and in vivo, but zinc caused a
modest increase in macrophage activity under the same conditions. The authors do not
attempt to explain this stimulation of activity by zinc, but the fact that it occurs with both
in vitro and in vivo experiments suggests it is not an artifact.

3. Miscellaneous Metals
In a rather old study, exposure of brown bullhead catfish to lead for up to 183 days was
reported to produce a reduction in spleen size but an increase in leukocyte number and
an especially large increase in the number of thrombocytes (Dawson, 1935). Crandall and
Goodnight (1963) obtained somewhat similar findings in guppies exposed to lead for at
 263

least 60 days. They also noted that lymphoid tissue in the head kidney was greatly reduced
in those exposed to lead. The same workers observed similar changes in guppies chroni­
cally exposed to zinc.
Chronic exposure of trout and carp to nickel, zinc, copper, or chromium was found to
suppress to a variable extent the primary humoral response to an intraperitoneal inocu­
lation of MS2 bacteriophage (O’Neill, 1981). O’Neill (1981; p. 329) notes that metals
may “...block the active sites of antibody molecules and disturb the metabolism, ionic
balance and cellular division of immunocompetent cells”.
Among the metals, manganese has relatively low toxicity to fish (Jones, 1964). It is
an essential trace element for various functions and stimulates NK cell activity in
mammals (Keen et al., 1985). Thus, it is interesting to see that it also stimulated NK cell
activity in carp based on both an in vitro and an in vivo test (Ghanmi et al., 1990). The
authors cite work on mammals which suggest that the stimulatory effect is due to factors
such as lymphokines or interferon in those species. This metal was also found to have a
“strong enhancing effect on phagocytosis” by carp macrophages when added to the test
medium in an in vitro test (Cossarini-Dunier, 1987), somewhat similar to what was
reported above for zinc.
When phagocytes undergo phagocytosis they show a burst of oxygen consumption
and chemiluminescence which can be measured using luminol as an amplifier. The
chemiluminescence response is therefore a measure of phagocytosis of a group of cells.
Using an in vitro preparation, Wishkovsky et al. (1989) found inhibition of this response
in macrophages from three species of estuarine fish when in the presence of tributyltin
in the test medium.
On the basis of one study (Roales and Perimutter, 1977), it appears that methylmercury
can severely inhibit at least some components of the immune system. Blue gourami
(Trichogaster, a tropical freshwater species) were exposed to methylmercury for 4 or 5
weeks and then tested for viral neutralizing or bacterial agglutination titers. Some inhi­
bition of the neutralization occurred and bacterial agglutination was non-detectable in
those exposed to mercury.
There has been some interest in the use of in vitro systems for testing various toxicants
because it requires fewer animals and does not require extensive facilities for exposing the
animals to the test chemical (Anderson et al., 1989). Elsässer et al. (1986) used this approach
with trout phagocytes and the chemiluminescent response mentioned above. They incu­
bated the phagocytes at a concentration of copper, aluminum, or cadmium equivalent to
1/10 of the lowest concentration that showed toxicity for a cultured indicator cell line.
Copper caused a strong inhibition of the phagocytic response, aluminum somewhat less so.
Cadmium, however, caused an initial stimulation followed by a variable decrease. These
findings are interesting but need validating in intact systems. A real problem with in vitro
studies is simulating the concentrations of test chemicals that might prevail in the intact
animal. Another potential confounding factor is the duration of the exposure which is
usually for only a few hours when in vitro, but when in vivo, it may be for days or weeks.
However, the in vitro approach could be useful for screening a wide variety of potential
immunomodulators which could then be tested in the intact fish.

B. ORGANIC POLLUTANTS
A major class of organic contaminants that fish may get exposed to is, of course,
pesticides. Given the extreme variety of pesticides used, it is somewhat surprising so little
is known about how they affect the immune systems of fish (Plumb and Areechon, 1990).
Zeeman and Brindley (1981) reviewed the earlier work, nearly all of which suggested
decreased disease resistance in fish exposed to various pesticides. They note, however,
that this may often be explained as a generalized stress effect (also see below), especially
given that the test concentrations used were usually quite large.
264

Atrazine, a triazine herbicide, and lindane, an organochlorine insecticide, were tested

for their direct effect on carp macrophage phagocytosis in vitro. No effect was found at
concentrations up to the limit of their solubility in water (Cossarini-Dunier, 1987).
Cossarini-Dunier cites other work in the same laboratory which showed no effect on
antibody production by these pesticides when given in the food to carp for 2.5 months.
There was no effect even though the lymphoid organs were highly contaminated, nor did
the pesticides have any effect on graft rejection times. Another organochlorine insecticide
(Mirex) was tested in trout via inclusion in the diet for a year at 50 ppm (Cleland and
Sonstegard, 1987; Cleland et al., 1988). This dietary exposure caused no effect on
humoral immune expression or on NK cell activity. Similar negative results were found
when humoral immune expression was measured in carp given food contaminated with
lindane (up to 1000 ppm) for a year (Cossarini-Dunier et al., 1987). When trout were
given a daily body dose (1 mg/kg) of lindane in their diet for 30 days, a decrease in
chemiluminescent response was observed but there was no effect on lymphocytic prolif­
eration and on the number of circulating B lymphocytes (Dunier et al., 1994). Taken
together, these findings suggest that organochlorine insecticides may have relatively little
effect on the fish immune system, at least when exposures are at low levels, even for long
times, but, if exposures produce a generalized stress response reflected in an elevated
serum cortisol level, then immunosuppression may occur as was seen when trout were
exposed to endrin for 30-60 days (Bennett and Wolke, 1987a,b).
From the very limited work that has been done, it appears that organophosphorus
insecticides may also exhibit relatively little immunotoxicity. Dunier et al. (1991) tested
the effect of trichlorofon and dichlorvos on lymphocytic proliferation and phagocytosis
in vitro using carp cells. The doses were high and caused suppression of these two
functions. This may have relevance where these pesticides are used for ectoparasite
treatments of fish in aquaculture. However, when dichlorvos was administered in the
ambient water (trichlorofon is rapidly converted to this molecule in water), no effect was
detected on the humoral response to Yersinia ruckeri.
Channel catfish exposed to malathion for 30 days exhibited suppression of antibody
agglutination. Two doses were used (0.5 and 1.75 mg/L), only the higher one caused
suppression (Plumb and Areechon, 1990). Because this organophosphorus insecticide
breaks down rapidly in the environment, it seems doubtful that concentrations this high
would persist for long.
The responses of fish to sediment contaminated by diesel fuel are complex and
difficult to interpret. Tahir et al. (1993) report that exposure of dab to this sediment for
4 weeks caused an increase in leukocyte number with low doses but a suppression with
high doses. Serum lysozyme activity and kidney phagocytic respiratory burst activity
were depressed at all doses whereas the number of antibody-secreting cells in the kidney,
serum bacteriocidal activity, and anti-protease activities tended to increase with exposure.
The authors speculate that the stimulatory effect on some immune functions may be a
compensatory response to suppression of others by immunotoxins in the sediment.

C. MISCELLANEOUS POLLUTANTS
Lymphocyte proliferation and antibody production were both suppressed in channel
catfish exposed to acidified waters (pH 4) for 2 weeks. The secondary antibody response
was also suppressed. The author (Morra, 1993) presents limited evidence that the mecha­
nism may have been the suppression of the ability of B cells to process the antigen.
The enzyme lysozyme is found in the lysosomes of neutrophils and macrophages and
is secreted into the blood by these cells. Mock and Peters (1990) found that fairly high
doses of ammonia can cause a depression in its activity in the blood. This may be a
generalized stress response because the same investigators found that the stress of
handling and transport also caused suppression of lysozyme activity.
 265

The finding of a suppression of lysozyme activity may only occur with acute stressors.
Secombes et al. (1991) found no effect on lysozyme activity of chronic exposures of fish
to sewage sludge, but they did get decreases in thrombocyte numbers and increases in
melanomacrophage centers. In a subsequent study Secombes et al. (1992) found that the
sludge caused a decrease in leukocyte bactericidal activity and in antibody-secreting cells
but no change in specific Ig or phagocytosis. In the same study, it was noted that
immunization overcame the inhibitory effect of sewage sludge exposure on leukocyte
bactericidal activity.

D. IMMUNE EFFECTS IN FISH FROM CONTAMINATED

NATURAL WATERS
The potential for using measurements of immune function in field monitoring programs
is probably large, however, they have not been used much (Wester et al., 1994). This may
be largely due to the shortage of easily applied procedures that would lend themselves to
routine monitoring. The usual approach has been to look for diseases in sampled fish
(Sindermann, 1979). For this purpose, O’Connor et al. (1987) developed an index of
disease conditions (primarily external) that may be induced by pollution in marine
teleosts and shellfish. Their publication also has a section to help non-professionals
interpret the findings and evaluate their importance.
A rather extensive disease study was carried out by Malins et al. (1988) in which they
sampled English sole in Puget Sound over a 5-year period in order to investigate the
etiological relationships between prevalences of disease (primarily hepatic neoplasms)
and concentrations of aromatic hydrocarbons in the sediment and their metabolites in the
fish bile. They obtained good correlations between concentrations of chemicals in sedi­
ment and tissues and prevalence of disease. However, Malins et al. (1988; p. 61) wisely
point out that “.. .such relationships cannot be interpreted as de facto evidence of specific
cause and effect.” This is because not all potential harmful chemicals can be identified
in the samples and even the ones measured may act through synergistic or antagonistic
interactions with others. Even with these caveats, it seems that disease surveys may be
a useful biomarker (among others) for environmental health (McCarthy and Shugart,
1990).
Increased occurrence of neoplasms in fish exposed to contaminants suggests suppres­
sion of a primary defense that vertebrates have against the spread of transformed or
malignant cells. Key players in this are the cytotoxic leukocytes often called NK cells.
Thus, it is interesting that Faisal et al. (1991) report a depression in NK activity in
Fundulus taken from a river in Virginia which passes through a heavily industrialized
urban area and the water and sediments contain a wide variety of chemicals including
polynuclear aromatic hydrocarbons and creosote. In another study, the chemotactic
response of fish macrophages was tested in two other species of fish from the same river
and these were compared with fish from another river that was relatively clean (Weeks
et al., 1986). A 30 to 40% decrease in macrophage chemotactic activity was found in
those fish from the contaminated river. When those fish were held in clean water for 3
weeks the chemotactic activity returned to normal, showing that the effect of the polluted
water was reversible. This raises the question (not asked by the authors) whether the
return to normal immune function was due to depuration of the toxic chemicals from the
tissues, or was it due to changes in hormonal status?
In the same laboratory that found suppression of macrophage chemotactic activity in
fish from a contaminated river, a stimulation of macrophage chemoluminescence was
observed in Fundulus from the same river (Kelley-Reay and Weeks-Perkins, 1994). This
apparent stimulation by the contaminants was further confirmed by observing
chemoluminescence stimulation of macrophages in fish transferred from the control river
to waters from the contaminated one. The mechanism for this remains obscure but may
266

involve increased lymphokines or a direct effect of some chemical constituent of the

waters.
Following a chemical spill (including chlorinated and heterocyclic hydrocarbons,
aromatic nitro, and heavy metal compounds) eels were found to have considerable
histopathological damage in the spleen (Spazier et al., 1992). In addition,
melanomacrophage centers were lacking in eels suffering from the aftermath of the
spill.
Melanomacrophage centers are fairly easy to assay using standard histological meth­
ods. Wester et al. (1994) suggest they may be a useful biomarker for immunotoxicology
in part because they are considered as the primitive analog of the mammalian lymph
follicle. Their density may decrease in fish from contaminated waters or along a pollution
gradient, although as was noted above, some chemicals may cause increased density of
the centers. Wester et al. (1994) suggest that the latter response may reflect accumulation
of cytotoxic waste.

III. HORMONAL MODULATION OF IMMUNE RESPONSE

The general adaptation response (GAS) of fish to any sort of stress is characterized by
elevations in cortisol and catecholamines (Pickering, 1981). While these hormones are
involved in the maintenance of homeostasis in the face of the stress, they can also have
a marked effect on numerous immunological functions.
The evidence is fairly strong now that cortisol suppresses several of the immune
functions in fish, even at concentrations that are only slightly higher than those found in
“unstressed” animals (Thomas and Lewis, 1987; Pickering and Pöttinger, 1989; Ainsworth
et al., 1991). These investigations were performed using fish in which cortisol was given
either in the food or via intraperitoneal injection, thus the confounding effects of contami­
nant chemicals or other stressors were eliminated. Moreover, in studies (Tripp et al.,
1987) carried out in vitro, physiological concentrations of cortisol suppressed the mito­
genic response of coho salmon lymphocytes so at least one molecular mechanism has
been revealed. The suppression of immune function by exogenous cortisol has also been
shown to make trout more susceptible (in a cortisol dose-dependent manner) to common
bacterial and fungal diseases (Pickering and Pöttinger, 1989).
The interrelations of cortisol and immune function may be more complex than a mere
suppression of the latter by the former. Schreck and Bradford (1990) have found that fish
lymphokines can suppress the secretion rate of cortisol from interrenal cells, thus exhib­
iting a form of negative feedback. This is, incidentally, the reverse of what occurs in
mammals (Sapolsky et al., 1987).
Although cortisol alone can have a considerable effect on a variety of immune system
activities, it is premature to conclude that all effects of pollution (or other stressors) are
due to this hormone (Maule and Schreck, 1990). In mammals, catecholamines which
become elevated during stress, mobilize blood granulocytes and macrophages (Weeks et
al., 1992) thereby causing their numbers to rise in the blood. This could be a form of
compensation for the effect of some contaminant on immune function. Clearly, we are
only beginning to touch the surface of this field of pollution physiology.

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B u ll, 76, 717, 1979.
Snieskzko, S. F., The effects of environmental stress on outbreaks of infectious diseases of fishes, J. F ish
B i o l , 6, 197, 1974.
Spazier, E., Storch, V. and Braunbeck, T., Cytopathology of spleen in eel A n g u illa a n g u illa exposed
to a chemical spill in the Rhine river, D is . A q u a t. O r g a n is m s , 14, 1, 1992.
Stolen, J. S., Fletcher, T. C., Anderson, D. P., Robertson, B. S. and van Muiswinkel, W. B., Eds.,
T e c h n iq u e s in F ish I m m u n o lo g y, Vols. 1-4, SOS Publications, Fair Haven, NJ, 1993-1994.
Stromberg, P. C., Ferrante, J. G. and Carter, S., Pathology of lethal and sublethal exposure of fathead
minnows, P im e p h a le s p r o m e la s , to cadmium: a model for aquatic toxicity assessment, J. T o x ic o l
E n v iro n . H e a lth , 11, 247, 1983.
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 Chapter
1 2

Behavior and Nervous

System Function
I. INTRODUCTION
In Chapter 1, where the levels of integration (complexity) spectrum were discussed, it was
pointed out that behavior is toward the end indicating a high level of integration. Thus,
behaviors are influenced by a multitude of factors around and within the animal; however,
it is safe to say that all of these operate through the nervous system. The sensory receptors
are constantly sampling the environment and feeding this information to the central
nervous system which integrates this with other external and internal information before
making a behavioral response. In addition, changes in hormonal levels and blood chem­
istry, whether caused by natural factors or pollutants, may alter activities of particular
areas of the brain, thereby influencing behavior.
Categories of behavior in fish (called “ethological units” by Smith, 1984) include
those behaviors associated with schooling, feeding, migration, aggression, fear, learning,
rheotropism, and attraction to or avoidance of a chemical or temperature. Even as broad
a classification as this has some overlap, but it does give a rough framework. (Some
workers include breathing frequency as a behavior category. In this book, breathing is
covered in Chapter 3, swimming capacity is reviewed in Chapter 8, and reproductive
behaviors are taken up in Chapter 13.)
For a good overview of “normal” fish behavior, the interested reader should see
Pitcher (1993). The effects of pollutants on fish behavior have received increasing
attention over the past decade or so, and several recent reviews have appeared (Atchison
et al., 1987; Beitinger, 1990; Henry and Atchison, 1991; Blaxter and Hallers-Tjabbes,
1992; Scherer, 1992; Birge et al., 1993; Little et al., 1993). The physiology of fish sensory
organs including vision, olfaction, audition, electrical, and taste are extensively reviewed
in Evans (1993). In an earlier review. Smith (1984) made a good attempt to relate altered
behavior to changes in function of specific brain regions in the fish, but it is clear that this
aspect of neurotoxicology of fishes remains in its infancy.
First, a few general comments about the brain of teleost fishes. These organisms have
the same major brain structures as the higher vertebrates, but the relative sizes of these
parts are quite different. Thus, among the structures that can be easily observed, fish have

271
272

relatively large cerebellums and optic and olfactory lobes, but usually a very small
cerebrum. (The latter is called the telencephalon in many references.) There are marked
differences in brain structure between different fish species based largely on their life
style (Gutherie, 1983). For example, those species which detect food primarily by vision
have large optic lobes whereas those that use olfactory sense for feeding have large
olfactory bulbs. While learning in higher vertebrates relies heavily on the cerebrum, fish
appear to have considerable capacity for learning in the cerebellum, as well.
In the vertebrates, muscular activity and equilibrium is largely coordinated by the
cerebellum. The integration of sensory information and the initiation of an appropriate
locomotor activity, to a large degree, depend on the reticulomotor (reticular) system. This
large group of neurons is located in the anterior end of the medulla oblongata and projects
into the mesencephalon. In fish, the portion of the reticular system called the ventrolateral
peduncular neuropil is the key to nearly all motor activity (Smith, 1984). For a more
extensive discussion of fish brain functional anatomy see Gutherie (1983), Northcutt and
Davis (1983), and Davis and Northcutt (1983). Smith (1984) provides a more general
summary of the brain regions of fish and the neurotransmitters and behaviors associated
with those regions. One of the generalizations to be drawn from these reviews is that there
is a good deal of functional overlap between the major portions of the brain of the fish,
which compounds the problems of physiological analysis.

II. LOCOMOTOR ACTIVITY

In a sense, nearly all behaviors of fish involve locomotor activity. Indeed, it is the
predictable body movements of an animal that the behavioral scientist observes and uses
to draw conclusions as to purpose, causality, etc. However, here we are referring to
seemingly random movements. Because the extent of muscular activity considerably
influences the energetic cost of existence for a fish, this topic is also taken up from a
different perspective in Chapter 8.

A. METHODS FOR MEASURING LOCOMOTOR ACTIVITY

Observations of changes in locomotor activity in response to the presence of a pollutant
tended in earlier studies to be anecdotal and often associated with other experimental
purposes. The fish were described as becoming “restless”, “excitable”, “lethargic”,
“dashing wildly”, etc. These subjective observations can be of some qualitative use, but
it is much better to quantify the extent of bodily activity. For this purpose, various devices
have been developed, some of considerable complexity and/or ingenuity.
As a fish moves through the water, it will cause small water currents that have the
potential of being detected if the fish is in a sufficiently small chamber. One of the earlier
schemes to measure fish movements by this means was that of Spoor (1946) who
suspended small paddles in the water which, when moved, caused an electrical contact
to close and record on a strip-chart recorder. A more recent development along this line
is that of Fisher et al. (1983) who used a movement transducer attached to the paddle to
detect its oscillations. Small water currents can also be detected by changes in the rate of
heat loss from a small thermoregulated chamber in the aquarium (Beamish and Mookherjii,
1964) or a thermistor in the water (LaBarbera and Vogel, 1976).
The small voltage changes produced by fish moving in the water can be detected with
electrodes in the water of a small aquarium (Spoor et al., 1971), but this method is not
especially quantitative. It mostly indicates whether the fish is moving around in the test
tank or sitting still. It detects the breathing movements quite well (Heath, 1972), however,
so it can be useful in measuring that physiological variable. These various methods offer
a way of quantifying fish locomotor activity while measuring some other physiological
 273

activity such as oxygen consumption or heart rate, where the fish must be rather severely
confined. However, when only the measurement of locomotor activity is desired, other
techniques discussed below are probably more suitable.
Perhaps the most elaborate of the devices for quantifying locomotor activity of fish is
that of Kleerekoper (1977). Briefly, this is a round steel tank either 200 or 549 cm in
diameter. The central open section (100- or 250-cm diameter) is surrounded by radial
dividers which separate the outer area into 16 equal chambers. These have photoelectric
“gates” which are used to detect when a fish enters a chamber and into which one it goes.
As a fish moves from one chamber to another, its movements are quantified by computer.
This gives an indication of the speed of movement, how much exploratory activity is
present, and the orientation taken when a fish exits a chamber. An improved version of
this system has been developed by Scarfe et al. (1985).
A video camera coupled to a minicomputer has been used to measure movement
patterns and chemical avoidance by bluegill (Lubinski et al., 1980). The fish were in a
tank 50 cm square with the camera viewing from above. The position of the fish was
recorded as a pair of coordinates every 4 s, and every 10 min behavioral parameters, such
as direction of turn and angular size of the turn, were calculated. This system is limited
to small fish in a relatively small tank. An array (1900) of photoconductive cells embed­
ded in the floor of a larger tank permits monitoring of fish in a more natural setting or the
use of larger fish (Kleerekoper, 1969). Fish locomotor patterns have also been monitored
with a set of infrared emitters and photoelectric sensors dividing the tank into eight equal
areas. Movement from one area to another activates a counter (Morgan, 1979).
Unfortunately, the above schemes are expensive and complicated to operate. A rela­
tively simple way of quantifying locomotor activity has been developed by Ellgaard et al.
(1977). Groups of fish are confined to a 10-gal aquarium. To run a test, they are moved
to one half of the tank with a partition and a separate partition containing four holes of
appropriate size is inserted to divide the aquarium in half. Then the original partition is
removed and the number of fish in the empty half is counted at intervals. The data are then
treated as an opposed first-order reaction to determine rate constants.
Another fairly simple although labor intensive technique is described by Little et al.
(1990). They used single small trout in a circular cylinder viewed from above with a video
camera. A fish was watched for a period of 2 min and with the aid of a stopwatch, the
duration of time in which the fish was moving was determined. This would appear to
work very well with trout, which are a relatively active species. It remains to be seen how
it would work with species that are not so active.

B. METALS
The effect of copper on locomotor activity appears to vary with the species. At low
sublethal concentrations copper stimulated locomotor activity in brook trout (Salvelinus
fontinalis) (Drummond et al., 1973), however, the external electrode technique which was
used for this work failed to detect a dose-dependent response. Instead of a stimulation of
activity as seen in the trout, bluegill exhibited a decreased level of activity which was
dependent both upon concentration and duration of exposure (Ellgaard and Guillot,
1988). Four marine teleost species were tested in the Kleerekoper apparatus and a
considerable species variation was noted in their response to copper (Scarfe et al., 1982).
Immediately upon being exposed to the metal (0.1 mg/L), the sea catfish {Arius felis) and
sheepshead (Archosargus probatocephalus) increased their activity while the pinfish
(Lagodon rhomboides) and 33% of the Atlantic croaker {Micropogon undulatus) became
hypoactive. Species differences also occurred in the data on angular orientation which,
according to the authors, is probably controlled by different neurophysiological mecha­
nisms. From a toxicological standpoint (i.e., LC50), the sea catfish was found to be the
274

most sensitive of the four species tested. Subsequent work with the sea catfish has shown
the response to be dose dependent with a reasonably well-defined threshold of 0.1 mg/L
(Steele, 1983).
Although copper produced hypoactivity in bluegill, the metals chromium, zinc, and
cadmium have been shown in the same laboratory to cause a dose-dependent increase in
locomotor activity in the same fish species (Ellgaard et al., 1978; See Table 1, Chapter 8).
The authors attributed the stimulatory effect of the metals to a direct effect on cellular
energy metabolism. However, this appears to me to be a confusion of cause and effect.
Metals stimulate some enzymes and inhibit others (see Chapter 9) and a primary deter­
minant of whole-animal energy metabolism is muscular activity, rather than the other way
around (see Chapter 8). Moreover, there is no particular reason for alterations in basal
energy expenditure, as might be produced by pollutants affecting the enzymes, to affect
spontaneous muscular activity.
Henry and Atchison (1979a,b, 1986) used a variety of body movements of bluegill as
indicators of sublethal concentrations of zinc, cadmium, or copper. Coughs, yawns, jerk
swimming, fin flickering, agonistic behaviors, and chafing against objects were counted
visually and found to increase in a dose-dependent manner. The social rank of a fish
influenced the response, especially in the copper where the most subordinate and most
dominant individuals were affected by the metal to the greatest extent. Agonistic behav­
iors by dominant individuals were increased by exposure to the metals (but see section
below on aggression). Concentrations as low as 21 |ig Cd/L, 99 |Lig Zn/L, and 34 |LLg Cu/L
were detectable with this method. These concentrations are roughly an order of magni­
tude below the 96-h LC50, so the behavioral measures proved quite sensitive to the three
metals tested.
In summary, many metals apparently stimulate activity in fishes which is then re­
flected in an elevated metabolic rate, a topic discussed at some length in Chapter 8. The
mechanism(s) for metal stimulation of activity in fish is largely open to conjecture. Smith
(1984) suggests they may act somewhat similar to organochlorine insecticides which are
known to enhance sensory and motor nerve activity. Increased exploratory activity
associated with avoidance behavior may be a key process, especially upon the initial
exposure to a metal (Scarfe et al., 1982). In other words, the metals probably act as a
physical irritant to a potentially wide assortment of external tissues of the fish. Where
suppressions of activity are seen (e.g., copper and bluegill), inhibition of some peripheral
sensory function is possible and, of course, effects on the central nervous system areas
cannot be ruled out.

C. MISCELLANEOUS CHEMICALS
High levels of petroleum hydrocarbons have been reported to cause initial hyperactivity
which then progresses to a general lethargy. If exposure continues, loss of equilibrium is
seen (Anderson, 1975). Lower concentrations may result in a dose-dependent depression
in muscular activity (Ellgaard et al., 1979). Overall, these changes have been likened to
those observed with general anesthesia in fish or acute alcohol intoxication in mammals.
The implication is that such behavioral alterations are due to disruption of nervous
activity in the reticular formation in the brain stem.
Organochlorine insecticides tend to cause hyperactivity in fish, at least upon initial
exposure to the chemical, but the quantitative measurement of this effect has generally
been indirect via determination of changes in the rate of oxygen consumption (Waiwood
and Johansen, 1974; Bansal et al., 1979). DDT (and possibly some other organochlorine
compounds as well) causes altered function of the lateral line organs (Anderson, 1971;
Bahr and Ball, 1971). Electrical recording from the lateral line nerves showed that a low
frequency pressure wave in the water normally caused a short burst of nerve impulses.
 275

Exposure to sublethal levels of DDT increased the duration of the burst, and the DDT
effect is especially dramatic at lower temperatures. This latter observation is interesting
in that DDT has been shown to be more toxic at lower temperatures (Cope, 1965) and fish
exposed to DDT actively avoid colder waters when given a choice (Ogilvie and Ander­
son, 1965). We should hasten to note here that the temperature effect on organochlorine
pesticide toxicity is not the same for all of them. For some of these pesticides, increased
temperature increases toxicity, for others it is decreased (see Cairns et al., 1975 for
review).
The hyperactivity induced by organochlorine insecticides would appear to be related
to an increased sensitivity to external stimuli, a phenomenon noted in mammals exposed
to pesticides of this group (Murphy, 1975). These substances could also cause altered
neurochemical activity. Fingerman and Russel (1980) did a combined study of the action
of the polychlorinated biphenyl Aroclor 1242 on locomotor activity and brain neurotrans­
mitters in killifish {Fundulus grandis). Activity was measured visually with a grid
chamber and Aroclor caused a roughly tenfold increase in activity on the first day which
gradually reduced to a fivefold elevation over controls by day three of exposure. Because
the solution was not changed, this decrease may reflect a declining Aroclor concentration.
The catecholamines norepinephrine and dopamine in the brains decreased after 24 h of
exposure. The interpretation of these findings is unclear because the function of these
neurotransmitters in the brains of fishes has received little experimental investigation.
Furthermore, discrete levels of the transmitter in specific brain regions were not mea­
sured. These may be more critical than the overall brain concentration.
While a number of investigators have seen stimulation of activity with organochlo-
rines (discussed in preceding paragraph), more prolonged exposure may produce an
inhibition. Little et al. (1990) observed a dose-dependent decrease in activity of juvenile
trout after 96-h exposures to chlordane. At a concentration one half the 96-h LC50 (the
highest dose tested), activity in the trout was reduced by approximately 50%. Pentachlo-
rophenol caused a similar effect. Part of this discrepancy in results between the various
studies may be due to the kinetics of the response. Ellgaard et al. (1977) tested bluegill
at several very low concentrations of DDT over a period of up to 16 days. There was a
dose-dependent stimulation in spontaneous activity, however, at concentrations in excess
of 10 |ig/L the stimulation was transient and an inhibition of activity was revealed after
several days. The higher the dose, the less the duration of hyperactivity before the
suppression.
The effect of organophosphorus insecticides on activity is contradictory; stimulation
and inhibition have both been observed (Bansal et al., 1979; Rath and Misra, 1979;
Johnson, 1978; Henry and Atchison, 1984; Little et al., 1990) (also see Chapter 8). The
particular preparation, fish species, duration of exposure, and dose used all may contrib­
ute to the disparity of responses. It is well known that these chemicals, along with the
carbamate insecticides, affect the central nervous system by inhibition of acetylcholines­
terase, the enzyme that prevents the buildup of acetylcholine (Zinkl et al., 1991). As
Smith (1984) points out, they inhibit acetylcholinesterase in peripheral tissues as well,
which could also affect locomotor activity.
Residual chlorine is a strong oxidizing agent that irritates gill tissue (Bass and Heath,
1977). In spite of this irritation, sublethal exposures of brook chair for up to 6 days caused
lethargy and an increase in thigmotaxis (Jones and Hara, 1988). In the same study it was
further noted that the aberrant behavior persisted in some individuals for at least 46 days
after being returned to non-chlorinated water. This long persistence is interesting as Bass
and Heath (1977) observed rapid recovery in gill function following fairly high but
intermittent (i.e., for less than 2 h) chlorine exposures. Thus, normal behavior recovered
more slowly than some other physiological functions.
276

III. AVOIDANCE OF OR ATTRACTANCE TO

WATERBORNE CHEMICALS
Many of the changes observed in locomotor activity of fish when they are exposed to
chemical pollutants probably reflect a simple attempt to get away from the irritant,
especially when test concentrations are high. In order to avoid low concentrations, the fish
must first be able to detect the chemical in the water, and then go down a gradient toward
“clean” water. Detection is probably the more critical factor here, and there have been
several ingenious devices built to quantify this ability in fishes. They fall roughly into two
types: the steep gradient chambers and the shallow gradient chambers (see Figure 1). In
the former, the fish is given a rather distinct choice while in the latter, it is exposed to a
range of concentrations over some defined distance. The usual procedure is to place one
or more fish into the test chamber and, before adding the chemical, allow them to adjust
to the new environment. Then, the test chemical is added to the water inflow on the dosed
side and observations are begun. Generally, the concentration is then raised in a stepwise
progression over a period of several hours. The data gathered may be the time spent in
the dosed water, the number of entries into that water, or distribution of the fish after some
time interval. In most instances, visual observation (often with the aid of a video camera)
is used, but photocells coupled to a computer have also been successfully adapted to this
sort of study (Kleerekoper et al., 1973).
There have been several reviews of fish avoidance behavior studies (Cherry and
Cairns, 1982; Giattina and Garton, 1983; Atchison et al., 1987; Beitinger, 1990). Of
considerable interest have been the attempts to define a threshold, or that concentration
of chemical which is required to cause a significant avoidance (or preference). Table 1
gives some values for metals that have been observed. Water hardness is included in the
table as this is known to greatly influence metal toxicity to fish (Sprague, 1985). With the

A) STEEP GRADIENT CHAMBERS

B) SHALLOW GRADIENT CHAMBERS

I.

Figure 1 Schematic diagram of several of the systems used to test for avoidance or attractance
of fishes to aquatic contaminants. (A) 1. Countercurrent flow chamber; 2. “rosette apparatus”; 3.
“Y” trough; 4. modified fluvarium; (B) 1. fluvarium; 2. linear gradient. (From Giattina, J. D. and
Garton, R. R., Residue Rev., 87, 43, 1983. With permission.)
 277

Table 1 Minimum Concentration of Waterborne Metal

Producing an Avoidance Response in the Laboratory
Concentration Hardness
Chemical (mg/^) (mg/^) Species
Copper 0.1 89.5 Rainbow trout
2.4 20 Atlantic salmon
5.0 5.4 Goldfish
6.4 25 Rainbow trout
70 112 Rainbow trout
Zinc 5.6 14 Rainbow trout
50 112 Rainbow trout
54 20 Atlantic salmon
Cadmium 50 112 Rainbow trout
Nickel 23.9 26 Rainbow trout
Lead 26 28 Rainbow trout
Mercury (attractance) 0.2 112 Rainbow trout

References and data in Giattina, J. D. and Garton, R. R., R e s id u e R ev ., 87, 43,

1983.

exception of cadmium, fish exhibited an avoidance (attractance for mercury) at concen­

trations well below the 96-h LC50. Indeed, it may be as low as the LOEC determined by
standard chronic tests (Atchison et al., 1987). Thus, they possess the ability to sense the
presence of some metals at quite low concentrations, and in some instances at a concen­
tration below that which causes reductions in reproduction. Unfortunately, it is not
possible to generalize this high ability to avoid all metals. Cadmium concentrations may
actually become lethal before fish avoid it in the water (Black and Birge, 1980; Chapman,
1978), or with some species, it is not detected at concentrations manyfold higher than
lethal, (Hartwell et al., 1989). Recently, McNicol and Scherer (1991) found that lake
whitefish exhibited a dichotomous response to cadmium. Some of the fish were attracted
to cadmium at nearly all test concentrations, others were repelled by the metal. If the
direction was ignored, it was also found that lake whitefish reacted to very low and very
high concentrations of cadmium, but virtually ignored the intermediate ones.
Selenium also appears to not be avoided by fish (Hartwell et al., 1989). Test conditions
contribute markedly both to the qualitative and quantitative data obtained in avoidance
studies. For example, goldfish in a shallow gradient actually were attracted to copper, but
the same concentrations in a steep gradient resulted in avoidance (Kleerekoper et al.,
1972; Westlake et al., 1974). Furthermore, high copper concentrations may attract rain­
bow trout, while low ones cause an avoidance behavior (Giattina et al., 1982).
Long-term acclimation to metals can affect tolerance (Anadu et al., 1989) and avoid­
ance behavior. Hartwell et al. (1987) tested fathead minnows with a blend of metals which
simulate the effluent from a fly ash settling pond of an electric power plant. Acclimation
to this blend composed of copper, arsenic, chromium, and selenium at a total concentra­
tion of 48 |ig/L for 3 months caused the fish to lose avoidance behavior to a concentration
(245 jig/L) that was clearly avoided by controls. Surprisingly, acclimation to twice the
previous level for 3 months resulted in an actual preference for elevated metals by the
fish. Acclimated fish mildly avoided concentrations 5 times the holding concentration
after 6 months, but by the end of 9 months of acclimation to the metals, they were not
responsive to concentrations as high as 10 times the exposure level. Thus acclimation to
a blend of metals caused the fish to largely lose the ability to avoid them. It seemingly
is not known whether acclimation to single metals would cause a similar loss of behav­
ioral sensitivity.
278

Just why under certain circumstances some metals are attractive while they are
avoided or ignored under other conditions is not at all clear. Currently, there is no
knowledge of the sensory modality involved in the detection of metals by fish (but see
next section on sensory receptors). Receptor organs, such as those around the eyes and
the gills, may be important in detection of metals in the water but no direct evidence
appears to be available.
Information on avoidance of DDT is somewhat variable, depending on species and
method of testing (Giattina and Garton, 1983). One of the more interesting observations
is that DDT-susceptible mosquitofish (Gambusia affinis) avoid DDT, but those resistant
do not (Kynard, 1974). Thus resistance which has been acquired to a pesticide, which may
make them able to tolerate massive tissue levels (Ferguson, 1967), may also cause a loss
in sensory sensitivity to that chemical. On the other hand, lest one think that the situation
is that straightforward, Kynards (1974) also tested different populations of mosquitofish
avoidance to parathion (an organophosphorus insecticide) and found the susceptible ones
exhibited an avoidance threshold of 200 p,g/L and the resistant ones 1000 g/L. This
sounds like the same general trend as with the DDT ; however, the 24-h LC50s were found
to be 20 and 2000 ftg/L, respectively. So the resistant fish avoided acutely toxic concen­
trations while those susceptible did not, thus selection may have favored the ones able to
avoid harmful concentrations of the parathion.
A variety of other chemicals has been shown to cause avoidance responses in fish.
These include pulp and paper mill effluents, the herbicide 2,4-D, and residual chlorine
(see Giatinna and Garton, 1983 for references). Chlorine has probably received more
attention in this regard than any other chemical. In freshwater, residual chlorine is
composed of two major constituents, “free” chlorine made up of hypochlorous acid and
hypochlorite ions; and combined chlorine made up mostly of monochloramine. Free
chlorine is more toxic than the combined form (Heath, 1977) and fish avoid it at lower
concentrations than they do the combined form (Cherry et al., 1979). They also avoid
sulfur dioxide, which is used as a dechlorination chemical (Hall et al., 1984). In general,
a wide variety of species, both marine and freshwater, have been found to avoid chlorine
at concentrations well below the lethal level, but temperature, salinity, body size, and time
of exposure all influence the results (Cherry et al., 1982; Hall et al., 1983). Temperature
is an especially critical factor as an elevated temperature can attract fish into concentra­
tions of chlorine that would otherwise be avoided. This, incidentally, is an aspect that
could be a complicating factor for a variety of substances in addition to chlorine, because
many effluents containing toxicants also have waste heat.
Not all chemicals are avoided by fish at concentrations below the lethal levels. These
include the already mentioned metals cadmium and mercury. Other chemicals that appear
to not be avoided at all or require acutely lethal concentrations to induce avoidance are
PCBs, phenol, parathion, the herbicides Garlón and Vision (Giattina and Garton, 1983;
Beitinger, 1990; Morgan et al., 1991), and probably some others that have not been tested.
It would seem reasonable to hypothesize that this apparent failure to respond is due to an
inability to detect the presence of the substance at low concentrations. However, using
classical conditioning methods, Hasler and Wisby (1950) showed that fish can detect
phenol at a concentration as low as 0.0005 mg/L, which is far below the lethal level. Thus,
failure to detect the chemical cannot be the only reason for this lack of response. From
a practical standpoint, it means that the predictive value of using this behavior for
estimating chronic toxicity is severely limited. Further, it cannot be assumed that fish will
avoid lethal levels of all chemicals in the environment and thereby provide some sort of
safety factor.
There are examples of contaminants acting as an attractant rather than a repellant. Hara
and Thompson (1978) found that whitefish (Coregonus clupeaformis) were attracted to
the detergent sodium lauryl sulfate (SLS) at all concentrations they tested except the
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highest (10 mg/L), which is above the 96-h LC50. Mercuric chloride may also be an
attractant for rainbow trout at a sublethal level (0.2 mg/L) (Black and Birge, 1980), and
mention was made above of how copper under some circumstances can act as an
attractant. However, these examples of attraction are clearly the exception.
Avoidance of chemicals can have considerable ecological importance. A habitat may
be effectively eliminated by this mechanism, and thus it could explain the absence of
some fish in polluted areas. For migratory species, such as salmon and eels, a polluted
stretch of river could block either upstream or downstream movement. Saunders and
Sprague (1967) reported that adult Atlantic salmon on their upstream migration actively
avoided areas contaminated with copper and zinc at concentrations that were clearly
sublethal. Based on both their field and laboratory data, they concluded that concentra­
tions above 38 |Lig Cu/L and 480 |ig Zn/L would effectively block migration.
The combination of a chemical that is avoided with one that is attracted can produce
some interesting problems for the fish, as well as the experimenters. Alanine is a
ubiquitous constituent of prey odor so many fish are attracted to it. Steele et al. (1990)
arranged experiments to test the effect of copper on that attractance behavior in zebrafish.
A concentration of copper of only 10 jiig/L was avoided by itself, but in the presence of
alanine it was not avoided. Perhaps more importantly, a copper concentration of only 1
jiig/L suppressed attraction to alanine! This concentration is well below the USEPA
criterion for copper (USEPA, 1986).
Determining a threshold concentration for avoidance behavior is important for pollu­
tion biologists, but extrapolations from laboratory determinations to the field may be
misleading as a host of conditions such as hardness, temperature, acidity, previous
exposure to the pollutant, and the sharpness of the gradients can have a large effect on the
response of the fish.

IV. SENSORY RECEPTORS

The sense organs are the way the central nervous system samples the environment. Most
of the locomotor and avoidance behaviors which have been discussed herein are actually
the result of some sensory input. In this section, the concern will be with the action of
pollutant chemicals on receptor function per se. Because of the technical difficulties of
investigating these physiological functions, the database is severely limited. Research on
the effects of pollutants has generally fallen into three areas: (1) histological lesions in
specific sensory receptor organs, (2) alterations in nerve impulse frequency as recorded
from sensory nerves during or following exposure to a test chemical, and (3) recording
of summed electrical activity directly from the olfactory organ or eye while stimulating
the organ with a chemical or light. The resulting recording is referred to as an electro-
olfactogram (EOG) and electroretinogram (ERG), respectively.
In addition to the usual senses that most people think of such as sight, touch,
temperature, etc., fish are especially well endowed with chemical senses (Kara, 1993).
They have olfactory receptors, generally located in the paired nostrils, which sense
material dissolved in the water and the gustatory sensors which are located in the roof of
the mouth, on the barbels (if present), gill rakers, and in some species, over the entire
body. These chemoreceptors serve for feeding, kin recognition, alarm communication,
reproduction, and navigation.
The olfactory receptors have received the bulk of attention from fish researchers
interested in pollution effects on sensory function. For example, the olfactory epithelium
has been examined with scanning electron microscopy before and after exposure to
benzene, a common petroleum hydrocarbon (Babcock, 1985). It was found that the
epithelium experiences physical damage from exposure to sublethal levels of this hydro­
carbon in the water.
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The lake whitefish is normally attracted to a food extract and it has been shown by
cauterizing the nares that they are attracted to the odor. This olfactory function for
detecting food is significantly reduced by chronic exposure to 50 |ag HgCl/L (Kamchen
and Kara, 1980). The well-known homing migrations of adult salmon have been shown
to depend on the olfactory sense (Stabell, 1992). Salmon follow one or more odors up the
stream until they find the home where they spawn. This olfactory homing is extremely
sensitive to “foreign” substances in the water as is exemplified by the observation that
rinsing one’s hands in the stream will cause the migrating adults to reverse direction and
retreat downstream (Smith, 1982).
When the olfactory bulb of a salmon or trout is perfused with a solution containing any
of several different amino acids (alanine is a popular one to use), a characteristic burst of
nerve impulses can be recorded from the olfactory nerves, or a distinct EOG can also be
detected. However, if the fish are first exposed to inorganic mercury (>0.10 mg/L) or
copper (>0.008 mg/L) there is a depression of the response within 2 h. These metal
concentrations are 3-5 times lower than the lethal levels. The degree of neurological
depression increased with time of exposure and concentration (Hara et al., 1976). Increas­
ing the calcium level in the water reduces the inhibitory effect of copper on the receptor
function (Bjerselius et al., 1993). Copper at concentrations as low as 0.044 mg/L have
been shown to change the attractiveness of homestream water to migrating salmonids
(Sutterlin and Gray, 1973), and this is well above the concentration causing inhibition of
olfaction sensitivity to amino acids. (Also see the above discussion on avoidance of
metals in the field and laboratory.)
The mechanism by which metals alter olfactory response of fish has received some
attention. In high doses of copper (0.5-5.0 mg/L) estuarine fishes exhibit histological
lesions of the olfactory mucosa (Gardner and LaRoche, 1973). At lower doses, Hara et
al. (1976) hypothesized the effect to be primarily due to the metals tying up sulfhydryl
and amino groups in the membranes of the receptor cells. Because they observed a
rather rapid response and quick recovery, it was suggested the effect may be limited to
the membrane, at least at the lower levels they used. Winberg et al. (1992) present
evidence that low concentrations of copper do not inhibit binding of odorants to the
receptor membrane. Instead, they propose that the copper inhibits the transduction
mechanism of the receptor cells (i.e., the conversion of the presence of odorant into
nerve impulses).
Further work on the mechanisms of inhibition has been done using methylmercury and
inorganic mercury. Baatrup et al. (1990) found that methylmercury accumulates mostly
in the olfactory receptor cells of the sensory epithelium whereas inorganic mercury
accumulates mainly along the borders of the receptor and in the supporting cells of the
epithelium. What was especially striking was how the methylmercury caused a more
permanent damage to the olfactory function in that rinsing with freshwater failed to
restore normal activity. Baatrup et al. (1990) suggest that the mercury ion acts on the
calcium gate of the olfactory receptors and inhibits the binding of amino acids to the
membrane. Methylmercury, on the other hand, has little effect on calcium channels for
diffusion but does affect active transport of calcium. Methylmercury further accumulates
in the cell more than the inorganic form thereby potentially causing effects on enzyme
function there (Baatrup et al., 1990).
In addition to the receptors for smell, the function of taste receptors is also blocked by
mercury and copper (Hikada, 1970; Sutterlin and Sutterlin, 1970) so these metals appear
to have a rather broad inhibitory effect on chemoreceptors. Recall that copper is avoided
at very low concentrations by fish whereas high concentrations or steep gradients may
actually cause attraction. This raises the interesting question of what is being detected in
the avoidance/preference responses. One could speculate that since copper and mercury
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are such effective chemosensory inhibitors, perhaps it is the inhibition of detection of one
or more substances (rather than the metal, per se) that is the effective stimulus when fish
are exposed to either of these elements.
As mentioned earlier, the detergent SLS is an attractant for whitefish (Kara and
Thompson, 1978). The ABS (“hard”) detergents have also been found to inhibit feeding
behavior in catfish (Bardach et al., 1965) and in the flagfish (Jordanellafloridae) (Foster
et al., 1966). Kara and Thompson (1978) have shown neurologically how SLS may also
affect feeding. They recorded nervous activity from the olfactory bulb of fish exposed to
this detergent and a standard food stimulus. The response to the food stimulus was
reduced almost immediately upon exposure to the detergent and the response was dose
dependent over a range of 0.1-10 mg/L. This is, of course, similar to the effects seen with
mercury and copper, however, the mechanism is undoubtedly different. With a detergent,
the observed depression of sensory function may involve removal of the thin mucous
layer over the receptor membranes. This layer is a source for the inorganic ions required
for the sensory transduction process (Kara, 1993).
The petroleum hydrocarbon naphthalene has been found to induce histological lesions
in the olfactory, gustatory, and lateral line receptors at concentrations that cause no effect
on other organs except gill epithelium (DiMichelle and Taylor, 1978), thus these sensory
receptors may be one of the first organs to suffer from this chemical insult. Other
components of petroleum appear to cause specific alterations in the olfactory organ
epithelium. According to Gardner (1978), whole crude oil causes hyperplasia, water
soluble components produce epithelial metaplasia, and water insoluble components in­
duce dilation and congestion of submucosal vasculature. Scanning electron microscopic
examination of the olfactory epithelium of sole following an 8-day exposure to the water-
soluble fraction of Prudhoe Bay crude oil “revealed degenerative changes in the
chemosensory cilia and a loss of the microridges that circumscribe the perimeter of the
epithelial cells surrounding the olfactory organs in six of eight fish” (Hawkes 1980;
p. 3230).
Rather specific lesions in olfactory tissue may also be characteristic of the action of
copper, mercury, and silver, at least in some species (Gardner, 1975). However, cadmium
does not seemingly affect olfactory tissue (Gardner, 1978). This last observation may also
relate to the finding that cadmium is not avoided by fish except at high concentrations (see
section on avoidance).
The lesions in the olfactory organs caused by metals such as copper and zinc appar­
ently are not permanent. Moran et al. (1987) noted that fish that had experienced nearly
complete loss of receptor epithelium from having been accidentally exposed to metals in
the water showed good regeneration within 8 days of being placed in non-contaminated
waters.
Lead and zinc may, in addition to affecting olfactory organs, cause histological effects
to the lateral line organs if the metal concentrations are fairly high (Haider, 1979). The
lateral line organs of fish serve primarily to detect water movements and transient
pressure changes and hydrodynamic interactions in the immediate vicinity of a fish
(Popper and Platt, 1993). Temperature has a marked effect on the neurogenic activity of
these receptor organs (Peters and Weber, 1977).
In trout the impulse frequency in nerves from the lateral line exhibits a rather steady
spontaneous rate which is directly proportional to temperature. Peters and Weber (1977)
investigated the action of DDT on the output from the lateral line organs and found that
the insecticide caused the impulse frequency from the receptors to take on a bursting
character at temperatures below 12°C. This altered pattern of activity may help explain
the changes in orientation and schooling activity observed in goldfish exposed to DDT
at sublethal levels (Weis and Weis, 1974).
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It has frequently been observed that fish exposed to DDT exhibit hyperexcitability to
mechanical stimuli, such as those produced by tapping on the side of an aquarium. This
is sensed by the lateral line so Peters and Weber (1977) tested the threshold of the lateral
line organs by providing them with a controlled mechanical stimulus and found they were
not altered by DDT, even though Anderson (1968) had earlier reported that DDT caused
the lateral line organs in trout to produce a more prolonged burst of activity following a
single mechanical stimulus. Thus, the DDT effect on reflex responses to external stimuli
(Anderson, 1970; Peters and Weber, 1977) must involve central nervous system changes,
perhaps in part brought about by the abnormal input from the bursting activity seen at
lower temperatures and more direct effects of DDT on neuronal function within the
nervous system (Narahashi and Haas, 1968).
There has been little work on the effects of pollutants on the eyes and associated
structures of fish. Copper has been reported to cause damage to the cornea of larval
striped bass (Bodammer, 1985). The visual system is quite sensitive to mercury poisoning
in mammals (Evans et al., 1975) and it may also affect that of fish. Panigrahi and Misra
(1978) reported blindness and exophthalmia (protrusion of the eyeball) in Indian catfish
exposed to mercury for periods of 4 weeks. They attribute the blindness to effects of the
mercury on the brain and optic nerves, although no direct evidence is provided. It is
perhaps noteworthy that methylmercury tends to concentrate in the mammalian brain
preferentially in the visual and motor areas (Shaw et al., 1975). Using a psychophysical
technique, Hawryshyn et al. (1982) found that rainbow trout injected with methylmercury
15 days previous to testing exhibited a loss of both rod and cone sensitivity. This is in
contrast to the finding for mammals that only the rod reception was affected by mercury
(Fox, 1979).

V. FEEDING AND PREDATOR-PREY BEHAVIOR

A wide variety of pollutants at low sublethal concentrations have been shown to depress
feeding rate. These include residual chlorine (Jones and Kara, 1988), copper (Drummond
et al., 1973; Waiwood and Beamish, 1978), petroleum components (Woodward et al.,
1987), detergents (Foster et al., 1966), dioxins (Mehrle et al., 1988), organophosphorus
and organochlorine insecticides (Little et al., 1990), and low pH water (Jones et al., 1985).
The mechanism for this suppression of feeding is largely unknown. Feeding and the
behaviors associated with it, such as predation, represent a high level of integration of
input from gustatory, lateral line, visual, olfactory, and internal visceral receptors. A
pollutant may act on any or all of these receptors as well as influence the central nervous
system directly by causing lesions in certain areas or changing the rate of neurotransmitter
production or breakdown. Regarding the latter, in a very interesting study, Pavlov et al.
(1993) noted reduced feeding in bream contaminated with an organophosphorus insecti­
cide and the suppression of food consumption correlated with reduced acetylcholines­
terase in the brain. They then followed this observation up with intraperitoneal injection
of cholinergic drugs and one which restores acetylcholinesterase. These treatments caused
a return to normal feeding thus providing strong evidence for the involvement of the
neurotransmitter acetylcholine in the control of feeding. The control of appetite by the
brain of fish (Fletcher, 1984) (as well as other animals) is poorly understood and
undoubtedly involves several areas with the ventrolateral peduncular neuropil probably
being the primary one.
The depression of feeding by acute exposures to toxicants may reflect some deficit in
peripheral sensory function rather than brain activity, particularly where the effect occurs
rapidly upon pollutant exposure (see preceding section on sensory effects). Elevations in
the hormone cortisol in response to stress could also suppress appetite because, at least
in some species, it causes a rise in blood glucose (see Chapter 8). Little et al. (1990)
 283

conclude from their work with juvenile rainbow trout and several agricultural pesticides
that inhibited motivation for feeding occurred at higher test concentrations; but at lower
concentrations, reduced feeding efficiency and fewer strike frequencies (at prey) pre­
dominated.
The behaviors associated with feeding have received considerable attention and there
has been some interest in the effect previous exposure to a toxicant has on the predator-
prey interaction. Essentially two experimental approaches have been used, which Giddings
(1981) refers to as the “mechanistic approach” and the “population approach”. In the
former, various aspects of the specific behaviors, such as reactive distance, handling time,
or capture success are measured. The population approach is done by enclosing prey in
the same chamber with the predator and counting survivors of the prey after some interval
of time. Refuge areas are often provided for prey and hiding spots for predators are
available in some systems. If the chemical causes altered behavior, such as jerks or
hyperactivity in the prey, this presumably will be reflected in a greater rate of predation.
The methodology involved in these studies has been discussed at some length by Giddings
(1981) and Little et al. (1985).
One of the first to use the predator-prey interaction in a toxicological test was
Goodyear (1972) who irradiated mosquitofish with ionizing radiation and then tested
their survival rate in groups with a single largemouth bass. Irradiation caused a much
more rapid loss of fish by predation due to these fish tending to wander out of the refuge
which was provided in the test chamber. Organophosphorus insecticides (Hatfield and
Anderson, 1972), mercury (Kania and Ohara, 1974), cadmium (Sullivan et al., 1978),
hydrazine (Fisher et al., 1980), pentachlorophenol (Brown et al., 1985), and methyl
parathion (Farr, 1978), all at low sublethal levels, have been shown to increase predation
rate on prey, or in some way alter the interaction. In a carefully conducted study, Sullivan
et al. (1978) showed that exposure to 50 |ig/L cadmium for 48 h actually decreased prey
vulnerability of fathead minnows. They attribute this to an example of “sufficient chal­
lenge” in which an organism apparently benefits from a small dose of some substance that
is harmful if prolonged or at a higher dose (Smyth, 1967). However, when the minnows
were exposed for 21 days to the same or lower concentration of cadmium, predation was
increased significantly. The actual behavioral differences observed in the prey were rather
subtle. This is a schooling species and the researchers noted that the cadmium-treated fish
often would briefly change direction in the school and/or orient at right angles to the
others for several seconds (they were branded so as to be recognizable among non-
exposed members). Holcombe et al. (1980) also observed a decline in schooling activity
in fathead minnows exposed to the herbicide 2,4,-D. The lateral line organs are important
in schooling behavior (Popper and Platt, 1993) so these findings suggest, but do not prove,
an effect of the test chemical on that sensory receptor. Because schooling is considered
to be of survival value to avoid predation (Taylor, 1976), it is easy to see how this altered
behavior of individuals would make them more vulnerable to predators.
Apparently the action of metals on schooling behavior differs with the metal, or
perhaps fish species. Koltes (1985) used a computerized video technique to examine the
effect of copper on Atlantic silverside (Medidia) schools. She observed that low concen­
trations of copper caused the schools to increase activity, individual fish decreased their
nearest neighbor distances and swam more in parallel orientation. In a sense, the schools
became more uniform although swimming at a higher speed. It is not clear whether this
would affect predation.
Most studies of predator-prey behavior use fish that have been raised in hatcheries or
kept in the laboratory for a considerable period of time. Weis and Khan (1991), on the
other hand, obtained mummichogs (F. heteroclitus) from a highly polluted estuary and
conspecifics from a pristine habitat. Those from the polluted habitat exhibited a reduced
ability to capture juvenile guppies.
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A rather novel type of predator-prey interaction was investigated by Fisher et al.

(1980) using the dorsal light response of bluegill {Lepomis macrochirus). In this case, the
predator was the test animal. Most fish will tilt the dorsal surface of the body toward a
strong light source when it is first turned on, and the degree of tilt increases if the fish sees
desirable prey. Indeed, the more “desirable” the prey, the greater the tilt and this can be
quantified using an appropriate chamber fixed with a protractor (Vinyard and O’Brian,
1975). In this study, the effect of hydrazine (a widely used reagent in jet fuels, herbicides,
pharmaceutical drugs, etc.) on the predator was measured by exposing the fish for only
10-15 min before testing. An artificial prey was used for which the bluegills were to
respond. A significant reduction in tilt occurred at a concentration of hydrazine approxi­
mately 1/10 the 96-h LC50 for this species. The authors wisely avoid drawing any
ecological implications from this study.
Exposing only the prey, or the predator, to the test chemical is actually a bit of an
artificial situation since, in nature, presumably both will be inhabiting the same water.
Thus, the work of Woltering et al. (1978) is of interest because they exposed both the
predator (largemouth bass) and prey (mosquitofish) to ammonia. At concentrations above
0.34 mg/L there was actually a lower rate of predation. When prey densities were raised,
the ammonia effect was greater and the prey even harassed the predator!
One of the most thorough predator-prey studies is that of Sandheinrich and Atchison
(1989). They used a mechanistic approach which yielded considerably more information
than merely the feeding rate. Bluegill were exposed for 4 days to 4 concentrations of
copper ranging from 5-1700 p.g/L. Prey used were two species of Daphnia, an amphipod,
and two sizes of zygoptera. Both copper-treated and untreated prey were tested. Prey
handling time was the most consistent parameter to be altered. It increased significantly
with increasing copper concentrations and this effect occurred with both treated and
untreated prey. This caused overall consumption rate of prey to go down in a dose-
dependent fashion, which, of course can be predicted to depress growth rate. Prey
handling time refers to the interval between when a prey item is taken in and the fish
begins to hunt another item. The mechanism by which copper, or anything else, changes
prey handling time is apparently unknown.
In summary, we see that the behaviors associated with predator-prey interactions are
extremely sensitive to a variety of chemicals in the water. Increased vulnerability to being
eaten could have population consequences for the prey, but if the same water is causing
the predator to feed less, for whatever reason, then the two effects may even cancel each
other out, or the predator may experience more of an effect than the prey. Sandheinrich
and Atchison (1990) make a strong plea for a more mechanistic approach to these sorts
of studies so the findings can be indicative of effects that might occur in natural
communities. They show how with the use of foraging theory and bioenergetics modeling
that predictions of effects on growth rate can be made based on things like alterations in
food consumption and increased cost of food handling by predators. These predictions
can then be field verified. The possibilities for future research on this seem promising.

VI. AGGRESSION
Here we are referring to intraspecific aggressive behaviors associated with territoriality
and social hierarchies. A particular species often shows characteristic aggressive displays
which can be quantified, although for the inexperienced observer, there would be a large
degree of subjectivity. Smith (1984) has reviewed the literature on the neurobiological
basis of aggression in fish. In short, it involves mostly the telencephalon portion of the
forebrain (which incidentally also serves for olfaction). Evidently, the telencephalon of
 285

the fish brain is analogous to the limbic system of higher vertebrates where most of the
emotional responses are integrated. There are lower brain nuclei in fish where the
behaviors may initiate, but they are then facilitated in the telencephalon.
Bluegill are prone to develop social hierarchies, at least in captivity. Henry and
Atchison (1979a) exposed this species for 15 days to several concentrations of a mixture
of cadmium and zinc. Aggression, as measured by number of nips, increased at the low
concentrations while it decreased at the higher ones, so the effect was dependent on dose
but not in a linear fashion. Henry and Atchison (1986) later observed that copper at only
34 |ig/L for 96 h caused dominant bluegill to increase their number of threats and nips.
Exposure of bluegill groups to methyl parathion at concentrations less than 1/10 the
96-h LC50 caused a reduction in aggressive behaviors with the greatest effect seen in the
dominant fish during the first 24 h (Henry and Atchison, 1984). This same organophos-
phorus insecticide caused male Siamese fighting fish to ignore each other after being
exposed for 5 days to a fairly high (1.0 mg/L) dose (Welsh and Hanselka, 1972).
Aggressive interactions can cause the “loser” to become more susceptible to a toxi­
cant. Sparks et al. (1972) found that the submissive bluegill of a pair was less able to resist
a lethal level of zinc than was the dominant one. However, if a shelter (overturned
flowerpot) was provided, the aggressive interactions were reduced and the differential
sensitivity to zinc was eliminated. The authors note that these sorts of behavioral hierar­
chies may contribute to variability in toxicity bioassays.
Because toxicants usually alter the frequency of aggressive encounters, rather than
their character, it has been suggested that the mode of action may be on the telencephalon,
because the same thing occurs when certain neural pathways there are severed (Smith,
1984). In general, it appears that aggressive behaviors may not be as sensitive to pollutant
exposures as other behaviors such as general locomotor activity and prey-predator
interactions.

VII. LEARNING
Laboratory studies of learning in fish are largely limited to conditioned responses. The
literature on conditioning in fishes has been reviewed by Marcucella and Abramson
(1978) in a chapter entitled, “Behavioral Toxicology and Teleost Fish”. Unfortunately,
that title is rather misleading as there is nothing on the action of environmental pollutants
included. There is a good discussion of the action of various drugs on conditioned
responses which can be useful in interpreting the neurogenic basis of a particular re­
sponse.
In classical conditioning studies, a stimulus, generally a light, is administered to the
fish at the same time it receives an electrical shock through the water. Of course, the fish
exhibits an immediate escape maneuver. The same procedure is repeated until the fish
“learns” to associate the light with the unconditioned stimulus and responds before the
shock is given. It is then deemed trained. Weir and Hine (1970) trained goldfish in this
manner and then exposed them to one of a variety of metals for either 24 or 48 h before
testing again. Therefore, this was an investigation of the effect of the metals on retention,
rather than learning. They observed a marked dose- and time-dependent impairment of
the conditioned response. The metals tested were arsenic, lead, mercury, and selenium,
and in all cases, significant impairment was observed at concentrations below the esti­
mated LC 1, so the behavioral criterion was quite sensitive to the metals in the water.
There has been a fair amount of interest in the effect of insecticides, especially DDT,
on fish learning, but the results have been unclear. When a mild stimulus is applied to the
guiar region of a fish, it will twist its tail in a propeller-like manner. Anderson and Prins
286

(1970) found that this reflex could be conditioned using light as the conditioned stimulus
and a mild electric shock applied to the gular area as the unconditioned stimulus. A 24-h
exposure to a sublethal dose of DDT caused a greatly reduced ability to “learn” this reflex
suggesting that DDT had some effect on the central nervous system to inhibit learning.
Using a more conventional type of conditioning setup, they also showed a reduction in
learning from DDT exposure (Anderson and Peterson, 1969), but when this was reevalu­
ated in the same laboratory with a modified procedure, no effect of the DDT was noted
(Jackson et al., 1970). The previous findings may have been due to an impairment of
performance, rather than learning, an important point to consider when doing “learning”
studies.
Several different experiments were performed by Hatfield and Johansen (1972) to
evaluate the action insecticides of different types have on both learning and retention. A
24-h exposure to the 96-h LC50 concentration caused a slight enhancement of learning
with DDT, no effect from methoxychlor, and a mild to severe retardation of learning with
the organophosphorus compounds Abate and sumithion. Retention was relatively unaf­
fected by the insecticides and the alterations in learning were apparently not permanent
as 7 days of recovery in non-contaminated water returned the fish to “normal”. Treatment
at 1/10 the 96-h LC50 with each of the chemicals caused no effect on learning, but
whether a chronic exposure would result in changes is unknown from these data.
The organophosphate parathion clearly inhibits both learning and retention in gold­
fish. Sun and Taylor (1983) found that 24-h exposure prior to training had a dose-
dependent effect on learning, and it is noteworthy that the lowest concentration found
effective (100 \igfL) was commonly observed in irrigation streams in California. Reten­
tion of the conditioned response was affected to approximately the same extent as
learning when the fish were exposed to parathion after training and prior to retention
testing. The mechanism(s) by which xenobiotics may affect learning are diverse. These
include motivation, attention, sensitivity of sense organs and, of course, the memory
process itself (Marcucella and Abramson, 1978).
Parathion is a strong anticholinesterase substance. Goldfish exposed to this insecticide
at a concentration the same as used by Sun and Taylor for 24 h exhibited a 62% decrease
in cholinesterase activity in the brain (Weiss, 1961). Anticholinesterase drugs in general
are known to suppress both learning and retention in a variety of animals (Deutsch, 1971),
so this is at least one of the probable mechanisms involved.
In concluding this section, it seems pertinent to note that since the early 1970s, there
has been rather little interest in studying the action of pollutants on learning processes in
fishes. It is not obvious just why this is so, but some tentative conjectures will be
attempted here:
1. The conditioned response is the only sort of “learning” thus far used and it appears that
short-term acute exposures have relatively little effect unless the concentrations of the
test substance is at the upper end of the sublethal range. Thus, it is not an especially
sensitive test for screening pollutants.
2. The conditioned response test is primarily a tool of the comparative psychologists so
most aquatic biologists and physiologists are not oriented toward its use.
3. It is difficult to draw ecological relevance from results of conditioned response studies.
Of course, these “reasons” are not mutually exclusive and should not mean that such
investigations may not prove fruitful. Because foraging behavior is rather sensitive to the
presence of contaminants, and in natural environments learning is an important compo­
nent of foraging (Hart, 1993), combining learning and foraging behavior studies might
prove useful.
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VIII. OPTOMOTOR RESPONSE

The optomotor response refers to the maintaining of an unchanging position relative to
a moving visual stimulus (Scherer and Harrison, 1979). Fish use this in schooling and in
maintaining position relative to the shore in a fast moving stream. In the laboratory it is
usually assessed by moving a background past fish around a small circular chamber and
determining how well the fish tracks the background. The optomotor response thus
involves visual function, integration of information by the central nervous system,
coordinated motor activity, and swimming capacity, all of which might be sensitive to
toxicants in the environment. The effects of pollutants on swimming capacity, which is
usually measured in swimming tunnels was discussed in Chapter 8. Here, the orientation
is more on the behavioral aspects of this response, but as we will see, the variables of
swimming capacity and optomotor response interact with each other.
Dodson and Mayfield (1979) determined that exposure of rainbow trout for 24 h to the
herbicides Diquat and Simazine (fenitrothion) at concentrations commonly seen in the
field caused loss of optomotor response such that it would probably cause downstream
displacement of the fish if they had been in a normally fast flowing trout stream. Their
procedure involved having the fish swim around the circle of a chamber as they attempted
to keep up with a rotating outer drum. Those exposed did not swim as well and some
tended to go in the opposite direction. However, using the same agricultural chemicals,
Peyster and Long (1993) observed an actual enhancement of optomotor response in
fathead minnows. They used a different procedure in which the drum was rotated slowly
in alternating directions during six 1-min periods to test response latency. This variable
decreased with chemical exposure so the fish were responding more quickly to the
stimulus. They also tested swimming capacity in a water tunnel and found the pesticides
caused a considerable decrease in that parameter. They speculate that the increased
response to the stimulus could be an example of hormesis, or it could reflect an increase
in sensitivity to stimuli, such as seen with substances that inhibit acetylcholinesterase
(McKim et al., 1987). Fenitrothion is a strong inhibitor of that enzyme; it is not known
if Diquat does the same. In the Dodson and Mayfield (1979) study, it seems to me that
they were actually measuring swim capacity more than optomotor reflex. At the risk of
sounding pedantic, one cannot help but note that it is a good example of how experimental
design can certainly affect results.
Using a different approach, but still with a rotating striped drum around the central fish
tank, Richmonds and Dutta (1992) tested bluegill and malathion, an organophosphorus
insecticide. Their procedure was to rotate the drum and count the frequency of turns either
in the direction of the drum or in the opposite direction (reversals). Exposures to the
insecticide were for 24 h and there appeared to be a sort of threshold concentration of
0.016 ppm. At that concentration a distinct increase in orientation toward the drum
rotation direction occurred. Reversals were relatively rare but these also increased at that
concentration. At a concentration of 0.032 ppm orientation was similar to control levels
and at 0.048 ppm the fish became quite lethargic. So at a concentration below that
producing distinct toxicity, a state of hypersensitivity seems to take place. In the same
laboratory (Dutta et al., 1992) the pesticide diazinon (another cholinesterase inhibitor)
was tested and it was found to decrease the optomotor response in a dose-dependent
fashion. They also observed a rather good correlation between inhibition of cholinesterase
in the brain and inhibition of optomotor response. However, surprisingly, they do not
comment on their seemingly contradictory findings in the two studies.
When trout fry emerge from the gravel in a stream they normally orient into the
current. Ostrander et al. (1990) exposed the eggs during late embryonic development to
288

benzo[a]pyrene, a component of petroleum and other things containing coal tars, at a

concentration of 25 ppm. The emerging fish appeared normal except they did not orient
into the current normally.

IX. ACETYLCHOLINESTERASE
This enzyme (AChE), frequently referred to as cholinesterase, plays an important role in
the regulation of nerve impulse transmission at cholinergic synapses. It hydrolyzes
acetylcholine, a common neurotransmitter, and thereby prevents it from accumulating in
and around a synapse. Blood plasma contains a second form of cholinesterase called
pseudocholinesterase, which has no known function, although in vitro, it hydrolyzes
acetylcholine. The measurement of this pseudocholinesterase is a commonly used diag­
nostic tool in human and veterinary clinical laboratories for detecting individuals who
have been exposed to organophosphate insecticides.
Inhibition of brain cholinesterase in fishes exposed to organophosphate or carbamate
insecticides in food or water has been reported by many workers (e.g., Weiss, 1961;
Holland et al., 1967; Post, 1969; Coppage, 1977; Malyarevskaya, 1979; Gantverg and
Perevoznikov, 1984; Rao and Rao, 1989; Heath et al., 1993). A review of the use of AChE
inhibition as a diagnostic tool for organophosphate or carbamate poisoning of fish in field
studies has been published by Zinkl et al. (1991).
Gibson et al., (1969) have pointed out that care must be taken in interpreting results
of AChE measurements in fish brain as different parts of this structure have markedly
different normal concentrations. It is remarkable how much of an inhibition of AChE that
a fish can tolerate before death. In general, it appears that around a 70-80% loss of
activity must take place before death of the fish (Coppage and Mathews, 1974; Gantverg
and Perevoznikov, 1984). Of course, less extensive inhibition is associated with a wide
range of behavioral effects such as hyperactivity and loss of equilibrium (discussed in
Zinkl et al., 1991), some of which may prove lethal for reasons other than direct poisoning
by the pesticide. There appears to be a considerable species difference in the degree of
AChE inhibition experienced with a given dose. When perch (Perea fluviatilis) and carp
(Cyprinus carpio) were exposed to the same concentration (0.75 mg/L) of Carbophos, the
brain AChE activity in the perch rapidly went to zero within 5 h whereas carp exhibited
only a mild transient reduction in AChE activity (Gantverg and Perevoznikov, 1984).
Indeed, the carp were able to tolerate 5 mg/L for at least 40 h with a 40-70% reduction
in AChE, but no obvious signs of toxicity. Gantverg and Perevoznikov (1984) propose
no mechanism for such a large species difference although several seem possible. They
include differences in rate of uptake of the pesticide into the body and brain, different
rates of detoxification, and different rates of activation. Regarding the latter, they do
mention that carbophos has no direct inhibitory effect on AChE but that oxidative
enzymes in the animal body convert it into malaoxon which is an anticholinesterase.
Thus, the perch could be doing this at a more rapid rate.
A variety of tissues besides brain also have AChE. The extent of methyl parathion
inhibition of this enzyme in these tissues was compared in tilapia which were exposed for
48 h to a dose one third the LC50 (Rao and Rao, 1984). Following this treatment, the
relative amount of AChE inhibition was found to be greatest in brain, followed by muscle,
gill, and liver in that order. This is the same order of degree of innervation of the tissue,
and the control levels of AChE show a good correlation with that pattern, as well. Thus,
the higher the AChE level in a tissue, the more susceptible it is to inhibition.
Small fish may show greater inhibition than larger ones to the same dose of poison
(Rath and Misra, 1981). Small fish may also recover more rapidly when placed in clean
water (Rath and Misra, 1981), however. Heath et al. (1993) found little recovery of AChE
activity in larval striped bass 10 days after being exposed to methyl parathion for 4 days
 289

Table 2 Molarity of Test Chemical Causing

50% Reduction of Muscle Acetylcholinesterase
Activity In V itro
Chemical I 50 Chemical I 50
Carbaryl 1.0 X 10-5 Dieldrin 2.2 X 10-5
Malaoxon 1.8 X 10-5 Methyl Hg 5.0 X 10-5
C u ^ -^ 1.6 X 10-4 Diazinon 5.0 X 10-5
PM 5.0 X 10^ Atropine 5.7 X 10-5
Cd2-^ 5.7 X 10^ Malathion 5.7 X 10-5
Tubocurarine 1.0 X 10-5 Cr^-^ 7.1 X 10-5
Hg2- 1.6 X 10-5 1.0 X 10-2

Data from Olson, D. L. and Christensen, G. M., E n v iro n . R es.,

21, 327, 1980.

at a low sublethal level. As a rule, recovery is much slower in fish than in mammals
(Wallace and Herzberg, 1988). Depending on the dose and exposure duration, it can take
days to weeks for recovery to take place (Weiss, 1961; van der Well and Welling, 1989;
Morgan et al., 1990; Heath et al., 1993).
Organophosphorus and carbamate insecticides clearly are potent AChE inhibitors, but
they may not be the only pollutants that cause this effect in fishes. In vitro studies have
shown that a variety of other substances exhibit a strong inhibitory action on this enzyme.
From the data in Table 2, it is interesting to note that carbaryl and malaoxon were the
strongest inhibitors, as might be expected, but several metals are also quite effective.
Indeed, they are even more potent than malathion, a widely used organophosphate
insecticide. Extrapolating from in vitro data such as these, to the intact animal is, of
course, fraught with uncertainties. As was pointed out in Chapter 9, the in vivo effect can
often be considerably different than that seen when the test chemical is merely added to
a reaction mixture in vitro. However, these findings do suggest that we should not ignore
the possibility that other chemicals, besides the well-studied pesticides, may inhibit
AChE. For example, Shaw and Panigraphi (1990) found as much as a 26% inhibition of
AChE in fish from a mercury-contaminated estuary. They also analyzed for mercury in
the brain of the fish and found a close correlation between the level of mercury and AChE
inhibition.
Even factors that are not necessarily anthropogenic may reduce AChE activity.
Malyarevskaya (1979) reported that the toxins of bluegreen algae or even environmental
hypoxia caused a severe drop in brain and liver AChE of perch. This finding of an
inhibitory effect of hypoxia (the level of hypoxia was not mentioned in the paper) is
interesting but I was unable to confirm it in my laboratory using trout and bluegill as
experimental animals. Nevertheless, it appears that considerable caution is needed in
concluding pesticide poisoning from inhibition of AChE alone.
Recently it has been revealed that injection of adrenaline into perch can produce an
increase (doubling) in brain AChE (Pavlov et al., 1994). Increased protein synthesis was
also observed and the authors note that this same thing occurs in mammals and is the
result of induction of adenylate and guanylate cyclases by adrenaline. When fish are
under stress, they typically exhibit elevations in adrenaline (discussed in Chapter 8).
Thus, it is tempting to conjecture that this mechanism may actually reduce the extent of
AChE inhibition in fish exposed to acute levels of pesticides that inhibit AChE.

X. CONCLUDING COMMENTS
In concluding this chapter, a few general points seem appropriate to emphasize:
290

1. Some fish behaviors (e.g., locomotor activity and avoidance) are extremely sensitive to
pollutant chemicals, whereas others (e.g., aggression) seem to be rather refractory.
2. Interpretations of a change in behavior from a mechanistic standpoint must consider the
fact that the test chemical will often simultaneously act on a variety of points in the
nervous system. These include the sensory receptors, specific neurons in the brain, rates
of neurotransmitter secretion, and rates of neurotransmitter inactivation. Thus, care
must be taken to avoid jumping to conclusions as to “mode of action” when other
additional and conceivably more important modes were not even examined.
3. Some behavioral alterations may be modulated by pollutant-induced changes in other
physiological functions such as osmoregulation, respiration, and metabolism of hor­
mones. Because behaviors involve such a large degree of integration in the organism,
understanding the mechanisms of their alterations in response to pollution will be an
especially difficult challenge.
4. Finally, there is a great need in future studies to try to close the gap in our knowledge
as to what extent, if any, a change in some behavior affects populations and community
structure. In some cases, for example social behavior, alterations in behavior may be an
artifact of the lab setup and have little or no effect on the ecology of the organisms.
Other variables such as predator-prey relationships may have profound effects on the
community structure but this needs quantifying.

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 Chapter
13
Reproduction
I. INTRODUCTION
The maintenance of a population of fishes is ultimately determined by the ability of
its members to reproduce. Thus, from a practical fisheries standpoint, this aspect of
physiology has profound ecological significance. In one sense, the other physiologi­
cal functions (the topic of most of this book) serve mainly in order to make it possible
for the organism to reproduce. When various homeostatic mechanisms are compro­
mised, as from pollutant exposure, the organism may not even survive to carry out
the ultimate function of reproduction. This chapter, however, deals with the action of
chronic levels of pollutants that do not necessarily affect survival but do impact the
process of reproduction. Fish have also been found to be useful models in biomedical
research on reproductive toxicology in a broader taxonomic sense (Motte and Landolt,
1987).

II. BRIEF OVERVIEW OF FISH REPRODUCTIVE PHYSIOLOGY

The physiology of reproduction in fish has been the topic of several reviews and symposia
(e.g., Sundararaj, 1981; Hoar et al., 1983; Redding and Patino, 1993). The tremendous
variety of aquatic habitats has made possible the evolution of an almost infinite variety
of reproductive modes among the some 30,000 species of fishes, so generalizations are
difficult. Here the discussion will be kept to a very broad level; the interested reader can
then pursue it further, if desired, with the aid of the references mentioned, for this is a
topic of considerable depth and breadth.
Reproduction involves several more or less distinct processes. These are (1) the
formation of gametes; (2) laying or release of eggs (which in some species are retained
internally); (3) fertilization, often associated with some complex spawning behavior; (4)
embryonic development in the egg; and (5) hatching and larval development. All of these
are often cued to particular seasons of the year and are under hormonal controls which
are modulated by environmental factors.

299
300

A. REPRODUCTIVE STRATEGIES
Most teleost fish species are seasonal breeders, however, a few may breed at any time the
sexes can get together. In the Northern hemisphere, temperate zone warm-water fishes
tend to spawn in spring and early summer; the salmonids and other cold water species are
often late summer and autumn spawners. An individual species, depending on local
conditions, can have very different spawning times. For example, the common carp
(Cyprinus carpió) spawns in April in Israel, summer in France, and both spring and
autumn in India (Sundararaj, 1981).
Physiological changes in preparation for spawning may require months and invoke a
considerable energy expenditure, especially in females. Photoperiod and water tempera­
ture are the most important environmental cues for reproductive functions, with tempera­
ture usually being the dominant one. In order to increase productivity of commercial
breeding programs, these cues are artificially altered to induce reproduction at times other
than the usual. The effects of environmental cues on reproduction are extensively re­
viewed in Munro et al. (1990).
The reproductive strategies of fishes determine the behavior after eggs are laid. Balón
(1975) has proposed a classification of these patterns that has been widely adopted. In this
system each species falls into one of three broad groups: those that do not guard their eggs
after they have been laid or shed into the water column; those that do guard them; and
those that are bearers who carry them in brood chambers or that bear young “alive”.
Within each group are subclassifications depending on the substrate used for the eggs,
how well they are hidden, whether a nest is constructed, etc.
In the context of this book, it should be mentioned here that the reproductive strategy
will influence the extent to which the eggs and larvae come in contact with certain
pollutants. For example, chemicals such as aromatic hydrocarbons will affect pelagic
eggs and larvae more easily than benthic ones, but crude oil that settles onto the bottom
may smother eggs laid there. Toxic chemicals in the sediments will affect those on the
bottom, as well. Finally, the chemical composition of the estuarine areas (which are
nurseries for many oceanic fish species) will be considerably different than the open
ocean.

B. HORMONAL CONTROL OF GAMETOGENESIS

Our understanding of hormonal control of gametogenesis in fish is based largely from
work on salmonids. The details may differ considerably in other species.
The formation of eggs and sperm, the first step in reproduction, is initiated by the
environmental cues mentioned (Figure 1). These work through the hypothalamus
which acts on the pituitary by way of gonadotropin-releasing hormones (GnRH). The
pituitary gland is then stimulated to release gonadotropins I and II (GtH I and II)
which stimulate gametogenesis in the sex organs. GtH I is comparable to follicle-
stimulating hormone in mammals and is the dominant GtH in fish undergoing active
growth and gametogenesis. GtH II is stimular to luteinizing hormone in mammals and
predominates during final maturation of the gonads and at spawning (Redding and
Patino, 1993).
Each ovum develops in an ovarian follicle which is composed of the central ovum
surrounded by follicular (granulosa) and thecal layers of cells that are instrumental in
forming the yolk. These cells also secrete the ovarian sex steroid hormones.
Yolk formation, termed vitellogenesis, involves both endogenous and exogenous
material. Vitellogenin, a lipophosphoprotein, is synthesized in the liver and transported
by the blood to the ovary where it is taken up into the oocytes. Estrogenic hormones
from the ovary stimulate vitellogenin production and its uptake into the ovum and are
 301

Light Cycles/remperature

Figure 1 Diagram of the major endocrine Secondary Sex

controls of reproduction in fish. See text for Characters
discussion. Spawning Behavior

stimulated by GtH I (Tytler, 1991). Progestins from the ovary may be important in final
oocyte maturation (Redding and Patino, 1993).
The sex steroids affect gametogenesis directly and indirectly via both positive and
negative feedback on the hypothalamus and pituitary. In seasonally immature fish, the
sex steroids generally promote GnRH and GtH release through positive feedback,
whereas negative feedback predominates in sexually mature fish (Redding and Patino,
1993).
In the males, the germ cells in the testes may be in synchronous or variable stages of
development, depending on the species. These spermatogonia are surrounded by Sertoli
and Leydig cells which provide support and regulation of spermatogenesis (Callard,
1991). The Leydig cells produce the androgens, primarily testosterone, ketotestosterone,
and androstenodione. In male brook trout, testosterone and 11-ketostetosterone levels rise
at spermiation and then fall rapidly thereafter (Sangalang and Freeman, 1974). “Female”
sex steroids are also present in the males, as is testosterone in the female. Sperm
production is a synchronous event in some teleosts whereas in others it is cyclic or
continuous.

C. SPAWNING
The sex hormones are all steroids and since their molecular structures and physiological
activities overlap, understanding the details of their functions has been a considerable
ongoing challenge. Estrogens such as estrone and estradiol control secondary sex char­
acteristics in female fish and cause serum calcium to rise.
The steroid hormones in the male and female are important determinants of sexual
behavior, thus they have effects on brain function. Prespawning female rainbow trout
show higher levels of testosterone than males, probably because this hormone is a
precursor for estradiol. In this species both testosterone and estradiol exhibit peaks
some months before spawning (Koivusaari et al., 1984; Dickhoff et al., 1989). In males,
but not females, the gonadotropin(s) from the pituitary may directly stimulate sexual
behavior. (See Stacey [1983] for review of primary literature on hormonal control of
behavior.)
302

Sex pheromones are secreted externally and affect conspecifics. Some actually serve
as hormones within the individual thus coordinating both gametogenesis and spawning
behavior. The pheromones may be steroids or prostaglandins and are detected by the
olfactory system (Stacey, 1991). Because a number of pollutant chemicals have been
shown to reduce the sensitivity of the olfactory system (see Chapter 12), this might be a
mechanism by which chemicals could reduce spawning success, although there is cur­
rently no experimental evidence on this.
The pituitary and gonadal hormone controls of reproduction are obviously quite
complex. Recently, there has been interest in an additional endocrine gland which appears
to be involved in reproduction, namely the thyroid (Leatherland, 1987). The thyroid
hormones cycle in parallel with the female sex steroids in trout (Cyr et al., 1988) and
oocytes selectively accumulate the thyroid hormone T3 although its function in the oocyte
is currently unclear (Weber, 1992). Thyroid hormones may act as permissive hormones
during somatic and gonadal growth and maturation (Leatherland, 1993).
The number of eggs laid, or released into the water, depends on the reproductive
pattern. Pelagic spawners must produce huge numbers to compensate for the high
mortality rate of the eggs and larvae, whereas those at the other extreme that lay protected
eggs need to lay relatively few. Of course, there is an almost infinite variety in between.
Fecundity refers to the number of eggs in a female fish, and there are several ways of
measuring this (Grim and Glebe, 1990). The larger members of a species produce more
and larger eggs than would be predicted merely by the size difference, but the effect of
this relationship on reproduction is not as straightforward as it appears because of
interactions of fecundity with other factors such as fertility (Bagenal, 1978).
Fertility refers to the actual number of young produced by a female, rather than the
number of eggs. Except in laboratory situations, this is extremely difficult to measure
because young disperse so rapidly. Yet it is really a better indicator of reproductive
success since some eggs may not be fertilized and eggs can be resorbed in the ovary and
thereby not be released at all. This latter condition (oocyte atresia) has been suggested as
a biomarker for xenobiotic effects on reproduction as it increases in fish subjected to a
variety of chemical stressors (Hinton et al., 1992). In general, there is also a direct
relationship between fecundity/fertility and food abundance.
Gamete viability may vary from female to female of the same species. It is also
influenced by non-specific stressors. For example, when rainbow trout were subjected to
the stress of approximately weekly 3-min lifts out of the water, egg sizes and survival of
progeny were reduced (Campbell et al., 1992).
The pelagic eggs of many marine teleosts have extremely high water content (-h90%),
which is responsible for their buoyancy because they are more dilute than seawater. The
water is taken into the maturing egg across the vitelline membrane into the yolk itself
shortly before release by the female. The amount of water taken in raises the volume of
the egg by a factor of 3-5 (Garik and Havey, 1984). This water uptake is a separate
process from the subsequent one of water hardening.
Most teleost eggs are enclosed by a tough membrane called the chorion. A sperm
enters through a single opening, the micropyle, which then closes after fertilization. The
chorion then swells by imbibition of water (water hardening) but remains permeable to
dissolved substances. The water that is imbibed forms the perivitelline fluid which is
retained between the chorion and the vitelline membrane surrounding the egg.

D. DEVELOPMENT
Many aspects of the developmental biology of fishes have been reviewed in Whitt and
Wourms (1981) and in Volume 11 of Hoar and Randall (1988). The rate of embryonic
development varies with the type of egg (i.e., its size and how much yolk is present) and
such environmental factors as temperature and salinity. As a general rule, the time
 303

between fertilization and hatching is much shorter in pelagic forms than those which
provide protection for the eggs. For example, the anchovy (JEngraulis) and striped bass
(Morone saxatilis) hatch out in 2-4 days after fertilization while some salmon may
require as much as 5 months of embryonic development during which time they remain
buried in the gravel of a streambed. These marked differences in incubation period could
influence the impact of a xenobiotic on embryonic development as those with the longer
periods would have a greater period of time for chemical uptake and toxicological effect.
Because the chorion is quite tough, the larvae develop “hatching glands” around the
head just before hatching. These secrete an enzyme (protease?) which softens the chorion
to permit escape from the egg.
When a larva emerges from the egg, it is not necessarily a free-swimming form.
Depending on the species and local environmental conditions, it may continue for a time
as a free embryo receiving its nutrition from the yolk sac. In salmon, these sac fry, or
alevins as they are frequently called when referring to salmonids, may stay buried in the
gravel of the streambed for a month or two.

III. ACTION OF POLLUTANTS ON REPRODUCTIVE PROCESSES

Originally, most of the studies on pollutant effects on reproduction were for the purpose
of estimating a maximum acceptable toxicant concentration (MATC), which could then
be used in establishing water quality criteria. The concept of the MATC is based on the
idea that certain stages in the life cycle will be more sensitive than others. Thus, breeding
members of a population of fish in the laboratory were exposed to a graded series of
concentrations of the toxicant and this was maintained through two generations. The
MATC is defined as the threshold between the concentration where a measured effect is
observed and the next lower one where no significant effect is observed.
McKim (1977) reviewed the results of 56 life-cycle tests done on 34 organic and
inorganic chemicals and concluded that in most cases, the embryo and larval stages are
as sensitive, or more so, than any of the other stages in the reproductive cycle. Criteria
used were mostly survival, however, growth, egg hatchability, and developmental abnor­
malities were also utilized. Woltering (1984) reviewed 173 partial or complete life-cycle
tests involving a wide assortment of metals and organic compounds and some 15 species
of fish. Survival of the fry was the most sensitive stage in roughly two thirds of the tests.
However, reproduction (fecundity) and hatchability and to a lesser extent growth of the
adult fish, were the most sensitive for some chemical/species combinations. Further
discussion of the MATC and methodologies for determining it can be found in Rand and
Petrocelli (1985).
Our interest in this chapter is primarily physiological. For that purpose, we will follow
the same sequence of events discussed in the section on general reproductive physiology
and the coverage will be largely limited to those studies where physiological processes
(other than survival) were investigated. For each physiological process, the action of
metal and other inorganic contaminants will be discussed first, followed by consideration
of organic pollutants. Some of the effects of environmental stressors in general, including
a few pollutants, on reproductive processes in fish have been reviewed by Donaldson
(1990).

A. HORMONAL CONTROLS
Prolonged cadmium exposure of male brook trout caused these hormones to continue to
rise after spermiation at which time they normally decline (Sangalang and Freeman,
1974). In spite of this high level of steroids in the blood, the testes exhibited
histopathological damage from cadmium. Thus, these abnormal hormonal elevations
could be due to an inhibition of the hepatic microsomal cytochrome P450 system which
304

is responsible for the normal metabolic breakdown of steroids (among other things). In
support of this hypothesis, cadmium has been shown to inhibit cytochrome P450 in
mammals and it could possibly do so in fish.
Steroid elevation from cadmium exposure may also occur by the metal acting on the
gonad directly. Kime (1984), using an in vitro preparation, observed stimulation by
cadmium of testosterone and IIB-hydroxytestosterone synthesis in testes of rainbow trout.
This does not rule out the possible suppression of what would normally be a rapid hepatic
metabolism of these hormones, but does indicate that cadmium can have a direct stimu­
latory effect on testosterone production as well as inhibiting the normal elimination of the
hormone by the liver. What effect this enhanced testosterone level has, if any, on sperm
production, behavior, etc., is not known. Because normal production of gonadotropin
hormones from the pituitary is probably under modulatory feedback control from the sex
steroids, a notable effect on other aspects of reproduction could occur.
Other metals may not act the same as cadmium. For example, Allen-Gil et al. (1993)
found a negative correlation between body burden of heavy metals (lead, mercury, and
nickel) in feral Arctic grayling (Thymallus articus) and plasma testosterone, so for these
metals in this species, inhibition rather than stimulation was seen. Estradiol in the females
was, on the other hand, unaffected by the heavy metals.
Copper (Schreck and Lorz, 1978) and possibly most other metals except cadmium
causes a large elevation in the hormone cortisol. Cortisol stimulates hepatic metabolism
of androgens (Hansson, 1981) which could reduce these steroids in the blood. The cortisol
response to cadmium stress is, on the other hand, essentially nil, even in fish dying of the
metal exposure (Schreck and Lorz, 1978; Thomas and Neff, 1985). This fits in with the
observed stimulation of androgen levels from this metal but suppression by others.
Mercury, both inorganic and organic forms, caused histological lesions in the Leydig
cells of the freshwater catfish Glorias (Kirubagaran and Joy, 1992). Exposures were for
up to 180 days during the period preparatory to spawning. Recall that the Leydig cells are
responsible for androgen synthesis. Decreases in plasma cholesterol (a steroid hormone
precursor) were also noted in this study. The decreased cholesterol could be due to a
slowing of the synthesis of this molecule or to a stimulation of the hepatic metabolism of
steroid molecules by the cytochrome P450 system. In the same study the gonadosomatic
index was observed to be depressed. While no measurements of testosterone were made,
it seems likely that this would be lower as would the rate of spermatogenesis in fish
exposed to mercury.
Cyanide and lead at quite low concentrations (10 jUg/L) cause marked suppression of
spermatogenesis in trout. The mechanism of this has been investigated and found to
involve a suppression of the transformation of spermatogonia to spermatocytes (Ruby et
al., 1993a). The effect is apparently not due to a direct effect on the testes, however,
instead it appears to involve a reduction in gonadotropin from the pituitary as selective
gonadotropin-secreting cells there are lost following 12-day exposures to these pollutants.
Ruby et al. (1993a) go on to note that others have shown elevations in the levels of brain
dopamine in response to cyanide. Because dopamine is a controlling factor for gonado­
tropin, this could provide a mechanism whereby the cyanide (and probably lead) work
their effect on spermatogenesis via the hypothalamic pituitary gonad axis.
Cyanide at 10 pg/L concentration for 12 days also caused several effects on female trout.
These included suppressions in plasma estradiol (E2), the thyroid hormone T3, vitellogenin,
and oocyte diameters (Ruby et al., 1993b). The latter effect was probably due to the reduced
hormones and vitellogenin. Estradiol was severely reduced (>50%) and the mechanism may
have been the same as seen with the effect on spermatogenesis, namely suppression of
pituitary release of gonadotropin via effects of the cyanide on the dopamine levels in the
hypothalamus. Vitellogenin synthesis depends on estradiol so the whole process of repro­
ductive suppression by cyanide apparently begins in the hypothalamus.
 305

As noted above, lead seems to inhibit spermatogenesis and androgen production by

suppressing gonadrotropin release in males. The same may be true for females. Thomas
(1988) found that chronic oral administration of lead to female Atlantic croaker caused
a reduction in gonadal growth and estradiol levels in the plasma. Using an in vitro
preparation, he was able to show that the capacity of the steroidogenic ovarian tissue was
unaffected by the lead. Thus, the decline in estradiol must be due to there being less tissue
available presumably due to reduced gonadotropin stimulation, although that was not
measured.
The plasma concentrations of pituitary gonadotropins and sex steroids of coho salmon
from various Great Lakes locations vary considerably depending on the location from
which they were taken. Salmon from some sites have low levels of all of these hormones
(Flett et al., 1992) and this is related to degrees of pollution in those areas. There are
probable multiple causes for these suppressions which could involve direct effects on the
hypothalamus (such as seen with cyanide and lead as discussed above) and/or there may
be increases in metabolism of hormones by the mixed function oxygenase system. The
hormone reductions are apparently not related to cortisol as this stress hormone showed
normal levels.
Petroleum hydrocarbons seem to cause a variety of effects on reproductive parameters.
Chronic exposure of marine fishes to petroleum resulted in decreased testicular growth
(Payne et al., 1978). However, at approximately the same time Whipple et al. (1978)
found accelerated gonadal maturation in starry flounder when exposed for up to 21 days
to water-soluble components of crude oil.
An acute 7- to 14-day exposure of mature landlocked salmon (Salmo salar sebago) to
crude oil caused depression in the plasma concentration of free + conjugated androgens
(Truscott et al., 1983). The 11-ketotestosterone was the more sensitive component of the
androgen pool. Truscott et al. (1983) present some evidence to indicate that the steroid
reductions may be due to enhanced metabolism of the steroids by the liver, for the oil
induced the hepatic aryl hydrocarbon hydroxylases as a detoxification process. However,
they are careful to point out that the definitive confirmation of this would be to find
changes in the amount of metabolites of the androgens in the bile following oil exposure,
and this was not done. They make the interesting point that depression of androgen levels
at spawning time has effects that are difficult to predict, as these steroids are elevated for
a considerable period of time before spawning and may be normally far higher than
required. Temporary inactivation by glucuronic acid (in the plasma) may also be a
complicating factor. Indeed, they showed that a major fraction of the total androgen in the
blood was conjugated to this acid.
Prolonged exposures of male winter flounder to very low levels of crude oil in
sediments caused no effect on spermatogenesis, testes weights, or on the plasma levels
of free testosterone and 11-ketotestosterone. However, there was a severe depression in
the levels of plasma conjugates testosterone and 11-ketotestosterone glucuronids (Truscott
et al., 1992). It is not clear whether this exposure regime had any reproductive effects on
the flounder which often come in physical contact with the sediment, however, the
authors cite work by others which suggests these conjugates may act as pheromones
during spawning. Thus, their suppression could affect spawning behavior, a hypothesis
that would probably be challenging to test.
Numerous studies have shown effects of pesticides on gonadal function in fish. The
effects may occur at several levels from the hypothalamus through the pituitary to the
gonadal cells directly. For example, the organochlorine insecticides have been shown to
inhibit gonadotropin synthesis by the pituitary (Singh and Singh, 1982). Some, such as
the organochlorine hexachlorocyclohexane (Singh et al., 1994), seem to have the poten­
tial to inhibit gonadotropin secretion and at the same time suppress the ability of the
gonadal cells to respond to the gonadotropin.
306

Figure 2 Changes in plasma estradiol in Anabas testudineus exposed to carbaryl (1.66 ppm)
for 90 days. Letters above points indicate statistical comparison with controls: a = p <0.05, b =
p <0.01, c = p <0.001, NS = not significant, and N = 5 per point. (From Choudhury, C. et al..
Environ. Biol. Fishes, 36, 319, 1993. With permission.)

Bagchi et al. (1990) demonstrated reduced steroidogenesis in catfish exposed to

organophosphorus insecticides and the effect was at least in part due to a suppression of
the gonadal enzymes responsible for the hormone synthesis. However, while the gonad
may be acted on directly by organophosphorus insecticides, Singh and Singh (1980)
observe reduced gonadotropin “potency” of the pituitary in fish exposed to malathion or
the organochloride endrin.
One of the axioms of toxicology is that the toxic effects are dependent on both dose
(concentration in aquatic systems) and duration of exposure. The latter point is empha­
sized in the study by Choudhury et al. (1993) where Anabas, the climbing perch, was
exposed to methyl parathion and carbaryl for 90 days but also sampled at several intervals
during this time. Figure 2 shows how plasma estradiol levels rose until day 15 after which
the exposed animals exhibited a marked decline while the controls continued to increase.
The concentrations used were 2 and 10% of the LC50 for methyl parathion carbaryl,
respectively.
Direct histological damage of the pituitary and gonads has been documented in several
studies involving pesticides (see Shukla and Pandey, 1986; Singh et al., 1993, 1994 for
citations). Other than seeing direct damage such as atresia of oocytes, it is often difficult
to tell to what extent the damage affects such functions as hormonal secretion or response
to a gonadotropin. When done, however, along with hormonal studies, histological
examination can help pin-point the mechanism(s) involved in hormonal suppression.
Bleached kraft mill effluent has a wide variety of effects on fish including actions on
reproductive processes. Extensive field investigations have shown that the effluent may
suppress white sucker plasma sex steroids in both sexes and cause reduced gonad size, and
these changes are associated with elevated mixed function oxygenase activity (McMaster
et al., 1991; Munkittrick et al., 1991). Similar findings were obtained with lake whitefish
(Coregonus) (Munkittrick et al., 1992). In addition to the elevated metabolism of plasma
steroids by the liver in exposed fish, the kraft mill effluent also suppressed gonadotropin
secretion by the pituitary and reduced ovarian steroid biosynthetic capacity (Van der Kraak
et al., 1992). The overall effect of the pulpmill effluent is a reduction in gonad size, delayed
maturation, and reduced expression of secondary sexual characteristics.
 307

A further mechanism of reduced steroid production in pulpmill effluent exposed fish

was revealed by work on carp. Exposures to phenol or sulfide, two components of
pulpmill effluent, caused an inhibition in the conversion of cholesterol by the gonad to
steroidal products (Mukherjee et al., 1991). This finding does not preclude additional
actions of these contaminants at other points in the hypothalamic pituitary gonadal axis.
Of course, not all pulpmills are alike. Gagnon et al. (1994) working with white sucker
found that local conditions can complicate the situation so that reductions in only some
hormones may occur and there may or may not be any effect of the effluent on gonad
development and fecundity.
One final note on pulpmill effluent: there appears to be one or more components in the
effluent that have the ability to alter the secondary sexual characters of certain fishes.
Kraft pulpmill effluent has been found to induce male secondary sex characters in female
mosquitofish, a livebearer (Bartone and Davis, 1994). This was noted in both field and
laboratory studies. Furthermore, when masculinized females from the field were placed
in non-contaminated water the male traits decreased. At present it is unclear if the effect
is due to androgenic or antiestrogenic stimuli, or both.
The PCB Clophen A50 has been found to produce a dose-dependent delay in spawning
of minnows (Phoxinus phoxinus). It also suppresses gonadal development in flounder
(Bengtsson, 1980). It thus is not suprising to note that PCBs have been shown to cause
a suppression of plasma testosterone, estrogen, and cortisol in both rainbow trout and carp
(Sivarajah et al., 1978). All three hormones decreased approximately the same amount
(30-60%) following 4 weeks of PCB injections. Cytochrome P450 and several microso­
mal enzymes were induced in the liver, presumably as a result of the PCB and these
enhanced catalytic activities were correlated with the lowered steroids, so they are a
possible cause of this phenomenon.
Koivusaari et al. (1984) have shown there are multiple forms of cytochrome P450 in
trout liver and some of these change in concentration depending on the state of spawning.
They found that the levels of most of the cytochrome P450 and monooxygenase activities
were lowest just prior to spawning, which may reflect a form of downregulation to avoid
metabolism of the sex steroids. This does, however, suggest the fish would be least able
to handle many foreign organic compounds.
The relationship of general stress to reproductive hormone levels is not at all clear. For
example, handling of trout can cause depressions in plasma testosterone and estradiol
(Magri et al., 1982; Barton and Iwama, 1991). Of course, the classic stress response is an
elevation in plasma cortisol from the interrenal tissue (Donaldson, 1981; Barton and
Iwama, 1991) so it is noteworthy that Foo and Lam (1993) showed that cortisol implants,
which raise the blood levels of cortisol, also lower testosterone levels in Tilapia. Plasma
cortisol also generally rises at spawning time (Billard et al., 1981) and catecholamines,
which are involved in the primary stress response (Mazeaud and Mazeaud, 1981) may
actually stimulate gonadotropin secretion by the pituitary (Chang et al., 1983).
The yolk precursor, vitellogenin, is in part under the control of estradiol. Injections of
estradiol induce this material within 6 days (Chen et al., 1984). Fish fed a diet for 6
months contaminated by PCB or the insecticide Mirex exhibited a greatly reduced
induction of vitellogenin by estradiol injection (Chen et al., 1984). A possible mechanism
has been revealed using an in vitro preparation. Anderson et al. (1993) found that when
cytochrome P450 (CYP 1A) was added to a hepatocyte cell culture, it depressed estradiol
induction of vitellogenin production. Elevated levels of CYP lA in liver cells are
commonly encountered in response to a wide variety of organic contaminants (see
Chapter 5).
Cadmium can also suppress vitellogenin production. (Poulsen et al., 1990). The
mechanism is apparently different, however. Cadmium is noted for altering calcium
regulation in fish causing a hypocalcemia in freshwater species (see Chapter 7). Calcium
308

binds to vitellogenin and is then transported to the developing oocytes. Thus it is

interesting that cadmium, in addition to the hypocalcemia, also causes a direct decrease
in vitellogenin binding to calcium (Haux et aL, 1988). Ghosh and Thomas (1993) further
showed that cadmium binds to vitellogenin and displaces zinc and calcium from the
protein. The ultimate effect is suppressed development of larvae from cadmium-exposed
females (Haux et al., 1988).
Exposures of trout to cyanide for 12 days caused a dramatic decrease in vitellogenin
synthesis after 6 days (Ruby et al., 1986). As was shown later in the same laboratory
(Ruby et al., 1993b), estrogen levels drop as a result of sublethal cyanide exposure and
this hormone is what stimulates vitellogenin synthesis. Thus, we see that vitellogenin
levels can be suppressed either by the toxic chemical acting directly on the liver or by
reducing estrogen concentration in the blood.
As discussed above, a wide variety of water pollutants cause suppression of vitellogenin
production. However, recent work has revealed that some pollutants can induce vitellogenin
production in male fish, where it is not normally found. When caged male trout were held
in the outflows from sewage treatment plants for two weeks, they produced vitellogenin
in their blood. This was also seen in fish even several kilometers downstream of the
outfalls (Anon., 1993). Several components in domestic sewage are weakly estrogenic.
These include breakdown products of several detergents and the urine of women on birth
control pills. Jobling and Sumpter (1993) have shown that these compounds bind to the
estradiol receptor in liver cells of trout.
One final endocrine gland that apparently is affected by pollution is the thyroid
(Leatherland, 1993). Field studies of salmon in the Great Lakes have revealed extensive
thyroid hyperplasia which is not due to iodide deficiency. Tentative candidates are
waterborne goitrogens of microbial origin that correlate with the degree of eutrophication
in the lakes. Whether this is a contributing factor to the decline in reproduction of the
Great Lakes salmon remains to be confirmed. Indeed, the thyroid hormone levels are
actually quite high in these fish (Leatherland, 1993).
From the above discussion, it is evident that pollutants can suppress the levels of sex
steroids and vitellogenin by a variety of mechanisms, both involving production and
clearance. The points of action associated with steroid production can be at the hypotha­
lamic control of the pituitary, the formation of one or both of the gonadotropins by the
pituitary, the sensitivity of the gonad to gonadotropin stimulation, the capacity of the
gonad to produce the steroid sex hormones, and the ability of the liver to produce
vitellogenin. In many studies only one or two of these points have been investigated so
we should not conclude that just because a given toxic compound suppresses a given
process (e.g., pituitary release of gonadotropin) that it is the only mechanism of action.

B. EGG PRODUCTION
There is not much information on the effects of pollutants on fecundity, in part because
it is difficult to induce some species to reproduce in captivity. Laboratory studies in which
brook trout were exposed through two or more generations to low levels of copper, zinc,
or lead suggested that this function is relatively insensitive to these metals (McKim and
Benoit, 1971; Holcombe et al., 1976, 1979). However, Mount (1968) using copper and
Brungs (1969) using zinc found that egg production in fathead minnows was severely
reduced in concentrations that had little or no effect on eggs, fry, or adults. Kumar and
Pant (1984) found that 2- to 4-month exposures of an Indian teleost {Puntius) to copper,
zinc, or lead caused histopathological changes in both testes and ovaries. Zinc and lead
were more harmful to testes than was copper, even though the latter had a greater toxicity
to the whole animal. All three metals caused disappearance of oocytes in the ovaries. The
authors suggest that this effect is due to a direct action of the metals on the gonads,
although altered gonadrotropin levels are not excluded.
 309

Field studies of white sucker from metal-contaminated sites compared with reference
sites revealed increased fecundity in one case and decreased fecundity in another (reviewed
in Munkittrick and Dixon, 1989a). In the case where increased fecundity was seen, there
was also a reduced spawning and those eggs released had abnormally soft shells, so the
fecundity measurement alone was misleading. It seems obvious that the effect of metals on
fecundity varies with the species and perhaps some other variables as well. These conflict­
ing results also point out the danger of extrapolating from one species of fish, especially
considering the extreme diversity of reproductive patterns among the teleosts.
From the standpoint of fecundity, probably more work has been done on acid stress
than any other form of pollution. This work has been reviewed in Chapter 10. A common
observation is the appearance of large numbers of atretic follicles (McCormick et al.,
1989). Cyanide appears to act in a manner rather similar to low pH in that yolk deposition
in the developing oocytes is inhibited when female trout are exposed to sublethal levels
(Lesnaik and Ruby, 1982), and this is associated with a depressed level of serum calcium
(Costa and Ruby, 1984).
Ovum development is an energetically expensive process so food availability would
seem to be an important variable that could have a considerable effect on fecundity. Thus,
field conditions where food might be limited by pollution or other variables could explain
some cases of decreased fecundity. Where that is not a factor, it seems safe to generalize
that fecundity will usually be depressed if estradiol is low because this causes a low
vitellogenin and thus yolk deposition is reduced. As was mentioned above, some toxic
chemicals also act directly on the liver to slow vitellogenin production.

C. TRANSFER OF CONTAMINANTS INTO EGGS FROM ADULTS

It was noted some time ago (Allison et al., 1963) that brook trout exposed to sublethal
levels of DDT exhibited no alteration in egg production at doses that did, however, cause
subsequent high mortality among the sac fry growing in non-contaminated water. Since
then considerable evidence has been accumulated that a variety of organic chemicals can
be transmitted from contaminated parent fish through the yolk lipids, and these then cause
direct mortality of the fry or increased sensitivity of the fry to stress (Landner et al., 1985;
Ankley et al., 1991; Hall and Oris, 1991).
The transfer of contaminants to eggs by the female fish has been further investigated
by analyzing the parent tissues and eggs in gravid fish taken from Lake Ontario and Lake
Erie (Niimi, 1983). Some 11 organic contaminants and mercury were found in the 5
species tested. They include the insecticides DDT, chlordane, Mirex, endrin, and dieldrin,
among others. In general, there were no large differences in concentration of the organics
between the whole-body of the parent and the eggs. However, little mercury appeared in
the eggs compared to the rest of the body. Assuming all the eggs would have been laid,
the percent of the total body burden for the organic contaminants which would have been
deposited in the eggs ranged from 5.5% for rainbow trout to 25.5% for yellow perch. The
percent lipid content in the fish and that in the eggs had a marked effect on this transfer
capacity. Miller (1993) has shown that the amount transferred into the eggs can be
predicted from body burden data on parents. Niimi (1983) reviewed several studies that
suggest that the levels of organics transferred into these eggs would have had detrimental
effects on subsequent development. Ankley et al. (1991) also provide evidence that PCBs
transferred into the egg from the mother reduces hatching success in chinook salmon. Of
course, this could be due to teratogenic effects on the developing embryo.
While mercury may not transfer readily from the mother into the eggs, some other
metals such as selenium and copper apparently do. Bluegills given dietary selenium at 4
doses for 60 days exhibited no effects of the metal on any reproductive index except
survival of the fry, which was severely reduced in those from parents receiving the highest
amounts of selenium (Coyle et al., 1993).
310

Munkittrick and Dixon (1989b) have found that metal transfer may actually make
white sucker larvae more resistant to subsequent copper exposure but this difference is
lost after yolk absorption is complete at 21 days posthatch. There was also copper uptake
into the eggs at egg activation time (water hardening), but the authors maintain the
metabolic impacts are different. They further point out that the increased tolerance
associated with the maternal metal transfer is not associated with metallothionein synthe­
sis so the basis of the increased metal resistance is not clear.

D. EFFECTS ON SPERM AND FERTILIZATION

Sperm would appear to have little protection from the insults of foreign chemicals so there
has been some interest in determining its sensitivity to toxicants. The assessment of
effects on sperm is generally done by observing motility under a microscope following
exposures to test solutions, or by determining subsequent fertilization success.
Anderson et al. (1991) reported that sperm was more sensitive to copper than was
either the embryo or larva of topsmelt (Atherinops affinis). Their tests were done in
seawater which may be important as work on freshwater species (Billard and Roubaud,
1985) suggests that this element has little effect on sperm, even at high concentrations.
Because the sperm of freshwater forms has a period of motility less than 2 min (Duplinsky,
1982), the opportunity for chemical exposure in natural field freshwater situations must
be extremely short.
Mercury presents some interesting problems for fertilization in Fundulus, an estuarine
species. Judith and Peddrick Weis and their students (reviewed in Weis and Weis, 1989)
have investigated two populations of this species that exhibit markedly different sensitivi­
ties to mercury. One population, designated Piles Creek, has experienced mercury expo­
sure for many generations and produces eggs and sperm that are much more tolerant of
methylmercury than a similar population from a clean area near Long Island. This
enhanced tolerance was not found, however, in larvae or juveniles so it is limited to the
gametes.
An increased tolerance to methylmercury was not without a cost. Somewhat surpris­
ingly, the Piles Creek fish produced gametes that were less tolerant to inorganic mercury
than the reference population (Khan and Weis, 1987). This phenomenon was further
investigated by Khan and Weis (1993) who found by scanning electron microscopy that
the micropyle is affected differently by the two mercury forms. Methylmercury caused
a reduced fertilization by inducing a premature or artificial activation of the egg due to
rupture of the cortical vesicles and this then caused blockage of the micropyle. Inorganic
mercury, on the other hand, caused a swelling of the micropylar lip and a decrease in
micropylar diameter. Just why enhanced tolerance to methylmercury causes a reduced
tolerance to inorganic mercury remains a mystery.

E. EGG ACTIVATION
The newly shed fish egg immediately undergoes activation, which is often called water
hardening and requires about 1-2 h in salmon. This process of water uptake and formation
of the perivitelline fluid presumably depends on long-chain macromolecules, such as
gelatins and collagens, which form moderately crosslinked polymers containing ionized
groups which attract water. Eddy and Talbot (1983) observed that strongly hydrated ions
such as metallic cations inhibit this uptake of water. Exposure of Atlantic salmon eggs to
1 mM aluminum (valence 3) caused a complete cessation of water hardening while
cations of lesser charge, such as zinc, also caused some reduction in water uptake when
tested at the same concentration but did not block the process entirely.
Often there is metal pollution in association with acidification so the observations of
Peterson and Martin-Robichaud (1982) are noteworthy. They found that a pH of 4.5 or
less inhibited water hardening, so the metals and acidity could act in concert on this
 311

process. While metals are quite inhibitory of water uptake by the egg, non-electrolytes,
even those that are hydrophilic (e.g., alcohol), have little if any affect on this process
(Eddy and Talbot, 1983).
Rosenthal and Alderdice (1976) reported reduced volume of Pacific herring eggs
when exposed to 0.1 mM cadmium prior to fertilization. The effect was also shown to
be dose dependent. The authors propose that cadmium may compete with calcium for
binding sites on the mucopolysaccharide coat surrounding the egg capsule. This could
in turn alter the permeability characteristics of this membrane. Whether other metallic
cations, which do not interact with calcium in quite the manner of cadmium, would
cause similar effects on water hardening of marine eggs is unclear. Eddy and Talbot
(1983) predict that compared to freshwater species, it would require very high concen­
trations of most polyvalent metal ions to inhibit water hardening in pelagic eggs of
marine fish.
An inhibition of water uptake might be particularly important for pelagic eggs which
adjust their volume in order to maintain buoyancy in the water column (Rosenthal and
Alderdice, 1976). In general, a pollutant that reduces water uptake would cause a
reduction in buoyancy and could then cause the eggs to sink into waters of lower oxygen
and/or salinity. Finally, it should be noted that egg activation involves the uptake of water
which will also bring into the egg whatever is dissolved in the water (Munkittrick and
Dixon, 1989b).

F. OOCYTE AND EMBRYO ENERGY METABOLISM

The mature oocyte before fertilization utilizes an energy metabolism based primarily on
glycolysis (Boulekbache, 1981), thus the oxygen demand is low at that stage. Akburak
and Eamshaw (1984) report that the addition of copper (0.1-1.0 mM) to unfertilized eggs
of Eurasian perch {Perea fluviatilis) causes an immediate dose-dependent stimulation in
oxygen consumption. The percent increase is as great as 2600% so the effect is quite
dramatic. In an effort to understand the mechanism of this response, they exposed eggs
to uncouplers of oxidative phosphorylation. These produced only a 50% increase in
oxygen consumption so the authors propose the interesting hypothesis that the egg’s
respiration is limited by a low oxygen permeability of the chorion, and exposure to copper
caused a removal of this limitation. In support of this they found that temperature had
little effect on oxygen consumption rate of the eggs in the absence of copper, but when
the metal was present, there was a nearly twofold increase for each 10°C increase (i.e.,
QIO = 1.86). That is all well and good, but if oxygen permeability is so severely limiting
for the unfertilized egg, there must be a tremendous increase in permeability following
fertilization. Otherwise, how can the embryo show a fourfold increase in oxygen con­
sumption during normal development (Boulekbache, 1981)? Another fascinating anomaly
is that zinc caused no effect on the oxygen consumption of perch eggs (Akburak and
Eamshaw, 1984).
When ATP production is inhibited by metabolic uncouplers such as dinitrophenol
(DNP), fish embryos frequently exhibit arrested differentiation and even dedifferentiation
(Rosenthal and Alderdice, 1976). This results in a variety of developmental abnormalities
which are similar to the “spontaneous” malformations which have been described in the
literature. Rosenthal and Alderdice (1976) review several studies on a variety of sub­
stances including metals, oil dispersants, DDT, etc., which showed the same sort of
effects as those produced by DNP. Even physical changes such as temperature shock or
severe environmental hypoxia cause the same thing. In other words, these developmental
abnormalities are non-specific and presumably occur in part as a result of a reduction in
ATP production in the tissues of the developing embryo.
While metabolic poisons can produce a variety of teratogenic effects, the first stage of
development before the formation of the blastula is quite insensitive to cyanide, a strong
312

metabolic inhibitor (Leduc, 1978). This is undoubtedly because the early embryo is
supported primarily by anaerobic glycolysis and would thus be insensitive to metabolic
poisons, unless they also inhibited glycolysis.
The heart rate of embryos decreases in response to a variety of environmental chemical
stressors (Rosenthal and Alderdice, 1976). While under some circumstances, this response
may be more sensitive than other measures such as survival to hatching. Sharp et al. (1979)
report that relatively high concentrations of petroleum hydrocarbons are required to produce
a slowing of heartbeat in Fundulus. They also found that oxygen consumption of 10-day-old
embryos is also changed very little in response to hydrocarbons.

G. CHANGES IN POLLUTANT SENSITIVITY DURING EMBRYONIC

DEVELOPMENT
The various stages of embryonic development show different sensitivities and even
different qualitative effects of a pollutant insult. In general, early embryonic development
before gastrulation has been completed is the most sensitive stage for various components
of petroleum and for mercury (reviewed in von Westemhagen, 1988). This differential
sensitivity appears to be due in part to a progressive reduction in permeability of the egg
membrane to hydrocarbons and perhaps to metals as the embryo develops.
The early cleavage stage is also the most sensitive one to conditions of low pH
(Peterson et al., 1982; Lee and Germing, 1980). Death occurs in acid waters due to a
corrosion of the ectodermal cells, and the thin tissue layers at this stage allow rapid
movement of electrolytes out of the egg. The older embryos, however, appear to be the
most tolerant of acidic pH of any of the life stages in fish, even more so than the adults.
Siddens et al. (1984) attribute this to the relative impermeability of the chorion to
hydrogen ions. Because there is a precipitous decline in hydrogen ion tolerance at
hatching when the organism is no longer protected by that membrane, the hypothesis has
apparent merit.
Part of the reason for the high sensitivity of the early embryo to chemical harm may
rest with the process of water hardening. As was mentioned above, this uptake of water
will also tend to bring into the egg some of the dissolved contaminants. However, lest one
conclude that the earliest stage is always the most susceptible to harmful chemicals, we
should recall the lack of sensitivity of this stage to cyanide and other metabolic inhibitors.
As the embryo develops, it probably becomes more tolerant of various toxicants, at
least in part due to a reduction in permeabilty of the capsule to them. For example,
Skidmore (1966) removed the outer egg membrane from zebrafish {Branchydanio rerio)
embryos and found their tolerance to zinc decreased markedly. Eddy and Talbot (1985)
provide evidence that the outer membrane and the perivitelline fluid together give some
protection from aluminum, zinc, or acidic water. Thus, the egg capsule appears to have
the ability of excluding some metallic ions while at the same time increasing its perme­
ability to oxygen as the embryo develops. Apparently mercury is not one of them as
M. Greeley (personal communication) has found that medaka embryos will readily
bioconcentrate this metal.
Lake trout eggs are rather resistant to high doses of selenium, up to 10 mg/L, but then
an interesting metal interaction occurs at concentrations ten times higher than this;
selenium produces a protective effect from mercury toxicity (Klaverkamp et al., 1983).
Whether this has any relevance to “real” situations is open to question.
Continuous exposure to a toxicant may produce some changes in developmental rate of
different embryonic stages. Figure 3 illustrates this phenomenon in herring eggs exposed to
zinc. Note that cleavage was prolonged but the process of segmentation and gastrulation
was shortened as a result of the zinc. Also at the higher concentrations, hatching time was
shortened primarily due to a shortening of the final period of development.
 313

384

336

288
Eye pigmentation

i/)
Otic capsule
a 240

UJ Length of embryo
i 192
H 1 com plete circle
Z
o
P 144
< ! Brain & Optic capsule
oo
3
Ü
Z 96
Segm entation
Gastrulation

38 Blastula

13

Cleavage
5
I
0
0.1 0 .5 2.0 6.0

PPM ZINC

Figure 3 Histogram showing the development stages of herring (Clupea harengus) eggs
incubated in different concentrations of zinc at a salinity of 21 %o and temperature 8°C. C = control
and H = 50% hatching. (From Somasundaram, B. et al., Aquat. Toxicol., 5, 167, 1984. With
permission.)

H. HATCHING TIME AND VIABLE HATCH

Hatching time could also be called incubation time and refers to the period between
fertilization and 50% hatch of a group of eggs. Assuming concentrations of a toxicant are
not so high as to block hatching altogether, the general trend is a reduction in hatching
time. This has been found for cadmium, (Rosenthal and Sperling, 1974), zinc,
(Somasundaram et al., 1984), silver in freshwater (Davies et al., 1978) and seawater
(Klein-MacPhee et al., 1984), and the PCB, Clophen A50 (Bengtsson, 1980). In general
the effect is dose dependent up to where there is a significant reduction in hatching
success. If that happens, it may take longer to reach 50% hatch because many of the eggs
fail to hatch. A shortening of the hatching time often produces larvae that are smaller than
normal so this does not necessarily represent a more rapid development, although that
possibility is not entirely excluded (Trojnar, 1977).
Cyanide again appears to be an exception to many other toxic substances in that it
caused a lengthening of the incubation period at all concentrations tested (Leduc, 1978).
A lowering of the environmental pH also has generally been reported to prolong hatching
time (Nelson, 1982). The hatching enzyme, which is used to soften the egg capsule just
before hatching, has a pH optimum well above 7 (Hagenmeier, 1974). Because the
perivitelline fluid where this enzyme does its work has a pH similar to the ambient water
(Peterson et al., 1980), there is a good probability that the delay in hatching in eggs in acid
water involves a low activity of this enzyme. Abnormalities of the developed embryo,
particularly of the skeletal structure, could also be a contributing factor (Peterson et al..
314

1982). Nelson (1982) found that raising the external calcium level partially reversed the
effect of low pH on hatching in rainbow trout.
Viable hatch refers to the number of larvae actually emerging. The extensive data on
this parameter has been reviewed by von Westemhagen (1988). He notes that where
exposures of eggs have occurred, cadmium or zinc has considerable effect at low
concentrations in freshwater but rather high (and unrealistic) concentrations of these
metals are required to cause reductions of viable hatch in seawater. Copper, on the other
hand, causes considerable effects on viable hatch at concentrations as low as 30-90 pg/L
in both freshwater and seawater.

I. LARVAE
Larvae that hatch from eggs that have been continuously exposed to some pollutant are
frequently smaller, so development must have been inhibited, or hatching occurred
prematurely. Several examples of reduced larval length in larvae from eggs exposed to
metals or petroleum and chlorinated hydrocarbons are cited by von Westemhagen (1988).
He makes the interesting speculation, however, that since smaller length is frequently
correlated with larger yolk sac, development may proceed normally (if there are no lasting
effects). Further, it is not entirely clear whether smaller body size per se at hatching
predicts reduced recruitment into the population. M. Greeley (personal communication)
has observed that smaller larvae at hatch often catch up with their larger siblings at yolk-
sac absorption.
Zinc caused some interesting dose-dependent effects on larval size at hatching in
herring. Somasundaram et al. (1984) found that at concentrations up to 2.0 mg/L the
length of the larvae increased whereas those at concentrations above 6.0 mg/L showed
decreasing lengths. These findings raise the question of to what extent a similar relation­
ship between concentration and larval size at hatching might take place with other
pollutants.
When eggs are exposed to various inorganic or organic contaminants, the resulting
larvae may suffer from a variety of teratogenic deformities. According to Rosenthal and
Alderdice, (1976, p. 2057) “...in general, malformations observable after hatching that
originated from tissue injury during earlier stages include (1) yolk-sac deformities,
including patches of necrotic tissue and incomplete yolk circulation; (2) various types of
eye deformation, generally involving reduction in size of one or both eyes, and as well,
disorganization of retinal tissue; (3) otic capsule defects, ranging from missing otoliths
to absence of otic capsules; (4) jaw anomalies, including stages from absence of the lower
jaw to its deformation or delaid formation; (5) fin defects; and (6) malformations of the
vertebral column.” Some of these problems of development are probably the result of
abnormal chromosome division of the fish embryos (Longwell et al., 1992). Many of
these same deformities are also produced by natural stressors, such as low oxygen, or
abnormal salinity and so do not necessarily implicate pollutants. However, studies in
which several low concentrations of a contaminant chemical are tested can be useful in
determination of a non-observed effect concentration.
Abnormally formed larvae will usually exhibit aberrant behavior, such as uncoordi­
nated and reduced swimming activity. Larvae that swim into areas of petroleum
hydrocabons tend to become narcotized and this can occur at very low concentrations
(von Westemhagen, 1988). Reduced swinuning activity would be expected to cause a
reduction in food consumption due to lower foraging rate and therefore result in a lower
growth rate. It has also been shown that narcotized larvae are more susceptible to
predation (Rosenthal and Alderdice, 1976).
Rainbow trout and coho salmon fry emerge from the gravel beds 2-4 months after
hatching but before exogenous feeding. Ostrander et al. (1990) exposed the eggs of
rainbow trout to a 24-h pulse of benzo[a]pyrene (25 mg/L) 1 week before hatching. When
 315

fry from exposed eggs emerged from the gravel they showed decreased ability to swim
upstream against the current either due to a reduced stamina or swimming speed, it is not
clear which. This study shows how a lipophilic substance can be absorbed, probably into
the yolk, over a short period but then affect the young fish 6-8 weeks later.
The ontogeny of various control systems in fish has received relatively little attention,
thus the study by Fu and Lock (1990) is interesting from both a toxicological and basic
physiology standpoint. They exposed tilapia eggs to cadmium and then used immunocy-
tochemical methods to detect activity of the pituitary cells responsible for prolactin
synthesis, a hormone involved in calcium regulation. Larvae from exposed eggs had
reduced calcium content but when put into non-contaminated water, this recovered in 288
h. The recovery in calcium content was concomitant with increased activity of the
prolactin cells, a phenomenon also observed in adult fish exposed to cadmium (Fu et al.,
1989).
It is well known that aluminum toxicity is a major cause of poor recruitment of many
fishes in soft, acid waters. Other metals may also become mobilized in acid waters and
therefore be a contributing factor for fishery declines (Spry et al., 1981). Thus it is
interesting that studies involving combinations at realistic concentrations of various trace
metals (aluminum, cadmium, copper, iron, lead, nickel, and zinc) indicate that some
combinations are far more toxic than others. Combinations involving either aluminum or
copper were the most harmful, with copper being worse than aluminum (Sayer et al.,
1991). These metal combinations also caused the fry to have reduced whole-body
calcium, sodium, and potassium content. When lead and/or zinc were combined with
aluminum or copper, they slightly reduced the harmful effect of the latter two metals.
Exposures of embryos to metals at a sublethal level can (but not necessarily always)
induce a greater tolerance to that metal in the subsequent larvae from those eggs. This was
found for cadmium in rainbow trout (Beatie and Pascoe, 1978) but not for chromium in
the same species (Stevens and Chapman, 1984). Flagfish (Jordanella floridae) embryos
acclimated to zinc produced larvae that could tolerate this metal better than controls
(Spehar et al., 1978). Weis and Weis (1983) found no enhancement of mercury tolerance
in Fundulus larvae by acclimating their eggs to this element. Instead, acclimation to
methylmercury produced a slight but significant decrease in the time to death of larvae
in a lethal concentration of this compound.
An improvement in tolerance to a metal in adult fish has been attributed to the
synthesis of the protein metallothionein, which serves to sequester the metal (see Chapter
5). Weis (1984) has found that some clutches of Fundulus embryos taken from unpolluted
sites are more tolerant of methylmercury or inorganic mercury than others. At the time
of hatching, these tolerant embryos had twice as much of a protein that co-migrates on
a gel with Fundulus adult metallothionein, but there was essentially no metallothionein
in the eggs at the time of deposition, which presumably is the most critical time for
subsequent teratogenic effects. Thus, the high levels of this protein in the resistant embryo
clutches may be a mere coincidence. Also, acclimation of embryos with low doses of
mercury did not induce this protein. Weis (1984) concludes that metallothionein has a
questionable role in embryonic and larval tolerance to mercury, which may also explain
why acclimation to mercury does not enhance tolerance to it. It was also noted that
tolerance to other toxicants such as PCBs or lead did not correlate with tolerance to
mercury. So the mechanism of mercury tolerance probably does not relate to chorionic
permeability either (Weis and Weis, 1989).
While acclimation to a pollutant may occasionally make the organism less sensitive
to that chemical, tolerance to other environmental factors such as temperature may be
adversely affected by the exposure. Thus, Middaugh et al. (1975) observed a reduction
in thermal maximum in larval spot following sublethal cadmium exposure. Heath et al.
(1994) exposed fathead minnow larvae to a synthetic pyrethroid insecticide for 24 h and
316

then tested their upper and lower lethal temperatures (critical thermal maximum and
minimum). Concentrations of the insecticide equivalent to 20 and 70% of the 96-h LC50
were used. The overall effect of the exposures was to reduce the critical thermal maxi­
mum and increase the critical thermal minimum, thus narrowing the thermal tolerance
limits of the larvae.
The size and shape of the yolk sac is sensitive to a variety of stressors including salinity
and temperature (Rosenthal and Alderdice, 1976). When exposed to a chemical pollutant
during embryonic development, the yolk sac of the emerging larvae may be smaller or
larger than normal and interactions with salinity are complex (see Rosenthal and Alderdice,
1976). The mechanisms of these alterations probably involve two processes which are
widely separated in time. On the one hand, changes in yolk volume can be produced by
alterations in yolk water content due to changes in the osmoregulatory processes of the
egg shortly before hatching (Alderdice and Forrester, 1974). The other major mechanism
functionally goes back to when vitellogenesis occurs since that is when the yolk is laid.
As was discussed earlier in this chapter, this process is under hormonal control so when
changes in yolk are observed, we may, in fact, be seeing the result of a pollutant acting
on the sex hormone picture of the adult fish some weeks or months before the ultimate
effect becomes evident in the larvae.
Posthatching encounters with a variety of pollutants have been shown to cause erratic
swimming and most particularly, a slowing of muscular activity in the larvae (Rosenthal
and Sperling, 1974; von Westemhagen et al., 1974; Dethlefsen et al., 1975). Activity of
larvae is difficult to quantify so the data are largely subjective. The effect of any change
in this on sac fry is somewhat unpredictable as their sole source of energy is the yolk. A
reduction in energy expenditure for muscular activity could actually leave more available
for growth, and this indeed seems to happen with trout exposed to low levels of cyanide.
Sac fry exposed to this chemical grow as fast or faster than controls (Leduc, 1978).
However, after the fry have absorbed their yolk sac, the effect of slower swimming would
be to reduce feeding ability as they would be unable to explore as much water volume for
prey.
Swimming capacity has rarely been examined in larval fish after exposure to pollu­
tants. Heath et al. (1993) used a simple system involving a petri dish with a smaller one
glued in the center of it, thus forming a circular “race track”. Six radial lines were placed
on the bottom of the track and the dish filled with water. Single larvae were then placed
in the outer track and chased with a glass rod and the number of lines crossed per minute
was determined. Newly hatched striped bass larvae that had been exposed for four days
to the insecticide methyl parathion or molinate (an herbicide) at a concentration less than
one half the LC50 exhibited a decreased swimming performance. This impaired perfor­
mance persisted even after the fish had been allowed to recover in non-contaminated
water for 10 days.
As the larva of salmon (and probably many other species) develops but before
exogenous feeding begins, the larva must absorb from the water approximately 65% of
the sodium, 45% of the potassium, and 75% of the calcium required for normal function.
The rest comes from the yolk. Rombough and Garside (1984) found that cadmium causes
a dose-dependent (0.47-300 |Xg/L) decrease in the uptake of potassium and calcium. The
uptake of sodium is, however, little affected by cadmium in the medium. Calcification of
skeletal elements is inhibited, probably because of the reduced body calcium. This results
in thin fin rays and an abnormal spinal column. The interference of calcium metabolism
by cadmium can also affect subsequent equilibrium of the fish because the otoliths and
otic capsules do not develop normally (von Westemhagen et al., 1974). Meteyer et al.,
(1988) have further shown that the effect of cadmium on calcium uptake can persist even
after larvae are returned to control water and they no longer have accumulated cadmium
in their bodies.
 317

Finally, the heart rate of fish larvae can often be measured rather easily as they are
nearly transparent, so the heart can be viewed under a microscope. Tortorelli et al. (1990)
measured this in newly hatched catfish {Plecostomus commersoni) fry exposed to six
different sublethal concentrations of paraquat, an herbicide. Exposure times were as long
as 60 h and a dose- and time-dependent inhibition of heart activity was recorded. Using
these data they were able to define a maximal acceptable toxicant concentration that was
within the recommended single application rate for this herbicide.

J. CONCLUDING COMMENT
Reproduction has so many fairly discrete steps that there are numerous points where
toxic contaminants could and do have impact. In carrying out studies on the effects of
pollutants on reproduction, care must always be taken to avoid attributing mechanisms of
toxic action to particular processes (e.g., teratogenic changes in embryo) as being the
primary cause of poor recruitment into a population, unless other processes have also
been examined (e.g., fecundity). Indeed, from a mechanistic standpoint, identifying the
most sensitive stage(s) in the whole reproductive process for particular species and
contaminants should be a challenging goal for the future. For those planning research in
this area, von Westemhagen (1988) provides some thoughtful insights into fruitful
approaches.

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in marine teleost species, M a r. E n v iro n . R e s ., 14, 422, 1984.
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ü se of Physiological and
Chapter
14
Biochemical Measures in
Pollution Biology
I. INTRODUCTION
Throughout this book, the emphasis has been on the study of physiological changes as a
means for understanding the responses of individual fish to the presence of various types
of pollution. Practical use of this information has been ignored, for the most part. Indeed,
there is no intrinsic reason why such investigations must have a practical use. The spirit
of curiosity has long been the driving force behind so-called “pure science”, and will
undoubtedly continue to have a primary impact in the future. Because humankind is
constantly changing the physical and biological conditions of ecosystems, including the
addition of pollutants, there will be a continued curiosity to understand the results of those
perturbations at several levels of biological organization so that broader theoretical
constructs can be formulated. Also, the specific biological levels that are investigated by
different workers complement and often depend on each other.
Having said that, it is obvious that the changes in fish that occur as a result of pollution
can also have real practical use. An organism makes a powerful integrator of the effect
of any number of changes in chemical and physical (e.g., temperature) factors in the
aquatic environment. The usual approach of performing chemical analysis and physical
measurements of natural waters may or may not detect changes that are affecting the fish
therein. In a real sense, “they” are the judge of that. Thus, water pollution control
authorities increasingly are looking for ways to use aquatic organisms to determine the
biological consequences of pollution.
Essentially, physiological measurements in organisms such as fish can be used in three
ways in pollution control: (1) as a supplemental aid in the formulation of water quality
criteria, from which standards can be established; (2) monitoring the health of populations
of fish in the field so predictions of harm can be made before serious changes in
population size occurs; and (3) as a biological sensor for detecting sudden changes in
water quality as might be produced by an accidental spill or intentional poisoning of a
water supply.

325
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II. WATER QUALITY CRITERIA

Numerous pieces of information are used in formulating water quality criteria for aquatic
organisms. These include traditional bioassays, where death is the “physiological” mea­
sure, all the way to field surveys (Alabaster and Lloyd, 1980; APHA, 1992; Lloyd, 1992).
Most pollution biologists would probably agree that life cycle tests in which fish are
exposed to several concentrations of a presumed toxicant for a year or more (allowing
time for reproduction) yield the most relevant information for the development of water
quality criteria. The working assumption is that certain stages of the life cycle are more
sensitive than others and therefore, a pollutant in concentrations above some threshold
would reduce a population by acting on that particular stage. Still, these tests are severely
limited in that not all important species will reproduce in the laboratory; they are
extremely expensive to carry out; the tests are conducted at a constant toxicant concen­
tration in the lab whereas in nature, this rarely occurs; and finally, only a small number
of toxicants could conceivably be evaluated. Consequently, complete life cycle tests have
not been used in recent years.
Because of the time and expense involved, there has been considerable effort ex­
pended to develop modified forms of fish and invertebrate life cycle tests that will yield
essentially the same information as one which is complete. In essence, that has meant
identifying the most sensitive stages and concentrating on those. McKim (1977) evalu­
ated 56 studies in which complete or partial life cycle tests were carried out and concluded
that the embryo-larval and early juvenile life stages were the most sensitive in most cases.
The biological measures of sensitivity were decreases in hatchability, survival and
growth, and deformities in the juveniles. More recently, Landner et al. (1985) have shown
that exposure of the adults before spawning to a mixed effluent can cause severe effects
on the subsequent embryos and larvae, which again emphasizes the sensitivity of the
young.
Woltering (1984) critically reviewed 173 tests involving a wide variety of chemicals
and concluded that the inclusion of measures of fry and/or juvenile growth rate adds little
significant information for the amount of work involved. Some (e.g., Birge et al., 1979)
have been successful in carrying out greatly abbreviated embryo-larval lethality tests of
only 4-8 days posthatch which avoids problems of feeding, not to mention all the
“Murphy’s Law” type of difficulties associated with long-term studies. Such abbreviated
life cycle tests can even be carried out in the field using natural receiving waters. The
procedures for carrying out acute and chronic toxicity tests with aquatic organisms are
now well standardized (APHA, 1992; Weber et al., 1989).
Aside from complete or partial life cycle tests, other physiological data may also be
utilized as supplemental information in development of criteria. These can range all the
way from measures of changes in behavior in the presence of the test chemical to
assessment of cellular enzyme activity. In order to have practical use for this purpose,
such tests must involve exposures of the test organisms to several concentrations of the
toxicant, preferably including one that causes no measurable effect on the parameter
being measured. In this manner, some indication of a threshold concentration is obtained.
This of course does not necessarily represent a “safe” concentration as other physiological
measures may have lower thresholds, but were not measured. Physiological changes that
are clearly associated with acute stress, such as elevated blood glucose, depressed liver
glycogen concentration, high cortisol levels, etc., are not of much aid for determinations
of water quality criteria, but along with other blood and tissue measures, they may prove
useful in biomonitoring (see below). A variety of biochemical or physiological measure­
ments applied to the younger stages might prove useful in reducing the usual time
involved in carrying out such studies. These could include measures of such things as
stress protein induction, changes in cellular enzyme activity, alterations in ventilation and
 327

heartbeat frequency, aberrant behavior, and examination for histological lesions. Care
must be taken to not rely on only one or two measures, because they will not be equally
sensitive to the presence of all chemicals.

III. BIOMONITORING OF FISH IN THE FIELD AND MESOCOSMS

Biomonitoring with aquatic organisms has experienced a tremendous explosion of infor­
mation since the early 1980s resulting in several book-length reviews (Hocutt and
Stauffer, 1980; Vemberg et al., 1981; J. Cairns et al., 1984; V. Cairns et al., 1984; Bayne,
1985; McCarthy and Shugart, 1990; Adams, 1990; Huggett et al., 1992). The major
reason for the interest in this field is that traditional measures of water quality based on
chemical analysis of effluents or receiving waters may not detect the most important
components, and interactions among chemicals and factors such as temperature in the
waterway may enhance or reduce water quality and alter the availability of chemicals to
fish and other aquatic organisms. Thus, the reasoning goes, the organisms themselves
should serve as sensors to supplement the chemical data. This means that other than death,
appropriate measurements must be devised to indicate whether the recepient organisms
are, in fact, being harmed by the quality of the water. Before discussing a few of the
physiological and biochemical approaches, which are becoming known as “biomarkers”,
some broader generalities will be considered.
Biomonitoring techniques are, more often than not, methods to measure stress in
organisms or ecosystems. In an individual organism, which is the topic here, it is
sometimes said that there are two levels of stress, the generalized and the specific (Bayne,
1985). The generalized responses are stereotypic thus they are the same irregardless of the
environmental stressor, while the specific ones may vary with the cause. This apparent
dichotomy often breaks down when examining the effect of chemical pollutants on fish.
For example, the typical “generalized” stress response involves an elevation in blood
sugar (hyperglycemia) mediated by a rise in the hormone adrenaline, whereas, as was
shown in Chapter 8, there are a number of cases where a toxicant causes hypoglycemia,
even in fish under considerable “stress” from the chemical. As for specific responses, a
major theme running through this entire book is that a given pollutant at sublethal
concentrations will generally cause a wide spectrum of effects at all levels in the animal,
from the cellular to the level of behavior. The case of a pollutant at sublethal concentra­
tions acting on a specific target function in the animal is a relative rarity, although certain
functions are generally more sensitive than others to a particular chemical or physical
“insult”, and this phenomenon can sometimes be useful for diagnostic purposes.
Most people dealing with stress in fish are reluctant to define it, perhaps wisely so. In
an American Fisheries Society Symposium on the subject (Adams, 1990), researchers
working on various levels of biological organization ranging from the molecular to the
ecosystem presented a variety of approaches for measuring stress, but none of them
presented a quotable definition. Most assumed they were seeing stress when one or more
variables that were being measured differed significantly from controls (Heath, 1990).
The problem with this assumption is that it does not incorporate biological significance.
A statistically signicant change in some physiological variable does not necessarily mean
the fish is unhealthy. For the purposes of biomonitoring in fish, it may be better to say
the organism is under stress when one or more physiological variables are altered to the
point where long-term survival may be impaired (but not necessarily due to failure of the
variable being measured, as it may be only a secondary indicator). In a sense, evaluating
long-term survival is an almost unattainable ideal because it is so difficult to make such
predictions, but it remains a useful goal. And long-term survival should also incorporate
into it the survival of not only the individual, but also the population.
328

Ecologists have long sought for a single all purpose method to assess enviromental
health or condition. As Cairns and van der Schalie (1980, p. 1180) so appropriately put
it: “ This is the contemporary version of the search for the Holy Grail and almost certainly
will be no more successful.” In other words, no single method will ever serve for all
situations. It seems safe to say that the same conclusion can be applied to the application
of physiological and biochemical measures to biomonitoring the health of fish in the field.
There will always be a need for a number of methods in order to be responsive to all types
of potential forms of degradation of water quality.
In choosing measures to use in a biomonitoring program, there are at least three criteria
that ought to be met. To some investigators these may seem self-evident but need
emphasis, nonetheless:
1. The method should have sufficient sensitivity that an alteration will be predictive of
death or reproductive impairment, should conditions continue unchanged. It does little
good, if the animal is almost dead before the factor being measured changes signifi­
cantly, for by that time, the population could be lost.
2. In order for a method to be widely used, it should be easy to perform on fish that have
been captured in the field. If the method requires expensive equipment and highly
trained scientists to perform it, financial constraints will probably prohibit its use unless
it has especially high sensitivity and relatively low inherent variability. Then, the small
number of samples required might make even an expensive technique applicable.
3. The measured factor must be relatively insensitive to the stress of capture. The usual
methods for capture of fish, such as electroshocking and netting, are highly stressful to
the organisms and cause immediate changes in a variety of physiological activities and
biochemical measurements (Wedemeyer, 1972, 1976; Schreck et al., 1976; Pickering
et al., 1982).
Even when control fish are captured in the same way, the handling associated with this
undoubtedly contributes to a great deal of variability which tends to mask more subtle
changes. The utilization of caged fish placed in areas to be monitored offers a partial
solution to this problem (Mitz and Giesy, 1985; Oikari et al., 1985; Bidwell and Heath,
1993). Some creative developments in devising methods of capture that minimized this
stress would be extremely valuable in biomonitoring as they would open up the possibili­
ties of using a much wider assortment of physiological/biochemical techniques.
Three additional points deserve mention:
1. Seasonal changes in virtually any biochemical/physiological measurement can often
be as large or larger than those produced by the presence of chemical stressors (e.g.,
Larsson et al., 1985; Bidwell and Heath, 1993), and climatic variations from year to
year are superimposed on the seasonal effects. Thus, any attempt to determine a
baseline at one time and then to make measurements of a sample population at some
other time, even at the same season but different year, must deal with this problem.
A better approach may be to sample control fish from a nearby similar area known
to be free of contaminant stress at approximately the same time as those exposed are
sampled.
2. Physiological compensation, often called acclimation, is a widespread phenomenon in
many kinds of organisms. Initially, upon exposure to an altered environmental factor
(e.g., temperature, chemical pollutant), a fish will exhibit some physiological response.
Upon continued exposure, if the degree of stress is mild, the physiological factor may
exhibit compensation (acclimation) in that it tends to return toward that of unexposed
controls (see Chapter 1, Section V). Thus, this potential for compensation must be
realized in water quality monitoring programs (Sastry and Miller, 1981).
 329

3. Biomarkers can measure exposure in the sense they assess the biological availability of
a toxicant to the organisms. The induction of proteins for detoxification (e.g., mixed
function oxidate [MFO] enzymes for organic contaminants and metallothionein [MT]
for metals) is clearly this type of biomarker and does not necessarily mean the fish are
under stress. Biomarkers also measure effects of environmental toxicants on feral fish.
These measurements may indicate failure of homeostasis (e.g., loss of plasma electro­
lytes) or a response by the fish to the stressor (e.g., elevation in plasma glucose).
The following are some biochemical/physiological measurements that may be suitable
for biomonitoring of fish in the field. While these have been suggested by various
investigators at different times, in most cases it remains to be ascertained to what extent
they meet all the criteria listed above. The theoretical bases behind them are to be found
in appropriate chapters of this book and in the references cited herein. The sequence
followed here is from the cellular to the whole-body but does not reflect any priority, it
is more a matter of convenience.

A. CELLULAR AND TISSUE CHEMISTRY

There are numerous enzymes and specialized proteins that could be assayed in tissues,
however, only a few are currently proposed for biomonitoring. If the fish are large, tissue
biopsies can be taken and the fish returned to the water (Harvey, 1990). For most
variables, rapid freezing or other preparation is necessary to prevent intolerable postmor­
tem changes in tissue constituents.
We start with the measurement of stress protein induction, a new but potentially
promising technique (Sanders, 1993). It requires very small amounts of tissue and is
unaffected much by the stress of capture, providing the fish are processed rapidly. Gill
and muscle may be especially appropriate tissues, liver much less so (B. Bradley, personal
communication). Stress protein analysis is not simple unless the laboratory has consid­
erable expertise in running and interpreting sodium dodecyl sulfate (SDS) gels.
Glutathione is a cellular peptide involved in a wide variety of cellular activités. The
overall concentration of glutathione generally rises in the presence of metals and organic
contaminants (Thomas and Wofford, 1984; Thomas and Juedes, 1992) so it can be
interpreted as a good indicator of exposure, not necessarily stress. The analysis is
moderately easy although some would argue that the ratio of reduced glutathione (GSH)
to the oxidized form (GSSH) should be determined (Stegeman et al., 1992). This com­
plicates the analysis somewhat.
MT is a cellular protein that is useful in indicating metal contamination. MT is induced
in several tissues, especially liver, by the presence of elevated metals (see Chapter 5)
(Roesijadi, 1992). MT induction has been successfully used to detect metal pollution in
natural waters (Roch et al., 1982). Because MT induction is also influenced by other
environmental factors, such as season, the use of appropriate reference animals is critical
(Endel and Brower, 1989).
The cytochrome P450 system (MFO) is induced by a number of organic xenobiotics.
The literature on the cytochrome P450 system is considerable (see Chapter 5); recent
reviews of the use of this method for field biomonitoring include Payne et al., 1987;
Melancon et al., 1988; and Stegeman and Hahn, 1994. A variety of methods to measure
various aspects of the cytochrome P450 system are being used including P450 mRNA
induction with the use of cDNA probes and monoclonal and polyclonal antibodies.
However, the more commonly used technique is to measure the catalytic activity of
ethoxyresorufin-O-deethylase (EROD) or of aryl hydrocarbon hydroxylase (AHH). The
catalytic assays are generally the easiest to carry out and, while usually done with liver, can
also be done with other tissues as well (Stegeman, 1989). Recently, a spectrophotometric
330

method has been proposed for rapid screening of samples and routine monitoring
(Lindstrom-Seppa et al., 1993).
Cytochrome P450 activity (often called MFO) is extremely sensitive to organic
pollutants and is induced fairly rapidly (few days) in exposed fish. It is insensitive to the
stress of capture so lends itself to use with feral organisms. MFO activity is also induced
in eggs and larvae so age is not an important variable (Binder and Lech, 1984).
Up to now, the factors mentioned have been, for the most part, adaptations to some
sort of stressor. We now consider some variables that reflect damage or loss of homeo­
stasis.
Vitamin C (ascorbate) is rapidly depleted in fish exposed to sublethal levels of several
examples of both inorganic and organic substances, although increases have been seen
with chlorinated phenolics (see Chapter 6). The determination of ascorbate in liver and
other tissues is relatively easy (Carr et al., 1983) and “natural” stressors such as a change
in temperature or salinity, or even handling, do not seem to have much effect on it
(Thomas and Neff, 1984). Still, care must be taken in selecting appropriate species of fish
as the ability of different species to synthesize this vitamin differs greatly, and, of course,
diet may have a considerable effect on ascorbate levels.
A tissue factor that is is especially easy to measure is the water content. All that is
required is a reasonably sensitive balance and an oven to dry the tissues. Sublethal levels
of waterborne copper (Heath, 1984), cadmium (McCarty and Houston, 1976), and a
mixture of metals simulating the effluent from a sulfide ore smeltery (Larsson et al., 1984)
have been shown to cause a small but consistent rise in water content of muscle and/or
liver of freshwater fish. In all likelihood, any chemical that affects osmoregulation in
marine or freshwater species (Chapter 7) will alter the percentage of water in a tissue,
except that estuarine fishes living in water nearly isotonic to the blood would probably
not exhibit any effect.
Using caged fish, Grippo and Dunson (1991) showed that whole-body sodium loss can
serve as a good indicator of acid and metal toxicity. This does require the use of an atomic
absorption spectrophotometer which not all laboratories have, but the procedures are
fairly straightforward, otherwise.
Acetylcholinesterase (AChE) is found in nearly all tissues. Because this enzyme is
associated with neuronal synapses, the concentration is proportional to the extent of
innervation of the tissue (Rao and Rao, 1984). AChE is inhibited by organophosphorus
and carbamate insecticides, and the extent of this inhibition has been used to diagnose fish
that are suffering from this type of poisoning (Zinkl et al., 1991). While inhibition of this
enzyme is considered a classical example of a biomarker specific for a particular group
of toxicants, in vitro studies implicate several metals as possible inhibitors as well (Olson
and Christensen, 1980). Shaw and Panigraphi (1990) found inhibition of AChE in fish
from a mercury-contaminated estuary, and the degree of inhibition was proportional to
the mercury body burden. These findings suggest caution in interpreting AChE declines
as being exclusively due to pesticide contamination.
Lysosomes are membrane-bound cellular organelles that contain acid hydrolases for
intracellular digestion and breakdown of necrotic tissue. The membrane of lysosomes
becomes less stable in response to a wide variety of environmental stressors. The stability
of lysosomes can be quantified and has been used to reveal sublethal effects of cadmium
in the laboratory (Versteeg and Giesy, 1985) and to detect a pollution gradient in the field
(Kohler, 1991).
One of the most biologically relevant outcomes of a deterioration in water quality is
a change in growth rate of the resident fish. Growth and energetics are intimately related
(see Chapter 8). When the energy content of the animal is compromised, either due to
reduced feeding or increased energy demand for tissue repair, growth may be reduced.
Protein and lipid concentrations are easily determined in body tissues (Busacker et al..
 331

1990) and are not influenced much by the stress of capture. Carbohydrate stores such as
plasma glucose and liver glycogen are much more variable and are extremely sensitive
to capture stress so are not as useful for field studies.
Growth per se is extremely difficult to quantify in routine surveys where body length
and weight are recorded, as it is often not clear as to whether the same population is being
sampled each time. Thus, the measurement of one or more biochemical variables that
reflect growth rate rather than absolute body size is needed. The measurement of the
concentration of tissue RNA and DNA (Chapter 8), so the RNA/DNA ratio can be
computed, has been used for assessment of starvation in larval fishes. This has not been
applied much for pollution investigations in the field, but has considerable promise. The
measurement of these nucleotides has relatively little intrinsic variability, are sensitive to
alterations in water quality, and are probably not affected by the stress of capture
(Passino, 1984; Busacker et al., 1990; Houlihan et al., 1993). Because of their much
greater growth rate, young fish are more likely to show measurable effects of pollutant
stress than are older ones on nucleotide concentration. Sensitive fluorometric enzyme
assays now make it possible to measure RNA and DNA in single fish larvae of quite small
size (Heath et al., 1993).
Houlihan et al. (1993) have pointed out that there is a close relationship between
protein synthesis, growth, and oxidative metabolism. Good correlations are obtained
when protein synthesis is related to the rate of oxygen consumption, and the latter
correlates well to the maximal rate of activity of the cellular enzymes citrate synthase,
cytochrome oxidase, and lactate dehydrogenase. Thus, measurements of the activity of
these enzymes from tissues (or whole-body extracts) from feral fish should yield indica­
tions of recent relative growth rate, although some sort of laboratory calibration probably
is needed before application in the field.

B. HISTOPATHOLOGY
The microscopic examination of tissues has long been used in pathology laboratories for
human and veterinary medicine both for disease diagnosis and toxicology investigation.
While strictly speaking it is not a biochemical/physiological technique, it is certainly
related to the biochemical changes that are observed, as they are frequently the result of
lesions that can be seen histologically. In a keynote address at a symposium on pollution
physiology, Sindermann (1985) made a strong plea for the inclusion of histopathology in
all future physiological/pollution investigations. Some might say that is overdoing things,
but it is clear that there is an unrealized potential for problems in aquatic toxicology to
be approached with these techniques. This is especially true for field studies as tissue
structure is quite sensitive to chemical insults and relatively unaffected by extraneous
factors such as season or the stress of capture. Hinton et al. (1993, p.l59) claim that, “No
other category of biomarker enables the researcher to examine so many potential sites of
injury so rapidly.”
Perhaps one of the greatest impediments to the greater use of histopathology in
biomonitoring is the fact that it is so labor intensive, and there are few technicians with
the requisite training for preparing and especially interpreting the microscope slides.
Hinton (1990) has prepared an excellent guide to the basic techniques involved in fish
histology and the use of this as a biomarker is discussed in considerable depth in Hinton
et al. (1993). Atlases on fish histology include Hibiya (1982) and Groman (1982).

C. HEMATOLOGY, IMMUNOLOGY, AND BLOOD CHEMISTRY

Under the topic of hematology here is included the measurement of hematocrit (packed
cell volume), hemoglobin concentration, erythrocyte count, and leukocyte count (or
volume). Hematological methods have been used for many years by biologists to assess
the general health of fish in hatcheries and research laboratories. The procedures are well
332

standardized (Blaxhall and Daisley, 1973; Wedemeyer and Yasatuke, 1977; Houston,
1990) and are easy to carry out, even in the field. Hematological measures are not immune
from the stress of capture but are influenced far less than some other measurements, such
as blood glucose (Larsson et al., 1985). In Chapter 4, studies were reviewed which
showed that some chemical pollutants induce anemia whereas others tend to cause an
excessively high hematocrit and/or hemoglobin concentration. Moreover, hematological
values are also influenced by the osmoregulatory status of the animal, which in turn is
frequently upset by pollution (Chapter 7).
More recently, Houston et al. (1993) developed a way of using hematology which
considerably increases its sensitivity. It is well known that a wide variety of environmen­
tal stressors cause changes in rates of erythrocyte synthesis. This results in more immature
cells, division of circulating juvenile cells, and karyorrhexis (fragmentation of chromatin
and breakdown of nuclei). These changes can have a considerable effect on respiratory
gas transport even though the hematocrit is normal. The procedure involves making blood
smears, which can be done in the field, and then making differential counts under a
microscope.
The leukocyte count is sensitive to metals (Larsson et al., 1985) and possibly other
pollutants as well. Since it is rather tedious to perform with the usual counting chamber,
McLeay and Gordon (1977) proposed the utilization of a procedure termed the “leukocrit”
which permits an estimate of the percent volume of leukocytes in the circulating blood.
Blood is collected in microhematocrit tubes and after centrifugation, the thickness of the
layer of leukocytes (on top of the erythrocytes) is measured using a low-power micro­
scope with an ocular micrometer. Thus, this measurement can be obtained from the same
tubes used for hematocrits with very little additional time expenditure. It remains to be
determined how sensitive the procedure is to subtle environmental changes. In general,
chemical and physical stressors cause a decrease in the leukocrit whereas infections
produce the opposite response, however, it is also possible to get elevations in granulo­
cytes concomitant with a decrease in lymphocytes, thereby yielding an unchanged leukocrit.
Thus, the method has its limitations for the detection of chronic stress (Wedemeyer et al.,
1983).
It is becoming more and more evident that the immune system of fish is sensitive to
many environmental insults (see Chapter 11). The general pattern is a suppression of
immune function at several points which then makes the fish more susceptible to disease.
Disease surveys are a useful technique that can be performed (O’Connor et al., 1987;
Goede and Barton, 1990). For those more interested in the actual immune system
function, several tests are now available which range from simple to do to those that
require considerable expertise and technology (Weeks et al., 1992). The potential for
future application of these immune tests for biomonitoring is probably quite good.
Blood chemistry measurements are usually performed on plasma or serum, although
the assay of erythrocytic delta-levulinic acid dehydratase (ALA-D) is used as a test for
environmental exposure to lead (Hodson et al., 1984) (see below). The various procedures
in blood chemistry range from the determination of blood clotting time, which is rela­
tively easy to perform, to the analysis of hormones such as cortisol or catecholamines,
which require elaborate laboratory facilities. The proliferation of automatic clinical
analyzers for the determination of a considerable list of factors in blood plasma has
generated some interest in their application to fish blood (e.g.. Miller et al., 1983).
However, the high cost of purchase and maintenance will probably limit their use for
biomonitoring. There are a number of test kits available designed for the clinical labora­
tory which do the same analyses as the clinical analyzers at a cost that is far more
reasonable when relatively small numbers of samples are to be run. Because the volume
of blood available in a sample from fish is generally quite small, the usual procedures
applicable to a clinical laboratory frequently have to be scaled down to accommodate
 333

plasma volumes in the range of 10-100 |liL. Then it becomes possible to use the plasma
when the hematocrit is prepared in microhematocrit tubes.
Some blood chemistry measurements that seem to offer promise in biomonitoring
include: chloride, osmolality, total protein, glucose, clotting time, and plasma enzyme
assays. The first two factors give essentially the same information so there is little reason
to measure both when doing routine biomonitoring, especially when sample volumes are
severely limited. If a vapor pressure osmomoter is available, osmolality is quite easy to
measure and requires only a small drop of blood. The trend for fish in freshwater that are
under stress will be a decrease in chloride or osmolality and the converse will occur in
seawater (Chapter 7).
Total protein concentration in the plasma gives an indication whether hemoconcentration
or dilution has occurred and it also shows the presence of nutritional stress. However, it
may be relatively insensitive to chemical pollutants in the water, unless there is a sizable
osmoregulatory alteration, which would be indicated by changes in osmolality or chlo­
ride. Plasma protein is extremely easy to measure with a refractometer, although colori­
metric procedures are probably more precise.
Blood glucose is one of the more commonly used factors for measuring acute stress.
It is technically easy to determine in small volumes (5-10 jiL) of plasma and there are
several test kits available on the market. The classic stress response is an elevation of
blood sugar in response to the hormones adrenaline and possibly cortisol (see Chapter 8),
however, hypoglycemia may occur with some pesticides (Lockhart and Metner, 1984;
Pant and Singh, 1983). Unfortunately, this factor is particularly sensitive to the stress of
capture and handling (Pickering et al., 1982) which limits its applicability. Furthermore,
the changes in response to pollution, while often quite large, also tend to be rather
transient so this may not be a good indicator of chronic stress.
There has been some interest in using plasma enzyme assays, especially for the
enzymes that are released from damaged liver (see Chapter 6) and heart tissues. The
primary enzymes of interest are serum glutamic oxalacetic transaminase, alkaline phos­
phatase, lactate dehydrogenase, and sorbital dehydrogenase. Lockhart and Metner (1984)
showed how some of these can exhibit rather large changes during chronic exposure to
a synthetic triaryl phosphate oil. Dixon et al. (1987) suggest that sorbital dehydrogenase
may be especially good for assessing liver damage from a wide variety of substances, both
metallic and organic. They found elevations in this enzyme in the plasma before histo­
logical lesions were evident in the liver. One potential problem with plasma enzymes in
fish is that there can be a large degree of variability (Miller et al., 1983) and this may be
in part due to the manner in which the analysis is often performed. Most of the assays are
carried out at temperatures above 30°C, yet trout and other coldwater species possess
enzymes that are adapted to function at temperatures well below that level. Thus, the
enzyme activity that is measured may not be truly representative of the fish.
The enzyme leucine amino naphthylamidase is released from lysosomes in damaged
tissues. Its appearance in the blood was, at one time, thought to be a good indicator of
tissue damage from toxic chemicals (Bouck, 1984), but Dixon et al. (1985) found that the
level of this enzyme in the blood is very sensitive to reproductive activity, salinity, stress
of capture, and nutritional level. This caused such large variability that its usefulness as
a biomarker for pollution was low.
Most blood enzymes that are assayed for clinical diagnosis are those that are released
from damaged tissues. An exception is ALA-D. It is found in the erythrocytes and
erythropoietic tissues and is easily measured. ALA-D is apparently inhibited by only lead,
other metals have little or no effect on this enzyme (Hodson et al., 1984), thus it seemed
to have promise as an assay for this element and is used diagnostically for suspected lead
poisoning in human medicine. However, Hodson et al. (1984) reviewed its use in fish
biomonitoring and noted that organic lead has little effect on it. Also, contamination by
334

inorganic lead is rarely severe enough to cause significant inhibition. Thus, while it may
have some utilization, it will probably have to be combined with analysis of levels of lead
in the blood. Then, combined with the enzyme assay, one can determine the extent of lead
contamination of the fish, and the type of lead compound. For example, a high blood lead
but little inhibition of ALA-D would indicate organic lead, whereas inhibition of the
enzyme would indicate the inorganic form. An extensive bibliography of fish blood
chemistry has been recently published by Folmar (1993).

D. CHALLENGE TESTS
The idea behind a challenge test is that a fish that has been exposed to an altered
environmental quality for a period of time (anywhere from days to years) may show a
reduced (or perhaps enhanced) capacity to tolerate a different stress (Wedemeyer et al.,
1984). The ones that have been suggested include tolerance to an elevated temperature,
hypoxia, reference toxicants, diseases, and crowding.
The easiest and quickest temperature tolerance test is the determination of the critical
thermal maximum (CTM). This is done by raising the temperature at a constant rate
(usually 1°C 3 min) until the test fish lose equilibrium (Bonin, 1981). The endpoint is
reasonably sharp, however, differences between pollutant-exposed fish and controls may
be small, depending on the pollutant (Paladino et al., 1980; McLeay and Howard, 1977).
Heath et al. (1994) found that sublethal exposure of fathead minnow larvae for 24 h to
the pyrethroid pesticide cyfluthrin caused a reduction in the upper lethal temperature and
an increase in the lower lethal temperature. Thus, the thermal limits of the fish were
effectively constricted.
Tolerance to hypoxia is customarily measured by merely sealing fish in a jar and
allowing them to deplete the oxygen until death occurs, then the residual dissolved
oxygen is measured. Generally, the residual levels are proportional to dose of toxicant to
which the fish has been exposed (Giles and Klaprat, 1979). Those chemicals that affect
respiratory gas exchange will probably cause the greatest effect on the resistance to
hypoxia (see Chapter 3).
There are a number of reference toxicants that have been suggested. One of the
original purposes was to compare stocks of fish from different sources in bioassay
investigations, but these chemicals could also be used as a challenge test. Some chemicals
that have been proposed are sodium pentachlorophenate, sodium chloride, phenol, and
sodium azide (Wedemyer et al., 1984). The time to death in a lethal concentration is the
variable most commonly measured, however, other factors such as blood osmolality
could also be used.
Theoretically, exposure to some pollutant should make the fish more sensitive to some
other different chemical stressor, but this does not always occur. Heath (1987) exposed
bluegill to copper or zinc for 7 days and then tested their resistance to a hypertonic NaCl
challenge. Surprisingly, the fish that had been exposed to the metals survived far longer
than did controls. Subsequent blood analyses revealed that the exposed fish were regu­
lating blood electrolytes better in the hypertonic solution than controls, apparently due to
a reduction in permeability of the gills to NaCl.
Challenge tests might be increased in sensitivity by using a somewhat less severe
challenge (i.e., sublethal) and measuring one or more of the physiological factors dis­
cussed above while or after the fish is challenged. Larsson et al. (1984) evaluated this
approach by exposing fish to a simulated sulfide ore smeltery effluent (containing several
metals) for 27 days, and then challenged them by removing them from the water for
exactly 3 min. They were then put back in water and sampled 2 or 4 days later. The stress
of this asphyxiation induced a condition of hyperglycemia, muscle glycogen depletion.
 335

elevated muscle water, and lowered electrolyte levels in the blood as well as some minor
hematological alterations. More importantly, the fish that had been previously exposed to
the effluent exhibited significantly greater physiological responses to the challenge stress
than did non-exposed controls.
Heath (1991) exposed bluegill to a sublethal concentration of copper which was then
followed by a hypoxic stress similar to what might be experienced in a pond going
hypoxic at night. Several physiological parameters were measured during the stress and
subsequent recovery. In general the copper caused a more severe response to the hypoxia
and delayed recovery.
A form of challenge test that has not been used much yet in pollution work is
swimming capacity (see Chapter 8). This generally requires an appropriate swimming
tunnel which can be costly to build. However, Heath et al. (1993) tested swimming
capacity of striped bass larvae utilizing a petri dish with a smaller one glued in the middle.
This creates a sort of circular “race track”. Radial lines are marked on the bottom of the
dish. Larvae that have been exposed to test waters for a period of time can then be tested
by placing them one at a time in the race track and then chasing with a glass rod. The
number of lines crossed in a minute is then counted.

E. BEHAVIOR TESTS
Most behavioral tests determine whether a stimulus (e.g., food) elicits an abnormal
behavioral response outside the normal range of variability. Behavior integrates changes
that may have occurred at lower levels of biological complexity, such as biochemical
alterations in the nervous system but is only beginning to be used in toxicological
investigations (Little, 1990). The best behavioral tests will indicate changes that might
have an impact on survival, growth, or reproduction (Beitinger, 1990).
Some of the more potentially useful behavior assays are feeding behavior, phototaxis,
predator avoidance, and spontaneous swimming activity (Beitinger, 1990; Birge et al.,
1993; Little et al., 1993; Henry and Atchison, 1991). None of these require especially
elaborate or expensive equipment nor is there a high level of expertise needed. Tests of
avoidance and attractance to contaminants or temperatures and spawning behavior would
be much more difficult to incorporate into field biomonitoring programs, although they
would certainly have high ecological relevance. A potential advantage of behavioral tests
is that they are relatively non-invasive so the organisms could be removed from their
resident waters, tested, and then returned.

F. CONCLUSIONS
There will probably always be the issue of how relevant a given variable is to the
ecological health of a body of water. This is a connection that is extremely difficult to
make and is a valid criticism of these methodologies. Thus, it should be emphasized here
that in any biomonitoring program using physiological measurements, reliance should
never be placed on only one or two variables. Rather, several should be used spanning
more than one level of biological organization. Then, if changes are seen in several
variables and especially if a pollution gradient is detected, a fairly high degree of
confidence in the findings can then be held. Examples of this approach are Adams et al.
(1988, 1992) and Hodson (1990). These papers and the one by Aldrich (1989) provide
useful discussions of theoretical and statistical aspects of the problems of monitoring
organisms in the field utilizing physiological methodologies.
The various techniques discussed above provide a sort of snapshot of physiological
condition in the test organisms. We now move to the matter of obtaining physiological
data in real time.
336

IV. EARLY WARNING SYSTEMS

As the name implies, an early warning system is a means to detect the presence of toxic
materials in water before the water gets to a place where greater harm may occur. Such
systems are also often referred to as automated biomonitors (Gruber et al., 1991).
Applications include the detection of a suddenly increased concentration of one or more
toxicants in effluents, waste spills, and as a monitor of waters to be used for drinking.
An important limitation of monitoring using chemical analyses alone for assessing
water quality is that an unexpected chemical may go undetected until considerable harm
has taken place. An early warning system uses fish or other organisms as a sensor. The
test fish are held in a continuous flow system receiving the waters to be monitored while
one or more physiological variables are measured more or less continuously. In general,
only short-term changes are detected with such systems as opposed to chronic or cumu­
lative effects.
Cairns and van der Schalie (1980) and Gruber et al. (1991) have discussed in some
detail the criteria that must be met for suitable early warning systems. The major ones are
1. The variable to be measured must be quantifiable and have the potential for automatic
recording with computer or other electronic equipment.
2. The physiological variable must give reliable detection with a short latent period. In
other words, such a system must operate on a real-time basis.
3. The system must be easy to operate by relatively untrained personnel.
4. The parameter to be measured must be sensitive to a wide variety of toxicants.
Obviously, these criteria eliminate a large number of physiological variables in fish from
consideration in early warning systems. For example, measures of tissue chemistry and
blood composition are unsuited for such a purpose. Even though cannulated fish can be
maintained for weeks, automation of the blood analyses is a nearly insurmountable
problem. This limits the potentially useful variables essentially to four: body movement,
oxygen consumption rate, ventilation/coughing frequencies, and neurological activity.
Various schemes for detecting body movement have been devised using photocells or
movement transducers (discussed in Chapter 12), but there has been only a limited
attempt to automate this kind of data gathering (Smith and Bailey, 1988). A different type
of bodily activity is the ability to maintain position in a slowly moving current of water
(sometimes referred to as rheotaxis). Automated systems have been developed utilizing
photocells at the rear of the swimming chamber to detect when the fish loses rheotaxis
in response to a toxicant entering the water (Poels, 1977). A 2-year test of a rheotaxis
system in The Netherlands successfully detected six alarms produced by increased
pollutants in the river and one false alarm (Balk et al., 1994).
The oxygen consumption of a fish can be monitored on a continuous basis but it is
sensitive to a variety of factors other than pollutants (see Chapter 8). One well-known fish
physiologist commented to the effect that one has only to think evil of a fish to double
its rate of oxygen consumption!
The application of changes in ventilatory activity has received the most development
and application in automated biomonitoring. It is obvously related to oxygen consump­
tion rate but changes in ventilation are generally more sensitive to the presence of
toxicants than is oxygen consumption alone. The delicate gill tissue is the first tissue to
be affected by a waterborne chemical and this can cause immediate and very marked
changes in ventilation and/or coughing.
The methods for measuring ventilation by fish have been reviewed by Heath (1972).
Even though that review is old, there have not been any significantly new methods
developed since then. The simplest technique, which is non-invasive and involves no
attached wires or tubes to the fish, is the use of electrodes at each end of a small test
 337

chamber. With a suitable amplifier, the potential changes (in the millivolt range) between
the electrodes produced by the breathing of the fish can be detected and the sine wave
displayed on a chart recorder (Spoor et al., 1971). Aberrant breaths such as coughs can
also be detected. This ventilatory recording technique has been interfaced with a com­
puter and is now available commercially for detecting the presence of toxic chemicals
coming into domestic drinking water supplies (Gruber et al., 1991). Baldwin et al.,
(1994a,b) evaluated this technique over 1-year trial periods. They found a low rate of false
alarms and a sensitivity using trout of between 10 and 250% of the LC50 (depending on
pollutant) with a response within 40 min. As expected, the system detects acutely toxic
concentrations much better than chronic changes.
The fish ventilation response has also been used to monitor water quality in trout
streams in Tennessee (Morgan et al., 1988). Data collection platforms equiped with
ventilation chambers, a water sampler, and pH and temperature sensors are placed beside
streams in remote locations. Then, with the use of hardwire or satellite transmission, real­
time data are transmitted to a central data processing facility. In this way, several streams
can be monitored simultaneously.
Finally, the weakly electric fish Gnathonemus petersi has been found to change the
character of its electric discharge in the presence of toxicants. These can be detected with
electrodes in the water and Geller (1984) interfaced such a system with a microcomputer
to automate it. Further evaluation of the sensitivity of this fish to detect pollutants was
done by Lewis et al. (1993). It would be interesting to compare the fish ventilatory system
with this one as to sensitivity, false alarms, etc. Then the next big hurdle will be to get
wider acceptance by municipalities, regulatory agencies, and industry.

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 Index

Acid phosphatase, 224-226

liver, 132
Acclimation, 234 Acid pollution, 239-257
to acid pollution, 248 comparative species sensitivities, acid
biomonitoring, 328 pollution, 240
energetics, physiological, 186 Acid rain. See Acid pollution
hypoxia, environmental, 36 Acrolein, respiratory response, 54
to metals, 277 Active metabolic rate, energetics,
Accumulation physiological, 174
of metals in different organs, Adenosine triphosphate, hypoxia,
xenobiotics, 91 environmental, 29
of organics, xenobiotics, 101 Adenylate energy charge, 232
Acetate, energetics, physiological, 194 liver, 126
Acetylcholine Adenylates, 232, 233
nervous system function, 282 hypoxia, environmental, 30
respiratory response, 58 Adrenalin. See also Epinephrine
Acetylcholinesterase hypoxia, environmental, 35
biomonitoring, 330 liver, 127
energetics, physiological, 199 osmoregulation, 143
hypoxia, environmental, 17 water pollution, 9
nervous system function, 282, 286-288 Adrenaline
Acid energetics, physiological, 178, 200-202,
acclimation to, acid pollution, 248 206, 207
effects of chronic exposures, acid hypoxia, environmental, 24
pollution, 247 nervous system function, 289
effects on swimming performance, acid respiratory response, 58
pollution, 251 Aerobic scope, energetics, physiological,
effects on ventilation and blood gases, 175, 195, 198, 200
acid pollution, 250 Aggression, nervous system function, 284
hematology, 73 Air breathing, hypoxia, environmental, 33
hormonal responses, acid pollution, Alanine, 279
249 hypoxia, environmental, 30
respiratory response, 50, 57, 61 nervous system function, 279
water pollution, 3 Alanine aminotransferase, 222
Acid exposure, acid pollution, 244 liver, 131

343
344

Alarm response, respiratory response, 55 Angiotensin, respiratory response, 61

Albumin, liver, 129, 131 Anoxia, hypoxia, environmental, 15, 16,
Aldicarb, 229 30, 31
excretion, xenobiotics, 112 Anthracene
Aldolase, 228 excretion, xenobiotics, 112
Aldosterone, osmoregulation, 143 hypoxia, environmental, 18
Aldrin respiratory response, 54
hematology, 74 xenobiotics, 112
water pollution, 3 Antigen processing, immunity, 260
Alkaline conditions, acid pollution, 249 Antioxidant, 231
Alkaline phosphatase, 219, 224, 226, 227 defenses, hematology, 70
liver, 131 Appetite
Allethrin, water pollution, 4 acid pollution, 252
Aluminum energetics, physiological, 175, 178, 179,
acclimation, osmoregulation, 156 193
acid pollution, 239, 240, 243, 247, 248, nervous system function, 282
251, 252 Aquatic surface respiration, hypoxia,
effect on swimming ability, energetics, environmental, 33
physiological, 196 Aquatic toxicology, water pollution, 5
effects on blood calcium, Arginine vasotocin, acid pollution, 250
osmoregulation, 156 Aroclor, liver, 127
effects on osmoregulation, Aroclor 1242, 275
osmoregulation, 156 Aromatic hydrocarbons
energetics, physiological, 181, 196 excretion, xenobiotics. 111
immunity, 263 immunity, 265
low assimilation by gut, xenobiotics, 86 reproduction, 300
osmoregulation, 156 Arsenic
reproduction, 310, 315 liver, 127
respiratory response, 54 nervous system function, 285
xenobiotics, 82, 86 respiratory response, 50
Amino acid water pollution, 2
energetics, physiological, 200 xenobiotics, 96
oxidase, energetics, physiological, 205 Arterial oxygen
Amitrol, water pollution, 4 respiratory response, 47
Ammonia, 230 tension, 56
effects on osmoregulation, Ascorbate, 231
excretion, acid pollution, 249 biomonitoring, 330
liver, 127 Ascorbic acid
nervous system function, 284 liver, 134
respiratory response, 50, 54, 60 xenobiotics, 109
water pollution, 3 Aspartate aminotransferase, 131, 219, 222
xenobiotics, 109 Assimilation
Anaerobic energy expenditure, energetics, efficiency
physiological, 174, 175 energetics, 173
Anaerobic metabolism xenobiotics, 86, 91
effect of temperature, hypoxia, energetics, physiological, 186
environmental, 29 intestine, xenobiotics, 81
Androgen, acid pollution, 241. See also Atrazine, immunity, 264
Testosterone Atrial natriuretic factor, osmoregulation,
Anemia 143
biological significance, hematology, 73 Automated biomonitors, biomonitoring,
hematology, 68, 70 336
 345

Avoidance Biomagnification, xenobiotics, 79

acid pollution, 252 Biomarker, 233
hypoxia, environmental, 34 acid pollution, 250
waterborne chemicals, 275 biomonitoring, 327, 329, 330, 331, 332
energetics, physiological, 182, 207
hematology, 72
B
immunity, 265, 266
B lymphocytes, immunity, 260, 261 reproduction, 302
Baseline, biomonitoring, 328 water pollution, 5
Behavior, 271-297. See also Specific xenobiotics, 107
behaviors Biomonitoring of fish in the field and
acid pollution, 252 mesocosms, 327
biomonitoring, 327 Biotelemetry, energetics, physiological,
effects of hypoxia, environmental, 190, 191
33 Biotransformation
reproduction, 301 effects on accumulation, xenobiotics,
tests, biomonitoring, 335 104
water pollution, 6 of organic contaminants, xenobiotics,
Benzaldehyde, respiratory response, 106
54 Biotransfusion in intestine, xenobiotics,
Benzene 110
energetics, physiological, 189, 205 Blood, 67
nervous system function, 279 affinity for oxygen, hypoxia,
Benzo[a]pyrene environmental, 22, 25
energetics, physiological, 189 oxygen transport, hypoxia,
excretion, xenobiotics, 112 environmental, 24
nervous system function, 287 Blood acid-base
reproduction, 314 acid pollution. See Arterial acid-base
xenobiotics, 110, 112, 113 hypoxia, environmental, 35
Benzophenone, energetics, physiological, Blood acidity
194 hypoxia, environmental, 34
Beryllium, respiratory response, 50 respiratory response, 56
Bile Blood cell count
immunity, 265 hematology, 68
liver, 129, 133 hypoxia, environmental, 35
metals in, xenobiotics, 96 Blood chemistries. See Individual
xenobiotics, 95, 98, 102, 111 constituents
Bilirubin, liver, 133 Blood clotting, effect of stress, liver, 129
Bimodal respiration, hypoxia, Blood electrolytes, effects of acid on, acid
environmental, 33 pollution, 244
Bioaccumulation, xenobiotics, 79 Blood glucose, hypoxia, environmental,
Bioassay, water pollution, 5 35
Bioavailability Blood liver, volume, 126
water pollution, 2 Blood oxygen
xenobiotics, 81, 86, 101 hematology, 71
Biochemical oxygen demand, water osmoregulation, 156
pollution, 1 respiratory response, 59, 60
Bioconcentration Blood pressure, acid pollution, 245
organic pollutants, xenobiotics, 101 Blood proteins, binding of organic
xenobiotics, 79, 94, 101 chemicals, xenobiotics, 88
Bioenergetics, 171-216, 171 Bluegill, hypoxia, environmental,
Biological organization, water pollution, 6 20
346

Bluegreen algae, nervous system function, importance of environmental levels in,

289 acid pollution
BOD. See Biochemical oxygen demand osmoregulation, 152, 156
Body burden, xenobiotics, 79 regulation of blood concentration,
Bohr effect osmoregulation, 144
acid pollution, 251 reproduction, 301, 308, 309, 315, 316
hypoxia, environmental, 25 xenobiotics, 82
Bone Calcium channels, respiratory response,
marrow, immunity, 260 59
xenobiotics, 95 Capture, biomonitoring, 333
Botanicals, water pollution, 3 Carbamate, 275
Brain, 224, 225, 229, 232, 233, 271 insecticides, 229
anatomy, 271 nervous system function, 275, 288
energetics, physiological, 177, 185, 206 water pollution, 3
hypoxia, environmental, 30, 31 Carbaryl
nervous system function, 271, 282, 288, energetics, physiological, 185
289 reproduction, 306
xenobiotics, 93, 98 respiratory response, 54
Burst speed, energetics, physiological, xenobiotics, 91
195 Carbofuran, 199, 233
Burst swimming, energetics, physiological, energetics, physiological, 199
199 liver, 135
water pollution, 4
Carbohydrates, energetics, physiological,
200
Cadmium, 69, 219, 220, 222, 231, 274 Carbon dioxide effect on ventilation,
avoidance of, 277 hypoxia, environmental, 23
biomonitoring, 330 Carbon tetrachloride, liver, 130, 131
effects on blood oxygen, respiratory Cardiac output, energetics, physiological,
response, 57 190
effects on calcium metabolism, Cardiovascular response, 47-66
osmoregulation, 152 Carp, hypoxia, environmental, 20
effects on hematology, 69 Catalase, 219, 231, 232
effects on immune function, immunity, Catecholamine, 275
261 acid pollution, 245, 247, 250, 251
effects on osmoregulation, 146 energetics, physiological, 207
effects on reproduction, 303 hypoxia, environmental, 22, 24-26, 35
energetics, physiological, 181-183, 191, immunity, 266
204 nervous system function, 275
immunity, 263 reproduction, 307
liver, 127, 132, 135 respiratory response, 58, 59, 61
nervous system function, 274, 281, 283, Catfish, hypoxia, environmental, 18, 20
285, 289 Caudal neurosecretory system, acid
osmoregulation, 162 pollution, 250
reproduction, 303, 304, 307, 311, Cell fragility, hematology, 70
313-316 Cell mediated immunity, immunity, 260
respiratory response, 50, 54, 57, 59, 61 Cellular enzymes, 228
water pollution, 2 Cerebellum, 272
xenobiotics, 79, 82, 86, 94, 97, 98, 108 nervous system function, 272
Calcitonin, osmoregulation, 144 Cerebrum, 272
Calcium nervous system function, 272
acid pollution, 240-248 Ceruloplasmin, xenobiotics, 91
 347

Challenge tests, biomonitoring, 334 reproduction, 315

Chattonella, respiratory response, 59 respiratory response, 54
Chemicals, energetics, physiological, 206 water pollution, 2
Chemoreceptors, nervous system function, xenobiotics, 82, 94
279 Circulatory physiology, respiratory
Chemotactic, immunity, 265 response, 58
Chlordane, 275 Citrate synthase
energetics, physiological, 185, 199 biomonitoring, 331
hematology, 74 energetics, physiological, 190, 191,
nervous system function, 275 195
water pollution, 3 Clay, respiratory response, 54
Chloride cell Clotting time
acid pollution, 242, 244, 248, 250 biomonitoring, 333
osmoregulation, 141 hematology, 72
proliferation, osmoregulation, 162 Coal dust, respiratory response, 54
xenobiotics, 97 Cobalt
Chlorinated hydrocarbons hematology, 72
hematology, 74 water pollution, 2
liver, 128 Collagen, liver, 134
xenobiotics, 91 Compensatory growth, energetics,
Chlorine, 275, 278 physiological, 193
effects on blood oxygen, respiratory Complement, immunity, 260
response, 56 Conditioned responses, nervous system
effects on hematology, hematology, function, 285
71 Conjugation reactions, xenobiotics, 109
liver, 127 Conversion efficiency, energetics,
nervous system function, 275, 278, 282 physiological, 186
osmoregulation, 161 Copper, 220, 221, 224, 225, 232, 233, 274,
respiratory response, 50, 54, 59 279
water pollution, 2 avoidance of, 277
Chlormaine, hematology, 71 biomonitoring, 330, 334, 335
Chlorothalonil effects on immune function, immunity,
hematology, 72 262
respiratory response, 50, 54 effects on liver histology, liver, 127
xenobiotics, 109 effects on locomotor activity, 273
Cholesterol effects on metabolic rate, 176-179, 176
energetics, physiological, 207 effects on osmotic and ionic regulation,
liver, 129, 132, 133 osmoregulation, 145
reproduction, 304, 307 effects on plasma enzymes, liver, 131
Chorion energetics, physiological, 176, 183, 191,
acid pollution, 242 197, 203
reproduction, 302, 303 hematology, 74
Chorionase, acid pollution, 242 hypoxia, environmental, 18
Chromaffin cells, energetics, physiological, immunity, 262
200 liver, 127, 131, 133, 134, 135
Chromium, 225, 274 nervous system function, 274, 279, 280,
effects on osmoregulation, 282-285, 289
osmoregulation, 155 osmoregulation, 149, 162
energetics, physiological, 181, 194 reproduction, 304, 308-311, 314, 315
hematology, 74 respiratory response, 50, 54, 57, 61
immunity, 262 water pollution, 2
nervous system function, 274, 289 xenobiotics, 81, 91, 95, 97
348

Copper uptake, xenobiotics, 85 energetics, physiological, 188, 190, 191,

Corpuscles of stannius, osmoregulation, 195
144 Cytochrome P450, 234, 304, 307, 329, 330
Corticosteroid, energetics, physiological, xenobiotics, 107
178 Cytoplasmic inclusions, liver, 128
Cortisol, 230
acid pollution, 247, 249, 250
energetics, physiological, 198, 200-205,
207 DDT, 274, 275, 278
hematology, 74 effect on amino acid uptake,
hypoxia, environmental, 35 osmoregulation, 158
immunity, 262, 266 effect on osmoregulation,
liver, 127, 133, 135 osmoregulation, 158
nervous system function, 282 liver, 133
osmoregulation, 143 nervous system function, 274, 275, 278,
reproduction, 307 281, 285
water pollution, 9 reproduction, 309
Coughing xenobiotics, 113
biomonitoring, 336 Dehydroabietic acid, hematology, 72
respiratory response, 54, 55 Delta-aminolevulinic acid dehydratase, 69
Creosote, immunity, 265 Delta-aminolevulinic acid dehydrase, 224
Cresol, xenobiotics, 111 Depuration, xenobiotics, 96
Critical oxygen tension Detergent, 278
acid pollution, 251 effects on osmoregulation,
hypoxia, environmental, 27, 36 osmoregulation, 157
Critical swimming speed, energetics, energetics, physiological, 205
physiological, 195, 196 excretion, xenobiotics, 112
Critical thermal maximum nervous system function, 278, 281,
biomonitoring, 334 282
hematology, 72 reproduction, 308
reproduction, 316 respiratory response, 50, 59
Crotisol, osmoregulation, 163 water pollution, 3
Crucian carp, hypoxia, environmental, 16, xenobiotics, 85, 90
31 Diazinon
Crude oil, respiratory response, 54 liver, 127
Cutaneous respiration, hypoxia, nervous system function, 287
environmental, 18 water pollution, 4
Cyanide Dibenzofurans, xenobiotics, 101
energetics, physiological, 188, 194, 195, Dichlorvos
198, 200 immunity, 264
liver, 131 water pollution, 4
reproduction, 304, 308, 309, 311, 313 Dieldrin, 230
respiratory response, 54, 59, 60 liver, 127
water pollution, 3 respiratory response, 59
Cypermethrin water pollution, 3
energetics, physiological, 207 xenobiotics, 91
respiratory response, 54 Differential leukocyte counts, immunity,
Cyprinid 261
hypoxia, environmental, 16, 19 Dimethoate, energetics, physiological,
larvae, hypoxia, environmental, 32 206
Cytochrome oxidase Dioxins, nervous system function, 282
biomonitoring, 331 Diquat, nervous system function, 287
 349

Disease Electroolfactogram, nervous system

biomonitoring, 332 function, 279
immunity, 265 Electroretinogram, nervous system
Dispersant, respiratory response, oil and, function, 279
54 Embryo-larval lethality tests,
Dissolved organic matter, xenobiotics, 90 biomonitoring, 326
Dissolved oxygen, energetics, Embryo i
physiological, 176 biomonitoring, 326 i
Dorsal light response, nervous system energetics, physiological, 189
function, 284 hypoxia, environmental, 17
Dose, water pollution, 7 Embryonic development, reproduction,
Dragonet, hypoxia, environmental, 20 302, 303, 312
Dursban Endosulfan, 229
liver, 127 energetics, physiological, 207
water pollution, 4 respiratory response, 54
Endrin
energetics, physiological, 206
heptachlor, water pollution, 4
Early warning systems, biomonitoring, 336 immunity, 264
Eels, hypoxia, environmental, 18 liver, 127, 132, 133
Effect osmoregulation, 158
of acid on, osmoregulation, 244 xenobiotics, 88
of acid on larvae, acid pollution, 243 Energetics, physiological, 171-216
on blood and urine chemistry, hypoxia, Energy
environmental, 34 budget, energetics, physiological, 174,
on enzyme activity, hypoxia, 187
environmental, 37 charge, hypoxia, environmental, 30
on gill histopathology, hypoxia, expenditure methods of measuring,
environmental, 36 energetics, physiological, 174
on kidney function, hypoxia, metabolism, energetics, physiological,
environmental, 35 173
on larvae, hypoxia, environmental, 27 Enterohepatic circulation, xenobiotics, 97
on muscle capillaries, hypoxia, Enzyme activity
environmental, 37 effects of metals on, 219-225
on organic chemical uptake, xenobiotics, use for estimating metabolic rate, 190
90 Enzyme methodology, 217
on temperature selection, hypoxia, Enzymes, 229
environmental, 34 Epinephrine, 230. See also Adrenalin
Effect of copper on, energetics, Erythrocyte, 224
physiological, 179 count, biomonitoring, 331
Egg hematology, 67
acid pollution, 240, 241, 242 nucleoside triphosphate, hypoxia,
activation, reproduction, 310 environmental, 36
capsule, reproduction, 312 synthesis, biomonitoring, 332
energetics, physiological, 189 Erythron, hematology, 69
hypoxia, environmental, 33 Erythropoiesis, hematology, 74
reproduction, 300, 302, 303, 309 Estradiol
Electric fish, biomonitoring, 337 acid pollution, 241
Electrobranchiogram, respiratory response, reproduction, 304
53 Estrogen, reproduction, 301, 307, 308
Electromyography, acid pollution, Estrogenic effluents, reproduction, 308
252 Estrogens testosterone, reproduction, 306
350

Ethanol Fight or flight, water pollution, 9

hypoxia, environmental, 34 Fish acute toxicity syndromes, water
metabolic pathway, hypoxia, pollution, 10
environmental, 31 Fly ash, 277
Ethological units, 271 nervous system function, 277
nervous system function, 271 Follicle-stimulating hormone,
Ethyl, energetics, physiological, 194 reproduction, 300
Excretion Foraging theory, nervous system function,
of metals, xenobiotics, 96 284
of organic contaminants, xenobiotics, Formalin, respiratory response, 50
no Free radical, hypoxia, environmental,
Exercise, acid pollution, 248 17
Exocrine pancreas, liver, 129 Fry
Extraction coefficient, xenobiotics, 82 acid pollution. See Larvae
Extraction efficiency, hypoxia, effects of cadmium on osmoregulation
environmental, 25 in, 153
Eye, nervous system function, 279, 282 osmoregulation, 153
energetics, physiological, 196
environmental factors, energetics,
physiological, 176
Fat hypoxia, environmental, 17
energetics, physiological, 203 osmoregulation, 160
liver, 126
xenobiotics, 106
Fecundity, reproduction, 302, 303, 308,
309 Gallbladder, xenobiotics, 97, 111
Feeding Gametogenesis, hormonal control,
effects on metabolic rate, energetics, reproduction, 300
physiological, 175 Garlón, 278
energetics, physiological, 188 Gill, 145, 157, 162, 222, 224
nervous system function, 282 acid pollution, 246
Fenitrothion, 199 control of blood circulation, hypoxia,
energetics, physiological, 199 environmental, 24
excretion PCB, excretion, xenobiotics, energetics, physiological, 177, 185
111 function recovery, respiratory response,
nervous system function, 287 56
respiratory response, 54 histopathology, 50, 197
xenobiotics. 111 respiratory response, 49
Fenvalerate, 233 hypoxia adjustments, hypoxia,
effects on osmoregulation, environmental, 24
osmoregulation, 158 microenvironment, xenobiotics, 85
energetics, physiological, 185 nervous system function, 288
respiratory response, 54 osmoregulation, 145, 162, 163
Fertility, reproduction, 302 respiratory response, 61
Fertilization structure, respiratory response, 47
acid pollution, 241 surface microenvironment, xenobiotics,
reproduction, 310 82
Fibrinogens, liver, 129 tissue metabolism: effects of metals,
Field measurements of energy expenditure, energetics, physiological, 182
energetics, physiological, 189 uptake, limiting factors, xenobiotics,
Field studies, effects on immune function, 88
immunity, 265 vasculature, respiratory response, 59
 351

xenobiotics, 93, 97, 99, 110 H

Gills, 153, 163
Glucagon, 143 Handling time, nervous system function,
energetics, physiological, 207 284
osmoregulation, 143 Hardness, xenobiotics, 84
Glucose, 223 Hatchability
acid pollution, 247, 250 biomonitoring, 326
biomonitoring, 333 reproduction, 303
energetics, physiological, 173, 179, 200, Hatching time, reproduction, 312, 313
201, 202, 204, 205, 207 Heart, 222
Glucose-6-phosphatase, 223, 230 hypoxia, environmental, 31
energetics, physiological, 205 Heart pacemaker, respiratory response, 58
Glucose-6-phosphate dehydrogenase, 219, Heart rate
222 biomonitoring, 327
Glutamate, synthetase, 227 effect of hypoxia, hypoxia,
Glutamate dehydrogenase, 223, 227, 230 environmental, 26
energetics, physiological, 295 energetics, physiological, 190
Glutamic oxaloacetic transaminase, liver, reproduction, 312, 317
130 respiratory response, 59, 61
Glutamic pyruvic transaminase, liver, Hematocrit
130 acid pollution, 245
Glutathine, 231 biomonitoring, 331
Glutathione, 231, 233 hematology, 68, 73, 74
biomonitoring, 329 hypoxia, environmental, 35
xenobiotics, 98, 107 Hematological methods, hematology, 68
Glyceraldehyde dehydrogenase, 225 Hematology, 67-78, 331, 332
Glycogen, 228 acid, 73
acid pollution, 252 aldrin, 74
energetics, physiological, 173, 188, 200, chlormaine, 71
202, 203, 204, 206, 295 chlorothalonil, 72
hypoxia, environmental, 29 chromium, 74
liver, 127 hematopoietic tissue, 67
Glycolysis, 223, 225 organochlorine pesticides, 72
Goldfish organophosphate insecticides, 74
effects of anoxia, hypoxia, tolerance, 72
environmental, 31 zinc, 74
hypoxia, environmental, 16, 31 Hemocytoblast, hematology, 67
Gonadotropin-releasing hormones, Hemoglobin
reproduction, 300 biomonitoring, 331
Growth hematology, 67
acid pollution, 241, 247 hypoxia, environmental, 25
biomonitoring, 326, 330, 331 Hepatocytes
energetics, physiological, 177, 182, 186, energetics, physiological, 201
188, 189, 191, 194, 197, 207 liver, 133
nervous system function, 284 Hepatosomatic index, liver, 126
reproduction, 303 Herbicides
water pollution, 6 water pollution, 3
Growth hormone, 143 xenobiotics, 101
energetics, physiological, 194, 207 Hexachlorocyclohexane, reproduction, 305
Growth rate, biomonitoring, 326 Hexokinase, 223, 226
Gut, 162 Histopathology, biomonitoring, 331
osmoregulation, 162 Homeostasis, water pollution, 8, 10
352

Hormesis Insulin diabetes, energetics, physiological,

energetics, physiological, 193, 199 204
nervous system function, 287 Insulin-like growth factor, 143
Hormonal controls, 143 osmoregulation, 143
Humoral immune expression, immunity, Interferon, immunity, 260
264 Intestine
Hydrazine, nervous system function, 283, uptake of organic chemicals,
284 xenobiotics, 90
Hydrocarbons, xenobiotics, 111 xenobiotics, 100
Hydrogen peroxide, 231 Iodine, water pollution, 2
Hyperoxia, hypoxia, environmental, Ionic fluxes, species differences, 142
15 Ionizing radiation, nervous system
Hypoxemia, 156 function, 283
osmoregulation, 156 lonoregulation, acid pollution, 242
respiratory response, 56, 61 Iron, 232
Hypoxia reproduction, 315
acclimation, 36 respiratory response, 50
avoidance, 34 water pollution, 2
blood, affinity for oxygen, 22, 25
crucian carp, 16, 31
effects on gill histopathology, 36
K
environmental, 17 Kidney, 152-154, 162, 224
noradrenaline, 24 acid pollution, 247
prostaglandins, 24 effects of cadmium, 152
swimming, speed, effect of hypoxia, osmoregulation, 152
31 energetics, physiological, 204, 205
trichlorobenzene, 17 excretion of pesticides, xenobiotics, 113
trout, 20 hematology, 71
UV light, 18 immunity, 260
ventilation, 20 osmoregulation, 152, 154
Hypoxic stress, biomonitoring, 335 xenobiotics, 92, 95, 97, 99, 113
Kraft pulpmill effluents. See Pulpmill
effluents
1
Kuppfer cells, liver, 127
Immune function, effects of pollutants on
260-265
Immune system, 259-269
biomonitoring, 332 Lactate
Immunoglobulins, immunity, 260 acid pollution, 251
Immunology, biomonitoring, 331 energetics, physiological, 173, 183
Immunotoxicology, immunity, 260 Lactate dehydrogenase, 224, 225, 229
Incubation time, reproduction, 313 biomonitoring, 331
Individual chemicals, energetics, energetics, physiological, 195
physiological, 206 Lactic acid
Insecticides energetics, physiological, 203, 205, 206
respiratory response, 59 hypoxia, environmental, 29, 30, 34, 35
water pollution, 3 respiratory response, 56
xenobiotics, 101 Lamellae, hypoxia, environmental, 20
Insulin, 143 Larvae, 314-317
energetics, physiological, 200, 203, 204, acid pollution, 241
206, 207 biomonitoring, 326, 335
osmoregulation, 143 effects of acid on, acid pollution, 242
 353

energetics, physiological, 189, 191, 194, zinc, 128

196 Locomotor activity, 272
hypoxia, environmental, 17 methods for measuring, 272
nervous system function, 288 nervous system function, 272
reproduction, 300, 308-310, 314 reproduction, 316
swimming performance, 199 Luteinizing hormone, reproduction, 300
energetics, physiological, 199 Lymph nodes, immunity, 260
Lateral line receptors, nervous system Lysosomes, 225, 229
function, 281 biomonitoring, 330, 333
Lead, 219, 220, 221, 224 liver, 132
biomonitoring, 333
effects on osmoregulation, 155
hematology, 70
M
nervous system function, 281, 285, Magnesium, 152
289 osmoregulation, 152
xenobiotics, 79, 85, 93, 96, 98, 100 Malaoxon, nervous system function, 288
Learning, nervous system function, 285 Malate dehydrogenase, 223, 224, 229
Leucine amino naphthylamidase, Malathion, 199, 226
biomonitoring, 333 energetics, physiological, 199, 207
Leukocrit, biomonitoring, 332 immunity, 264
Leydig cells, reproduction, 304 liver, 135
Life cycle tests, biomonitoring, 326 nervous system function, 287, 289
Lindane respiratory response, 50
immunity, 264 water pollution, 4
water pollution, 4 Manganese
Lipid effects on osmoregulation, 157
biomonitoring, 330 osmoregulation, 157
energetics, physiological, 173, 198, 200, immunity, 262
202, 207 water pollution, 2
liver, 132 Maximal acceptable toxicant
Lipid peroxidation concentration, reproduction, 317
hematology, 70 Mercury, 220-225
liver, 136 attractance to, 277
xenobiotics, 99 nervous system function, 277
Liver, 125-139, 125-140, 126, 132, 222, effect on mucus production, 161
224, 232, 233 effects on osmoregulation, 153
acid phosphatase, 132 Metabolic rate
arsenic, 127 effects of metals on 176-186
ascorbic acid, 134 effects of pesticides 184-189
blood liver, volume, 126 energetics, physiological, 185
brian, 134 Metabolic scope, energetics, physiological,
cholesterol, 129, 132, 133 175, 177
collagen, 134 Metallothione, biomonitoring, 329
endrin, 127, 132, 133 Metallothionein, 231, 232, 234
energetics, physiological, 177, 200 reproduction, 315
exocrine pancreas, 129 Metaloids, water pollution, 2
histology, 125 Metals, 239
interrenal, 135 acclimation to, xenobiotics, 100
mercury, 127, 131, 132 accumulation in different organs,
petroleum hydrocarbons, 126, 127, 135 xenobiotics, 91
plasma proteins, 129 bioavailability, xenobiotics, 81
sorbitol dehyrogenase, 130 effect on MFO activity, xenobiotics, 108
354

mechanisms of uptake, xenobiotics, hypoxia, environmental, 31

81 nervous system function, 288
uptake in food, xenobiotics, 86 xenobiotics, 92
water pollution, 2
xenobiotics, 81
N
Metasystox, 226, 228
Methemoglobin, 160 Na,K ATPase, 145, 150, 151, 154, 155,
hematology, 71 156, 157, 158, 162, 163, 225
osmoregulation, 160 acid pollution, 250
Methlmercury, reproduction, 310 energetics, physiological, 173
Methoxychlor osmoregulation, 145, 150, 151,
energetics, physiological, 185 154-158, 162, 163
water pollution, 4 Naphthalene, 159
Methyl parathion, 199 nervous system function, 281
energetics, physiological, 185, 199 osmoregulation, 159
nervous system function, 283, 285, respiratory response, 54
288 xenobiotics, 90
reproduction, 316 Natural killer cells, immunity, 260
respiratory response, 54 Nervous system function, 278
water pollution, 4 acetylcholine, 282
Methylmercury, 154, 224 diazinon, 287
hematology, 71 Dieldrin, 289
immunity, 263 feeding, 282
nervous system function, 280 fenitrothion, 287
osmoregulation, 154 hydrazine, 283, 284
reproduction, 315 hypoxia, 289
xenobiotics, 83, 85, 91, 97, 100 lead, 281, 285, 289
Mg ATPase, 157, 158 liver, 288
Micropyle, reproduction, 302, 310 naphthalene, 281
Mill effluent, water pollution, 4 olfactory, 281
Mirex, immunity, 264 petroleum hydrocarbon, 279
Mitochondria, 229 predator-prey behavior, 283
Mitochondrial calcium, xenobiotics, 99 silver, 281
Mode of action, water pollution, 10 telencephalon, 284
Molinate, 199 temperature, 281
energetics, physiological, 193, 199 zinc, 281, 285, 289
reproduction, 316 Neutrophils, immunity, 260, 261, 262
water pollution, 4 Nickel
Monocytes, immunity, 259, 260 hematology, 74
Mucus, 161 immunity, 262
acid pollution, 250, 251 reproduction, 304, 315
immunity, 259 respiratory response, 50
importance in osmoregulation, 161 Nitrate
osmoregulation, 161 hypoxia, environmental, 18
osmoregulation, 161 water pollution, 3
respiratory response, 50 Nitrite, 161
xenobiotics, 82, 85, 95 effects on hematology, hematology, 72
importance for metal uptake by gills, effects on osmoregulation, 160
82 osmoregulation, 160
Muscle, 232 hematology, 72
energetics, physiological, 185, 193, osmoregulation, 161
200 water pollution, 3
 355

nervous system function, 278

reproduction, 307, 309, 315
Octanol/water partition coefficients, P-cresol, energetics, physiological, 194
xenobiotics, 87 Pentachlorophenol, 226, 228, 275
Oil energetics, physiological, 187, 198
dispersant, 157 hematology, 74
osmoregulation, 157 liver, 135
spill dispersant, respiratory response, 59 nervous system function, 275, 283
Olfactory bulbs, 272 water pollution, 4
nervous system function, 272 xenobiotics, 102
Olfactory receptors, nervous system Percentage utilization, hypoxia,
function, 279 environmental, 25
Olfactory system, reproduction, 302 Permethrin, respiratory response, 50
Oocyte, reproduction, 302 Pesticides
Operant conditioning, acid pollution, 252 effects on immune function, immunity,
Optic lobes, 272 263
nervous system function, 272 effects on locomotor activity, 275
Optomotor response, nervous system effects on metabolic rate, 184-189
function, 287 energetics, physiological. See also
Organic chemical, mechanisms of uptake, Individual chemicals
xenobiotics, 87 hematology, 74
Organic contaminant excretion, water pollution, 3
xenobiotics, 110 Petroleum
Organochlorine insecticides, 294 reproduction, 312
effects on cellular respiratory response, 50
energetics, physiological, 185. See also water pollution, 4
Individual insecticides Petroleum hydrocarbons, 225, 274
nervous system function, 282, 294 effect of salinity on
reproduction, 305 osmoregulation
Organophosphorus effect on liver, 126
nervous system function, 282 effect on metabolic rate, 225
xenobiotics, 104 energetics, physiological, 189
Organotin, xenobiotics, 95 effect on osmoregulation, 159
osmoregulation, 159
energetics, physiological, 193, 198, 295
liver, 126, 127, 135
Paraquat, 230 nervous system function, 274
reproduction, 317 reproduction, 305, 314
respiratory response, 50, 59 Petroleum hydrocarbons accumulation,
Parathion, 226, 228, 278 xenobiotics, 104
nervous system function, 278, 286 Phagocytes, immunity, 263
water pollution, 4 Phase I reactions, xenobiotics, 107
xenobiotics, 91 Phase II reactions, xenobiotics, 109
Parquat, water pollution, 4 Phenol, 278
Parr-Smolt transformation, 143 effects on osmoregulation,
effect of copper on, 150 osmoregulation, 158
osmoregulation, 143 excretion, xenobiotics. 111
Partition coefficient, xenobiotics, 101 hypoxia, environmental, 18
Pasteur effect, hypoxia, environmental, 30 liver, 127, 131, 133
PC. See Critical oxygen tension nervous system function, 278
PCBs, 231, 278 respiratory response, 50
energetics, physiological, 192, 198 xenobiotics, 109
356

Phenoxyacetic acid herbicides, xenobiotics, liver, 132

113 Psychological stress, water pollution, 8
Pheromones, reproduction, 302 Pulpmill effluent, 278
Phosphamidon, 226, 229 effect on energy stores, energetics,
Phosphatase, 229 physiological, 202
Phosphoenol-pyruvate carboxykinase, 222 effect on growth, energetics,
Phosphorylase, 228, 230 physiological, 193
Plaice, hypoxia, environmental, 18 effect on hematology, hematology, 72
Plasma calcium, 153 energetics, physiological, 188, 193, 196,
Plasma clearance by the liver of specific 202
dyes, liver, 130 hematology, 72
Plasma copper, xenobiotics, 95 liver, 133
Plasma enzyme assays, biomonitoring, nervous system function, 278
333 reproduction, 306, 307
Plasma enzymes of liver origin, liver, 130 respiratory response, 54
Plasma proteins, liver, 129 Pyrethroid, 158
Plasma skimming, hematology, 69 biomonitoring, 334
Pollutants 200-207 energetics, physiological, 185
Pollution control, biomonitoring, 325 reproduction, 315
Polyaromatic hydrocarbons, xenobiotics, Pyrethrum, water pollution, 4
91
Polychlorinated dibenzo-p-dioxins,
Q
xenobiotics, 101
Polychlorineated biphenyls, water Quinalphos, 206, 226
pollution, 4
Polycyclic aromatic hydrocarbons,
xenobiotics, 81
Polyhalogenated biphenyls, xenobiotics, Ram gill ventilation, hypoxia,
101 environmental, 23
Postprandial effect, energetics, Regulation of metal concentration,
physiological, 175 xenobiotics, 96
Potassium, 152, 155 Renin, 160
osmoregulation, 152 osmoregulation, 160
Predator-prey behavior, nervous system respiratory response, 60
function, 283 Reninangiotensin system, 143
Prey, nervous system function, 284 Reproduction, 299-323
Progestins, reproduction, 301 strategies, 300
Prolactin, 143, 144, 153, 163 water pollution, 6
acid pollution, 250 Reproductive hormones, effects of
osmoregulation, 143, 153 pollutants on, reproduction, 303
reproduction, 315 Residual dissolved oxygen, biomonitoring,
Prolonged swimming speed, energetics, 334
physiological, 195 Residue analysis, xenobiotics, 110
Propionate, hypoxia, environmental, 30 Resin acids, water pollution, 4
Prostaglandins Respiration, energetics, physiological,
hypoxia, environmental, 24 174
reproduction, 302 Respiratory conformity, hypoxia,
Protein environmental, 28
acid pollution, 247 Respiratory poisons, energetics,
biomonitoring, 330, 331, 333 physiological, 198
energetics, physiological, 173, 194, 200, Respiratory regulation and conformity,
202-205, 207 hypoxia, environmental, 27
 357

Respiratory response, 47-66 liver, 130

aluminum, 54 triglycerides, energetics, physiological,
arterial oxygen tension, 56 207
cadmium, effects on blood oxygen, 57 Sevin, 228
chlorine, 50, 54, 59 water pollution, 4
coal dust, 54 Sewage sludge, immunity, 265
Cypermethrin, 54 Silt, water pollution, 2
formalin, 50 Silver, 220, 224
heart rate, 59, 61 nervous system function, 281
maximum acceptable toxicant reproduction, 313
concentration, 55 xenobiotics, 81
oil spill dispersant, 59 Silvex, water pollution, 4
surfactants, 54 Skin
zinc, 50, 54, 56, 57, 59 excretion of organics, xenobiotics,
effects on blood gases, 56 113
Reticular system, 272, 274 hypoxia, environmental, 18
nervous system function, 272, 274 uptake of organic chemicals,
Reticuloendothelial cells, xenobiotics, xenobiotics, 90
106 xenobiotics, 95, 96, 97, 111, 113
Rheotaxis, biomonitoring, 336 Smoltification, acid pollution, 247
RNAase, 219 Social rank of a fish, 274
RNA/DNA ratio nervous system function, 274
biomonitoring, 331 Somatostatin, 143
energetics, physiological, 193, 194 osmoregulation, 143
Root effect, hypoxia, environmental, 25 Sorbital dehydrogenase, biomonitoring,
Rotenone 333
energetics, physiological, 187 Spawning
water pollution, 4 effects of acid 240-241
Routine metabolic rate, energetics, reproduction, 300, 301
physiological, 175 Specific immune response, immunity,
260
Sperm, reproduction, 310
Spermatogenesis, reproduction, 301, 304,
Salinity 305
effect on PCP accumulation, Spinal deformities, liver, 134
xenobiotics, 103 Spleen
energetics, physiological, 176 hematology, 74
Schooling, nervous system function, 283 immunity, 260, 262, 266
Seasonal effects, biomonitoring, 328 Spontaneous activity, 199
Secondary blood system, xenobiotics, effects of metals on, energetics,
90 physiological, 181
Selenium, 155, 277 energetics, physiological, 181, 182, 185,
effect on mercury toxicity, 155 187, 199
osmoregulation, 155 Squalane, xenobiotics, 106
nervous system function, 277, 285 Standard metabolic rate, energetics,
osmoregulation, 155 physiological, 174
reproduction, 309, 312 Starvation, 219
water pollution, 2 Stress
xenobiotics, 95 biomonitoring, 327, 333
Sensory receptors, nervous system energetics, physiological, 200, 201,
function, 279 202
Serum. See also Plasma immunity, 264
358

reproduction, 307 Tin, 157

water pollution, 9 osmoregulation, 157
Sturgeon, hypoxia, environmental, 20 water pollution, 2
Succinic dehydrogenase, 223, 224, 228, xenobiotics, 95
229 Tissue oxygen tension, hypoxia,
Sugar, energetics, physiological, environmental, 25
202 T-lymphocyte, immunity, 261
Sumithion, 228 Toadfish, hypoxia, environmental,
Superoxide, 231 16
Surfactants Tolerance, hematology, 72
energetics, physiological, 205 Toluene, 159
respiratory response, 54 excretion, xenobiotics. 111
Suspended solids, water pollution, liver, 132
2 osmoregulation, 159
Sustained swimming capacity, energetics, respiratory response, 54
physiological, 200 xenobiotics. 111
Swimming Toxaphene
acid pollution, 248 liver, 134
reproduction, 316 water pollution, 4
speed, effect of hypoxia, hypoxia, Toxic action, water pollution, 10
environmental, 31 Toxicokinetic models, xenobiotics,
Swimming capacity 106
biomonitoring, 335 Toxicology, water pollution, 5
reproduction, 316 Trace metals, water pollution, 2
Swimming performance Transfer of contaminants into eggs from
acid pollution, 251 adults, reproduction, 309
energetics, physiological, 195 Transport by blood of pollutants,
xenobiotics, 91
Tributyltin, 157
effect on MFO activity, xenobiotics, 108
Target organ, water pollution, 10 immunity, 263
Taste receptors, nervous system function, osmoregulation, 157
230 water pollution, 3
Telencephalon, 272 xenobiotics, 95
nervous system function, 272, 284 Trichlorobenzene, hypoxia, environmental,
Temperature, 222, 275 17
nervous system function, 275 Trichlorofon, immunity, 264
Testosterone, reproduction, 301, 304, 305, Triclopyr, energetics, physiological,
307 185
Thermal pollution, water pollution, 5 Trout, hypoxia, environmental, 20
Thigmotaxis, acid pollution, 252
Thiotox, liver, 135
Ü
Threshold, water pollution, 7
Thrombocytes Ucrit, energetics, physiological, 195, 196,
hematology, 67 197, 198
immunity, 260, 262 Ultimobranchial bodies, 144
Thymus, immunity, 260 Uptake efficiencies, xenobiotics, 87
Thyroid, 143, 229 Urea nitrogen, liver, 131
acid pollution, 250 Uric acid, liver, 133
energetics, physiological, 188, 193 Urinary excretion, xenobiotics, 97
osmoregulation, 143 Urine
reproduction, 302, 304, 308 acid pollution, 247
 359

xenobiotics, 97 water quality criteria, 5

Urotinsins, 143 zinc, 2, 11
osmoregulation, 143 Water quality criteria, water pollution,
Utilization efficiency 5
respiratory response, 48 Wood fibers
xenobiotics, 82 energetics, physiological, 196
water pollution, 2
Wood pulp, respiratory response, 54
V
Ventilation
X
acid pollution, 250
biomonitoring, 326, 336, 337 Xanthine oxidase, 219, 223
changes, mechanisms, respiratory energetics, physiological, 205
response, 55 Xenobiotics, 79-123
Ventrolateral peduncular neuropil, aluminum, 82, 85, 86
272 arsenic, 96, 97
nervous system function, 272, 282 bioconcentration, 79
Viable hatch, reproduction, 313, 314 body burden, 79
Vitamin E, xenobiotics, 109 chromium, 82, 93, 94, 96
Vitellogenesis dibenzofurans, 101
acid pollution, 240 fenitrothion. 111
reproduction, 300, 316 gill uptake, limiting factors, 88
Vitellogenin hardness, 84
acid pollution, 241 lead, 79, 82, 85, 86, 93, 94, 96-100
reproduction, 304, 307, 308 metals, acclimation to, 100
mucus, 82, 84, 85, 93, 95, 97
organophosphorus, 104
w
petroleum hydrocarbons, 104
Water content, biomonitoring, 330 reticuloendothelial cells, 106
Water hardening urine, 97, 111
acid pollution, 242 zinc, 82, 84, 85, 91, 95-97, 100
reproduction, 302, 310, 312
Water hardness
acid pollution, 244
effect on copper toxicity, Yolk
osmoregulation, 145 proteins, acid pollution, 240
Water pollution, 1-13 sac, reproduction, 316
aquatic toxicology, 5
biochemical oxygen demand, 1
carbofuran, 4
cortisol, 9 Zinc, 150, 162, 221, 224, 274, 279
diquat, 4 avoidance of, 277
fish acute toxicity syndromes, 10 effects on blood calcium, 151
insecticides, 3 effects on blood gases, respiratory
manganese, 2 response, 56
mode of action, 10 effects on osmoregulation, 150
organophosphate, 3 nervous system function, 274, 279, 281,
physiological mode of death, 8 285, 289
resin acids, 4 osmoregulation, 150
stress, 9 water pollution, 2, 11
tin, 2 xenobiotics, 82, 85, 95, 97