Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

www.elsevier.com/locate/etap

Review article

Fish bioaccumulation and biomarkers in environmental risk

assessment: a review
Ron van der Oost a,!, Jonny Beyer b, Nico P.E. Vermeulen c
a
Department of Environmental Toxicology, OMEGAM Environmental Research Institute, PO Box 94685, 1090 GR Amsterdam, The Netherlands
b
Department of Marine Environment, RF-Rogaland Research, Stavanger, Norway
c
Department of Molecular Toxicology, Vrije Universiteit, Amsterdam, The Netherlands

Accepted 19 August 2002

Abstract

In this review, a wide array of bioaccumulation markers and biomarkers, used to demonstrate exposure to and effects of
environmental contaminants, has been discussed in relation to their feasibility in environmental risk assessment (ERA). Fish
bioaccumulation markers may be applied in order to elucidate the aquatic behavior of environmental contaminants, as
bioconcentrators to identify certain substances with low water levels and to assess exposure of aquatic organisms. Since it is
virtually impossible to predict the fate of xenobiotic substances with simple partitioning models, the complexity of bioaccumulation
should be considered, including toxicokinetics, metabolism, biota-sediment accumulation factors (BSAFs), organ-specific
bioaccumulation and bound residues. Since it remains hard to accurately predict bioaccumulation in fish, even with highly
sophisticated models, analyses of tissue levels are required. The most promising fish bioaccumulation markers are body burdens of
persistent organic pollutants, like PCBs and DDTs. Since PCDD and PCDF levels in fish tissues are very low as compared with the
sediment levels, their value as bioaccumulation markers remains questionable. Easily biodegradable compounds, such as PAHs and
chlorinated phenols, do not tend to accumulate in fish tissues in quantities that reflect the exposure. Semipermeable membrane
devices (SPMDs) have been successfully used to mimic bioaccumulation of hydrophobic organic substances in aquatic organisms. In
order to assess exposure to or effects of environmental pollutants on aquatic ecosystems, the following suite of fish biomarkers may
be examined: biotransformation enzymes (phase I and II), oxidative stress parameters, biotransformation products, stress proteins,
metallothioneins (MTs), MXR proteins, hematological parameters, immunological parameters, reproductive and endocrine
parameters, genotoxic parameters, neuromuscular parameters, physiological, histological and morphological parameters. All fish
biomarkers are evaluated for their potential use in ERA programs, based upon six criteria that have been proposed in the present
paper. This evaluation demonstrates that phase I enzymes (e.g. hepatic EROD and CYP1A), biotransformation products (e.g.
biliary PAH metabolites), reproductive parameters (e.g. plasma VTG) and genotoxic parameters (e.g. hepatic DNA adducts) are
currently the most valuable fish biomarkers for ERA. The use of biomonitoring methods in the control strategies for chemical
pollution has several advantages over chemical monitoring. Many of the biological measurements form the only way of integrating
effects on a large number of individual and interactive processes in aquatic organisms. Moreover, biological and biochemical effects
may link the bioavailability of the compounds of interest with their concentration at target organs and intrinsic toxicity. The
limitations of biomonitoring, such as confounding factors that are not related to pollution, should be carefully considered when
interpreting biomarker data. Based upon this overview there is little doubt that measurements of bioaccumulation and biomarker
responses in fish from contaminated sites offer great promises for providing information that can contribute to environmental
monitoring programs designed for various aspects of ERA.
# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Fish; Environmental risk assessment (ERA); Bioaccumulation; Biomarkers

! Corresponding author. Present address: Environmental Toxicology Division, DWR Water Management and Seweage Service, PO Box 94370,
1090 GJ Amsterdam, The Netherlands. Tel.: "/31-20-597-6712; fax: "/31-20-597-6777
E-mail address : [email protected]; future E-mail address [email protected] (R. van der Oost).

1382-6689/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 2 - 6 6 8 9 ( 0 2 ) 0 0 1 2 6 - 6
58 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

1. Introduction is generally performed by predictive methods, the

interest in the assessment of pollution that began in
The environment is continuously loaded with foreign the past and may have ongoing consequences in the
organic chemicals (xenobiotics) released by urban com- future is increasing. These so-called retrospective ERAs
munities and industries. In the 20th century, many are primarily concerned with establishing the potential
thousands of organic trace pollutants, such as poly- relationship between a pollutant source and an ecologi-
chlorinated biphenyls (PCBs), organochlorine pesticides cal effect caused by exposure of organisms to the
(OCPs), polycyclic aromatic hydrocarbons (PAHs), pollutant (Suter, 1993).
polychlorinated dibenzofurans (PCDFs) and dibenzo- The ability of various pollutants (and their deriva-
p -dioxins (PCDDs) have been produced and, in part, tives) to mutually affect their toxic actions complicates
released into the environment. Since the early sixties the risk assessment based solely on environmental levels
mankind has become aware of the potential long-term (Calabrese, 1991). Deleterious effects on populations are
adverse effects of these chemicals in general and their often difficult to detect in feral organisms since many of
potential risks for aquatic and terrestrial ecosystems in these effects tend to manifest only after longer periods of
particular. The ultimate sink for many of these con- time. When the effect finally becomes clear, the destruc-
taminants is the aquatic environment, either due to tive process may have gone beyond the point where it
direct discharges or to hydrologic and atmospheric can be reversed by remedial actions or risk reduction.
processes (Stegeman and Hahn, 1994). The presence of The sequential order of responses to pollutant stress
a xenobiotic compound in a segment of an aquatic within a biological system is visualized in Fig. 1
ecosystem does not, by itself, indicate injurious effects. (modified from Bayne et al., 1985). Such scenarios
Connections must be established between external levels have triggered the research to establish early-warning
of exposure, internal levels of tissue contamination and signals, or biomarkers, reflecting the adverse biological
early adverse effects. Many of the hydrophobic organic responses towards anthropogenic environmental toxins
compounds and their metabolites, which contaminate (Bucheli and Fent, 1995). Biomarkers are measurements
aquatic ecosystems, have yet to be identified and their in body fluids, cells or tissues indicating biochemical or
impact on aquatic life has yet to be determined. There- cellular modifications due to the presence and magni-
fore, the exposure, fate and effects of chemical con- tude of toxicants, or of host response (NRC, 1987).
taminants or pollutants in the aquatic ecosystem have Effects at higher hierarchical levels are always preceded
been extensively studied by environmental toxicologists. by earlier changes in biological processes, allowing the
Ecological or environmental risk assessment (ERA) is development of early-warning biomarker signals of
defined as the procedure by which the likely or actual effects at later response levels (Bayne et al., 1985). In
adverse effects of pollutants and other anthropogenic an environmental context, biomarkers offer promise as
activities on ecosystems and their components are sensitive indicators demonstrating that toxicants have
estimated with a known degree of certainty using entered organisms, have been distributed between tis-
scientific methodologies (Depledge and Fossi, 1994). sues, and are eliciting a toxic effect at critical targets
ERA has become increasingly important since environ- (McCarthy and Shugart, 1990). In this respect, it is also
mental scientists as well as the general public have interesting to study the development and application of
learned that chemicals which are not toxic to humans sensitive laboratory bioassays, based upon the responses
can have deleterious effects on natural resources which of biomarkers, such as the CYP1A responses in EROD
are generally valued, e.g. DDTs jeopardizing predatory or CALUX assays with hepatoma cell lines (Sawyer and
birds and fish, death of fish and other aquatic organisms
due to acid deposition in poorly buffered lakes which
also contributes to the die-back of forests (Bascietto et
al., 1990). The risk assessment process can be divided
into a scientifically oriented risk analysis and a more
politically oriented risk management . Risk analysis is a
process, which comprises some or all of the following
elements: hazard identification, effect assessment, ex-
posure assessment and risk characterization (Van Leeu-
wen and Hermens, 1995). Environmental risk
management deals with regulatory measures based on
risk assessment (Van Leeuwen and Hermens, 1995).
Risk management and risk analysis, are closely related
but different processes: in risk analysis the risk of a Fig. 1. Schematic representation of the sequential order of responses
certain situation is determined, whereas risk manage- to pollutant stress within a biological system. Modified from Bayne et
ment examines solutions to the problem. Although ERA al. (1985).
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 59

Safe, 1982; Murk et al., 1996). Bioassays offer many biochemical parameters) that have been used in order to
advantages for comparing the relative toxicity of specific assess exposure to and effects of environmental con-
chemicals or specific effluents. However, toxicity tests taminants. Emphasis will be placed on the biochemical
also have serious limitations for biological monitoring responses to organic trace pollutants, and the feasibility
(BM) because most do not account for the effect of of these markers as early-warning signals for environ-
chemical specification in the environment, kinetics and mental hazards. An extensive summary with the overall
sorption of chemicals to sediment, accumulation conclusions of this review will be presented in Section 7,
through food chains and modes of toxic action which and the perspectives will be given in the final Section 8.
are not readily measured as short-term effects The scope of this review will be to give an overview of
(McCarthy and Shugart, 1990). Depledge and Fossi fish bioaccumulation and biomarker studies and to
(1994) suggested the use of biomarkers in toxicity tests discuss the advantages and limitations of applying these
as an attempt to link biomarker responses to effects on parameters in the assessment of environmental risks in
life-history characteristics (e.g. survival and reproduc- aquatic ecosystems. At present, ERA processes are
tion), which will provide a further foundation for the use mainly based upon the determination and prediction
of biomarkers in environmental assessment. of contaminant levels in the various ecosystem compart-
For several reasons, fish species have attracted con- ments, and upon comparison of these concentrations
siderable interest in studies assessing biological and with legislative threshold values or environmental safety
biochemical responses to environmental contaminants standards. However, there is a growing awareness that
(Powers, 1989). Monitoring species should be selected focusing on chemical data alone is insufficient to reliably
from an exposed community on the basis of their assess the potential risks of the complex mixture of
relationship to the assessment endpoint as well as by contaminants in the aquatic environment. There is an
following some practical considerations (Suter, 1993). increasing trend to use the behavior of pollutants
For the assessment of the quality of aquatic ecosystems, (bioavailability, bioaccumulation, and biotransforma-
both criteria are met for numerous species of fish. Fish tion) as well as pollution-induced biological and bio-
can be found virtually everywhere in the aquatic chemical effects on aquatic organisms to evaluate or
environment and they play a major ecological role in predict the impact of chemicals on aquatic ecosystems.
the aquatic food-webs because of their function as a Emphasis in this review will, therefore, be placed on the
carrier of energy from lower to higher trophic levels use of bioaccumulation and biomarker responses in fish
(Beyer, 1996). The understanding of toxicant uptake, as monitoring tools for the assessment of the risks and
behavior and responses in fish may, therefore, have a hazards of environmental pollutants for the aquatic
high ecological relevance. Most of the general biomar- ecosystem, as well as on its limitations.
ker criteria appear to be directly transferable to certain
fish biomarkers (Stegeman et al., 1992). Between
different fish species, however, considerable variation 2. Biomarkers
in both the basic physiological features and the respon-
siveness of certain biomarkers towards environmental Several definitions have been given for the term
pollution may become apparent. Despite their limita- ‘biomarker’, which is generally used in a broad sense
tions, such as a relatively high mobility, fish are to include almost any measurement reflecting an inter-
generally considered to be the most feasible organisms action between a biological system and a potential
for pollution monitoring in aquatic systems. hazard, which may be chemical, physical or biological
In Section 2, general information on different types of (WHO, 1993). A biomarker is defined as a change in a
biomarkers will be discussed, together with criteria and biological response (ranging from molecular through
properties for valid biomarkers in environmental field cellular and physiological responses to behavioral
research. The process of ERA will be elucidated in changes) which can be related to exposure to or toxic
Section 3. The monitoring of aquatic pollution, which is effects of environmental chemicals (Peakall, 1994). Van
the most important aspect of the ERA process with Gastel and Van Brummelen (1994) redefined the terms
respect to biomarkers, will be discussed in Section 4. The ‘biomarker’, ‘bioindicator’ and ‘ecological indicator’,
processes governing the bioaccumulation of organic linking them to different levels of biological organiza-
trace pollutants in fish, as well as the use of fish as a tion. They considered a biomarker as any biological
bioconcentrator to identify certain substances with low response to an environmental chemical at the sub-
water levels will be reviewed in Section 5. Biota- individual level, measured inside an organism or in its
sediment accumulation data will be presented and products (urine, faeces, hair, feathers, etc.), indicating a
discussed with regard to the potential use of fish body deviation from the normal status that cannot be
burdens in the assessment of exposure to various groups detected in the intact organism. A bioindicator is
of organic pollutants. In Section 6 an overview will be defined as an organism giving information on the
presented of virtually all fish biomarkers (biological and environmental conditions of its habitat by its presence
60 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

or absence or by its behavior, and an ecological tion, so biochemical responses may be similar in a large
indicator is an ecosystem parameter, describing the variety of organisms. Good biomarkers are sensitive
structure and functioning of ecosystems. indices of both pollutant bioavailability and early
According to the NRC (1987), WHO (1993), biomar- biological responses. Biomarkers may be used after
kers can be subdivided into three classes: exposure to dietary, environmental or occupational
sources, to elucidate cause #/effect and dose#/effect
. biomarkers of exposure: covering the detection and
relationships in health risk assessment, in clinical
measurement of an exogenous substance or its
diagnoses and for monitoring purposes. Generally,
metabolite or the product of an interaction between
biomarker responses are considered to be intermediates
a xenobiotic agent and some target molecule or cell
between pollutant sources and higher-level effects (Suter,
that is measured in a compartment within an organ-
ism; 1990). When these compensatory responses are acti-
. biomarkers of effect: including measurable biochem- vated, the survival potential of the organism may already
ical, physiological or other alterations within tissues have begun to decline because the ability of the organism
or body fluids of an organism that can be recognized to mount compensatory responses to new environmental
as associated with an established or possible health challenges may have been compromised (Depledge and
impairment or disease; Fossi, 1994). The most compelling reason for using
. biomarkers of susceptibility: indicating the inherent biomarkers is that they can give information on the
or acquired ability of an organism to respond to the biological effects of pollutants rather than a mere
challenge of exposure to a specific xenobiotic sub- quantification of their environmental levels. Biomarkers
stance, including genetic factors and changes in may provide insight into the potential mechanisms of
receptors which alter the susceptibility of an organ- contaminant effects. By screening multiple biomarker
ism to that exposure. responses, important information will be obtained about
organism toxicant exposure and stress. A pollutant stress
The subdivision of biomarkers in the literature is situation normally triggers a cascade of biological
rather diffuse since biomarkers of exposure and those of responses, each of which may, in theory, serve as a
effect are distinguished by the way they are used, not by biomarker (McCarthy et al., 1991). Above a certain
an inherent dichotomy (Suter, 1993). The responses of threshold (in pollutant dose or exposure time) the
biomarkers can be regarded as biological or biochemical
pollutant-responsive biomarker signals deviate from
effects after a certain toxicant exposure , which makes
the normal range in an unstressed situation, finally
them theoretically useful as indicators of both exposure
leading to the manifestation of a multiple effect situation
and effects . Biomarkers of exposure can be used to
at higher hierarchical levels of biological organization
confirm and assess the exposure of individuals or
(Fig. 2, modified from McCarthy et al., 1991). Improper
populations to a particular substance (group), providing
application or interpretation of biomarker responses,
a link between external exposure and internal dosimetry.
however, may lead to false conclusions as to pollutant
Biomarkers of effect can be used to document either
stress or environmental quality. Certain responses
preclinical alterations or adverse health effects due to
external exposure and absorption of a chemical. Bio- established for one species are not necessarily valid for
markers of susceptibility help to elucidate variations in other species. Moreover, ecotoxicological data obtained
the degree of responses to toxicant exposure observed in laboratory studies can be difficult to translate into
between different individuals. The bioaccumulation of accurate predictions of effects that may occur in the field
certain persistent environmental contaminants in animal (ECETOC, 1993). Since both overestimation and under-
tissues may be considered to be a biomarker of exposure estimation of effects may occur, laboratory observations
to these chemicals (NRC, 1987; WHO, 1993). According on biomarkers must always be validated with field
to the definitions given by Van Gastel and Van research. Biomarkers applied in both the laboratory
Brummelen (1994), however, body burdens are not and the field, can provide an important linkage between
considered to be biomarkers or bioindicators since laboratory toxicity and field assessment. For field
they do not provide information on deviations related samples, biomarker data may provide an important
to ‘health’. In order to avoid confusion, in this review index of the total external load that is biologically
the analytical #/chemical indicators (body burdens) will available in the ‘real world’ exposure.
be referred to as bioaccumulation markers, while all In order to evaluate the strength and weaknesses of
biological (biochemical, physiological, histological and fish biomarkers objectively, we propose six criteria
morphological) indicators measured inside an organism comprising the most important information that should
or its products will be referred to as biomarkers. be available or has to be established for each candidate
In general, phenomena are more universal on a biomarker (based upon the criteria formulated by
cellular level than at higher levels of biological organiza- Stegeman et al., 1992):
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 61

Fig. 2. The principal scheme of responses in organisms to the detrimental effects of pollutant exposure. Modified from McCarthy et al. (1991).

. the assay to quantify the biomarker should be reliable understanding of biomarker responses and gradually
(with quality assurance (QA)), relatively cheap and expand that understanding. In time the biomarkers will
easy to perform; thus become a routine, well-characterized and scientifi-
. the biomarker response should be sensitive to pollu- cally and legally defensible tool for monitoring and
tant exposure and/or effects in order to serve as an assessing environmental pollution. Based on the magni-
early warning parameter; tude and pattern of the biomarker responses, the
. baseline data of the biomarker should be well defined environmental species offer the potential of serving as
in order to distinguish between natural variability sentinels demonstrating the presence of bioavailable
(noise) and contaminant-induced stress (signal); contaminants and the extent of exposure, surrogates
. the impacts of confounding factors to the biomarker indicating potential human exposure and effects and
response should be well established; predictors of long-term effects on the health of popula-
. the underlying mechanism of the relationships be- tions or the integrity of the ecosystem (McCarthy and
tween biomarker response and pollutant exposure Shugart, 1990).
(dosage and time) should be established;
. the toxicological significance of the biomarker, e.g.
the relationships between its response and the (long-
term) impact to the organism, should be established. 3. Environmental or ecological risk assessment (ERA)
In addition to these criteria it has been suggested that
Risk assessment can be defined as the process of
biomarkers should preferentially be non-invasive or
assessing magnitudes and probabilities to the adverse
non-destructive, to allow or facilitate environmental
effects of human activities or natural catastrophes
monitoring of pollution effects in protected or endan-
(Suter, 1993). The development of risk assessment has
gered species (Fossi and Marsili, 1997). Responses of
been driven by the need to allocate scarce resources to
‘ideal’ biomarkers correlate with the health or fitness of
estimate human-related risks (Power and McCarty,
the organism. With regard to the test organism, its basic
1997). Risk assessment clearly separates the scientific
biology and physiology should be known so that sources process of estimating the magnitude and probability of
of uncontrolled variation (growth and development, effects (risk analysis) from the process of choosing
reproduction, food sources) can be minimized (Stege- among alternatives and determining acceptability of
man et al., 1992). risks (risk management). The entire risk assessment
Fish biomarkers may be useful tools in several steps process consists of eight steps, which are defined as
of the risk assessment process: effect, exposure and follows (Van Leeuwen and Hermens, 1995):
hazard assessment, risk characterization or classifica-
tion, and monitoring the environmental quality of . Hazard identification is the identification of the
aquatic ecosystems. The authors believe that phased adverse effects, which may be caused by chemicals.
increases in the extent and complexity of environmental The likelihood of harm due to exposure distinguishes
monitoring scenarios will test and confirm previous risk from hazard.
62 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

. Effect assessment is the estimation of the relationship ERA has been employed primarily to deal with
between dose or level of exposure to chemicals and chemicals. In the last decade much research was devoted
the incidence and severity of an effect. Most of the to ERA, mainly by international bodies like the World
experiments are carried out in order to determine a no Health Organization (WHO), the Organization for
effect level (NEL), which can be converted to a Economic Co-operation and Development (OECD)
predicted no effect level (PNEL) or a predicted no and the European Centre for Ecotoxicology and Tox-
effect concentration (PNEC) for other species. icology of Chemicals (ECETOC). The objective of risk-
. Exposure assessment is the estimation of concentra- based environmental regulation is to balance the degree
tions or doses to which human populations or of permitted risk against the cost of risk reduction and
environmental compartments are or may be exposed. against competing risks. Ecological risk assessment has
For existing chemicals exposure can be assessed by several advantageous properties in environmental deci-
measuring concentrations, while for new chemicals a sion-making (Suter, 1993). It provides a quantitative
predicted environmental concentration (PEC) can be basis for comparing and prioritizing risks as well as a
estimated. systematic means of improving the understanding of
. Risk characterization is an integration of the first risks. In addition, it estimates clear consistent endpoints.
three steps of the risk assessment process in order to Whilst Suter (1990) criticized the ambiguous endpoints
estimate the incidence and severity of the deleterious of other approaches (such as ‘ecosystem integrity’) as
effects likely to occur due to actual or predicted being too vague to be subject to formal quantitative
exposure to chemicals. For newly developed chemi- analysis, Cairns and McCormick (1992) successfully
cals the PEC/PNEC ratio, i.e. the risk quotient , can refuted the accusation by pointing out that most of
be determined. The risk quotient, combined with the components that are encompassed by the term (such
uncertainty factors, links the risk analysis to the risk as species diversity, population dynamics, nutrient
management by quantifying the hazards and risks for cycling rates, etc.) are indeed quantifiable and can be
specific situations (Duke and Taggart, 2000; Jager et used to measure ecosystem well-being. Ecological risk
al., 2001). assessors analyze the effects of human actions on the
. Risk classification is the evaluation of risks in order natural environment. Although risk assessment is widely
to decide if risk reduction is required. Generally, risk used, consensus on an acceptable, comprehensive deci-
classification is performed using two risk levels, in sion-making framework that clearly establishes the role
which the upper limit is the maximum permissible of policy and science in formulating environmental
level (MPL) and the lower limit is the negligible level management principles has not emerged, according to
(NL). Power and McCarty (1997). Power and McCarty (1998)
. Risk #/benefit analysis is the drawing up of a balance carried out a comparative analysis of seven representa-
sheet of the respective risks and benefits of a tive frameworks for ERA/risk management.
proposed risk-reduction action. Like other risk assessment processes, ERA is primar-
. Risk reduction is taking measures to protect man and/ ily concerned with predictive assessments (e.g. PEC and
or the environment from the risks identified. A risk PNEC) that estimate the nature, probability and mag-
reduction may be achieved by defining safety stan- nitude of effects of proposed actions (Suter, 1990).
dards , such as the acceptable daily intake (ADI). However, emphasis has been shifting to retrospective
. Monitoring is a repetitive observation for defined assessments , i.e. assessments of human actions that were
purposes of one or more chemical or biological initiated in the past and may have ongoing consequences
elements according to a prearranged schedule over in the future, such as waste sites, acid rain and existing
time and space, using comparable and standardized pesticides (Suter, 1993). Retrospective assessment falls
methods (see Section 4). Monitoring is essential in into three categories with respect to the direction of
several stages of the ERA process (De Zwart, 1995). inference (Fig. 3). Source-driven assessments begin with
During a problem formulation chemical and BM of an existing pollution source, such as a spill or an
ambient waters may indicate deviations from the effluent, and attempt to determine the nature of the
normal (alarm and trend function), thus triggering effects. Exposure-driven assessments are prompted by
problem recognition. During the risk analysis stage evidence of exposure without prior evidence of a source
chemical monitoring (CM) of receiving waters as well or effects. Effects-driven assessments begin with an
as selected effluents can help in exposure character- observed effect such as a declining animal population
ization, while BM may be used to predict ecological or a fishless lake, and attempt to determine a cause. In
effects (instrument function and early-warning). Dur- all cases, the logical link between sources and effects is
ing the risk management stage monitoring will help exposure (Suter, 1993). The retrospective assessment of
by verifying control strategy results and in checking hazards and risks of existing chemicals may be estab-
compliance (control function). lished by actual measurements of concentrations and
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 63

Fig. 3. The relationship among the components of the risk characterization stage of retrospective assessments based on the process of ecological
epidemiology, including their respective environmental monitoring methods. Based on Suter (1993), Henderson et al. (1989), De Zwart (1995).

effects in the field, using bioaccumulation and biomar- formal, usually quantitative, expression of the results of
kers. toxicity testing or monitoring of an indicator is a
Any risk assessment must have defined endpoints. An measurement endpoint, which is the toxicological or
assessment endpoint is a formal expression of the biological input to risk assessment. The most useful
environmental values to be protected (Suter, 1993). measurement endpoints for risk assessments are multi-
Defining an assessment endpoint involves two steps: dimensional descriptive models such as concentration #/
(1) identifying the valued attributes of the environment response functions, rather than a single number, such as
that are considered to be at risk, and (2) defining these provided by 50% lethal concentration (LC50) tests.
attributes in operational terms. Suter (1993) proposed
five criteria that any endpoint should satisfy:
4. Monitoring aquatic pollution
1) societal relevance (understood and valued by public
and decision-makers); Monitoring is a repetitive observation for defined
2) biological relevance (important to a higher level of purposes of one or more chemical or biological elements
the biological hierarchy); according to a prearranged schedule over time and
3) unambiguous operational definition; space, using comparable and standardized methods
4) accessibility to prediction and measurement; (according to the definition of the United Nations
5) susceptibility to the hazardous agent(s). Environmental Program (UNEP)). This last step in the
risk management process, which is most relevant with
Since assessment endpoints are often vaguely defined respect to biomarkers, may serve a number of purposes:
or undefined, it has not always been clear what should the control function to verify the effectiveness of risk
be measured in retrospective ERAs (Suter, 1990). reduction or to ensure that previously formulated
Population- and ecosystem-level measures are relevant standards are being met, the signal or alarm function
to assessment endpoints but, due to compensatory and to detect sudden adverse changes in the environment, the
adaptive mechanisms, they are often resistant to effects trend function to enable the prediction of future devel-
and are not diagnostic for pollutants impacts. Sub opments and the instrument function for the recognition
organism-level measures are potentially much more and clarification of underlying processes. It is important
diagnostic and sensitive to pollutants, but the relevance that environmental monitoring programs should only be
of a biochemical or histological response to population undertaken if the objectives clearly state what the data
and community level assessment endpoints is poorly are going to be used for (Peakall and Walker, 1994). The
defined. Organism-level measures are intermediate in five environmental monitoring methods which may be
relevance, sensitivity and diagnostic utility. The para- performed in order to assess risks of contaminants for
meters that are measured in a monitoring study or organisms and to classify the environmental quality of
toxicity test are generally referred to as indicators. The ecosystems are listed below (see also Fig. 3):
64 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

. chemical monitoring (CM): exposure assessment by III) Populations (bioindicators): At this level, effects are
measuring levels of a selected set of well-known manifested as changes in the genetic structure, the
contaminants in abiotic environmental compart- age structure or the abundance of a population.
ments; IV) Ecosystems (ecological indicators): At this level,
. bioaccumulation monitoring (BAM): exposure assess- changes in species composition, abundance and
ment by measuring contaminant levels in biota or diversity may be indicative of the effects of pollution
determining the critical dose at a critical site (bioac- on communities.
cumulation);
A proper understanding of the relationships between
. biological effect monitoring (BEM): exposure and
biomarker responses and survival, growth or reproduc-
effect assessment by determining the early adverse
tion is generally considered to be a prerequisite for the
alterations that are partly or fully reversible (bio- use of biomarkers in ERA (Van Gastel and Van
markers); Brummelen, 1994). It is more and more acknowledged
. health monitoring (HM): effect assessment by exam- that biomonitoring (in addition to CM) is necessary for
ining the occurrence of irreversible diseases or tissue a reliable ERA. In the following sections a wide range of
damage in organisms; bioaccumulation markers (Section 5) and biomarkers
. ecosystem monitoring (EM): assessment of the integ- (Section 6) will be discussed with regard to their
rity of an ecosystem by making an inventory of, for feasibility in ERA processes.
instance, species composition, density and diversity.

A regular, systematic use of living organisms to

evaluate changes in environmental or water quality, as 5. Fish bioaccumulation markers and ERA
in BAM, BEM, HM and EM, is called biological
monitoring (BM) or biomonitoring (De Zwart, 1995). Exposure assessment has to provide information on
The term ‘toxicity monitoring’ is used to describe steady-state concentrations of potentially toxic xenobio-
measurements on the direct biomolecular and physiolo- tics in a selected environmental compartment. Methods
gical responses of individual organisms towards toxi- for assessing exposure to a chemical fall into two
cants in an experimental setup, including bioassays and categories (WHO, 1993):
biological early-warning systems, e.g. in BEM and HM.
. measurement of levels of chemical agents and their
A so-called integrated monitoring program is a study
metabolites and/or derivatives in cells, tissues, body
consisting of coordinated monitoring activities compris-
fluids or excreta, i.e. BAM;
ing both chemical and biological measurements in a
. measurement of biological responses such as cytoge-
variety of environmental media or compartments. An
netic and reversible physiological changes in the
example of an effect-based integrated monitoring pro-
exposed individuals, i.e. BEM, which will be dis-
gram is the so-called Triad approach, consisting of cussed in Section 6.
analyses of chemical contamination (CM), assessment of
toxicity using bioassays (BEM) and determination of the Measurement of covalent adducts formed between
in faunal community structure (EM), which is described chemical agents and cellular macromolecules (protein,
in detail by Chapman (1990). Analyses of Triad data in DNA) or their excretion products have characteristics of
order to determine the pollution status can involve both categories. In evaluating exposure, a distinction is
comparisons of Ratio-to-Reference (RTR) values, rank- made between the external dose, defined as the amount
ing, multivariate analyses and Mantel’s test on disease of a chemical agent in environmental contact with the
clustering (Mantel, 1967). Van Gastel and Van Brum- organism as determined by CM, and the internal dose,
melen (1994) proposed a stepwise integrated ERA of which is the total amount of a chemical agent absorbed
chemicals, consisting of four different biomonitoring by the organism over a period of time as determined by
levels using biomarkers, bioassays, bioindicators and BAM. Bioaccumulation markers and biomarkers of
ecological indicators: exposure will reflect the distribution of the chemical or
its metabolites, respectively, throughout the organism
I) sub-organismal (biomarkers): At the level of bio- (Fig. 3). Theoretically, this distribution can be tracked
chemical and physiological processes, deviations through various biological levels (e.g. tissue, cell, etc.) to
from the normal situation (‘health’) can be mea- the ultimate target (WHO, 1993).
sured using biochemical techniques. When released into the environment substances will
II) Organisms (bioassays): Survival, growth and repro- be subject to transport and transformation processes.
duction of individuals are chosen as endpoints of These processes together with emission patterns, envir-
the classic laboratory ecotoxicity tests. onmental parameters and physicochemical properties of
the substances, will govern their distribution and con-
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 65

centration in environmental compartments such as

water, air, soil, sediment and biota (ECETOC, 1993).
Contaminant distributions over different trophic levels
in the food web may provide a means to determine
structure in aquatic communities (Russell et al., 1999).
Organic trace contaminants, such as PCBs, OCPs,
PAHs, PCDFs and polychlorinated dibenzo-p -dioxins
(PCDDs) are ubiquitously present in the aquatic envir-
onment. The partition behavior of these hydrophobic
chemicals in sediment, water and biota is mainly
determined by lipid and organic carbon contents; the
Fig. 4. Bioaccumulation model for aquatic organisms. KOC: sorption
more hydrophobic a compound, the greater the parti-
coefficient; BCF: bioconcentration factor; BSAF: biota-sediment
tioning to these phases (Meador et al., 1995). accumulation factor; BMF: biomagnification factor. C refers to a
concentration and k to a rate constant. The subscripts S, W, F, B,
EXC and MET refer to sediment, water, food, biota, excretion and
metabolism, respectively. The digestible sediment fraction is consid-
5.1. Bioaccumulation ered to be part of the food. Adapted from Van der Oost et al. (1996a).

Persistent hydrophobic chemicals may accumulate in 5.2. Bioconcentration

aquatic organisms through different mechanisms: via
the direct uptake from water by gills or skin (biocon- The bioconcentration factor (BCF) of a chemical is
centration), via uptake of suspended particles (ingestion) the ratio of its concentrations in the organism and in
and via the consumption of contaminated food (bio- water during steady state or equilibrium (Opperhuizen,
magnification). Even without detectable acute or 1991). For the partitioning of chemicals between water
chronic effects in standard ecotoxicity tests, bioaccumu- and the lipid phases of organisms, the steady state BCF
lation should be regarded as a hazard criterion in itself, is defined as:
since some effects may only be recognized in a later
phase of life, are multi-generation effects or manifest kW CB
BCF $ $ (2)
only in higher members of a food-web, e.g. impact of kB CW
PCBs on the hatching success of eggs (Tillitt et al.,
Uptake of chemicals in organisms from water prob-
1992). Bioaccumulation of chemicals in biota may be a
ably follows a passive diffusion mechanism analogous to
prerequisite for adverse effects on ecosystems (Franke et
that of oxygen uptake (Thomann and Connolly, 1984).
al., 1994).
The linear relationship demonstrated between HCB
Contaminant levels in biota are determined primarily
exposure and tissue residues in clams supported a
by the uptake and elimination kinetics, which are typical
bioenergetics-based model which indicated that de-
for both chemicals and organisms (Gobas et al., 1988).
creased oxygen levels in water result in a more rapid
A model of the processes governing bioaccumulation
uptake and an increased body burden of hydrophobic
(uptake and clearance) in aquatic organisms is presented
chemicals (Boese et al., 1988). Yang et al. (2000)
in Fig. 4. According to this model, the concentration of
demonstrated a significant correlation between the
a chemical in biota (CB) over time (t) can be expressed
uptake constant (kW) of organochlorine compounds
by:
and fish oxygen consumption, regardless of fish size
dCB and species. Moreover, they demonstrated a significant
$[kw Cw "kF CF ]%kB CB
dt relationship between the depuration rate constant (KB)
and fish oxygen uptake. The uptake rate depends on the
$[kw Cw "kF CF ]%[kEXC "kMET ]CB (1)
water concentrations, which will generally be higher for
Here, C refers to a concentration; k to a rate less hydrophobic compounds (Gobas et al., 1993). It has
constant; and the subscripts W, F, B, EXC and MET been suggested that the rate of uptake of hydrophobic
to water, food, biota, excretion and metabolism, respec- chemicals in fish increase with a higher lipid content of
tively. Uptake of organic pollutants in fish may be direct the biological membranes (Spacie and Hamelink, 1982).
via exchange with the water phase (kWCW) or indirect The time required reaching a steady state between the
via the consumption of contaminated food (kFCF) water and fish concentrations can be determined by
(Thomann, 1989). Although biotransformation of or- caging uncontaminated fish in polluted areas and
ganic trace pollutants (kMETCB) has been reported for measuring pollutant tissue levels after different exposure
fish, clearance mainly occurs by simple release from the times. For rainbow trout, the estimated equilibrium
(lipoid) gill membranes and via faecal excretion into the times ranged between 15 and 256 days for various PCB
surrounding water (kEXCCB) (Brown, 1994). congeners (Vigano et al., 1994) and between 56 and 275
66 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

days for persistent OCPs (Galassi et al., 1996). The BCF with higher KOW values. The shallow slopes of the
is usually derived from parameters assessed in uptake log BSAF/log KOW regression lines in the study of
and elimination experiments, and is presented as a single Connor indicated a high affinity of both fish tissue
value without statistical analyses. Bailer et al. (2000) and sediment organic matter (OM) for organic com-
presented a strategy for obtaining standard errors, a pounds. According to the equation of Connor (1984) the
measure of precision, confidence intervals and a statis- BSAF increases from 0.05 to 1.80 for chemicals with
tical test for the BCF, which they considered to be a log KOW values between 4 and 8. In response to Breck
biological endpoint of great ecotoxicological value. (1985), Connor combined the non-linear relationships
The fate of chemicals is largely determined by between BCF and KOW (Mackay, 1982) and between
sorption to suspended particulates and sediments (Loo- KOC and KOW (Karickhoff, 1984) and postulated that
nen et al., 1994a). Sorption depends on the character- the BSAF was 0.077, independent of the KOW. Van der
istics of both the sediments and the chemicals involved Kooij et al. (1991), however, assumed the BSAF (i.e. the
(see Section 5.4). If sorption of hydrophobic chemicals is BCF/KOC ratio) of chlorinated hydrocarbons to be
considered as a partitioning between water and the approximately 2 (independent of KOW) and postulated
organic fraction of sediment, then the equilibrium that steady-state BSAF values varied between 1 and 4.
sorption coefficient (KOC) can be expressed as: In various field studies, however, higher BSAF values
were observed, especially for compounds with log KOW
kW CS
KOC $ $ (3) values higher than 6 (e.g. Van der Oost et al., 1996a;
kS CW Leadly et al., 1998). Tracey and Hansen (1996) reviewed
data on PCB, PAH and OCP BSAFs from various
Here the subscripts S and W refer to sediment and
laboratory and field experiments with fish and inverte-
water, respectively. If the processes of bioconcentration
brates. The analyses revealed similar BSAF values for
and sorption on sediments (i.e. the upper part of the
various benthically-coupled species, both within and
model in Fig. 4) have both reached equilibrium, then
among different habitat groups, and indicated that the
Eqs. (2) and (3) can be combined to define the biota-
sum of total exposures from all routes is similar across
sediment accumulation factor (BSAF):
species. Belfroid et al. (1996) assumed that a competitive
CB BCF process between sediment and biomass for the hydro-
BSAF $ $ (4) phobic compound would result in an optimum accumu-
CS KOC
lation at a certain point, but that in most cases
Both the BCF for the partitioning of chemicals bioaccumulation based on bulk soil and sediment
between water and the lipid phases of organisms and concentrations is independent of the hydrophobicity
the KOC for the partitioning between water and the (KOW) of the compound.
organic fraction of the sediment depend upon the
hydrophobicity of the chemicals (Connor, 1984). Var-
ious relationships have been derived between the log- 5.3. Biomagnification
transformed values of the 1-octanol/water partition
coefficient (log KOW) and the BCF of organic chemicals. Biomagnification is the ratio between the uptake of
Devillers et al. (1996) compared seven linear and non- the chemicals from food and their clearance (Sijm et al.,
linear BCF models with a dataset of 436 experimental 1992). During steady state, the biomagnification factor
BCF values recorded for 227 chemicals, in order to (BMF) can be defined as:
estimate their accuracy. For chemicals with a kF EF
log KOW B/6 all models yielded equivalent results, but BMF $ $FF (5)
kB kB
for the highly hydrophobic chemicals (log KOW !/6) a
bilinear model proved to be superior to the other models Here, FF refers to the amount of food transported
considered. Hawker and Connel (1985) derived an through the intestines per gram of fish per day and EF to
equation that related the KOW to the time needed to the efficiency of uptake of the chemical from food. Since
approach equilibrium between water and biota levels. bioaccumulation of persistent and extremely hydropho-
This allowed them to predict that only compounds with bic compounds cannot always be explained satisfactorily
log KOW B/6 attain equilibrium within 1 year. Further- by simple partitioning processes between sediment,
more, Thomann (1989) demonstrated that the excretion water and fish (Van der Oost et al., 1988; Thomann,
rate of hydrophobic chemicals from aquatic organisms 1989), it is likely that the uptake via contaminated food
was inversely related to the log KOW of the compounds. (biomagnification) contributes significantly to the bioac-
These studies are indicative of the relevance of the KOW cumulation of these contaminants in fish. If a primary
to assess the bioaccumulation potential. Among others, source of chemical input to an aquatic ecosystem is slow
Connor (1984) demonstrated a tendency toward higher release from polluted sediments then it is plausible that
BSAF values for chlorinated aromatic hydrocarbons uptake by benthic organisms followed by predation by
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 67

larger organisms such as fish may be a significant source relative gill ventilation volumes decrease with size while
of bioaccumulation (Farrington, 1991). Thomann relative feeding rates are almost equal (Opperhuizen,
(1989) demonstrated, by comparing predicted levels of 1991). When biomagnification is an important uptake
a food chain bioaccumulation model with field observa- route, individual and site-specific variations in bioaccu-
tions, that biomagnification was not significant for mulation patterns may, at least partly, be due to
chemicals with log KOW values up to approximately 5. differences in the fish diet (Van der Oost et al., 1996a).
For chemicals with log KOW values between 5 and 7, the This hypothesis may be investigated by determining the
observed bioaccumulation factors (BAFs) in feral fish levels of d15N in the contents of the gastrointestinal tract
indicated significant elevations (up to two orders of of fish, since this stable isotope is reported to be an
magnitude) above the calculated BCF values. LeBlanc integrative measure of trophic position, increasing with
(1995), however, demonstrated that significant biomag- higher trophic levels (Cabana and Rasmussen, 1994).
nification is only observed for chemicals with
log KOW !/6.3. He suggested that trophic-level differ- 5.4. Bioavailability
ences in bioaccumulation might be due higher BCFs as a
consequence of decreased chemical elimination efficien- When measuring bioaccumulation behavior the bioa-
cies of organisms occupying increasing trophic levels. vailability of the substance considered is a crucial
Russell et al. (1999) provided clear evidence for bio- parameter for valid results (Franke et al., 1994). In a
magnification of chemicals with log KOW values greater review by Belfroid et al. (1996), the bioavailability was
than 6.3, some evidence for biomagnification of chemi- defined as the fraction of the bulk amount of the
cals with log KOW values between 5.5 and 6.3, and no chemical present in soil/sediment and (interstitial) water
evidence for biomagnification for chemicals with log - that can potentially be taken up during the organism’s
KOW values less than 5.5. Fisk et al. (1998) demonstrated lifetime into the organism’s tissues (excluding the
that the persistent organochlorines with a log KOW of digestive tract). When the concentration in fish is not
approximately 7 have the greatest potential for food related to the real bioavailable concentration in the
chain accumulation in fish. water, this might result in underestimation of the
The mechanism of biomagnification and food chain bioconcentration potential (Kristensen and Tyle,
accumulation of organic chemicals can be explained 1991). Deviations in BSAF values predicted with the
with a fugacity-based hypothesis (Gobas et al., 1988), partitioning models may thus partly be due to differ-
which has been validated by experimental findings ences in bioavailability of the chemicals, possibly
(Gobas et al., 1993). Fugacity is equivalent to chemical resulting in a pronounced site-specific variation in
activity or chemical potential as it pertains to the bioaccumulation profiles of certain contaminants (Van
tendency of a chemical to escape from a phase, such der Oost et al., 1996a).
as water or food (Clark et al., 1988). A difference in Sediment characteristics such as particle size and OM
fugacity provides a driving force for net passive content may be important factors in determining the
chemical transport from high to low fugacity phases. bioavailability of hydrophobic chemicals. Conflicting
Food digestion in the gastrointestinal tract was found to results have been reported on the influence of particle
increase the chemical fugacity in the food 4#/5-fold by size on bioavailability (Belfroid et al., 1996). Usually,
altering the fugacity capacity, while an additional 2#/3- organisms preferentially ingest the smaller sediment
fold increase in the chemical concentration and fugacity particles (enriched in OM), which results in an increased
is caused by a reduction of the food volume due to contaminant uptake. Normalization of the contaminant
absorption from the gastrointestinal tract (Gobas et al., concentrations to OM or OC only partially accounts for
1993). Food digestibility and absorption were found to this increased uptake. OM is the main determinant of
be critical factors controlling BMFs and dietary uptake sorption of hydrophobic compounds to soils and sedi-
efficiencies under laboratory and field conditions (Go- ments (Belfroid et al., 1996). Therefore, the bioavail-
bas et al., 1999). Based upon diet information, bioener- ability of these chemicals generally decreases with
getics modeling and determinations of PCBs in both increasing soil or sediment OM contents (Landrum
predator and prey fish, Madenjian et al. (1998a,b) and Faust, 1991). These differences are taken into
estimated that the lake trout and the coho salmon in consideration when bioaccumulation is studied with
Lake Michigan, USA, retained respectively, 80 and 50% the OM normalized BSAF value, which is also referred
of the PCBs that are contained within their food. to as the bioavailability index (Rifkin and LaKind,
As a result of biomagnification, experimentally de- 1991). However, since OM cannot be considered con-
rived BCF or BSAF values of very hydrophobic stant, normalizing sorption constants to OM content
substances may deviate significantly from those pre- may be less accurate than generally assumed. The OM
dicted with partitioning models (Thomann, 1989; Van composition may have an additional influence on
der Oost et al., 1996a). Biomagnification may be more sorption characteristics (Belfroid et al., 1996). It was
important for larger fish than for smaller fish, since suggested that not only the sediment OM content should
68 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

be taken into account, but that the sedimentary soot compound, which may be either beneficial or harmful
phase also has a significant impact on bioavailability of to the organism. In case of a detoxication reaction the
organic trace pollutants like PAHs (Gustafsson et al., toxicity of the compound is reduced while the excretion
1997). Environmental behavior of PAHs could be is generally elevated. In case of bioactivation, however,
quantitatively explained using the soot #/carbon-normal- the compound is transformed into a reactive metabolite,
ized partition coefficient. Belfroid et al. (1995) indicated which is more toxic than the parent compound. The
that the sorption and bioavailability of a chemical were biotransformation process may thus be important in
also affected by the residence time in soil and sediments, determining the activity of a compound, the duration of
also referred to as aging. This generally means that with that activity and the half-life of the compound in the
a longer residence time in sediments the bioavailability body (Vermeulen, 1996).
of certain compounds, e.g. PAHs, decreases due to a An increased clearance of contaminants through
decreased desorption rate (White et al., 1999). For effective biotransformation (metabolism) may cause
several other factors, such as clay content, moisture severe deviations in BSAF values as predicted with
content and presence of oil or metals, influences on partitioning models. However, when uptake rates are
sorption and bioavailability are known to be expected significantly higher than metabolic clearance rates
(Belfroid et al., 1996). bioaccumulation can still occur even though the sub-
It has been demonstrated that the bioaccumulation of stance is readily biodegradable (Franke et al., 1994).
PCBs and chlorobenzenes (Schrap and Opperhuizen, Pollutant concentrations in tissues and differences in
1990), pesticides (Muir et al., 1994), polychlorinated excretion of metabolites can be a function of tissues and
dioxins and dibenzofurans (Loonen et al., 1994a) and conditions controlling the activity of biotransformation
PAHs (Haitzer et al., 1999) can be affected by the enzymes (Farrington, 1991). These conditions include
presence of particles in the aquatic phase, such as spawning, nutritional status, conditions and duration of
sediment, humic acids and other dissolved organic exposure to organic pollutants and life cycle stage of the
matter (DOM). In these studies it was suggested that a animal. Another important example involves the inter-
reduction of the uptake of hydrophobic chemicals was active effects of one chemical pollutant on another
caused by a reduced bioavailability of the compounds (Farrington, 1991). Simultaneous exposure of fish to
due to sorption on particles. On the one hand, it was PCBs and PAHs, for instance, significantly influences
demonstrated that distinct decreases in bioaccumulation the extent of uptake and metabolism of each (Stein et
of very hydrophobic contaminants due to DOM with a al., 1984). Biotransformation reactions of xenobiotic
high binding capacity occurred at environmentally organic chemicals in fish have been extensively reviewed
representative DOM concentrations (Haitzer et al., by Sijm and Opperhuizen (1989). Fish tissue levels of
1999). Sediment-bound chlorobenzenes, on the other chemicals that are easily biotransformed (e.g. low
hand, were bioavailable to benthic deposit feeders, chlorinated PCBs, PAHs, non-2,3,7,8-substituted
indicating that ingestion of sediment particles was a PCDD/Fs) are most likely not suitable as bioaccumula-
significant uptake route for these hydrophobic pollu- tion markers for exposure assessment, since their tissue
tants (Boese et al., 1990). Pollutant uptake via sediments levels do not reflect levels in the surrounding environ-
will only contribute significantly to the body burden of ment (Van der Oost et al., 1996a).
organisms which are able to digest (parts of) the
sediment (Opperhuizen, 1991). This uptake route, which 5.6. Bioaccumulation models
is probably of minor importance in fish, is represented in
the bioaccumulation model of Fig. 4 as a dotted arrow One of the most important steps in the risk assessment
between sediment and food. Digestible sediment is thus process is the determination of potential exposure.
considered as part of the food in this model. Typical exposure estimation involves combining pre-
dicted concentrations for target chemicals with certain
5.5. Biotransformation assumptions about the environmental fate of these
chemicals and the activity patterns of the receptors
An organism has two major ways of eliminating a (Valberg et al., 1996). Subsequently, the results of the
chemical: it is either excreted in its original form (the exposure assessment are combined with toxicity infor-
parent compound) or it is biotransformed by the mation to provide a quantitative estimate of risk.
organism. Biotransformation generally leads to the Although some linear correlations have been demon-
formation of a more hydrophilic compound which is strated between the octanol/water partition coefficient
more easily excreted than the parent compound (Ver- (log KOW) and the BCF (e.g. Mackay, 1982), their
meulen, 1996). The organ most commonly involved in relationship has been proved to be very poor for many
the biotransformation of foreign compounds is the liver, types of chemicals. It cannot be expected that the KOW
because of its function, position and blood supply. will be a good model of the bioaccumulation behavior of
Biotransformation may also alter the toxicity of a all organic chemicals in fish, because many factors
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 69

influencing accumulation are not taken into considera- . Van der Kooij et al. (1991) designed a partitioning
tion (Franke et al., 1994), including: model for toxic chemicals between water, organic
matter (OM) of sediments and particulate matter and
. phenomena of active transport; biota lipids. The BSAF was estimated to be approxi-
. the influence on the diffusion behavior through cell mately 2 for organic chemicals, independent of their
membranes; log KOW value.
. different rates of metabolism in various organisms . Sijm et al. (1992) proposed a life-cycle biomagnifica-
and the accumulation behavior of the metabolites; tion model for hydrophobic organic chemicals, using
. accumulation in specific organs and tissues (also by lipid content, growth, uptake and elimination ki-
adsorption to biological surfaces like gills and skin); netics, as well as reproduction and biotransforma-
. special structural properties (e.g. amphiphilic sub- tion. Most parameters were derived from long-term
stances, dissociating substances leading to multiple laboratory experiments with guppies.
equilibrium processes); . Thomann et al. (1992), Gobas (1993) both developed
. uptake and depuration kinetics, remaining level of rather similar models for predicting chemical residues
the substances or of metabolites after depuration. in aquatic food webs, which have gained general
scientific acceptance and are being used for both
In addition, it is important to consider the interactive
scientific and regulatory applications. Both models
effects of one chemical pollutant on the accumulation
incorporate the cumulative results of research origi-
behavior of another (Farrington, 1991; Van der Oost et nating from the early 1980s on bioaccumulation
al., 1991a). Despite these limitations, it is generally processes in aquatic food webs. Both models contain
accepted that substances with log KOW values higher rate equations for estimation of steady-state condi-
than or equal to 3 have the potential to bioaccumulate tions but also treat some chemical distributions as
(Franke et al., 1994). The log KOW may be more suitable equilibrium partitioning. Burkhard (1998) compared
for priority ranking for testing and assessing dangerous both models and observed that the BAFs of the
substances than for predicting reliable BCFs for un- Gobas model were slightly better in agreement with
known new substances (Franke, 1996). measured BAFs (determined from Lake Ontario
The best-known and most applied model to predict data). The KOW and the sediment #/water column
body burdens of contaminants in aquatic organisms is chemical concentration quotient were the dominant
the equilibrium partitioning theory (EPT). In the EPT it sources of uncertainties for predicted BAFs by both
is assumed that the concentration of a chemical in the models. Both models can be used in ERA studies for
organism is solely determined by the concentration in assessing the risks of bioaccumulative chemicals to
the water phase and the lipid content of the species (e.g. aquatic organisms and to organisms that consume
Van der Kooij et al., 1991). In the past decades there has them, including wildlife and humans.
been substantial progress in all aspects of biogeochem- . Hendriks (1995b) developed a model to predict non-
ical research related to the issues of bioavailability and equilibrium concentrations of trace contaminants in
disposition of toxic organic chemicals: solubility, sorp- biota by relating the main non-steady state para-
tion, uptake, metabolism, retention, release and excre- meter, the outflow rate, to the KOW and the size of the
tion. Predictive equations have been derived or have species. This intermediately complex model allowed
evolved empirically that relate molecular structural estimations for fairly unknown substances and spe-
characteristics or properties to biogeochemical behavior. cies.
Some predictive models for the bioaccumulation of . Park and Erstfeld (1997) developed a kinetic model to
xenobiotic organic chemicals in fish are listed below: simulate bioaccumulation of toxic chemicals under
natural conditions, using sediment sorption/deso-
. Thomann and Connolly (1984) constructed a model rption, bioavailability, uptake and elimination ki-
for PCBs in the Lake Michigan food chain, using netics. Biomagnification was not taken into account.
growth, respiration or metabolic rate, assimilation The model was validated in the laboratory with
efficiency of food (biomagnification), and bioener- chlordane in goldfish.
getics. The model was validated in the field with . Luk and Brockway (1997) designed a bioenergetics-
alewife and lake trout. based pollutant accumulation model, including age-
. McCarty (1987) used the interrelationship between dependencies for diet composition and energy den-
KOW, BCF and toxicity, in combination with first- sities of prey and consumer. Sensitivity analysis
order, one-compartment assumptions, to estimate indicated metabolic and growth-related parameters
toxicant kinetic parameters. The proportional rela- to be most critical. The model was fitted to the Lake
tionship could be used to convert kinetics data from a Ontario water and fish PCB concentrations.
bioconcentration basis to a toxicity basis and vice . Morrison et al. (1997) developed a mathematical
versa. model that estimates chemical concentrations in
70 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

phytoplankton, zooplankton, filter-feeding and det- et al., 1998). A poor comparability, on the other hand,
rivorous benthic invertebrates and fish. A validation was observed between organochlorine levels in SPMDs
experiment illustrated that 95% of observed concen- and those in feral carp and sauger or feral and caged
trations in invertebrates and fish were within a factor channel catfish in the Mississippi river (USA), which was
2 of model-predicted concentrations. most probably due to the metabolism and depuration of
these compounds by fish (Ellis et al., 1995).
5.7. Bioaccumulation assessment using SPMDs Although SPMDs do not fully fall within the scope of
this review, they are briefly described here since they
Passive sampling via SPMDs, introduced by Huckins may be used as an alternative for fish in order to
et al. (1990), is a so-called biomimetic technique used to investigate certain aspects in ERA studies. SPMDs can
simulate body residues and target concentrations of be applied to investigate bioavailability of nonpolar
complex organic mixtures. SPMDs usually consist of compounds (Huckins et al., 1990; Petty et al., 1995;
low-density polyethylene (LDPE) lay-flat tubing, filled Sabuliunas et al., 1998) and temporal trends in the levels
with either natural lipids or the model lipid triolein of waterborne contaminants (Bergqvist et al., 1998;
(1,2,3-tri[cis -9-octadecenoyl]glycerol). Thus far, SPMDs McCarthy et al., 2000) and to evaluate the significance
have been used for sampling organic contaminants in of point and non-point contaminant sources (McCarthy
water, sediments, air and soil. A wide range of nonpolar et al., 2000). Integrated research with SPMD sampling
to moderately polar chemicals, including PAHs, PCBs, and bioassay testing of the extracts may be a valuable
OCPs, PCDDs, PCDFs, and chlorophenols #/anisoles#/ approach for the assessment of levels and effects of
veratroles has been sampled successfully with SPMDs bioavailable hydrophobic pollutants.
(Booij et al., 1998). Other recently developed passive
samplers are diffusive gradients in thin films (DGTs) for 5.8. Exposure assessment using contaminant
metal sampling (Zhang et al., 1998) and the solid phase bioaccumulation in fish
micro-extraction (SPME) fibres to estimate the bioac-
cumulation potential in effluents and surface waters (De Although many studies have been carried out to
Maagd, 2000; Verbruggen et al., 2000). All these investigate the accumulation of organic trace pollutants
methods are designed to mimic aquatic animals as in aquatic organisms, generally, no standardized meth-
bioconcentrators of low level environmental pollutants. ods were used. Since the levels of hydrophobic con-
In order to predict water concentrations of contami- taminants in the water phase are usually too low for
nants by levels accumulated in the SPMDs, more has to reliable quantification, it is difficult to study bioconcen-
be known about the uptake kinetics (Rantalainen et al., tration in the field. Compared with the water column,
2000). Uptake of chemicals depends upon their chemical sediment is a more appropriate environmental compart-
and physical properties (notably KOW) but also upon ment to be related to levels of pollutants in biota
temperature, turbulence, flow rate of the water column (Connor, 1984; Rifkin and LaKind, 1991). Sediment
and biofouling of the membrane surface. Booij et al. characteristics change slowly, which makes it easier to
(1998) described a method to estimate the uptake kinetics collect representative samples, which, for example, are
in both laboratory and in field situations by spiking the less dependent on seasonal variations. In addition,
SPMDs, prior to exposure, with a number of ‘perfor- sediments may serve as a storage compartment for
mance reference compounds’ that do not occur in the long-term release, reflecting the history of discharges
environment. The release rate of these compounds is a to an area (Connor, 1984). There are, however, some
measure for the exchange kinetics between the SPMD factors, such as sediment composition, bioturbation,
and water. A fairly good agreement was found between sediment erosion, patchiness, degradation processes, etc.
SPMD-derived water concentrations of organochlorine which have to be taken into account in designing the
compounds and measured analyte values of tangential- proper sampling strategy (Kelly et al., 1994). The
flow ultrafilter permeates (Ellis et al., 1995) values affinity of a non-polar compound for sediments depends
obtained by using a resin column water sampler primarily on its hydrophobicity. Since the sorption of
(Rantalainen et al., 1998). In several studies the uptake hydrophobic contaminants is, among other factors,
rates in SPMDs were compared with those of aquatic determined by the sediments’ OM content (Landrum
invertebrates and fish. Meadows et al. (1998) demon- and Faust, 1991), sediment pollutant levels normalized
strated that the uptake rate constants (kU) of PCBs on an OM basis provide more information on the
estimated in SPMDs were similar to those in brown trout, bioavailable fraction. Since the capacity of an organism
with kU values for SPMDs ranging from one to two times to accumulate hydrophobic contaminants is highly
those of the fish. The pattern of congener uptake in the dependent upon their tissue lipid weight (LW), biota
fish and SPMDs was also similar. PCB and PCDD/F levels should be expressed on a LW basis. For compara-
congener profiles obtained in SPMDs reflected those in tive research it is, therefore, important to study bioac-
feral longnose suckers in a Canadian river (Rantalainen cumulation using the LW/OM-based BSAFs. Despite
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 71

the standardized BSAF method, one should realize that due to the primitive analytical methods which were used
bioaccumulation in field experiments will always be at that time. For completeness, however, these values
affected by site-specific differences in bioavailability are still listed in the Table 1. If bioaccumulation is
(Van der Oost et al., 1996a). Sometimes the biota determined using the same dimensions, less variation is
suspended solid accumulation factor (BSSAF) is also observed in BSAF values. When the LW:OM-normal-
used to quantify bioaccumulation (e.g. Hendriks et al., ized data are considered, BSAF values are generally
1998; Burkhard and Lukasewycz, 2000). higher than those predicted by EPT (Van der Kooij et
Laboratory derived BCFs of 227 organic chemicals al., 1991), indicating that bioaccumulation cannot be
have recently been reviewed by Devillers et al. (1996). explained solely by partitioning. Since log KOW values
Laboratory testing of bioaccumulation with high water of most PCB congeners are higher that 5. It is most
concentrations may lead to low BCF values, conse- likely that biomagnification through trophic transfer is
quently signaling low risk. Since the BCF is being used the primary mechanism governing the accumulation of
for classifying and labeling dangerous substances and in these compounds in fish, as was confirmed by numerous
ERA, low BCFs due to high test concentrations may be field surveys (e.g. Van der Oost et al., 1988, 1996a;
misleading and underestimate the risk (Franke, 1996). Zaranko et al., 1997; Metcalfe and Metcalfe, 1997).
To explore the validity of laboratory derived bioaccu- There is a trend towards higher BSAF values for higher
mulation ratios to field conditions, Hendriks (1995a) chlorinated PCB congeners, which might be due to
collected values from experiments and compared these biotransformation of lower chlorinated congeners, an
with ratios observed in field surveys. LW-normalized elevated biomagnification of the higher chlorinated
concentrations of persistent organics in fish were about congeners, or both (Lake et al., 1995). It was demon-
twice as high as those expected from laboratory studies, strated in three-spined sticklebacks that higher chlori-
while LW-normalized concentrations of less persistent nated PCB congeners generally showed higher BMFs
organics in fish were more than 1000 times lower than than the lower chlorinated congeners (Vanbavel et al.,
expected (Hendriks, 1995a). 1996). Despite the fact that BSAF values are reported to
An extensive monitoring program in the Dutch rivers be independent of the KOW (Ankley et al., 1992; Belfroid
revealed that the largest contribution to the overall et al., 1996), a significant negative correlation between
organic microcontaminant burden in aquatic organisms the BSAFs of extremely hydrophobic PCBs (seven or
came from traditionally monitored chemicals, such as more Cl-substituted) and the log KOW was observed in
PCBs, PAHs and OCPs (Hendriks et al., 1998). An striped mullet and spotted sea trout (Maruya and Lee,
overview of bioaccumulation studies using sediment and 1998), which supports the hypothesis that the highly
fish levels of PCBs, OCPs, PAHs, PCDDs and PCDFs is chlorinated congeners are less efficiently transferred in
presented in Tables 1#/4. Comparison between different the food web due to restricted membrane permeability
studies is difficult because both fish and sediment levels (Kannan et al., 1998).
are expressed on the basis of various dimensions (dry Indications for biotransformation of lower chlori-
weight [DW], fresh weight [FW], lipid weight [LW] or nated PCB congeners in fish have been demonstrated in
organic matter or carbon [OM or OC]). Only a few several studies (De Boer et al., 1993; Elskus et al., 1994;
studies reported both LW-based fish tissue levels and Sijm et al., 1992; Brown, 1992; Sijm and Opperhuizen,
OM-based sediment levels. Bioaccumulation character- 1989). In field research, a comparison between PCB
istics of different analyte groups are discussed in the levels in fish and sediments may be used to assess
following paragraphs, emphasis being placed on the use selective depletion of certain PCB congeners, indicating
of pollutant tissue levels as bioaccumulation markers for a selective metabolic clearance of those compounds by
exposure assessment. It will be discussed below whether fish (De Boer et al., 1993). Strong indications for a
or not the BSAFs of the compounds are consistent with metabolic degradation of the non-ortho-substituted
the partitioning model of Van der Kooij et al. (1991), congeners CB 77 and 126 were accordingly observed in
which predicted LW:OM-normalized BSAF values for yellow eel, as opposed to observations in other fish
organic chemicals ranging from 1 to 4, independent of species, e.g. pike-perch, cod, sole, dab and flounder (De
their log KOW values. Boer et al., 1993). In a long-term elimination study in
which PCB-exposed eel were transferred to a relatively
5.8.1. Polychlorinated biphenyls (PCBs) clean lake, it was demonstrated that elimination half-
Biota-sediment accumulation ratios (BASFs) of dif- lives of tetra- and penta-CBs ranged from 340 to 1450
ferent PCB congeners in fish are listed in Table 1. The days, while for most hexa-, hepta- and octa-CBs no
BSAF values range from 0.1 for CB 77 in channel measurable elimination was observed at all (De Boer et
catfish (Gale et al., 1997) to 2286 for total PCB in lake al., 1994).
trout (Veith et al., 1977). The results of Veith et al. Although large variations in PCB BSAF values were
(1977), as well as other results of PCBs analyses reported observed between different species of fish and different
before 1985, however, should be regarded as unreliable sampling sites (Table 1), PCB tissue levels seem to be
 72
Table 1
Biota-sediment accumulation factors (BSAFs) of polychlorinated biphenyls (PCBs) in fish

Species Mean fish sediment concentration ratios of congeners Dimensions Reference

CB 28 CB 52 CB 77 CB 101 CB 105 CB 118 CB 126 CB 138 CB 153 CB 180 Sum PCB

Atlantic cod Gadus morhua 119 72 106 72 LW:DW Beyer et al., 1996
Barbel Barbus barbus 61 163 172 156 123 DW:DW Galassi et al., 1994
Bleak Alburnus alburnus 83 187 214 232 156 DW:DW Galassi et al., 1994
2 #/4 2 #/15 3 #/27 4 #/24 2 #/38 2 #/53 4 #/66

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
Brown bullhead Ameiurus nebulosus B1 DW:DW Leadly et al., 1998
Brown trout Salmo trutta 1000 LW:DW Mowrer et al., 1982
4 FW:FW Niimi and Oliver, 1989
Burbot Lota lota 958 LW:DW Mowrer et al., 1982
1243 LW:DW Veith et al., 1977
Channel catfish Ictalurus punctatus 0.1 #/0.6 0.9 #/3.6 1.0 0.1 #/0.4 LW:OM Gale et al., 1997
2 LW:OM Tracey and Hansen, 1996
Chub Leuciscus cephalus 49 128 135 154 105 DW:DW Galassi et al., 1994
Coho salmon Oncorhynchus kisutch 3 #/8 FW:FW Niimi and Oliver, 1989
Croacker Micropogonias undulatus 1 LW:OM Tracey and Hansen, 1996
Cyprinid Chondrostoma soetta 38 85 96 106 77 DW:DW Galassi et al., 1994
Eel Anguilla anguilla 2 3 3 4 8 7 LW:OM Van der Oost et al., 1988
13 #/16 LW:OM Van der Oost et al., 1991a
3 #/45 4 #/18 5 #/17 7 #/28 16 #/43 8 #/40 6 #/42 LW:OM Van der Oost et al., 1996a
1 7 7 8 12 22 9 LW:DM!! Hendriks, 1995a
2 14 12 8 18 32 19 LW:OM!! Hendriks et al., 1998
English sole Parophrys vetulus 7 #/14 FW:FW Stein et al., 1992
Fathead minnow Pimephales promelas 2 LW:OM Tracey and Hansen, 1996
Flounder Platichthys flesus 4 #/11 LW:OM Vethaak et al., 1996
Gudgeon Gobio gobio 292 LW:DW Mowrer et al., 1982
Killifish Fundulus heteroclitus 1 #/275 DW:DW Elskus and Stegeman, 1989
4 #/33 DW:DW Lake et al., 1995
Lake trout Salvelinus namaycush 2286 LW:DW Veith et al., 1977
16 FW:FW Niimi and Oliver, 1989
Medaka Oryzias latipes 4 LW:OM Tracey and Hansen, 1996
Perch Perca fluviatilis 65 170 182 175 132 DW:DW Galassi et al., 1994
55 LW:DW Brevik et al., 1996
542 LW:DW Mowrer et al., 1982
Pike Esox lucius 18 #/35 LW:OM Van der Oost et al., 1991a
6 33 LW:OM Järnberg et al., 1993
Rainbow trout Salmo gairdneri or 2 #/9 FW:FW Niimi and Oliver, 1989
Oncorhynchus mykiss
Roach 6 #/12 LW:OM Van der Oost et al., 1991a
Rutilus rutilus 5 #/37 LW:OM Van der Oost et al., 1994b
4 12 12 10 10 17 7 LW:OM!! Hendriks, 1995a
Rutilus pigus 34 72 78 72 61 DW:DW Galassi et al., 1994
Rutilus rubilio 42 96 110 97 79 DW:DW Galassi et al., 1994
Rock sole Lepidopsetta bilineata 5 #/12 FW:FW Stein et al., 1992
Rudd Scardinius erythrophthalmus 57 150 172 168 124 DW:DW Galassi et al., 1994
Scup Stenotomus chrysops 2 LW:OM Tracey and Hansen, 1996
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 73

indicative of the exposure of aquatic animals to these

chemicals and may thus be used as bioaccumulation

Tracey and Hansen, 1996

Tracey and Hansen, 1996

Tracey and Hansen, 1996
Tracey and Hansen, 1996

Tracey and Hansen, 1996

Maruya and Lee, 1998 markers for exposure assessment in ERA studies.

Maruya and Lee, 1998

Galassi et al., 1994 Devault et al. (1996) observed that trends in lake trout

Hellou et al., 1999

Elskus et al., 1994
Stein et al., 1992
PCB concentrations in Lakes Michigan and Superior
reasonably mimic those in the water column over the
Dimensions Reference

long term.

Symbols and abbreviations !. DW, dry weight; FW, fresh weight; LW, lipid weight; OM, organic matter or organic carbon. !!, suspended solids instead of sediments.
5.8.2. Organochlorine pesticides (OCPs)
DW:DW

DW:DW
LW:OM

LW:OM

LW:OM
LW:OM
LW:OM
LW:OM

LW:OM
FW:FW

FW:FW BSAFs of different OCP compounds in fish are listed

in Table 2. The BSAF values range from 0.02 for
chlordane in goldfish (Park and Erstfeld, 1997) to 88 for
CB 138 CB 153 CB 180 Sum PCB

HCB and p ,p?-DDT in eel (Van der Oost et al., 1996a)

61

3 #/10

8 #/40
1

3
3
4
1

and p ,p ?-DDE in spotted gar (Ford and Hill, 1991).

These differences, which are less dramatic than those
found for PCBs but still cover more than two orders of
79
1

magnitude, may be due partly to differences in the

dimensions on the basis of which biota and sediment
levels where expressed.
1 #/3
82

LW:OM-based BSAF values of HCHs, drins and

DDD in eel (Van der Oost et al., 1996a) and drins in
70

1 #/3

roach (Van der Oost et al., 1994b) were within the

ranges of the partitioning model of Van der Kooij et al.
(1991), indicating a distribution mainly governed by a
CB 118 CB 126

bioconcentration mechanism. The higher BSAFs found

for DDE, DDT and HCB in eel (Van der Oost et al.,
1996a) and for HCB, HCHs and DDTs in roach (Van
Mean fish sediment concentration ratios of congeners

2 #/7

der Oost et al., 1994b) indicated that both bioconcen-

tration and biomagnification mechanisms were involved
in the accumulation of these compounds. Indications for
CB 101 CB 105

a biomagnification of toxaphene and DDT congeners

were also found in various fish species from different
trophic levels (Ford and Hill, 1991), while laboratory
studies revealed that dry weight-based BSAF values for
36

HCB in fathead minnows ranged from 12 to 15

(Schuytema et al., 1990).
CB 28 CB 52 CB 77

Biotransformation has been reported to influence the

accumulation of several OCP compounds in fish (Sijm
and Opperhuizen, 1989). DDT can be partly metabo-
lized to DDE (Sijm and Opperhuizen, 1989; Streit,
1992), which is in line with the high DDE BSAFs found
in most species of fish (Table 2). High metabolic
clearance rates have been reported for HCH congeners
Sea trout (spotted) Cynoscion nebulosus

Summer flounder Paralichthys dentatus

in fish (Butte et al., 1991), although BSAF values found

Starry flounder Platichthys stellatus

for summed HCHs are generally rather high (Table 2).

White perch Morone americana

Epoxidation to heptachlor epoxides is found to be the

Stripet mullet Mugil cephalus

major biotransformation route of heptachlor (Fendick

Weakfish Cynoscion regalis
Spot Leiostomus xanthurus

Pleuronectes americanus

et al., 1990).
Sheatfish Silurus glanis

Although large variations in BSAF values were

Table 1 (Continued )

observed for OCPs in different species of fish and

Winter flounder

between different sampling sites (Table 2), most OCP

tissue levels seem to be indicative of the exposure of
Species

aquatic animals to these chemicals and may thus be used

as bioaccumulation markers for exposure assessment.
 74
Table 2
Biota-sediment accumulation factors (BSAFs) of organochlorine pesticldes (OCPs) in fish

Species Mean fish-sediment concentration ratios (range) of compounds: Dimensions! Reference

HCB Lindane Sum HCH Sum Sum hepta- Chlordane Toxaphene Mirex pp- pp- pp- sum- Sum-
drins chlor DDE DDD DDT DDT OCP

Barbel Barbus barbus 1 73 DW:DW Galassi et al.,

1994
Bleak Alburnus 0.3 78 DW:DW Galassi et al.,

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
alburnus 1994
Bowfin Amia calva 0.7 23 26 27 18 25 FW:FW Ford and Hill,
1991
Brown bullhead 2 6 #/26 DW:DW Leadly et al.,
Ameiurus 1998
nebulosus
Brown trout Salmo 0.2 2 4 FW:FW Niimi and Oli-
trutta ver, 1989
Carp cyprinus carpio 0.5 26 60 38 9 49 FW:FW Ford and Hill,
1991
Channel catfish Icta- 0.1 #/0.6 0.9 #/3.6 4 LW:OM Tracey and
lurus punctatus Hansen, 1996
Chub Leuciscus cepha- 0.4 49 DW:DW Galassi et al.,
lus 1994
Coho salmon Oncor- 0.2 1 #/6 3 #/7 FW:FW Niimi and Oli-
hynchus kisutch ver, 1989
Cottonmouth Agkis- 0.3 0.3 18 0.3 B1 11 FW:FW Ford and Hill,
trodon piscivorus 1991
Croacker Micropogo- 3 LW:OM Tracey and
nias undulatus Hansen, 1996
Cyprinid Chondrosto- 0.2 38 DW:DW Galassi et al.,
ma soetta 1994
Eel Anguilla 28 #/42 LW:OM Van der Oost et
anguilla al., 1991a
3 #/88 1 #/9 1 #/5 2 #/23 12 #/66 1 #/5 7 #/88 6 #/23 LW:OM Van der Oost et
al., 1991a
3 20 5 #/20 10 20 10 1 LW:OM!! Hendriks,
1995a
7 19 8 6 1 15 12 3 LW:OM!! Hendriks et al.,
1998
Fathead minnow Pi- 12 #/15 DW:DW Schuytema et
mephales promelas al., 1990
3 LW:OM Tracey and
Hansen, 1996
Goldfish Carassius 0.02 FW:FW Park and Erst-
auratus feld, 1997
Killifish Fundulus het- 1 #/140 Elskus and Ste-
eroclitus geman, 1989
Table 2 (Continued )

Species Mean fish-sediment concentration ratios (range) of compounds: Dimensions! Reference

HCB Lindane Sum HCH Sum Sum hepta- Chlordane Toxaphene Mirex pp- pp- pp- sum- Sum-
drins chlor DDE DDD DDT DDT OCP

Lake trout Salvelinus 0.8 13 15 FW:FW Burkhard and

namaycush Lukasewycz,
2000
Mosquitofish Gambu- 0.17 2 5 4 1 5 FW:FW Ford and Hill,

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
sia affinis 1991
Perch Perca fluviatilis 0.1 66 DW:DW Galassi et al.,
1994
1.5 1 0.1 0.1 0.2 LW:DW Brevik et al.,
1996
Pike Esox lucius 32 #/35 LW:OM Van der Oost et
al., 1991a
Rainbow trout Salmo 0.2 #/0.4 2 #/7 2 #/6 FW:FW Niimi and Oli-
gairdneri or Oncor- ver, 1989
hynchus mykiss
Roach Rutilus 14 #/19 LW:OM Van der Oost et
rutilus al., 1991a
15 #/22 16 #/18 1 #/2 18 #/32 LW:OM Van der Oost et
al., 1991b
3 5 3 #/7 10 20 9 1 LW:DW!! Hendriks,
1995a
Rutius pigus 0.3 31 DW:DW Galassi et al.,
1994
Rutilus rubilio 0.1 43 DW:DW Galassi et al.,
1994
Rudd Scardinius ery- 0.1 75 DW:DW Galassi et al.,
throphthalmus 1994
Scup Stenotomus chry- 3 LW:OC Tracey and
sops Hansen, 1996
Sheatfish Silurus glanis 0.2 29 DW:DW Galassi et al.,
1994
Smallmouth buffalo 0.7 46 65 46 36 59 Ford and Hill,
Ictiobus bubalus 1991
Spot Leiostomus 3 LW:OM Tracey and
xanthurus Hansen, 1996
Spotted gar Lepisos- 0.7 23 88 79 21 80 FW:FW Ford and Hill,
teus oculatus 1991
Water snakes Nerodia 0.3 3 10 1 B1 7 FW:FW Ford and Hill,
spp . 1991
Weakfish Cynoscion 5 LW:OM Tracey and
regalis Hansen, 1996
White perch Morone 3 LW:OM Tracey and
americana Hansen, 1996

Symbols and abbreviations !, DW, dry weight; FW, fresh weight; LW, lipid weight; OM, organic matter or organic carbon; !!, suspended solids instead of sediments.

75
 76
Table 3
Biota-sediment accumulation factors (BSAFs) of polycyclic aromatic hydrocarbons (PAHs) in fish

Species Mean fish-sediment concentration ratios (range) of compounds Dimensions! Reference

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
Fluorene Phenanthrene Fluoranthene Pyrene Chrysene Sum Sum Sum PAH Sum PAH Sum PAH Sum PAH
PAH 2!! PAH 3!! 4!! 5!! 6!!

Antarctic fish 0.24 #/1.25 DW:DW McDonald et

Notothenia al., 1995
gibberifrons
Brown bull- 2 #/10 2 #/6 2 #/5 1 #/2 1 #/6 FW:DW Baumann and
head Ictalurus Harshbarger,
nebulosus 1995
0.01 #/0.10 LW:OM Van der Oost et
al., 1991a
Eel Anguilla 0.24 #/2.9 0.09 #/1.6 0.01 #/0.4 0.003 #/0.06 0.02 #/0.20 0.04 #/0.56 LW:OM Van der Oost et
anguilla al., 1994a
Killifish Fun- 0.05 #/ 0.004 #/0.05 0.0005 #/0.001 0.0003 #/ 0.0001 #/0.001 0.001 #/0.012 DW:DW Elskus and Ste-
dulus heterocli- 0.36 0.001 geman, 1989
tus
Lake trout 0.00011 0.00016 0.00710 0.00033 LW:OM Burkhard and
Salvelinus na- Lukasewycz,
maycush 2000
Pike Esox 0.02 #/0.09 LW:OM Van der Oost et
lucius al., 1991a
Roach Rutilus 0.02 #/0.13 LW:OM Van der Oost et
rutilus al., 1991a
0.5 #/2.3 0.1 #/0.6 0.02 #/0.14 0.01 #/0.06 0.01 #/0.13 LW:OM Van der Oost et
al., 1994a
Sunfish 0.00001 #/0.8 LW:OM Thomann and
Lepomis Komlos, 1999
macrochirus

Symbols and abbreviations !; DW, dry weight; FW, fresh weight; LW, lipid weight; OM, organic matter or organic carbon; !!, number of aromatic rings.
Table 4
Biota-sediment accumulation factors (BSAFs) of polychlorinated dibenzodioxins and dibenzofurans (PCDD/Fs) in fish

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
Species Mean fish-sediment concentration ratios (range) of congeners Dimensio- Reference
ns!
2378- 12378- 123678- 1234678- OCDD Sum 2378- 12378- 123678- 1234678- OCDF Sum
TCDD PCDD HxCDD HpCDD PCDD TCDF PCDF HxCDF HpCDF PCDF

Carp cyprinus 0.270 0.060 0.035 0.005 0.060 0.037 0.003 LW:OW Kuehl et
carpio al., 1987
Channel 0.15 #/0.48 0.19 #/0.31 0.06 #/0.28 0.01 #/0.71 0.01 #/0.86 0.01 #/0.72 0.01 #/0.19 0.004 #/0.21 0.01 #/0.04 0.001 #/0.07 0.001 #/0.07 0.003 #/0.17 FW:OM Gale et al.,
catfish Icta- 1997
lurus
punctatus
Eel Anguilla 0.220 0.002 0.020 0.001 0.001 0.001 #/0.13 0.020 0.001 0.005 0.002 0.001 #/0.13 LW:OM Van der
anguilla Oost et al.,
1996a
Guppy Poeci- 0.155 0.080 0.024 0.014 0.003 0.014 0.002 0.021 0.016 LW:OM Loonen et
lia reticulata al., 1994a
Lake trout 0.41 0.33 0.06 0.002 0.0004 0.43 0.65 0.07 0.006 FW:FW Endicott
Salvelinus and Cook,
namaycush 1994
White sucker 0.49 LW:OM Muir et al.,
Catostomus 1992a,b
commersoni
Winter 1 #/1.5 FW:FW Hellou et
flounder al., 1999
Pleuronectes
americanus

Symbols and abbreviations !; DW, dry weight; FW, fresh weight; LW, lipid weight; OM, organic matter or organic carbon.

77
78 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

5.8.3. Polycyclic aromatic hydrocarbons (PAHs) more detail in Section 6.4 (biotransformation products)
The bioaccumulation of PAHs by various marine and Section 6.10 (genotoxic parameters), respectively.
organisms has been extensively reviewed by Meador et
al. (1995). BSAFs of different PAH compounds in fish 5.8.4. Polychlorinated dibenzo-p -dioxins and
are listed in Table 3. The BSAF values range from 0.01 dibenzofurans (PCDD/Fs)
for total PAH in eel (Van der Oost et al., 1991a) to 10 BSAFs of different PCDD and PCDF compounds in
for phenanthrene in brown bullhead (Baumann and fish are listed in Table 4. The BSAF values range from
Harshbarger, 1995). All LW:OM-based BSAF values 0.0004 for OCDD in lake trout (Endicott and Cook,
were much lower than the values predicted with the EPT 1994) to 0.86 for OCDD in channel catfish (Gale et al.,
model of Van der Kooij et al. (1991), indicating a 1997). Generally, BSAF values of PCDFs and PCDDs
reduced uptake or an increased clearance of these are about three orders of magnitude lower than those of
compounds. BSAF values of PAHs in sunfish declined PCBs and OCPs (Rifkin and LaKind, 1991; Loonen et
with increasing KOW, probably due to low gut assimila- al., 1994a; Van der Oost et al., 1996a), and generally
tion efficiency and increased metabolism (Thomann and lower than those predicted with the partitioning model
Komlos, 1999). Partitioning of combustion-derived of Van der Kooij et al. (1991). In view of the log KOW
PAHs between water and sediment may be much less values of the PCDF/Ds, which range from 6 to 8 (Sijm et
than predicted, possibly because associations with al., 1989b; Loonen et al., 1994b), it was anticipated that
particles are much stronger than expected (Meador et their partitioning between different phases of the aquatic
al., 1995; Lamoureux and Brownawell, 1999). De environment would be similar to that of the highly
Maagd (1996) demonstrated site-specific PAH sorption chlorinated PCB congeners. Several reasons have been
coefficients, even after normalization on organic carbon discussed in the literature to explain this apparent lack
content. of bioaccumulation, namely biotransformation, reduced
PAH congeners in the aquatic environment can be lipid solubility, reduced membrane transport and re-
transformed by chemical (photo) oxidation or biological duced bioavailability (Opperhuizen and Sijm, 1990;
transformations (Neff, 1985). PAH biotransformation Loonen et al., 1994a,b). In the bioaccumulation model
occurs in many aquatic organisms, but it is most of Fig. 4, this may be demonstrated by a reduced uptake
effective in the liver of fish. PAHs are easily metabolized or an increased metabolic clearance.
by the phase I enzymes of the mixed function oxygenase Uptake rate constants of PCDFs and PCDDs from
system (MFO) to more hydrophilic products like water to fish are comparable to those of other chlori-
phenols, dihydrodiols, quinones and epoxides (Sijm nated hydrocarbons (Opperhuizen and Sijm, 1990;
and Opperhuizen, 1989; Lech and Vodicnik, 1985). Loonen et al., 1994b). The lower uptake rates of the
Reported half-lifes of parental PAHs in rainbow trout hepta- and octachlorinated congeners may result from
range from 1 day for acenaphtylene to 9 days for reduced membrane permeability due to a larger effective
phenanthrene (Meador et al., 1995). Some of the PAHs cross-sectional diameter of the molecules (Opperhuizen
can be excreted directly as unconjugated polar metabo- and Sijm, 1990). Rifkin and LaKind (1991) reported
lites in bile (via the gallbladder), but most PAH will be that fish accumulate dioxins by ingestion (biomagnifica-
excreted after conjugation by phase II enzymes (Ver- tion), rather than by bioconcentration. In a study with
meulen et al., 1992). guppies it was determined that both the BMF and the
Since the elimination of PAHs is generally very uptake efficiencies of the PCDFs and PCDDs were low
efficient in fish, no bioaccumulation of these compounds compared with those found for other hydrophobic
has generally been demonstrated. PAH fish tissue levels compounds (Loonen et al., 1991). Muir et al. (1992a,b)
are, therefore, not indicative of the levels to which the demonstrated that PCDF/Ds in the water phase were
animals were exposed and cannot be used as bioaccu- not readily bioavailable, since they were almost entirely
mulation markers for exposure assessment. In order to associated with small-sized particles or with dissolved
assess the exposure of fish to PAHs it is more appro- organic carbon. A congener-specific reduction in bioa-
priate to determine PAH metabolite levels in bile or vailability (Loonen et al., 1994a) might explain the
tissue DNA adduct levels (Tuvikene, 1995; Meador et al. decreasing BSAF values with an increasing number of
1995). Metabolite levels in an organism or in its excreta chlorine substituents in both dibenzofurans and dioxins
are the result of a clear interaction of a chemical with the found in most studies (Table 4).
biological matrix and often reflect the induction of The lack of accumulation of non-2,3,7,8-substituted
enzymatic reactions. Metabolite levels could, therefore, PCDF/D congeners has been attributed to selective
be considered to be biomarkers. Similarly, the formation biotransformation (Sijm et al., 1989a, 1993). Endicott
of DNA adducts after exposure to mutagenic and and Cook (1994) suggested that the low fresh weight-
carcinogenic chemicals in the field must be considered based biota-sediment ratios of PCDFs and PCDDs in
as a biomarker (Van Gastel and Van Brummelen, 1994). feral lake trout were due to extensive metabolism of the
PAH metabolites and DNA adducts will be discussed in compounds. However, since the biological half-lives of
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 79

the PCDF/Ds in trout range from 46 to 105 days (Van meulen, 1996). The toxicity of a foreign compound may
den Berg et al., 1994), this explanation does not seem be affected by metabolism, which can be either bene-
satisfactory. The toxicokinetics and metabolism of ficial (detoxication) or harmful (bioactivation) to an
PCDDs and PCDFs, and their relevance for toxicity, organism. The various pathways of toxication and
have been extensively reviewed by Van den Berg et al. detoxification and the possible consequences of bio-
(1994). Despite the low BSAF values of PCDD and transformation of xenobiotics are illustrated in Fig. 5.
PCDF congeners, fish tissue levels seem to be exposure- Toxic effects may manifest themselves when the parent
related (Van der Oost et al., 1996a). However, since their compound or its metabolites bind to cellular macro-
extremely low BSAF values cannot be explained satis- molecules, which may ultimately lead to membrane
factorily, the PCDD/F fish tissue levels should not be disruption, cell damage and or genotoxic effects that
regarded as valid bioaccumulation markers for exposure subsequently can lead to development and progression
assessment. of diseases (e.g. cancer). Metabolism is, therefore, an
important determinant of the activity of a compound,
the duration of that activity and the half-life of the
6. Fish biomarkers and ERA compound in the body (Timbrell, 1991).
Xenobiotic chemicals may be biotransformed in the
It is virtually impossible to monitor all contaminants liver according to the simplified mechanism of route I in
of anthropogenic (predominantly halogenated hydro- Fig. 6, which can be subdivided into phases I, II and III.
carbons) and natural origin (heavy metals and most Phase I is a non-synthetic alteration (oxidation, reduc-
PAHs) which form a potential threat to the environ- tion or hydrolysis) of the original foreign molecule,
ment. In order to assess the overall quality of the aquatic which can then be conjugated in phase II and catabo-
environment, however, a more promising approach is to lized in phase III (Commandeur et al., 1995). The phase
examine biochemical responses reflecting the potential III type enzymes (e.g. peptidases, hydrolases and b-
of contaminants to impair physiological processes in the lyase) that catalyze the catabolism of conjugated meta-
exposed organisms (McCarthy and Shugart, 1990). bolites to form easily excretable products fall beyond the
BEM by determining early adverse alterations (partially scope of this review. Many environmental contaminants
or fully reversible) is often necessary for a reliable study (or their metabolites) have been shown to exert toxic
of bioavailable aquatic pollution. A biomarker can be effects related to oxidative stress (Winston and Di
defined as a biological response, which can be related to Giulio, 1991). Defence systems have evolved to combat
exposure to or toxic effects of environmental chemicals oxyradical formation, using antioxidant enzymes. Stu-
(Section 3). Biomarkers can be used to assess the health dies on the induction of these antioxidant enzymes by
status of organisms and to obtain early-warning signals contaminant-generated increases in oxyradicals have
of environmental risks (Payne et al., 1987). Since many often been inconclusive (Winston and Di Giulio, 1991).
of the biomarkers are short-term indicators of long-term It is possible to analyze the impact of toxic xenobio-
adverse effects, these data may permit intervention tics on fish with various types of exposure and effect
before irreversible detrimental effects become inevitable biomarkers, most of which are included in the overview
(McCarthy and Shugart, 1990). Various biochemical below. The respective sections in which the different
parameters in fish have been tested for their responses to biomarker groups are discussed in more detail are given
toxic substances and their potential use as biomarkers of in parenthesis.
exposure or effect. Biomarkers, which have been in-
vestigated most extensively, are enzymes involved in the . Biotransformation enzymes: Generally, the most sen-
detoxication of xenobiotics and their metabolites (bio- sitive effect biomarkers are alterations in levels and
transformation enzymes, antioxidant enzymes). In fish, activities of biotransformation enzymes. In fish, the
the liver is the organ most commonly involved in the activity of these enzymes may be induced or inhibited
detoxication of foreign compounds. This fact makes it upon exposure to xenobiotics (Bucheli and Fent,
the target organ upon which this Section of the review 1995). Enzyme induction is an increase in the amount
will be mainly focused. or activity of these enzymes, or both. A two-phase cyt
Biotransformation or metabolism can be defined as P450 induction was, for instance, observed in PCB-
an enzyme-catalyzed conversion of a xenobiotic com- exposed rainbow trout: the first phase induction
pound into a more water-soluble form, which can be consisted of activation of existing enzymes, while
excreted from the body more easily than the parent the second phase included de novo enzyme synthesis
compound (Lech and Vodicnik, 1985). Biotransforma- (Sijm and Opperhuizen, 1989). It is generally assumed
tion of xenobiotic chemicals often involves enzymes that that de novo protein synthesis is the most important
have a relatively low degree of substrate specificity when enzyme induction process (Stegeman and Hahn,
compared with enzymes involved in the metabolism of 1994). Inhibition is the opposite of induction. In
constitutive compounds (Melancon et al., 1992; Ver- this case, enzymatic activity is blocked, possibly due
80 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

to a strong binding or complex formation between . Stress proteins, metallothioneins and multixenobiotic
the enzyme and the inhibitors. Two major types of resistance (Section 6.5): The stress proteins (also
enzymes involved in xenobiotic biotransformation called heat-shock proteins , HSP) comprise a set of
are distinguished: abundant and inducible proteins involved in the
#/ Phase I enzymes (Section 6.1); protection and repair of the cell against stress and
#/ Phase II enzymes and cofactors (Section 6.2); harmful conditions (Sanders, 1993). Special groups of
. Oxidative stress parameters (Section 6.3): Many stress proteins are the metallothioneins (MTs), which
environmental contaminants (or their metabolites) are inducible by both essential and toxic heavy metals
have been shown to exert toxic effects related to (Stegeman et al., 1992; Viarengo et al., 2000), and the
oxidative stress (Winston and Di Giulio, 1991). P -glycoproteins of the multixenobiotic resistance
Oxygen toxicity is defined as injurious effects due to (MXR) mechanism, which may be induced or in-
cytotoxic reactive oxygen species (ROS), also referred hibited by a wide variety of chemicals (Bard, 2000).
to as reactive oxygen intermediates (ROIs), oxygen . Haematological parameters (Section 6.6): Several
haematological parameters in fish are potential effect
free radicals or oxyradicals (Di Giulio et al., 1989a).
biomarkers. The leakage of specific enzymes (e.g.
Of particular interest are the reduction products of
transaminases) into the blood may be indicative of
molecular oxygen which may react with critical
the disruption of cellular membranes in certain
cellular macromolecules, possibly leading to enzyme
organs (Moss et al., 1986). Although less specific,
inactivation, lipid peroxidation (LPO), DNA damage
other haematological parameters, like hematocrit,
and, ultimately, cell death (Winston and Di Giulio, hemoglobin, protein and glucose, may be sensitive
1991). The activities of the antioxidant enzymes, to certain types of pollutants as well. In addition, the
which defend the organisms against ROS, are criti- blood levels of specific steroid hormones or proteins
cally important in the detoxification of radicals to normally induced by these hormones may be indica-
non-reactive molecules. tive for certain reproductive effects due to endocrine
. Biotransformation products (Section 6.4): Another disruption (see Section 6.8).
type of biomarker is the elevation in levels of . Immunological parameters (Section 6.7): A large
biotransformation products, such as metabolite levels number of environmental chemicals have the poten-
in body fluids or the amount of covalent adducts tial to impair components of the immune system.
formed between metabolites of biodegradable chemi- Both antibody- and cell-mediated immunity may be
cals and cellular macromolecules (proteins, RNA, depressed by certain pollutants, as reviewed by Vos et
DNA) (Melancon et al., 1992). These biomarkers al. (1989). Although most research on this system has
may have the characteristics of both exposure and been performed on mammalian species, it may be
effect assessment categories. considered a promising field to search for new fish
biomarkers (Wester et al., 1994).
. Reproductive and endocrine parameters (Section 6.8):
The impact of xenobiotic compounds on reproduc-
tive and endocrine effects has attracted growing
interest in recent years. Since a decreased reproduc-
tive capability in feral fish may in the long run
threaten the survival of a large number of susceptible
species, these parameters certainly deserve thorough
examination. Hormone regulation may be impaired
as a consequence of exposure to environmental
pollutants (Spies et al., 1990).
. Neuromuscular parameters (Section 6.9): With respect
to neuromuscular functions, recent studies indicated
that the ‘old’ biomarker acetylcholinesterase
(ACHE), which is sensitive to organophosphate
(OP) and carbamate pesticides, may be responding
to low levels of contaminants in the environment
Fig. 5. Possible toxication and detoxification pathways of xenobiotic (Payne et al., 1996).
compounds: (1) direct toxic effect (A); (2) metabolic activation; (3) . Genotoxic parameters (Section 6.10): The exposure of
formation of a stable metabolite which may cause a toxic effect (C); (4) an organism to genotoxic chemicals may induce a
detoxification. The reactive metabolite formed by bioactivation (2)
may cause a toxic effect (B) through reaction with critical targets (5) or cascade of events (Shugart et al., 1992): formation of
be detoxified through reaction with a protective agent (6). Adapted structural alterations in DNA, procession of DNA
from Timbrell (1991), slightly modified. damage and subsequent expression in mutant gene
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 81

Fig. 6. Simplified presentation of the fate of xenobiotic compounds in the liver cell. Route I, a possible mechanism for detoxification or toxication,
and route II, a possible mechanism for enzyme induction. AhR, aryl hydrocarbon receptor; HSP90, 90 kDa heat shock protein; ARNT, Ah receptor
nuclear translocator; DREs, dioxin responsive elements; cyt P450s, cytochrome P450 isozymes; GSTs, glutathione S -transferases; UDPGTs, UDP-
glucuronyl transferases.

products, and diseases (e.g. cancer) resulting from the enzymes and cofactors (Tables 7 and 8), antioxidant
genetic damage. The detection and quantification of enzymes (Tables 9 and 10) and various other types of
various events in this sequence may be employed as commonly used biomarkers (Tables 11 and 12).
biomarkers of exposure and effects in organisms When laboratory experiments were carried out with
exposed to genotoxic substances in the environment. different pollutant doses, or after different exposure
. Physiological and morphological parameters (Section times, the most prominent of the reported effects are
6.11): The actual measurement of adverse effects or presented (Tables 5, 7, 9 and 11). The strongest observed
of the consequences of those effects may also be used effects are also presented for multiple-site field studies or
as biomarkers. Determination of adverse effects can field caging studies with different exposure times, i.e.
be performed histopathologically, by investigating generally those at the most polluted site and after the
lesions, alterations or tumour formation (neoplasms) longest exposure time, respectively (Tables 6, 8, 10 and
in fish tissues. 12). The relative frequencies of the responses in various
biological and biochemical parameters, as presented in
Since the main objective of this review is to examine Tables 5 #/12, are illustrated in the graphs of Figs. 7 #/10.
the use of fish biomarkers in classifying the water In these graphs, the percentages of negative or positive
quality for ERA, emphasis will be placed on the most pollutant-induced biomarker responses for all fish
susceptible parameters, which may be used as early- species, as reported in the literature considered for this
warning systems for pollutant stress. Some of the most review, are visualized for both laboratory and field
commonly measured biomarker responses in fish, i.e. the studies. In the following paragraphs, the function and
biotransformation enzymes and the products of bio- responses of the parameters presented in the Tables and
transformation, will therefore, be discussed in more Figures will be discussed, together with those of other
detail, while other potential biomarker responses will biological parameters (see overview).
only be discussed briefly in the following sections. The
discussion will focus on the feasibility of various 6.1. Phase I enzymes
parameters as biomarkers for ERA. An overview of
the literature on pollutant-induced responses in fish is The first phase of metabolism, unmasking or adding
presented in Tables 5 #/12. In these tables, a separation is reactive functional groups, involves oxidation, reduction
made between four groups of biomarker responses, each or hydrolysis (Goeptar et al., 1995). For the majority of
group being divided into laboratory and field studies: xenobiotic compounds the phase I reactions are cata-
phase I-related enzymes (Tables 5 and 6), phase II lyzed by microsomal monooxygenase (MO) enzymes,
82 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

also known as the mixed-function oxidase (MFO) complex then binds to aryl hydrocarbon nuclear trans-
system (i.e. cytochrome P450 [cyt P450], cytochrome ferase (ARNT, a.k.a. Ah receptor nuclear translocator)
b5 [cyt b5], and NADPH cytochrome P450 reductase and migrates to the cell nucleus. In the nucleus of the
[P450 RED]). Most oxidative phase I biotransforma- cell ARNT binds to a DNA recognition sequence
tions in fish are catalyzed by these cytochrome P450- upstream of the cyt P450 genes, also known as the
dependent MOs. Cytochromes P450, comprising a large xenobiotic regulatory element (XRE) or dioxin respon-
and still expanding family of heme proteins, are sive element (DRE). Transcription factors now have
membrane-bound proteins which predominantly are ready access to the promotor region of the CYP1A gene.
located in the endoplasmic reticulum of the liver (Stege- Consequently, messenger RNA (mRNA) synthesis is
man et al., 1992; Bucheli and Fent, 1995). To a lesser increased, resulting in elevated protein levels (Stegeman
extent, they have also been found in various other fish and Hahn, 1994).
organelles and tissues (Celander, 1993). The cyt P450 Since the MFO system is sensitive to certain environ-
reactions can be grouped according to the type of mental pollutants, its activity may serve as a biological
substrate and can be divided largely into the synthesis monitor for exposure to certain classes of xenobiotic
and degradation of endogenous substrates and the chemicals (Sijm and Opperhuizen, 1989; Bucheli and
metabolism of xenobiotic substrates (Stegeman et al., Fent, 1995). Induction of cyt P450 1A (CYP1A), the
1992; Goksøyr and Förlin, 1992). The biochemistry and best studied biomarker for environmental contamina-
molecular biology of MOs, including current perspec- tion in aquatic ecosystems, has been reviewed by
tives on forms, functions and regulation of cytochrome Goksøyr and Förlin (1992), Bucheli and Fent (1995).
P450 in aquatic species, have been extensively reviewed There is strong evidence that the content and activity of
by Stegeman and Hahn (1994). induced CYP1A in fish is related to levels of aromatic
The most important feature of the MFO system is its and polychlorinated aromatic hydrocarbons in the
ability to facilitate the excretion of certain compounds animals and the environment in a dose-dependent
by phase I metabolism, as it transforms lipophilic manner (Stegeman and Lech, 1991; Stegeman and
xenobiotics to more water-soluble compounds (Bucheli Hahn, 1994). In general, the structural features asso-
and Fent, 1995). Xenobiotic phase I biotransformation ciated with CYP1A induction in fish are similar to those
via the MO system follows a reaction cycle which can be in mammals (Stegeman and Hahn, 1994). Even for the
divided into several steps (Stegeman and Hahn, 1994; best-known groups of inducers (i.e. PAHs and HAHs),
Bucheli and Fent, 1995; Goeptar et al., 1995). In the first however, our understanding is not sufficient to identify
step, the substrate binds to prosthetic heme ferric iron the most important contributors to environmental
(Fe3") of the enzyme. Following substrate binding, the induction. Presumably, structure/activity relationships
iron is reduced by electron transfer from the flavopro- (SARs) for induction of P450s are at least partly a
tein NADPH cytochrome P450 reductase (P450 RED). reflection of receptor-binding SARs. These relationships
Subsequently, O2 is bound; a critical point at which have not been directly examined in aquatic species,
catalysis may proceed or be interrupted, resulting in although it was demonstrated that the correspondence
release of active oxygen (superoxide). The next steps between AhR presence and CYP1A inducibility in fish is
involve the addition of a second electron, usually via nearly perfect (Stegeman and Hahn, 1994).
cytochrome b5 (cyt b5), and the formation of a peroxide, An overview of the responses of phase I-related
followed by cleavage of the O !/O bond, the formation of enzymes to environmental pollutants in both laboratory
a substrate radical, the hydroxylation of that radical and and field studies is presented in Tables 5 and 6,
the release of the product. respectively. Since phase I responses in laboratory
Hydrophobic organic micropollutants may induce the studies were extensively reviewed in 1989 by Sijm and
activity of the xenobiotic metabolizing enzyme systems Opperhuizen, this review focused on the most recent
in many species, including fish (Kleinow et al., 1987). studies.
The mechanism of enzyme induction has been studied
most extensively for cyt P450 isozymes. Years ago, 6.1.1. Total cytochrome P450 (cyt P450)
Knudson and Poland (1982) proposed a model to Xenobiotic phase I biotransformation in fish is mainly
describe the mechanism by which TCDD and related mediated by the cyt P450-dependent MO or MFO
compounds regulate gene expression through the aro- system (Fig. 6, route I). Although most cytochrome
matic hydrocarbon receptor (AhR). An updated rendi- P450 proteins do not show any response to pollutants,
tion of this model (according to Bucheli and Fent, 1995; the strong and selective induction of some P450
Safe, 2001) is shown in Fig. 6 (route II). Enzyme isoenzymes may cause a significant elevation in total
induction is initiated by the binding of a specific cyt P450 levels. Generally, this response is less sensitive
xenobiotic to a protein complex that comprises the Ah than that in the levels or activities of selected isoenzymes
receptor and the heat-shock protein 90 (HSP 90), the (Bucheli and Fent, 1995). It was demonstrated that
latter being subsequently released. The Ah receptor single xenobiotic compounds can act as inducers of
Table 5
Laboratory studies on responses of organic trace pollutants on fish hepatic phase l-related enzymes

Species Pollutants cyt P450! CYP1A! AHH! EROD! cyt b5! P450 RED! Others!! Reference

Antarctic fish Notothenia gibberifrons PAH (BaP) "" McDonald et al., 1995
Arctic charr PAH (BaP) $ "" CND: "" Wolkers et al., 1996
Salvelinus alpinus T6bH: $
Atlantic cod Gadus morhua Crude oil (PAHs) "" Aas et al., 2000
Atlantic salmon PAH (BNF) "" Goksøyr et al., 1991a
Salmo salar PAH (BNF) " "" "" $ " ECOD: "" Goksøyr and Larsen, 1991

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
Crude oil " Gagnon and Holdway, 2000
PAHs "" "" mRNA: "" Stagg et al., 2000
Atlantic tomcod Microgadus tomcod PAH (BNF, BaP), PCB 77 or mRNA: "" Courtenay et al., 1999
2378-TCDD
Barbel Barbus plebejus PCB (Aroclor 1260) " "" ECOD: "" Hugla and Thome, 1999
Blenny PAH (BNF, heavy gas oil) " " "" mRNA: " Celander et al., 1994
Zoarces viviparus PAH (BNF) " "" Förlin and Celander, 1993
Blue-striped grunt Haemulon sciurus PAH (BNF) " " "" Stegeman et al., 1990
Brook trout PCDD (2,3,7,8,-TCDD) "" Cormier et al., 2000b
Salvelinus fontinalis PAH (BaP) " Padros et al., 2000
Brown bullhead Ameiurus nebulosus PAH (BNF) $ "" Hasspieler et al., 1994
Bullhead Cottus gobio Organotins (TBT, TPT) %% %% % Fent and Bucheli, 1994
Califormia killifish Fundulus PAH (BaP) " Von Hofe and Puffer, 1986
parvipinnis
Carp PCDD (2,3,7,8-TCDD) " "" Van der Weiden et al., 1994
Cyprinus carpio PCDD (2,3,7,8-TCDD) "" "" Van der Weiden et al., 1992
PAH (BaP, chrysene) "" "" Van der Weiden et al., 1993
PCBs (Aroclor 1254) "" Melancon and Lech, 1983
PAH (BNF) " "" "" PROD: "" Riviere et al., 1990
Deltamethrin $ % APDM: % Banca et al., 1997
PCB (Aroclor 1254) " "" "" $ $ ECOD: " Ueng et al., 1992
PAH (BNF) "" "" Agradi et al., 2000
17a-ethynylestradiol $ % % " b5RED: $ Sole et al., 2000
Channel catfish PCB (Aroclor 1254) $ "" "" $ $ APDM: $ Ankley et al., 1986
Ictalurus punctatus BKME $ "" Mather-Mihaich and Di Giu-
lio, 1991
PAH (BNF) $ "" Hasspieler et al., 1994
2,4-D"picloram $ " Gallagher and Di Giulio, 1991
2-aminocanthracene (AA) % Watson et al., 1995
PAH (BaP) " " Ploch et al., 1998
PCB 126 "" "" Rice and Roszell, 1998
PCOD/F " Fiedler et al., 1998
Chinook salmon Oncorhynchus tsha- PAH (BNF) "" mRNA: "" Campbell and Devlin, 1996
wytscha
Cockscomb prickleback Anoplarchus BNF or oiled sediment "" Woodin et al., 1997
purpurescens
Cod PAH (BNF) " Goksøyr, 1991
Gadus morhua PAH (BNF " Goksøyr et al., 1991a
PCDD (2,3,7,8-TCDD) " "" Hektoen et al., 1994

83
 84
Table 5 (Continued )

Species Pollutants cyt P450! CYP1A! AHH! EROD! cyt b5! P450 RED! Others!! Reference

Crucian carp Carassius auratus PCDDs and metals "" Chen et al., 1998
Dab PCB (CB 77) "" " Sleiderink and Boon, 1996
Limanda limanda PAH (BNF) " "" Förlin and Celander, 1993
PAH (3MC) "" Lemaire et al., 1996
PAH (BaP) and PCB (CB 126) " Van Schanke et al., 2002
Eel Anguilla anguilla PAH (BaP) $ "" "" $ Lemaire-Gony and Lemaire,
1992

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
PAH (DNOC) $ "" Braunbeck and Völkl, 1991
Organotins (TBT, TPT) %% %% % Fent and Bucheli, 1994
PAH (BaP, BNF) "" Pacheco and Santos, 1997
PAH (BaP, BNF) " Pacheco and Santos, 1998
PAH (BNF) " Fenet et al., 1998
PAH (BaP) "" Rotchell et al., 1999
PAH (BaP) "" Rotchell et al., 2000
PAH (BNF) "" "" Agradi et al., 2000
PAH (BNF or BaP), PCB "" "" Schlezinger and Stegeman,
2000
English sole PAH (BaP) "" "" Varanasi et al., 1986
Parophrys vetulus PCB (Aroclor 1254), PAH "/"" EH: $ Collier and Varanasi, 1991
(BaP)
Fathead minnow Pimephales promelas PCB (CB 77) "" "" Lindström-Seppä et al., 1994
Flounder PCB (Clophen A50) $ " Besselink et al., 1998
Platichthys flesus PCB (CB 156), PAH (BaP) " "" AE: $ Beyer et al., 1997
PCBs " Besselink et al., 1998
PAH (BaP) "" Rotchell et al., 1999
PAH (BaP) " Rotchell et al., 2000
Fourhorn sculpin Myoxocephalus BKME "" Förlin et al., 1985
quadricornis
Gizzard shad PAH (BaP) "" Levine et al., 1994
Dorosoma cepedianum PAH (BaP) "" mRNA: "" Levine and Oris, 1997
Guppy Poecilia reticulata Phenobarbital (PB) " % $ Varanasi et al., 1987
Killifish Fundulus heteroclitus PAH (BNF) " "" "" " mRNA: "" Kloepper-Sams and Stegeman,
1992
PAH (BNF) or 3,3,7,8-TCDF "" "" mRNA: "" Bello et al., 2001
Lake sturgeon Acipenser fulvescens PCDF (2,3,7,8-TCDF) " Palace et al., 1996
Lake trout PAH (BNF) "" "" Förlin and Celander, 1993
Salmo trutta Propiconazole " " Egaas et al., 1999
Mountain whitefish Prosopium wil- PAH (BNF) " "" "" $ Kloepper-Sams and Benton,
liamsoni 1994
Mudfish Clarias anguillaris PCBs, OCPs " "" Gadagbui and Goksøyr, 1996
Mullit Chelon labrosus PCB (Phenoclor DP 6) " " APDM: " Narbonne and Gallis, 1979
Plke Esox lucius PAH (BNF) " "" Förlin and Celander, 1993
Perch Perca fluviatilis PCB (Clophen A50), PAH " "/"" Förlin and Celander, 1993
(BNF)
Plaice Pleuronectes platessa PCB (Clophen A40) " " $ Boon et al., 1992
Polar cod Boreogadus saida Crude oil "" "" mRNA: "" George et al., 1995
Table 5 (Continued )

Species Pollutants cyt P450! CYP1A! AHH! EROD! cyt b5! P450 RED! Others!! Reference

Rainbow trout PCB (Aroclor 1254), PAH "/"" PROD: $ Addison et al., 1987
Salmo gairdneri or Oncorhynchus my- (BNF)
kiss Phenobarbitone $ Addison et al., 1987
PAH (BNF) "" EH: $ Andersson et al., 1985
PCB (Clophan A50) "" EH: $ Andersson et al., 1985
OCP"OPP (endosulfan/disul- $ = Arnold et al., 1995
foton)

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
PAH (BNF) $ "" "" $ E2H: $ Celander and Förlin, 1991
PAH (BNF) " "" " "" mRNA: " Celander et al., 1993
PCB (CB 153) "" $ Da Costa and Curtis, 1995
Atrazine $ $ AE: $ Egaas et al., 1993
PCB (CB 126 and sediment "" Engwall et al., 1994
extracts)
Organotins (TBT, TPT) %% %% $ Fent and Bucheli, 1994
PCB (Clophen A50), PAH "" "" Förlin and Celander, 1993
(BNF)
PAH (BNF) "" Goksøyr et al., 1991a
PAH (BNF) "" Goksøyr, 1991
PAH (BNF) "" "" mRNA: "" Haasch et al., 1993a
PAH (BNF, ISF) " "" ECOD: "" Haasch et al., 1994
PCDD (2378-TCDD) " "" AE:$ Hektoen et al., 1994
PCB (Clophen A50) "" Holm et al., 1994
PCB (CBs 77 and 126) "" "" "" Huuskonen et al., 1996
OCP (endosulfan) " " " $ AE: " Jensen et al., 1991
BKME "" Lehtinen et al., 1990
OCP (HCB) $ /" Lindström-Seppä et al., 1996
BKME "" Martel et al., 1995
PCB (Aroclor 1254, CB 77) " "" ECOD: "" Melancon and Lech, 1983
PCDF (2378-TCDF) "" Muir et al., 1992a,b
PCDD (2378-TCDD) "" Newsted and Giesy, 1993
PCB (33?44?-TCB) "" Otto and Moon, 1995
BKME containing sediments "" " Otto et al., 1994
OCP (HCB) $ $ Roy et al., 1995
PCB (CB 118) " " "" $ AE: $ Skaare et al., 1991
PAH (engine exhaust extract) "" $ $ EH: $ Tjärnlund et al., 1996
PCB (CB 77) " "" ECOD: "" Tyle et al., 1991
PCOD (2378-TCDD) "" "" Van der Weiden et al., 1992
PCDD (2378-TCDD) "" "" Van der Weiden et al., 1992
PAH, PCB in sediment extr. " " APDM: " Vigano et al., 1995
PCB (33?44?-TCB) "" mRNA: "" Otto et al., 1996
PCB (CB 169) " " mRNA: " Donohoe et al., 1999
PCDDs "" Parrott et al., 1995
PCB (22?44?55?-HCB) " "" mRNA: " Foster et al., 1998
PAH (BNF) " Fenet et al., 1998
2378-TCDD "" Machala et al., 1998

85
 86
Table 5 (Continued )

Species Pollutants cyt P450! CYP1A! AHH! EROD! cyt b5! P450 RED! Others!! Reference

creosote containing sediments "" Hyotylainen and Oikari, 1999

PAH (BaP) "" "" mRNA: "" Levine and Oris, 1999
PAH (BNF) "" ADH: $ Lemaire et al., 1996
PCB (33?44?-TCB) "" Blom and Förlin, 1997
PAH (retene) "" Fragoso et al., 1999
Propiconazole "" $ mRNA: "" Levine et al., 1999
PCB (33?44?-TCB) "" mRNA: "" Otto et al., 1997

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
Redfish scianops ocellatus Brevetoxin $ " PROD: $ Washburn et al., 1994
Roach PAH (BNF) "" O’Hare et al., 1995
Rutilus rutilus BKME "" Aaltonen et al., 2000b
Safi fish Siganus canaliculatus PAH (BNF) " " " $ $ ECOD: " Raza et al., 1995
Sand flathead Platycephalus bassensis Treated waste water $ $ Mosse et al., 1996
Scup Stenotomus chrysops PCDF (2,3,7,8-TCDF) " "" "" $ mRNA: "" Hahn and Stegeman, 1994
PBB (33?44?-TCB) "" "" " " mRNA: "" White et al., 1997
Triphenyltin $ % % $ % CYP2/3A: $ Fent et al., 1998
Sea bass Dicentrarchus labrax PAH (3MC) "" ADH: " Lemaire et al., 1996
Skate Raja erinacea PCB 126 "" "" Hahn et al., 1998
Southern flounder Paralichthys lethos- PAH (BaP) "" Little et al., 1984
tigma
Speckled sanddab Citharichthys stig- PAH (BaP) " Von Hofe and Puffer, 1986
maeus
Squirrelfish Holocentrus PAH (BNF) $ " $ Stegeman et al., 1990
Starry flounder PAH containing sediments " Collier et al., 1992
Platichthys stellatus PAH (BaP) " Varanasi et al., 1986
Stickleback Gasterosteus aculeatus PCB (Clophen A50) "" P6bH: "" Holm et al., 1994
Striped mullet Mugil cephalus Crude oils " " " B5RED: $ Chambers, 1979
Sturgeon Acipenser naccarii PAH (BNF) "" "" Agradi et al., 2000
Sunfish Lepomis macrochirus PAH (BNF, BaP) "" Oikari and Jimenez, 1992
Tilapia Oreochromis niloticus PCB (Aroclor 1254) " "" "" " $ ECOD: "
Turbot Scophthalmus maximus PAH (BaP) " " Peters et al., 1997
Whitefish Coregonus lavaretus BKME "" Soimasuo et al., 1995
BKME "" PROD: "" Soimasuo et al., 1998b
Yellow perch Perca flavescens PBB (CB 126) %/" %/" Dabrowska et al., 2000
Zebrafish PCDD (2,3,7,8-TCDD) "" "" Buchmann et al., 1993
Brachydanio rerio PAH (BNF) or 2,3,7,8-TCDD " " Troxel et al., 1997
PCBs " Orn et al., 1998

Symbols and abbreviations: %%, strong inhibition ( B 20% of control); %, inhibition; $ , no (significant) response; ", induction; "", strong induction ( ! 500% of control); !, cyt P450,
cytochrome P450; CYP1A, cytochrome P450 1A isozyme; AHH, aryl hydrocarbon hydroxylase; EROD, ethoxyresorufin O -deethylase; cyt b5, cytochrome b5; P450 RED, NAD(P)H cytochrome
P450 reductase. !!, mRNA, cytochrome P450 1A ‘messenger’ RNA; CND, caffeine N -demethylase; T6bH, testosterone 6b-hydroxlase; P6bH, progesterone 6b-hydroxylase; EH, epoxide hydroxylase;
E2H, estradiol 2-hydroxylase; PROD, pentoxyresorufin O -dealkylase; APDM, aminopyrine N -demethylase; b5RED, cytochrome b5 reductase; AE, aldrin epoxidase; ADH, aldehyde dehydrogenase.
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 87

specific isoenzymes, but inhibit others. This may result consequences of CYP1A induction and the use of
in a considerable alteration of isoenzyme levels, whereas immunochemical techniques for CYP1A detection as a
the amount of total cyt P450 is not always affected biomarker in environmental monitoring. Although the
(Miranda et al., 1990). The total amount of cyt P450 liver is the most important organ with respect to
proteins is generally determined by scanning the differ- cytochromes P450, immunohistochemical staining re-
ence spectrum between the oxidized and the reduced vealed that CYP1A was expressed in other tissues as
forms from 400 to 500 nm (Omura and Sato, 1964). well. Route-specific cellular expression of CYP1A was
An increase in the total cyt P450 levels was observed observed in mummichog following exposure to the
in laboratory studies with various species of fish exposed aqueous and dietary BaP (Van Veld et al., 1997).
to organic trace pollutants (Table 5). Notably PAHs, Aqueous BaP exposure resulted in higher CYP1A levels
PCBs and PCDFs caused a significant increase in total in gills, heart, liver and blood vessels, while dietary BaP
cyt P450 levels, while PCDDs caused a very strong exposure resulted in high levels in gut epithelium.
increase (!/500% of control). These results are con- Elevation of the CYP1A protein levels due to exposure
firmed by many field studies, which reported significant to environmental pollutants is preceded by an increase
increases of hepatic cyt P450 levels in fish from polluted in CYP1A mRNA levels (Stegeman and Hahn, 1994).
environments (Table 6). A strong and significant Measurement of CYP1A mRNA by Northern blots is
decrease in cyt P450 levels was observed in bullhead, becoming an integral part of investigations on CYP1A
eel and rainbow trout exposed to organotins (Table 5). regulation, while several field trials have shown its
In feral brown bullhead and roach from polluted suitability as a biomarker (Bucheli and Fent, 1995).
environments cyt 450 was significantly decreased (Table Population differences in CYP1A mRNA inducibility
6). The cyt P450 responses for all fish species from 39 were observed in Atlantic tomcod from polluted and
laboratory studies and 35 field studies are summarized reference sites (Courtenay et al., 1999). Since differences
in Fig. 7A. A significant increase in cyt P450 levels was were also observed between the responsiveness to PAHs
observed in 53% of the laboratory studies and 51% of and halogenated aromatic hydrocarbons (HAHs), the
the field studies, while strong increases (!/500% of authors suggested that the CYP1A transcription in the
control) were observed in 3 and 6% of the laboratory tomcod was modulated by more than one molecular
and field studies, respectively. mechanism. A similar reduction in the sensitivity to
Although a positive response in cyt P450 levels was AhR agonists was observed in killifish from the heavily
observed in more than 50% of the reported laboratory contaminated New Badford Harbor (NBH) in the US
and field studies, its value as a biomarker for ERA in (Bello et al., 2001). It was suggested that this reduction
aquatic ecosystems is limited since the responses of was caused by an alteration in the AhR signal transduc-
individual isoenzymes (notably CYP1A, Section 6.1.2) tion pathway in NBH fish, most probably due to
are more specific and sensitive. Total cyt P450 determi- chronic exposure to high levels of HAHs. Other CYP
nations may, however, be useful in testing the condition isoenzymes are generally less responsive to environmen-
of subcellular microsomal fractions for EROD analyses, tal pollutants (Celander, 1993), although elevated
since degradation of cyt P450s during microsome CYP3A levels have been demonstrated in harp seals,
isolation or storage may be identified as an increased most probably due to toxaphene exposure (Wolkers et
peak in the difference spectrum at 420 nm. Moreover, al., 2000). Since CYP3A is associated with steroid
levels and activities of CYP1A isoenzymes may be metabolism (notably 6b-testosterone hydroxylation),
normalized to a cyt P450 basis, in order to obtain so- this may be important with respect to reproductive
called ‘turnover’ values (e.g. EROD/P450). toxicity (Section 6.8, Celander et al., 1989).
Numerous studies have demonstrated an increase in
6.1.2. Cytochrome P450 1A (CYP1A) the hepatic CYP1A protein levels in various species of
In fish, the class of cyt P450 isozymes which is fish after exposure to organic trace pollutants (Table 5).
responsible for the biotransformation of a myriad of Notably, PAHs, PCBs, PCDDs and PCDFs caused a
xenobiotic compounds (PAHs, PCBs, dioxins, etc.) is significant or a very strong increase ( !/500% of control)
the CYP1A subfamily, comprising two genes, CYP1A1 in CYP1A content. These results are confirmed by many
and CYP1A2 (Goksøyr and Förlin, 1992; Stegeman and field studies in which a strong and significant increase of
Hahn, 1994). The CYP1A protein levels can be deter- hepatic CYP1A protein levels was observed in many
mined immunologically, using mono- or polyclonal species of fish from polluted environments (Table 6). At
antibodies with ELISA, Western-blotting or histochem- environmentally relevant doses, organotins such as
ical techniques (Bucheli and Fent, 1995). Goksøyr and tributyltin (TBT) intensified the PCB-induced CYP1A
Husøy (1998) reviewed biochemical and toxicological induction in channel catfish, while a decrease of induc-
aspects concerning the cytochrome P450 system, with a tion was observed at higher TBT doses (Rice and
more detailed description of CYP1A induction re- Roszell, 1998). A study with liver microsomes from
sponses in fish. They also discussed the ecotoxicological scub indicated that 3,3?,4,4?-TCB inactivated CYP1A by
 88
Table 6
Field studies on responses of organic trace pollutants on fish hepatic phase I-related enzymes

Species Pollutants cyt P450! CYP1A! AHH! EROD! CYT b5! P450 RED! Others!! Reference

Atlantic cod Gadus morhua PCBs, PAHs " " Goksøyr et al., 1994
Atlantic/Baltic salmon BKME "" "" George et al., 1992b
Salmo salar Halogenated xenobiotics " " " Pesonen et al., 1999
Oil spill (PAHs) "" "" Stagg et al., 2000
Antarctic rockcod Notothenia coriiceps PAHs "" McDonald et al., 1995
Barbel PCBs and PAHs "" ADPM:" Vigano et al., 1998

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
Barbus plebejus PCBs and PAHs " Flammarion and Garric, 1997
Blue-striped grunt Haemulon sciurus PCBs, PAHs $ " $ Stegeman et al., 1990
Bream PCBs "" ECOD:"" Jedamski-Grymlas et al., 1995
Abramis brama BKME " "" "" " Lindström-Seppä and Oikari,
1991
BKME " Kantoniemi et al., 1996
Brown bullhead Ictalurus nebulosus Paint, organic solvents % $ $ Gallagher and Di Giulio, 1989
etc.
PCBs, DDE " Otto and Moon, 1996b
PAHs " Arcand-Hoy and Metcalfe, 1999
Bullhead Cottus gobio BKME " Bucher et al., 1993
Burbot Lota lota BKME " Kloepper-Sams and Benton,
1994
PCBs $ $ $ Lockhart and Metner, 1992
Carp BKME (PCDD/Fs) "" ECOD: $ Ahokas et al., 1994
Cyprinus carpio PHAHs, PAHs " "" "" " Curtis et al., 1993
PHAHs, PAHs " "" " $ Van der Oost et al., 1998
PCBs and PAHs "" ADPM:" Vigano et al., 1995
Channel catfish PAHs, PCBs $ " "" ECOD:" Haasch et al., 1993b
Ictalurus punctatus PCDDs "" "" PROD:" Ronis et al., 1992
Chub Various pesticides " Vindimian et al., 1993
Leuciscus cephalus WWTP effluent " Kosmala et al., 1998
PCBs and PAHs " Flammarion and Garric, 1997
Cockscomb prickleback Anoplarchus pur- Oil spill "" Woodin et al., 1997
purescens
Cod Oil spill "" Goksøyr et al., 1991b
Gadus morhua PAHs, PCBs, DDTs " " Beyer et al., 1996
Comber PAHs, PCBs, PCDDs " Burgeot et al., 1994
Serranus cabrilla PAHs " Narbonne et al., 1991
PAHs " Burgeot et al., 1996
Serranus hepatus PAHs " Burgeot et al., 1996
Dab PAHs, PCBs " " Sleiderink, 1995a
Limanda limanda PAHs, PCBs, PCDDs " Burgeot et al., 1994
PAHs, PCBs, DDTs $ "" Goksøyr et al., 1991b
Unknown " " Förlin and Celander, 1993
PCBs, OCPs "" Eggens et al., 1992
PAHs, PCBs " " " Sleiderink et al., 1995b
PAHs, PCBs " "" Sleiderink and Boon, 1995
Unknown "" mRNA:" Renton and Addison, 1992
Table 6 (Continued )

Species Pollutants cyt P450! CYP1A! AHH! EROD! CYT b5! P450 RED! Others!! Reference

Oil spill " Kirby et al., 1999a

Dragonet Callionymus lyra PAHs, PCBs, PCDDs " Burgeot et al., 1994
Eel Anguilla anguilla PCBs, OCPs, PAHs, $ "" "" " " PROD:" Van der Oost et al., 1996b
PCDF/Ds
PCBs, OCPs, PAHs, " " PROD:" Van der Oost et al., 1991b
PCDF/Ds
PAHs " Fenet et al., 1998

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
BKME " Pacheco and Santos, 1999
Pesticides " " Agradi et al., 2000
PAHs and PCBs "" "" Schlezinger and Stegeman, 2000
English sole PAHs and others " EN:$ Collier et al., 1992
Parophrys vetulus PAHs, PCBs " "" "" Collier et al., 1995
PAHs, PCBs "" "" Stein et al., 1992
Flounder PCBs, OCPs, PAHs " "" ECOD: $ Goksøyr et al., 1991b
Platichthys flesus PCBs, PAHs $ $ Eggens et al., 1995
PCBs, PAHs, DDTs " "" Beyer et al., 1996
Domestic/industrial " Weber and Karbe, 1995
waste
PCBs, PAHs $ "/ $ mRNA:" Vethaak et al., 1996
PCBs, HCB " "" "" Stegeman et al., 1988
PCBs, HCB " "" Addison and Edwards, 1988
PAHs and PCBs " Kirby et al., 1999b
Golden ide Leuciscus idus PAHs, DDTs " ECOD:" Jedamski-Grymlas et al., 1994
Grayling Thymallus thymallus PCBs "" " "" " ECOD:"" Monod et al., 1988
Grey mullet OCPs "" Rodriguez-Ariza et al., 1993
Mugil sp. PAHs, PCBs, OCPs "" ECOD:" Rodriguez-Ariza et al., 1994
Gudgeon Various pesticides " Vindimian et al., 1993
Gobio gobio PCBs, PAHs " Flammarion and Garric, 1997
Killifish PCBs, OCPs, PAHs $ " " $ AE:$ Elskus and Stegeman, 1989
Fundulus heteroclitus PCBs " " mRNA:" Haasch et al., 1993b
Oil spill, pesticides AE:" Burns, 1976
Largemouth bass Unknown " $ Schlenk et al., 1996
Micropterus salmoides PCBs " mRNA:" Haasch et al., 1993b
Longnose sucker Catostomus catostomus BKME " Kloepper-Sams and Benton,
1994
Minnow Phoxinus phoxinus Various pesticides " Vindimian et al., 1993
Mountain whitefish Prosopium williamsoni BKME $ "" "" $ Kloepper-Sams and Benton,
1994
BKME "" Kloepper-Sams and Owens,
1993
Nase PCBs " " "" % ECOD:" Monod et al., 1988
Chondrostoma nasus Unknown " "" Masfaraud et al., 1990
PCBs and PAHs "" ADPM:" Vigano et al., 1995
Nile tilapia Oreochromis niloticus PCBs, HCS "" " $ Bainy et al., 1996
Northern squawfish Ptychocheilus orego- PHAHs, PAHs $ $ $ $ Curtis et al., 1993
nensis

89
 90
Table 6 (Continued )

Species Pollutants cyt P450! CYP1A! AHH! EROD! CYT b5! P450 RED! Others!! Reference

Perch Perca fluviatilis BKME "" " PROD: " Huuskonen and Linström-Sep-
pä, 1995
BKME " Lindström-Seppä et al., 1992
PHAHs, PAHs " Balk et al., 1996
BKME $ " " $ Lindström-Seppä and Oikari,
1991
BKME " Förlin and Celander, 1993

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
BKME "" Förlin et al., 1995
BKME "" Kloepper-Sams and Owens,
1993
BKME " PROD: $ Karels et al., 1998
BKME " Kantoniemi et al., 1996
BKME " Förlin et al., 1995
PAHs and heavy metals $ % Tuvikene et al., 1999
Pike PCBs, OCPs, PAHs " " PROD: " Van der Oost et al., 1991b
Esox lucius PCDDs, PCDFs " Förlin et al., 1992
Plaice PCBs, OCPs, PAHs " " ECOD: " Goksøyr et al., 1991b
Pleuronectes platessa PCBs, PAHs $ $ Eggens et al., 1995
PCBs, OCPs " Galgani et al., 1991
Oil spill " Kirby et al., 1999a
Rainbow surfperch Hypsurus caryi Petroleum seep "" Spies et al., 1996
Rainbow trout Salmo gairdneri or BKME " Lindström-Seppä et al., 1992
Oncorhynchus mykiss PCBs $ Otto et al., 1996
BKME $ " "" $ Lindström-Seppä and Oikari,
1990
Petrochemical waste $ ECOD: " Nikunen, 1985
Oil "" Payne and Penrose, 1975
PAHs " Fent et al., 1998
PAHs and heavy metals $ % Tuvikene et al., 1999
PAHs "" "" Senchez-Hemandez et al., 1998
Red mullet PAHs, PCBs, PCDDs " Burgeot et al., 1994
Mullus barbatus PAHs " Burgeot et al., 1996
Redbreast sunfish PAHs, PCBs, phenols "" Jimenez et al., 1990
Lepomis auritus PCBs, Hg " " " " Adams et al., 1990
PCBs " Ham et al., 1997
Roach PCBs, OCPs, PAHs % $ $ % $ Van der Oost et al., 1994a
Rutilus rutilus PCBs, OCPs, PAHs " " Van der Oost et al., 1991b
BKME $ " "" $ Lindström-Seppä and Oikari,
1991
PCBs " "" "" $ ECOD: " Monod et al., 1988
Various pesticides " Vindimian et al., 1993
BKME " PROD: " Karels et al., 1998
BKME " Kantoniemi et al., 1996
PAHs and heavy metals $ % Tuvikene et al., 1999
Rock sole PAHs, PCBs " " " Collier et al., 1995
Lepidopsetta bilineata PAHs, PCBs " " Stein et al., 1992
Table 6 (Continued )

Species Pollutants cyt P450! CYP1A! AHH! EROD! CYT b5! P450 RED! Others!! Reference

Rubberlip surfperch Rhacochilus toxotes Petroleum seep " Spies et al., 1996
Ruffe Gymnocephalus cernua Domestic/industrial " ECOD: " Weber and Karbe, 1995
waste
Sand flathead Platycephalus bassensis PAHs "" ECOD: " Holdway et al., 1994
Sardine Sardina pilchardus PAHs, PCBs, DDE $ Peters et al., 1994
Seabream Diplodus annularis Oil, metals and others $ " Bagnasco et al., 1991
Shorthorn sculpin Myoxocephalus scorpius PAHs " Stephensen et al., 2000

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
Speckled sanddab Citharichthys stigmaeus PCBs, PAHs, DDTs "" Rice et al., 1994
Spoonhead sculpin Cottus ricei BKME "" Gibbons et al., 1998
Spot Leiostomus xanthurus PAHs " "" "" Van Veld et al., 1990
Squirrelfish Holocentrus rufus PCBs, PAHs " $ $ Stegeman et al., 1990
Starry flounder PAHs and others " EH: $ Collier et al., 1992
Platichthys stellatus PAHs, PCBs " "" " Collier et al., 1995
PAHs, PCBs, OCPs " Spies et al., 1988
PAHs, PCBs " " Spies et al., 1996
Stone loach Noemacheilus barbatulus WWTP effluent " Kosmala et al., 1998
Tilapia PCBs, OCPs " "" Gadagbui and Goksøyr, 1996
Oreochromis niloticus unknown " "" "" " Bainy et al., 1999
Sewage, industrial waste $ " "" Ueng et al., 1992
Toadfish Opsanus tau PAHs $ $ $ Collier et al., 1993
White sucker BKME " " Van den Heuvel et al., 1995
Catostomus commersoni BKME "" Hodson et al., 1992
BKME "" "" Munkittrick et al., 1992
Whitefish Coregonus muksun BKME $ "" "" $ Lindström-Seppä and Oikari,
1989
BKME " mRNA: " Soimasuo et al., 1998a
Winter flounder oil spill $ $ $ b5RED: $ Payne et al., 1984
Pseudopleuronectes americanus PAHs "" Payne et al., 1987
PCBs " " mRNA:" Elskus et al., 1992

Symbols and abbreviations: %%, strong inhibition ( B 20% of control); %, inhibition; $ , no (significant) response; ", induction; "", strong induction ( ! 500% of control); !, cyt P450,
cytochrome P450; CYP1A, cytochrome P450 1A isozyme; AHH, aryl hydrocarbon hydroxylase; EROD, ethoxyresorufin O -deethylase; cyt b5, cytochrome b5; P450 RED, NAD(P)H cytochrome
P450 reductase. !!, mRNA, cytochrome 450 1A ‘messenger’ RNA; AE, aldrin epoxidase; PROD, pentoxyresorufin O -dealkylase; ECOD, ethoxycoumerin O -deethylase; b5RED, cytochrome b5
reductase.

91
92 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

Table 7
Laboratory studies on responses of organic trace pollutants on fish hepatic phase II enzymes and cofactors

Species Pollutants GSH! GSSG! GST! UDPGT! Others!! Reference

Carp OPP (dichlorvos) $ Hai et al., 1995

Cyprinus carpio PAH (BNF) $ $ Riviere et al., 1990
17a-ethynylestradiol $ $ Sole et al., 2000
Catfish OPP (diclorvos) $ Hai et al., 1995
Ictalurus nebulosus
Channel catfish Icta- BKME (short term, 1 day) % Mather-Mihaich and Di Giu-
lurus punctatus lio, 1991
BKME (long term, 2 weeks) " Mather-Mihaich and Di Giu-
lio, 1991
PCB (Arochor 1254) $ $ Ankley et al., 1986
OCP (picloram and 2,4-D) $ Gallagher and Di Giulio, 1991
PAH containing sediments " " Di Giulio et al., 1989a,b
Cod PCDD (2,3,7,8-TCDD) $ Hektoen et al., 1994
Gadus morhua
Crucian carp Carassius PCDDs and metals in sedi- " " Chen et al., 1998
auratus ment
Dab PAH (3MC) $ $ DTD:$ Lemaire et al., 1996
Limanda limanda PAH (BaP) and PCB (CB $ Van Schanke et al., 2002
126)
Eel PAH (DNOC) "" ARST:" Braunbeck and Völkl, 1991
Anguilla anguilla PAH (BaP) $ Lemaire-Gony and Lemaire,
1992
PAH (BNF) $ Fenet et al., 1998
English sole PAH (BaP) $ Collier and Varanasi, 1991
Parophrys vetulus
Flounder PCB (CB 156), PAH (BaP) $ $ Beyer et al., 1997
Platichthys flesus
Killifish Fundulus het- PAH (BNF) or 2,3,7,8- " $ Bello et al., 2001
eroclitus TCDF
Lake sturgeon PCDF (2,3,7,8-TCDF) $ Palace et al., 1996
Acipenser fulvescens
Lake trout Propiconazole " Egaas et al., 1999
Salmo trutta
Mudfish PCBs, OCPs " " Gadagbui and Goksøyr, 1996
Clarias anguillaris
Plaice PCB (Aroctor 1254) " Clarke et al., 1992
Pleuronectes platessa PCB (Clophen A40) " Boon et al., 1992
Rainbow trout Salmo PCB (Clophen A50), PAH " " Andersson et al., 1985
gairdneri or Oncor- (BNF)
hynchus mykiss
OCP"OPP (endosulfan/di- " Arnold et al., 1995
fussion)
PAH (BNF) " " Celander et al., 1993
PCB (CB 153) $ Da Costa and Curtis, 1995
Atrazine $ Egaas et al., 1993
PCB (Clophen A50) " " DTD:$ Förlin et al., 1996
PAH (3MC) $ $ DTD:" Förlin et al., 1996
PCDD (2,3,7,8-TCDD) % Hektoen et al., 1991
PCB (CBs 77 and 126) " " Huuskonen et al., 1996
OCP (endosulfan) $ Jensen et al., 1991
OCP (HCB) " $ /" Lindström-Seppä et al., 1996
PCB (3,3?,4,4?-TCB) " " " "" GSH/GSSG: $ Otto and Moon, 1995
BKME containing sedi- " $ $ " Otto et al., 1994
ments
HCB $ $ Roy et al., 1995
PAH (engine exhaust ex- $ DTD:$ Tjärnlund et al., 1996
tract)
PAH, PCB in sediment ex- $ " Vigano et al., 1995
tracts
PCB (33?44?-TCB) $ Otto et al., 1996
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 93

Table 7 (Continued )

Species Pollutants GSH! GSSG! GST! UDPGT! Others!! Reference

PAH (BNF) $ Fenet et al., 1998

TCDD, PCB, DDE " Machala et al., 1998
PAH (BNF) $ " DTD:$ Lemaire et al., 1996
PCB (33?44?-TCB) $ $ Blom and Förlin, 1997
Redfish Brevetoxin $ Washburn et al., 1994
Sciaenops ocellatus
Scup PBB (33?44?-TCB) " White et al., 1997
Stenotomus chrysops
Sea bass PAH (3MC) % $ DTD:$ Lemaire et al., 1996
Dicentrarchus labrax
Seabream PCB (Arocolor 1254) " Pedrajas et al., 1995
Sparus aurata OCP (deielderin), OPP (ma- % Pedrajas et al., 1995
lathion)
Starry flounder PAH containing sediments $ Collier et al., 1992
Platichthys stellatus
Sunfish PAH (BaP) % Oikari and Jimenez, 1992
Lepomis macrochirus
Viviparous blenny PAH (BNF) $ " Celander et al., 1994
Zoarces viviparus
Whitefish BKME % Soimasuo et al., 1995
Coregonus lavaretus
Zebrafish PCDD (2,3,7,8-TCDD) " Baumann et al., 1993
Brachydanio rerio

Symbols and abbrevations: %%, strong inhibition (B 20% of control); %, inhibition; $ , no (significant) response; ", induction; "", strong
induction ( ! 500% of control) !, GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione-S -transferease, UDPGT, UDP

uncoupling the catalytic cycle, resulting in formation of 6.1.3. Ethoxyresorufin O -deethylase (EROD) and aryl
ROS within the active site (Schlezinger et al., 1999). The hydrocarbon hydroxylase (AHH)
ROS formed by CYP1A may contribute to the toxicity Next to CYP1A protein or mRNA levels, a common
of planar HAHs. The CYP1A responses for all fish method to examine the responses of the CYP1A
species from 60 laboratory studies and 48 field studies isoenzyme is to determine its catalytic activity. The first
are summarized in Fig. 7B. A significant increase in CYP1A related activity to be proposed as an indicator
CYP1A levels was observed in 91% of the laboratory of pollutant exposure was the aryl hydrocarbon hydro-
studies and 85% of the field studies, while strong xylase (AHH) activity (Payne, 1976). This activity is
increases (!/500% of control) were observed in 43 and usually measured by determining the hydroxylation of
39% of the laboratory and field studies, respectively. benzo[a]pyrene (Collier et al., 1995). The activity of
In all fish species considered, hepatic CYP1A protein ethoxyresorufin O -deethylase (EROD), however, ap-
levels seem to be a very sensitive biomarker of exposure peared to be the most sensitive catalytic probe for
to PAHs and HAHs, which will certainly be feasible in determining the inductive response of the cyt P450
ERA procedures. Levine and Oris (1999) suggested that system in fish (Goksøyr and Förlin, 1992). The EROD
CYP1A expression due to exposure to rapidly metabo- activity is measured by following the increase in
lized substances should preferably be measured in fluorescence of the reaction product resorufin (Burke
tissues that make direct contact with the environment, and Mayer, 1974). Recently, an extensive review was
such as the gill and intestine. CYP1A determinations published, compiling and evaluating existing scientific
may be used in various steps of the ERA process, such as information on the use, limitations, and procedural
quantification of impact and exposure of various considerations for EROD activity in fish as a biomarker
organic trace pollutants, environmental monitoring of of chemical exposure (Whyte et al., 2000).
organism and ecosystem ‘health’, identifying subtle early Increases in both AHH and EROD activities have
toxic effects, triggering of regulatory action, identifica- been observed in many species of fish after exposure to
tion of exposure to specific compounds, toxicological organic trace pollutants (Table 5). Notably, PAHs,
screening and the research on toxic mechanisms of PCBs, PCDDs and PCDFs caused very strong increases
xenobiotics (Stegeman et al., 1992). The CYP1A re- ( !/500% of control) in CYP1A catalytic activities. It
sponse has been validated for use in ERA monitoring was recently observed that substances such as nitrated
programs (Bucheli and Fent, 1995), assuming that all polycyclic aromatic hydrocarbons (NPAHs) and N-
potential variables that may affect this parameter are heterocyclic aromatic hydrocarbons (azarenes), which
considered in the experimental design. are ubiquitous in the environment, may contribute
94 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

Table 8
Field studies on responses of organic trace pollutants on fish hepatic phase II enzymes and cofactors

Species Pollutants GSH! GSSG! GST! UDPGT! Others!! Reference

Atlantic/Baltic salmon BKME $ $ George et al., 1992b

Salmo salar halogenated xeno- " GSH/GSSG:- Pesonen et al., 1999
biotics
Barbel Barbus plebejus PCBs and PAHs $ " " Vigano et al., 1995
Bream PCBs " Jedamski-Grymlas et al., 1995
Abramis brama BIKME " " " Lindström-Seppä and Oikari,
1991
BIKME $ $ Kantoniemi et al., 1996
Brown bullhead Ameiurus PCBs, DDE % " $ Otto and Moon, 1996b
nebulosus
Bullhead Cottus gobio BKME % Bucher et al., 1993
Carp PHAHs, PAHs % " " " GSH/GSSG:- Van der Oost et al., 1998
Cyprinus carpio PCBs and PAHs $ $ " Vigano et al., 1995
Chub (Leuciscus cephalus ) Heavy metals " Lenartova et al., 1997
Cod (Gadus morhua ) PAHs, PCBs, DDTs " Beyer et al., 1996
Eel Anguilla anguilla PCBs, PAHs, $ " " " Van der Oost et al., 1996b
OCPs, PCDD/Fs
PAHs $ Fenet et al., 1998
English sole PAHs and others $ Collier et al., 1992
Parophrys vetulus PAHs, PCBs "" Stein et al., 1992
Flounder PAHs, PCBs, DDTs $ Goksøyr et al., 1991b
Platichthys flesus PCBs, PAHs, $ Beyer et al., 1996
DDTs,
Domestic/industrial $ Weber and Karbe, 1995
waste
Grey mullet Mugil sp. OCPs " Rodriguez-Ariza et al., 1993
Nase Chondrostoma soetta PCBs, PAHs $ $ " Vigano et al., 1995
Nile tilapia Oreochromis ni- PCBs, HCHs $ Bainy et al., 1996
loticus
Perch Perca fluviatilis BKME $ " Huuskonen and Linström-Seppä,
1995
BKME " $ $ Lindström-Seppä and Oikari,
1991
BKME " Förlin et al., 1995
BKME $ $ Kantoniemi et al., 1996
BKME % $ Tuvikene et al., 1999
PAHs and heavy
metals
Plaice Pleuronectes platessa PCBs, OCPs $ Goksøyr et al., 1991b
Rainbow trout PCBs $ $ % $ Otto et al., 1996
Salmo gairdneri or Oncor- BKME $ $ Lindström-Seppä and Oikari,
hynchus mykiss 1990
BKME " Oikari and Kunnamo-Ojala,
1987
BKME % Oikari et al., 1985
Petrochemical waste " Nikunen, 1985
PAHs $ Fent et al., 1998
PAHs and heavy % $ Tuvikene et al., 1999
metals
Red mullet Mullus barbatus PAHs " DTD:$ Burgeot et al., 1996
Rock sole Lepidopsetta bili- PAHs, PCBs " Stein et al., 1992
neata
Ruffe Gymnocephalus cernua Domestic/Industrial $ Weber and Karbe, 1995
waste
Roach PCBs, PAHs, OCPs $ $ % Van der Oost et al., 1994a
Rutilus rutilus BKME " $ $ Lindström-Seppä and Oikari,
1991
BKME $ $ Kantoniemi et al., 1996
PAHs and heavy % $ Tuvikene et al., 1999
metals
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 95

Table 8 (Continued )

Species Pollutants GSH! GSSG! GST! UDPGT! Others!! Reference

Seabream Diplodus annularis oil, metals and $ % DTD:$ Bagnasco et al., 1991
others
Shorthorn sculpin Myoxo- PAHs $ DTD:$ Stephensen et al., 2000
cephalus scorpius
Starry flounder PAHs and others $ Collier et al., 1992
Platichthy stellatus PAHs, PCBs " Stein et al., 1992
Tilapia Oreochromis niloti- PCBs, OCPs " " Gadagbui and Goksøyr, 1996
cus
White sucker Catostomus BKME % Hodson et al., 1992
commersoni
Whitefish Coregonus muk- BKME $ /" Lindström-Seppä and Oikari,
sun 1989

Symbols and abbrevations: %%, strong inhibition (B 20% of control); %, inhibition; $ , no (significant) response; ", induction; "", strong
induction ( ! 500% of control);!, GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione S -transferease, UDPGT, UDP
glucuronyl transferase; !!, GSH/GSSG, thiol:disulfide ratio; DTD, DT diaphorase.

significantly to fish CYP1A induction in PAH-contami- ERA processes. Although certain chemicals may inhibit
nated environments (Jung et al., 2001). Generally, a EROD induction or activity, this interference is gen-
good correlation will be observed between CYP1A erally not a drawback to the use of EROD as a
protein levels and EROD activity (e.g. Van der Oost et biomarker (Whyte et al., 2000). Together with levels of
al., 1996b). Numerous field studies demonstrated a CYP1A protein and mRNA, the induction of CYP1A
strong and significant increase of hepatic CYP1A catalytic activities may be used both for the assessment
protein levels and activity in many species of fish from of exposure and as early-warning sign for potentially
polluted environments (Table 6). A strong and signifi- harmful effects of many organic trace pollutants.
cant decrease in EROD activities, however, was ob- Research on mechanisms of CYP1A-induced toxicity
served in bullhead, eel and rainbow trout exposed to suggests that EROD activity may not only indicate
organotins, while a decreased AHH activity was ob- chemical exposure, but may also precede effects at
served in phenobarbital-exposed guppies (Table 5). The various levels of biological organization (Whyte et al.,
PCB-induced EROD induction in channel catfish was 2000). Certain confounding variables, which may affect
inhibited by low doses organotins, although EROD the enzyme activities, however, will have to be consid-
activity was still elevated compared with reference fish ered when interpreting the responses in these para-
(Rice and Roszell, 1998). Studies of Fent et al. (1998) meters.
with scub exposed to triphenyltin (TPT) indicated a
degenerative effect of organotins on the fish microsomal
MO system, although some differences are observed 6.1.4. Cytochrome b5 (cyt b5)
between organotins, and between species. EROD activ- Cyt b5 is involved in the cyt P450-mediated biotrans-
ity and CYP 1A protein were also reduced in carp, after formations through electron donation by NADH via
i.p. administration of 17a-ethynylestradiol (Sole et al., cytochrome b5 reductase (Timbrell, 1991). It is unknown
2000). AHH responses for all fish species from 23 whether or not the AhR-mediated mechanism of induc-
laboratory studies and 33 field studies are summarized tion is also involved in the induction of cyt b5 levels in
in Fig. 7C. A significant increase in AHH activity was fish inhabiting polluted environments.
observed in 88% of the laboratory studies and 90% of Cyt b5 levels were measured in a limited number of
the field studies, while strong increases (!/500% of laboratory and field studies only. Increased cyt b5 levels
control) were observed in 43 and 39% of the laboratory were observed in laboratory studies with PCB exposed
and field studies, respectively. The EROD responses for mullet and striped mullet exposed to crude oils (Table
all fish species from 137 laboratory studies and 127 field 5). Two field studies, with eel and Nile tilapia from
studies are summarized in Fig. 7D. A significant polluted environments, demonstrated a significant in-
increase in EROD activities was observed in 88% of crease in cyt b5 levels, while a significant decrease was
the laboratory studies and 90% of the field studies, while observed in roach from a polluted site (Table 6). The cyt
strong increases (!/500% of control) were observed in b5 responses for all fish species from 12 laboratory
69 and 37% of the laboratory and field studies, studies and ten field studies are summarized in Fig. 7E.
respectively. A significant increase in cyt b5 levels was observed in
Both AHH and EROD activities in fish liver are very 33% of the laboratory studies and 50% of the field
sensitive biomarkers, and may thus be of great value in studies, while strong increases (!/500% of control) were
96 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

Table 9
Laboratory studies on responses of organic trace pollutants on fish hepatic antioxidant enzymes

Species Pollutants SOD! GPOX! GRED! CAT! Others!! Reference

Carp OPP (paraquat) $ " $ Gabryelak and Klekot, 1985

Cyprinus carpio OPP (paraquat) " Vig and Nemcsok, 1989
OPP (paraquat) $ $ $ LPOX:% Hai et al., 1995
17a-ethynylestradiol $ $ $ Sole et al., 2000
Catfish Ictalurus nebulo- OPP(dichlorvos) $ $ $ LPOX:" Hai et al., 1995
sus
Channel catfish Ictalurus BKME $ $ " LPOX:$ Mather-Mihaich and Di Giulio,
punctatus 1991
PAH containing sediments " Di Giulio et al., 1989a,b
OCP (2,4-D and picloram) $ Gallagher and Di Giulio, 1991
Dab Limanda limanda PAH (3MC) $ $ $ /% Lemaire et al., 1996
Eel Anguilla anguilla PAH (DNOC) % Braunbeck and Völkl, 1991
Lake sturgeon Acipenser PCDF (2,3,7,8-TCDF) " $ $ LPOX:"" Palace et al., 1996
fulvescens
Rainbow trout BKME containing sediments % $ Otto et al., 1994
Salmo gairdneri or On- PAH"PCB in sediments $ $ Vigano et al., 1995
corhynchus mykiss extract
OPP (Paraquat) % TBARS:"" Pedrajas et al., 1995
PCB (Aroclor 1254), OCP $ TBARS:" Pedrajas et al., 1995
(dieldrin)
HCB contaminated food " " " Roy et al., 1995
PCB (3,3?,4,4?-TCB) $ " "" % Otto and Moon, 1995
OCP"OPP (endosulfan/dis- $ G6PDH: $ Arnold et al., 1995
ulfoton)
PCB (Clophen A50) " Förlin et al., 1996
PAH (3MC) $ Förlin et al., 1996
PAH, PCB (engine exhaust " % G6PDH: $ Tjärnlund et al., 1996
extract)
DDE " Machala et al., 1998
PAH (BNF) $ $ $
Red mullet Mullus barba- PCB (Arcolor 1254) $ " % " LPOX:" Rudneva-Titova and Zherko,
tus 1994
Scorpion fish Scorpoena PCB (Arcolor 1254) $ " " " LPOX:" Rudneva-Titova and Zherko,
porcus 1994

Sea bass Cd and Cu % LPOX:" Romeo et al., 2000

Dicentrarchus labrax PAH (3MC) "/$ %/" $ Lemaire et al., 1996
Viviparous blenny PAH (BNF, heavy gas oil) $ Celander et al., 1994
Zoarces viviparus

Symbols and abbrevations: %%, strong inhibition (B 20% of control); %, inhibition; $ , no (significant) response; ", induction; "", strong
induction ( ! 500% of control);!, SOD, superoxide dismutase; GPOX, glutathione peroxidase; GRED, glutathione reductase, CAT, catalase; !!,
LPOX, lipid peroxidation; G6PDH, glucose-6-phosphate dehydrogenase; TBARS, thiobarbitutric acid reactive substances.

observed in none of the reviewed laboratory and field therefore, regarded as a typical MO enzyme in fish liver
studies. (Braunbeck and Völkl, 1991).
Although cyt b5 levels may be elevated in some fish Some studies reported an increased hepatic P450
species after exposure to organochlorine compounds, its RED activity in fish after exposure to PAHs and crude
value as a biomarker in ERA procedures remains oil, but most studies could not demonstrate any
questionable. Further research is required to elucidate significant alterations (Table 5). These results are
the mechanism of cyt b5 induction. confirmed by field studies, although significant increases
of hepatic P450 RED activity were observed in salmon,
bream, carp, eel, sunfish and grayling from polluted
6.1.5. NADPH cytochrome P450 reductase (P450 RED) environments. In most cases no significant differences
Cyt P450 activity depends on reduction of the heme could be observed between fish from control and
iron by electron transfer from the flavoprotein P450 polluted sites (Table 6). A significant decrease in P450
RED, and in some cases from cyt b5 (Kloepper-Sams RED activities was observed in bullhead, eel, scup and
and Stegeman, 1992; Goeptar et al., 1995). P450 RED is, rainbow trout exposed to organotins (Table 5), and in
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 97

Table 10
Field studies on responses of organic trace pollutants on fish hepatic antioxidant enzymes

Species Pollutants SOD! GPOX! GRED! CAT! Others!! Reference

Atlantic/Baltic salmon Salmo sal- Halogenated xenobio- $ " Pesonen et al., 1999
ar tics
Barbel Barbus plebejus PCBs and PAHs $ $ Vigano et al., 1995
Brown Bullhead Ameiurus nebu- PCBs, DDE " $ $ Otto and Moon, 1996b
losus
Bullhead Cottus gobio BKME $ $ G6PDH:$ Bucher et al., 1993
Carp Cyprinus carpio PHAHs, PAHs " $ $ $ Van der Oost et al., 1998
Channel catfish Ictalarus puncta- BKME " Mather-Mihaich and Di Giulio,
tus 1991
Chub heavy metals $ " $ TBARS:$ Lenartova et al., 1997
Leuciscus cephalus PCBs and PAHs $ $ Vigano et al., 1995
Dab Limanda limanda PAHs " " Livingstone et al., 1993
Eel Anguilla anguilla PCBs, PAHs, OCPs, $ $ $ Van der Oost et al., 1996b
PCDD/Fs
Grey mullet Mugil sp. OCPs " " $ " LPOX:- Rodriguez-Ariza et al., 1993
Nase Chondrostoma soetta PCBs and PAHs $ $ Vigano et al., 1995
Nile tilapia Oreochromis niloticus PCBs, HCHs " $ % % G6PDH:" Bainy et al., 1996
Rainbow trout Salmo gairdneri PCBs % $ Otto et al., 1996
Red mullet Mullus barbatus PAHs " % " Burgeot et al., 1996
Sardine Sardina pilchardus PAHs, PCBs, DDE " " Peters et al., 1994
Seabream Diplodus annularis Oil and others % $ G6PDH:" Bagnasco et al., 1991
Shorthorn sculpin Myoxocepha- PAHs $ " " Stephensen et al., 2000
lus scorpius
Spot Leiostomus xanthurus PAHs " Roberts et al., 1987

Symbols and abbrevations: %%, strong inhibition (B 20% of control); %, inhibition; $ , no (significant) response; ", induction; "", strong
induction ( ! 500% of control); !, SOD, superoxide dismutase; GPOX, glutathione peroxidase; GRED, glutathione reductase; CAT, catalase; !!,
G6PDH, glucose-6-phosphate dehydrogenase; LPOX, lipid peroxidation; TBARS, thiobarbitutric acid reactive substances.

nase from a PCB-polluted environment (Table 6). The (GA)) to the molecule (Commandeur et al., 1995;
P450 RED responses for all fish species from 24 Mulder et al. 1990). Some xenobiotic compounds
laboratory studies and 18 field studies are summarized possess the requisite functional groups (such as !/
in Fig. 7F. A significant increase in P450 RED activity COOH, !/OH or !/NH2) for direct metabolism by
was observed in 25% of the laboratory studies and 33% conjugative phase II enzyme systems, while others are
of the field studies, while a strong increase (!/500% of metabolized by an integrated process involving prior
control) was only observed in a laboratory study with action of the phase I enzymes (George, 1994; Lech and
eel exposed to PAHs (Braunbeck and Völkl, 1991). Vodicnik, 1985; Sijm and Opperhuizen, 1989). Phase II
Although the hepatic P450 RED activity in a limited enzymes can play an important role in homeostasis as
number of fish species appears to be affected by well as in detoxification and clearance of many xeno-
environmental contaminants, it should not be consid- biotic compounds. The major pathway for electrophilic
ered as a valid biomarker for ERA since most studies compounds and metabolites is conjugation with GSH,
could not demonstrate any significant effects (Fig. 7F). while for nucleophilic compounds conjugation with GA
is the major route (George, 1994). Other pathways play
6.2. Phase II enzymes and cofactors a minor role in fish and are the preferred route for only a
few compounds. Sulphatation is a competitive pathway
The so-called second phase of metabolism involves a to glucuronidation for PAH metabolites, but it is only
conjugation of the xenobiotic parent compound or its effective at very low substrate concentrations (George,
metabolites with an endogenous ligand (Fig. 6). Con- 1994).
jugations are addition reactions in which large and often Relatively little is known about the enzymology and
polar chemical groups or compounds such as sugars and molecular biology of piscine phase II systems, the
amino acids are covalently added to xenobiotic chemical current knowledge being confined to those catalyzing
compounds and drugs (Lech and Vodicnik, 1985). The glucuronidation and GSH conjugation (George, 1994).
majority of the phase II type enzymes catalyze these In addition to the phase I CYP1A genes, the Ah gene
synthetic conjugation reactions, thus facilitating the battery also comprises phase II genes like NADPH
excretion of chemicals by the addition of more polar menadione oxidoreductase, aldehyde dehydrogenase,
groups (e.g. glutathione (GSH) and glucuronic acid UDPGT and GST (Nebert et al., 1990; Celander,
 98
Table 11
Laboratory studies on responses of organic trace pollutants on fish PAH related parameters, serum transaminases and gross indices

Species Pollutants Bile MET! DNA add! ALT! AST! LSI! CF! Reference

Antarctic fish, Notothenia gibberifrons PAHs "" Yu et al., 1995

Atlantic cod, Gadus morhua Curde oil (PAHs) "" "" Aas et al., 2000
Atlantic salmon PAH (BNF) $ Goksøyr and Larsen, 1991
Salmo salar Crude oil "" Gagnon and Holdway, 2000
Bluegill sunfish, Lepomis macrochirus PAH (BaP) " $ Oikari and Jimenez, 1992
Brown bullhead PAH (BaP) "" Sikka et al., 1990

R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
Ictalurus nebulosus PAHs "" Leadly et al., 1999
PAH (BaP) " Ploch et al., 1998
Brook trout, Salvelinus fontinalis PAH (BaP) "" Padros et al., 2000
Brown trout Domestic waste water: acute " " Bucher and Hofer, 1990
Salmo trutta Domestic waste water: chronic $ $ Bucher and Hofer, 1990
Califormia killifish, Fundulus parvipinnis PAH (BaP) " Von Hofe and Puffer, 1986
Carp Atrazine $ $ Neskovic et al., 1993
Cyprinus carpio Pyrethroid (deltamethrin) " Balint et al., 1995
Channel catfish Ictalurus punctatus OCP (2, 4-D and pidoram) $ - Gallagher and Di Giulio, 1991
PAH (BaP) " Ploch et al., 1998
Dab PCB (CB 77) $ Sleiderink and Boon, 1996
Limanda limanda PAH (BaP) & PCB (CB 126) "" "" Van Schanke et al., 2002
English sole PAH (BaP) "" "" Varanasi et al., 1987
Parophrys vetulus PAH (BaP) "" Varanasi et al., 1986
PAH (BaP) "" Varanasi et al., 1989b
PAH (BaP) "" Collier and Varanasi, 1991
"" Stein et al., 1993
Flounder PCB (clophen A50) " $ Besselink et al., 1998
Platichthys flesus PCB (CB 156) $ $ $ Beyer et al., 1997
PAH (BaP) "" "" $ $ Beyer et al., 1997
PAH (BaP) "" Hylland et al., 1996
PAH (BaP) "" Malmström et al., 2000
Killyfish, Fundulus heteroclitus PAH (BNF) - Kloepper-Sams and Stegeman, 1992
Mudfish Clarias anguillaris PCBs, OCPs " $ Gadagbui and Goksøyr, 1996
Pike, Esox lucius B(a)P "" Ericson et al., 1999a
Rainbow trout Salmo gairdneri or Oncorhynchus mykiss PAH, PCB in sediment extracts " Vigano et al., 1995
BKME containing sediments $ $ Otto et al., 1994
PAH (BaP) " Potter et al., 1994
PCDF (2, 3, 7, 8-TCDF) $ Muir et al., 1992b
BKME " Lehtinen et al., 1990
PCDD (2, 3, 7, 8-CDD) " $ Newsted and Giesy, 1993
PCDD (2, 3, 7, 8-CDD) $ Van der Weiden et al., 1992
PAH (BNF, ISF) $ Celander and Förlin, 1991
PCB (3, 3?, 4, 4?-TCB) " Otto and Moon, 1995
PCB (CB 153) $ Da Costa and Curtis, 1995
OCP"OPP (endosulfan/disulfo- " $ Arnold et al., 1995
ton)
PAHs (effluent discharges) " Sagelsdorff, 1995
PAH (engine exhaust extract) " Tjärnlund et al., 1996
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 99

Symbols and abbreviations: %%, strong inhibition ( B 20% of control); %, inhibition; $ , no (significant) response; ", induction; "", strong induction ( ! 500% of control); !, bile MET,
metabolites in bile; DNA add, DNA adducts (liver); ALT, alanine transaminase (serum); AST, aspertate transaminase (serum); LSI, liver sometic index (liver); CF, condition factor (whole body).
Hyotylainen and Oikari, 1999 1993). The mechanism of induction for most forms of
phase II enzymes is, therefore, probably regulated via

Von Hofe and Puffer, 1986

the Ah-receptor as well (Sutter and Greenlee, 1992;
Hayes and Pulford, 1995). As compared with phase I

Hsu and Deng, 1996

Celander et al., 1994
Brumley et al., 1998
Fragoso et al., 1999

systems, the induction responses of phase II enzymes are

Adams et al., 1990
Mosse et al., 1996

Peters et al., 1997

Holm et al., 1994
Chambers, 1979
generally less pronounced (Andersson et al., 1985;

Orn et al., 1998

George, 1994), so that they may be masked by natural
Reference

variability factors (such as sex, maturity, nutrition,

season, temperature, etc.). Still, even slight alterations
of the phase II activity may be harmful to an organism.
The levels of phase II cofactors may also be affected
CF!

after exposure to environmental pollutants (Stegeman et

al., 1992; Lindström-Seppä and Oikari, 1991; Van der
Oost et al., 1996b). The responses of fish phase II
LSI!

$
$
"

"

enzymes and cofactors in laboratory and field studies

-

are listed in Tables 7 and 8, respectively.

AST!

6.2.1. Reduced and oxidized glutathione (GSH and

ALT!

GSSG)
Reduced GSH, a tripeptide consisting of g -glutamine,
cysteine and glycine, can be conjugated in the initial step
DNA add!

of mercapturic acid formation (George, 1994; Comman-

deur et al., 1995). Among its functions are two
""

""

contrasting roles in detoxifications, as a key conjugate

"

of electrophilic intermediates, principally via GST

activities in phase II metabolism, and as an important
Bile MET!

antioxidant (Stegeman et al., 1992; Commandeur et al.,

1995). GSH reacts with electrophilic compounds and
""
""

""
$

replaces hydrogen, chlorine, nitro-groups and all kinds of

other so-called leaving groups. The mercapturic acids are
Creosote containing sediments

formed after catabolism of the GSH conjugate with

peptidases and acetylases (Lech and Vodicnik, 1985;
PCDF (2, 3, 7, 8-TCDF)

Commandeur et al., 1995). Urinary excretion of mer-

PAH (heavy gas oil)
Chlorinated phenols

PCB (Clophen A50)

Treated waste water

capturic acids is a useful tool in the assessment of

toxicologically relevant internal doses in persons exposed
PAH (retene)

to electrophilic chemicals, and may be applied as a non-

PAH (BaP)

PAH (BaP)

PAH (BaP)
Pollutants

PCBs, Hg

Crude oil

invasive biomarker in human occupational and environ-

mental toxicology (De Rooij et al., 1997). A microplate
PCBs

assay for total GHS and GSH disulfide contents, based

upon a first order recycling reaction resulting in the
formation of 5,5?-dithiobis 2-nitrobenzoic acid (TNB),
was described by Vandeputte et al. (1994).
Influxes of oxyradical-generating compounds may
alter GSH status and/or metabolism in several ways
Speckled sanddab, Citharichthys stigmaeus

(Stegeman et al., 1992; Otto and Moon, 1995). Increased

Viviparous blenny, Zoarces viviparus

fluxes of oxyradicals can impose a drain on intracellular

Redbreast sunfish, Lepomis auritus

Stickleback, Gasterosteus aculeatus

reducing equivalents with potentially profound conse-

Turbot, Scophthalmus maximus
Striped mullit, Mugil cephalus

quences on a variety of metabolic processes. The

Scup, Stenotomus chrysops

consumption of GSH due to the direct scavenging of

Platycephalus bassensis

oxyradicals or as a cofactor for glutathione peroxidase

Table 11 (Continued )

(GPOX) activity may represent such a drain: NADPH

Brachydanio rerio

must be oxidized to maintain GSH levels via glutathione

Sand flathead

reductase (GRED) (Di Giulio et al., 1995). More

Zebrafish

indirectly, oxidative stress can impose a drain on the

Species

reductant pool as a consequence of the energetic costs of

mounting a defence against an increased flux of
100 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

Table 12
Field studies on responses of organic trace pollutants on fish PAH related parameters, serum transaminases and gross indices

Species Pollutants bile MET! DNA add! ALT! AST! LSI! CF! Reference

Antarctic fish PAHs " McDonald et al., 1995

Notothenia coriiceps
Barbel PAHs $ Kurelec et al., 1989
Barbus barbus PCBs and PAHs $ $ Flammarion and Garric, 1997
Bream PCBs, PAHs " Slooff et al., 1983
Abramis brama PCBs $ Jedamski-Grymlas et al., 1995
PAHs $ Kurelec et al., 1989
BKME " Kantoniemi et al., 1996
Brook trout PAHs " Ray et al., 1995
Salvelinus fontinalis
Brown bullhead PAHs "" Dunn et al., 1987
Ictalurus nebulosus PCBs, DDE $ " Otto and Moon, 1996b
PAHs "" Leadly et al., 1999
PAHs "" Dunn et al., 1990
PAHs " Arcand-Hoy and Metcalfe,
1999
PCBs, OCPs and " Leadly et al., 1998
PAHs
Brown trout Salmo trutta domestic waste $ Bucher and Hofer, 1990
water
Carp PAHs $ Kurelec et al., 1989
Cyprinus carpio PCBs, OCPs, " $ " $ $ Van der Oost et al., 1998
PCDDs
Chub PHAs $ Kurelec et al., 1989
Leuciscus cephalus WWTP effluent " Kosmala et al., 1998
PCBs and PAHs $ $ Flammarion and Garric, 1997
Cod PAHs, PHAHs "" Ericson et al., 1996
Gadus morhua PAHs, PCBs, "" $ " $ Beyer et al., 1996
DDTs
Dab Limanda limanda PAHs, PCBs, " " Goksøyr et al., 1991b
DDTs
PAHs, PCBs $ Sleiderink and Boon, 1995
PAHs $ Poginsky et al., 1990
PAHs $ Chipman et al., 1992
Eel Anguilla anguilla PCBs, PAHs, " "" Van der Oost et al., 1994a
OCPs, PDDD/Fs
PCBs, PAHs, $ $ $ $ Van der Oost et al., 1996b
OCPs, PDDD/Fs
English sole PAHs, PCBs "" Varanasi et al., 1987
Parophrys vetulus PAHs and others "" Collier et al., 1992
PAHs "" "" Varanasi et al., 1989a
PAHs, PCBs $ " Stein et al., 1992
PAHs " Poginsky et al., 1990
PAHs and PCBs " " Kirby et al., 1999a,b
Flounder PAHs "" Baan et al., 1994
Platichthys flesus PAHs, PCBs, "" "" $ $ Beyer et al., 1996
DDTs
PAHs, PCBs "" " $ $ Vethaak et al., 1996
PAHs, PCBs, $ $ Goksøyr et al., 1991b
DDTs
Killifish PCBs, OCPs, PAHs $ Elskus and Stegeman, 1989
Fundulus heteroclitus
Mountain whitefish Proso- BKME $ Kloepper-Sams and Owens,
pium williamsoni 1993
Mugil PAHs $ Kurelec et al., 1989
Mugil auratus
Oyster toadfish PAHs "" "" Collier et al., 1993
Opsanus tau
Perch Perca fluviatilis BKME " " Huuskonen and Linström-
Seppä, 1995
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 101

Table 12 (Continued )

Species Pollutants bile MET! DNA add! ALT! AST! LSI! CF! Reference

BKME " Kloepper-Sams and Owens,

1993
creosote "" Ericson et al., 1999b
BKME (chiorophe- "" Karels et al., 1998
nolics)
BKME " Kantoniemi et al., 1996
Plaice PAHs, PCBs $ Eggens et al., 1995
Pleuronectes platessa PAHs, PCBs, " $ Goksøyr et al., 1991b
DDTs
Rainbow surfperch Petroleum seep " Spies et al., 1996
Hypsurus caryi
Rainbow trout PCBs $ $ Otto et al., 1996
Salmo gairdneri or Oncor- BKME $ $ Lindström-Seppä and Oikari,
hynchus mykiss 1990
Petrochemical $ Nikunen, 1985
waste
Red mullet PAHs " Burgeot et al., 1996
Mullus barbatus
Redbreast sunfish BKME % $ Adams et al., 1992
Lepomis auritus
Roach PCBs, PAHs, OCPs $ $ $ Van der Oost et al., 1994b
Rutilus rutilus BKME % % Jeney et al., 1996
BKME (chlorophe- " Karels et al., 1998
nolics)
BKME " Kantoniemi et al., 1996
Rock sole PAHs, PCBs $ $ Stein et al., 1992
Lepidopsetta bilineata
Rubberlip surfperch Petroleum seep $ /" Spies et al., 1996
Rhacochilus toxotes
Shorthorn sculpin PAHs "" "" " Stephensen et al., 2000
Myoxocephalus scorpius
Starry flounder PAHs and others "" Collier et al., 1992
Platichthys stellatus PAHs, PCBs $ " Stein et al., 1992
Tilapia PCBs, OCPs % $ Gadagbui and Goksøyr, 1996
Oreochromis niloticus
White sucker BKME $ McMaster et al., 1994
Catostomus commersoni BKME " % Hodson et al., 1992
PAHs " Cormier et al., 2000a
PAHs " El Adlouni et al., 1995
Winter flounder PAHs " Varanasi et al., 1989a
Pseudopleuronectes ameri- PAHs " Gronlund et al., 1991
canus
Whitefish Coregonus muksun BKME $ /" $ Lindström-Seppä and Oikari,
1989

Symbols and abbreviations: %%, strong inhibition ( B 20% of control); %, inhibition; $ , no (significant) response; ", induction; "", strong
induction (! 500% of control); !, bile MET, metabolites in bile; DNA add,DNA adducts [liver]; ALT, Alanine transaminase [serum]; AST,
Aspertate transaminase [serum]; LSI, liver sometic index [liver]; CF, condition factor [whole body].

oxyradicals, i.e. biosynthesis of antioxidants (Winston al., 1983). In the healthy cell GSH:GSSG ratios are
and Di Giulio, 1991). typically very high, greater than 10:1 (Stegeman et al.,
Perhaps the most obvious direct effect of certain 1992). In rainbow trout it was observed that EROD
pollutants is a decrease in thiol status, i.e. the ratio of induction (due to PCB exposure) was intensified in
reduced to oxidized glutathione (GSH:GSSG), due to GSH-supplemented fish tissues, while the reverse was
either direct radical scavenging or increased peroxidase observed in GSH deficient fish (Otto et al., 1996, 1997).
activity (Stegeman et al., 1992; Otto and Moon, 1995). Strong indications were found that the tissue thiol status
Alternatively, normal GSH:GSSG ratios can be main- modulates Ah receptor inducible CYP1A gene expres-
tained due to increased activities of GRED or increased sion and catalytic activity, indicating a ‘cross-talk’
GSH synthesis. In mammals, GSH synthesis is consid- between the GSH and cytochrome P450 systems.
ered to be tightly regulated via feedback inhibition by Both GSH and GSSG levels have only been measured
GSH on a rate-limiting synthetic enzyme (Lauterburg et in a limited number of laboratory and field studies.
102 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

Increased GSH levels were observed in laboratory 1989). Most studies determine the total GST activity
studies with fish exposed to BKME, PAHs, PCBs and using the artificial substrate 1-chloro-2,4-dinitrobenzene
HCB (Table 7). Some field studies, e.g. with bream, (CDNB), which is conjugated by all GST isoforms with
perch and roach from BKME-polluted environments, the exception of the q-class enzymes (George, 1994; Van
demonstrated a significant increase in GSH levels, while der Aar et al., 1996).
significant decreases were observed in bullhead and carp The toxicity of many exogenous compounds can be
from polluted sites (Table 8). Two laboratory studies, modulated by induction of GSTs. Effects of inducing
with channel catfish and rainbow trout exposed to agents on total hepatic GST activity, measured by
PAH-containing sediments and PCBs, respectively, CDNB conjugation, have been observed in several fish
reported elevated hepatic GSSG levels (Table 7). Ele- species (George, 1994). As for CYP1A, the mechanism
vated GSSG levels in the field was only observed in one of induction for most GSTs in mammals is regulated via
study with eel from a polluted site (Table 8). The GSH the Ah-receptor (Pickett and Lu, 1989; George, 1994).
responses for all fish species from ten laboratory studies An additional form of GST induction which functions
and 17 field studies are summarized in Fig. 8A. A independently of the Ah-receptor has been elucidated
significant increase in GSH levels was observed in 60% and requires metabolism of the compound before
of the laboratory studies and 35% of the field studies. transcriptional activation of the respective subunit
The GSSG responses for all fish species from three gene can take place (Rushmore and Pickett, 1990).
laboratory studies and four field studies are summarized Due to the role that GSTs play in conjugating reactive
in Fig. 8B. A significant increase in GSSG levels was epoxide species and other electrophiles, induction of
observed in two of the laboratory studies (67%) and two these enzymes must be considered to be beneficial,
of the field studies (50%). Strong increases (!/500% of although metabolic activation of halogenated xenobio-
control) in hepatic GSH levels were only observed in tics by GST is also well recognized (Armstrong, 1990;
English sole from a site which was heavily polluted with Commandeur et al., 1995). There is a limited amount of
PAHs and PCBs (Stein et al., 1992), while no strong information regarding sexual, seasonal and develop-
increases in GSSG levels were observed in any of the mental differences in GST activity in fish (Stegeman et
reviewed laboratory and field studies. al., 1992).
Due to the limited number of observations on the An increase in hepatic GST activity has been reported
responses in GSH and GSSG levels, these parameters in several studies after exposure of fish to PAHs, PCBs,
cannot yet be considered as valid biomarkers for ERA OCPs and PCDDs, but most studies did not demon-
purposes. The key role played by GSH in detoxifications strate any significant alterations (Table 7). Attempts to
and the responsiveness of this system to xenobiotics, detect chemically induced activities of GSTs in free-
however, motivates continued research on its feasibility living fish also yielded conflicting results. Several studies
as a biomarker. Although it seems that pollutant- reported GST activities to be significantly increased, but
induced effects on GSH levels are restored by feed- in most cases no significant differences were observed
back-mechanisms, the hepatic GSH:GSSG ratio may be between fish from control and polluted sites (Table 8). A
a potential biomarker for oxidative stress (Van der Oost significant decrease in GST activities was observed in
et al., 1996b). rainbow trout, sea bass, seabream and sunfish exposed
to PCDDs, pesticides or PAHs (Table 7), and in some
6.2.2. Glutathione S -transferases (GSTs) fish species in polluted environments (Table 8). The
The conjugation of electrophilic compounds (or phase GST responses for all fish species from 43 laboratory
I metabolites) with GSH is catalyzed by the glutathione studies and 39 field studies are summarized in Fig. 8C. A
S -transferases (GSTs), a multigene superfamily of significant increase in GST activity was observed in 33%
dimeric, multifunctional, primarily soluble enzymes. of the laboratory studies and in 33% of the field studies,
Apart from their essential functions in intracellular while no strong increases ( !/500% of control) were
transport (heme, bilirubin and bile acids) and the reported in any of the laboratory and field studies
biosynthesis of leukotrienes and prostaglandins, a considered.
critical role for GSTs is obviously defence against Hepatic total GST activity in fish does not seem to be
oxidative damage and peroxidative products of DNA feasible as a biomarker for ERA, since increased
and lipids (George, 1994). The susceptibility of different activities are only observed in a limited number of fish
fish species to chemical carcinogenesis may be modu- species. In addition, the exposure to pollutants like
lated by the activity of GST (Varanasi et al., 1987). On PCDDs and PAHs may cause both induction and
the basis of substrate specificity, immunological cross- inhibition of the enzyme activity. However, more
reactivity and protein sequence data, the soluble GSTs research on this parameter, which is of paramount
have been grouped into four classes: a, m, p and q importance for major detoxification processes, may
(George, 1994). These enzymes are mainly located in the elucidate specific isoenzymes that have a more sensitive
cytosolic fraction of the liver (Sijm and Opperhuizen, and selective response to pollutants. Hepatic cytosolic
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 103

GST activity toward ethacrynic acid appeared to be pheromones, acting as stimulants of ovulation and
induced in rainbow trout exposed to PCB 153 or p ,p?- sperm production and also enhancing vitellogenesis in
DDE, but was not induced after 2,3,7,8-TCDD expo- many species (George, 1994). A decreased UDPGT
sure (Machala et al., 1998). This observation indicates activity can result in bilirubin accumulation (Oikari
that this parameter may be a suitable biomarker for and Nakori, 1982).
exposure to nonplanar PCBs and organochlorines that Hepatic UDPGT activities were reported to be
do not induce CYP1A activity. increased in most laboratory studies with fish exposed
to PAHs, PCBs, OCPs, PCDDs and BKME-contami-
6.2.3. UDP-glucuronyl transferases (UDPGTs) nated sediments (Table 7). These results are in line with
The synthesis of glucuronides by microsomal UDP- a number of field experiments in which an increased
glucuronyl transferases (UDPGTs) is a major pathway UDPGT activity was found in fish from polluted sites
for the inactivation and subsequent excretion of both (Table 8). Significant decreases in UDPGT activities
endogenous and xenobiotic organic compounds (Lech were only observed in BKME exposed whitefish and
and Vodicnik, 1985; Mulder et al., 1990; George, 1994). rainbow trout caged in a BKME-polluted environment
GA conjugation first requires synthesis of uridine 5?- (Table 8). The UDPGT responses for all fish species
diphosphoglucuronic acid (UDPGA). UDPGT cata- from 27 laboratory studies and 26 field studies are
lyzes the transfer of UDPGA to a wide variety of summarized in Fig. 8D. A significant increase in
acceptor substrates (aglycones) to form O-, N-,S- and C- UDPGT activity was observed in 52% of the laboratory
glucuronides (Kasper and Henton, 1980; Mulder et al., studies and in 42% of the field studies, while strong
1990), the majority being O-glucuronides (George, increases (!/500% of control) were only reported in two
1994). As with most enzymes that exhibit a broad laboratory studies, with eel exposed to dinitro-o-cresol
specificity for structurally diverse compounds, multiple (Braunbeck and Völkl, 1991) and rainbow trout exposed
isoenzymes belonging to a number of multigene families to PCB (Otto and Moon, 1995).
are found (George, 1994). The different UDPGT Although not as sensitive as phase I enzymes, the
isoenzymes are generally named after their acceptor UDPGT activity appears to be the phase II parameter
substrates, e.g. bilirubin, steroid and phenol UDPGTs. which is most responsive to pollutant exposure. It,
While the liver is quantitatively the most important site therefore, seems to be valid as a biomarker in certain
for glucuronidation of xenobiotics in fish, significant ERA monitoring programs, although more research will
activities have also been detected in extrahepatic tissues, be required to investigate the potential pollution-in-
including kidney, gills and intestine (George, 1994). duced responses of the various UDPGT isoenzymes.
In mammals, it was found that the UDPGT iso-
enzymes display a differential induction by xenobiotic 6.3. Oxidative stress parameters
compounds, and they also fall into four developmental
clusters, which suggests differential regulation during Many pollutants (or their metabolites) may exert
development (Burchell and Coughtrie, 1989). In a study toxicity related to oxidative stress. For instance, Win-
by Clarke et al. (1992), it was demonstrated that ston and Di Giulio (1991) found elevated rates of
multiple UDPGT isoforms with differing substrate idiopathic lesions and neoplasia among fish inhabiting
specificities were also present in fish. The UDPGT polluted environments to be related to the increased
isoform, which preferentially conjugates planar phenols, oxidative stress associated with pollutant exposure.
is induced by PAHs, most probably via an Ah receptor- Oxygen toxicity is defined as injurious effects due to
dependent mechanism (Nebert et al., 1990; George, cytotoxic reactive oxygen species (ROS), also referred to
1994). UDPGT activity in fish is reported to be as reactive oxygen intermediates (ROIs), oxygen free
influenced by sex, season, pH and temperature differ- radicals or oxyradicals (Di Giulio et al., 1989a; Halliwell
ences (Stegeman et al., 1992). Due to the ease of the and Gutteridge, 1999; Winzer, 2001). These reduction
assay, most studies have utilized 4-nitrophenol as the products of molecular oxygen (O2) are the superoxide
acceptor substrate. anion radical (O2%+ ), hydrogen peroxide (H2O2) and the
Although an increased UDPGT activity normally hydroxyl radical (OH+ ), an extremely potent oxidant
results in elevated detoxification of the inducing agent, capable of reacting with critical cellular macromole-
undesirable effects may also occur (Stegeman et al., cules, possibly leading to enzyme inactivation, lipid
1992). As is the case for cyt P450, UDPGT enzymes peroxidation (LPOX), DNA damage and, ultimately,
metabolize endogenous substrates such as steroid hor- cell death (Winston and Di Giulio, 1991).
mones. Induced UDPGTs may, therefore, seriously alter Numerous endogenous sources of oxyradical produc-
steroid metabolism, possibly with profound effects on tion exist, but of more immediate interest with respect to
the reproductive success of an organism (Stegeman et environmental biomarkers is the ability of a number of
al., 1992). Steroid hormone glucuronides formed in the structurally diverse compounds to enhance intracellular
testis and seminal vesicles of fish appear to be important oxyradical production through the process of redox
104 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

Fig. 7. Frequencies of pollutant-induced responses of phase I-related enzymes in fish: (A) cytochrome P450 (cyt P450); (B) cytochrome P450 1A
(CYP1A); (C) aryl hydrocarbon hydroxylase (AHH); (D) ethoxyresorufin O -deethylase (EROD); (E) cytochrome b5 (cyt b5); (F) cytochrome P450
(c) reductase (P450 RED). %/%/, strong decrease ( B/20% of control); %/, decrease; $/, no (significant) response; "/, increase; "/"/, strong increase ( !/
500% of control).

Fig. 8. Frequencies of pollutant-induced responses of phase II enzymes and cofactors in fish: (A) reduced glutathione (GSH); (B) oxidized
glutathione (GSSG); (C) glutathione S-transferase (GST); (D) UDP-glucuronyl transferase (UDPGT); --: strong decrease ( B/20% of control); %/:
decrease; $/: no (significant) response; "/: increase; "/"/: strong increase (!/500% of control).
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 105

Fig. 9. Frequencies of pollutant-induced responses of antioxidant enzymes in fish: (A) superoxide dismutase (SOD); (B) catalase CAT; (C)
glutathione peroxidase (GPOX); (D) glutathione reductase (GRED). %/%/, strong decrease ( B/20% of control); %/, decrease; $/, no (significant)
response; "/, increase; "/"/, strong increase ( !/500% of control).

Fig. 10. Frequencies of pollutant-induced responses of fish biotransformation products, serum transaminases and morphological parameters: (A)
fluorescent PAH metabolites in bile (bile FAC); (B) DNA adducts; (C) alanine transaminase (ALT) in plasma; (D) aspartate transaminase (AST) in
plasma; (E) liver somatic index (LSI); (F) condition factor CF. %/%/, strong decrease ( B/20% of control); %/, decrease; $/, no (significant) response;
"/, increase; "/"/, strong increase ( !/500% of control).
106 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

cycling. Redox-active compounds include aromatic diols tathione dependent peroxidases (GPOXs). SODs are
and quinones, nitroaromatics, aromatic hydroxyla- considered to play a pivotal antioxidant role; their
mines, bipyridyls and certain transition metal chelates importance is indicated by their presence in all aerobic
(Winston and Di Giulio, 1991). In the redox cycle, the organisms examined (Stegeman et al., 1992). Addition-
parent compound is typically first enzymatically reduced ally, the rate of SOD-catalysed O2%+ dismutation
by a NADPH dependent reductase (such as cyt P450 approximates the diffusion limit, making it one of the
RED) to yield a xenobiotic radical. This radical donates most active enzymes described (Fridovich, 1986). Most
its unshared electron to molecular O2, yielding O2%+ and techniques for the measurement of SOD activity are
the parent compound. Thus, at each turn of the cycle, indirect assays in which an indicating scavenger com-
two potentially deleterious events have occurred: a petes with endogenous SOD for O2%+ . A unit of SOD
reductant has been oxidized and an oxyradical has activity is defined as the amount that causes 50%
been produced (Winston and Di Giulio, 1991; Goeptar inhibition of the reduction of the scavenger under
et al., 1995). specified conditions (Sazuka, 1989; Stegeman et al.,
Oxidant-mediated effects with a potential suitability 1992). With the use of certain inhibitors the activities of
as biomarkers include either adaptive responses, such as SODs with different metal centers can be distinguished.
increased activities of antioxidant enzymes and concen- Some laboratory studies have reported an increased
trations of non-enzymatic compounds, or manifesta- SOD activity in fish exposed to paraquat, 2,3,7,8-TCDF
tions of oxidant-mediated toxicity such as oxidations of or HCB-contaminated food, but most studies could not
proteins, lipids and nucleic acids, as well as perturbed demonstrate any significant alterations (Table 9). In
tissue redox status (Winston and Di Giulio, 1991; Filho, eight of the 11 field studies considered, however, a
1996). Defence systems that tend to inhibit oxyradical significant increase of hepatic SOD activity was ob-
formation include the antioxidant enzymes such as served, i.e. in brown bullhead, carp, dab, grey mullet,
superoxide dismutase (SOD), catalase (CAT), glu- Nile tilapia, red mullet, sardine and spot from polluted
tathione-dependent peroxidase (GPOX) and glutathione environments (Table 10). Significant decreases in SOD
reductase (GRED). SOD, CAT and GPOX are critically activity were only observed in rainbow trout exposed to
important in the detoxification of radicals to non- BKME-containing sediments or paraquat (Table 9). The
reactive molecules. Numerous low-molecular-weight SOD responses for all fish species from 18 laboratory
antioxidants, such as GSH, b-carotene (vitamin B), studies and 11 field studies are summarized in Fig. 9A.
ascorbate (vitamin C), a-tocopherol (vitamin E) and A significant increase in SOD activity was observed in
ubiquinol10 have been described (Stegeman et al., 1992; 22% of the laboratory studies and 73% of the field
Lopez-Torres et al., 1993). In aquatic ecosystems, studies, while a strong increase (!/500% of control) was
dissolved oxygen and temperature are environmental not observed in any of the laboratory or field studies.
variables that are likely to influence oxidative processes More information is required before the hepatic SOD
(Parihar et al., 1997). These variables must be carefully activity in fish can be considered a valid biomarker for
controlled in laboratory experiments examining oxida- ERA. The literature survey revealed a notable difference
tive stress, and similarly considered in field studies between responses observed in laboratory studies and in
including this phenomenon in aquatic animals (Winston the field. A significant SOD induction was observed in
and Di Giulio, 1991). Differences in antioxidant capa- most of the reported field surveys, while most of the
cities were observed between rainbow trout that were laboratory studies did not report any significant re-
adapted to seawater or freshwater (Kolayli and Keha, sponses (Table 9).
1999). Species differences in the efficiency of antioxidant
defenses may partly explain prevalence of pathological 6.3.2. Catalase (CAT)
lesions observed in certain species of fish (Vigano et al., CATs are hematin-containing enzymes that facilitate
1998). In experiments with hepatocytes of male and the removal of hydrogen peroxide (H2O2), which is
female flounder it was demonstrated that many re- metabolized to molecular oxygen (O2) and water. Unlike
sponses to oxidative stress were sex-related (Winzer et some peroxidases that can reduce various lipid peroxides
al., 2001). The pollutant-induced responses of fish as well as H2O2, CATs can only reduce H2O2 (Stegeman
antioxidant enzymes in laboratory and field studies are et al., 1992; Filho, 1996). It was demonstrated that
listed in Tables 9 and 10, respectively. peroxisome-proliferating compounds (a class of non-
genotoxic carcinogens) induce both the activities of
6.3.1. Superoxide dismutase (SOD) H2O2-generating fatty acid oxidases and CAT in rodents
The SODs are a group of metalloenzymes that (Reddy and Lalwani, 1983; Halliwell and Gutteridge,
catalyse the conversion of reactive superoxide anions 1999). Since CATs are localized in the peroxisomes of
(O2%+ ) to yield hydrogen peroxide (H2O2), which in most cells and are involved in fatty acid metabolism,
itself is an important ROS as well. H2O2 is subsequently changes in activities may often be difficult to interpret
detoxified by two types of enzymes: CATs and glu- (Stegeman et al., 1992). Therefore, CAT activities in
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 107

erythrocytes may be a more appropriate marker for few field experiments reported, showed a significant
oxidant exposures in vertebrates. A transitory increase increase in hepatic GPOX activity only in grey mullet
in erythrocyte CAT activity was observed in crucian and chub from polluted sites (Table 10). A significant
carp exposed to paraquat (Gabryelak and Klekot, decrease in GPOX activity was observed in three field
1985). A commonly employed assay for the measure- experiments, i.e. with rainbow trout, red mullet and
ment of CAT activity follows the disappearance of seabream exposed at contaminated sites (Table 10). The
exogenous H2O2 spectrophotometrically (Sazuka, 1989; GPOX responses for all fish species from 14 laboratory
Stegeman et al., 1992). studies and 14 field experiments are summarized in Fig.
Increases in hepatic CAT activity were only observed 9C. A significant increase in GPOX activity was
in some experiments with fish exposed to PCBs, BKME observed in 43% of the laboratory studies and 14% of
or PAH-containing sediments, but most laboratory the field studies, while a strong increase (!/500% of
studies could not demonstrate any significant alterations control) was not observed in any of the laboratory or
(Table 9). Increased hepatic CAT activity was, however, field studies considered.
demonstrated in six of the 11 field studies considered The available information suggests that GPOX activ-
(Table 10). A significantly decreased CAT activity was ity in animals may be less responsive to pro-oxidants
observed some lab studies after PAH, DNOC, 3,3?,4,4?- than AsPOX in plants (Stegeman et al., 1992). More
TCB, engine exhaust extracts or cadmium exposure research is required to determine the potential utility of
(Table 9) and in a field study with Nile tilapia (Table GPOX activity in fish liver as a biomarker for ERA
10). The CAT responses for all fish species from 20 purposes.
laboratory studies and 11 field experiments are summar-
ized in Fig. 9B. A significant increase in CAT activity 6.3.4. Glutathione reductase (GRED)
was observed in 20% of the laboratory studies and 55% Although perhaps not involved in antioxidant defence
of the field studies, while a strong increase (!/500% of in the same way as the enzymes previously described,
control) was not observed in any of the laboratory or GRED merits attention because of its importance in
field studies considered. Similar as for SOD, more CAT maintaining GSH/GSSG homeostasis under oxidative
responses were observed in the field than in lab studies. stress conditions (Winston and Di Giulio, 1991). GRED
In general, CAT activity cannot be considered a valid catalyses the transformation of the oxidized disulfide
biomarker for ERA since both induction and inhibition form of glutathione (GSSG) to the reduced form (GSH),
are observed after exposure to environmental pollutants. with the concomitant oxidation of NADPH to
More research is required to elucidate the mechanism NADP". GRED activity can be measured spectro-
behind these effects on selected fish species. metrically by following the decrease in NADPH levels
(Worthington and Rosemeyer, 1974).
6.3.3. Glutathione peroxidase (GPOX) The responses of fish GRED to pollutants have
Peroxidases (POXs) are enzymes that reduce a variety apparently received little attention. An increased
of peroxides to their corresponding alcohols. While GRED activity was observed in laboratory experiments
CAT employs one molecule of H2O2 as donor in the with fish exposed to PCBs, PAHs, DDE and HCB-
reduction of another H2O2 molecule, peroxidases em- contaminated food (Table 9). A significant increase in
ploy other reductants. The principal peroxidase in fish is hepatic GRED activity was observed in two of the
a selenium-dependent tetrameric cytosolic enzyme that reported field experiments, i.e. with Atlantic salmon and
employs GSH as a cofactor. GPOX catalyses the shorthorn sculpin (Table 10). Significant decreases in
metabolism of H2O2 to water, involving a concomitant GRED activity were only observed in laboratory studies
oxidation of reduced GSH to its oxidized form (GSSG). with red mullet exposed to PCBs (Rudneva-Titova and
GPOX is considered to play an especially important role Zherko, 1994), and in a field experiment with Nile
in protecting membranes from damage due to LPOX. tilapia from a contaminated site (Bainy et al., 1996). The
This observation led to the view that the major GRED responses for all fish species from 11 laboratory
detoxification function of GPOX is the termination of studies and 11 field experiments are summarized in Fig.
radical chain propagation by quick reduction to yield 9D. A significant increase in GRED activity was
further radicals (Lauterburg et al., 1983). The use of observed in 55% of the laboratory studies and in 18%
peroxidases in plants may be used to detect early of the field studies, while a strong increase (!/500% of
oxidant responses, but the use of GPOX received control) was only observed in a laboratory study with
relatively little attention as a biomarker in animals PCB-exposed rainbow trout (Otto and Moon, 1995).
(Stegeman et al., 1992). The ratio between hepatic levels of reduced and
An increased GPOX activity was observed in experi- oxidized glutathione (GSH:GSSG) has been suggested
ments with fish exposed to paraquat, PAH (3MC), as a potential fish biomarker (Section 6.2.1). Research
PCBs and HCB-contaminated food, while a decrease on altered GRED activity may, therefore, be important
was only observed after 3MC exposure (Table 9). The in this context. The feasibility of GRED activity as a
108 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

biomarker in ERA processes, however, remains ques- bluegill sunfish exposed to anthracene and UV-light
tionable since the enzyme does not seem to be responsive (Choi and Oris, 2000). New trends in the demonstra-
to contaminant exposure in 82% of the considered field tion of LPOX by measurement of degradation
studies. products such as aldehydes, acetone and malondial-
dehyde have been described by De Zwart et al.
6.3.5. Non-enzymatic antioxidants (1997). LPOX appears to have considerable potential
The potential use of levels of reduced and oxidized as a biomarker for ERA (Stegeman et al., 1992; Hai et
glutathione (GSH and GSSG) as biomarkers has been al., 1995), although it can occur as a consequence of
discussed under the phase II cofactors (Section 6.2.1). cellular damage due to a variety of insults other than
Ascorbate (vitamin C) is an important water-soluble exposure to xenobiotics causing oxidative stress
antioxidant, and may additionally serve as a cofactor for (Kappus, 1987).
enzymes involved in collagen biosynthesis or neuro- . A recently developed analytical method for measur-
transmitter conversions (Stegeman et al., 1992; Lopez- ing and quantifying the capability of biological
Torres et al., 1993). Studies addressing the feasibility of samples to neutralize ROS is the so-called total
ascorbate as a biomarker are very scarce. The utility of oxyradical scavenging capacity (TOSC) assay (Win-
ascorbate as a biomarker is limited to plants and ston et al. 1998; Regoli et al., 2000). By generating
animals that can synthesize it. In a large-scale field different ROS at a constant rate, the assay provides
study in Sweden, perch inhabiting waters contaminated an index of specific biological resistance to various
with BKME consistently displayed higher ascorbate types of ROS. Since the relative efficiency of anti-
concentrations than fish from a reference site (Anders- oxidants may considerably vary towards different
son et al., 1988). a-Tocopherol (vitamin E) is a lipid- ROS, the TOSC assay has been standardized for
soluble antioxidant that is synthesized by plants, but measuring the scavenging capacity of cellular anti-
required in the diets of animals (Stegeman et al., 1992). oxidants with respect to various ROS (Regoli and
This compound appears to play a major role in Winston, 1999; Winzer et al., 2001).
protecting membranes from LPO. Its direct application . DNA oxidation may be the result of OH+ attack at
as a biomarker for ERA is probably restricted to plants; various sites of DNA bases, thus generating hydro-
however, as is the case for ascorbate, measurements of xylated bases (Stegeman et al., 1992; Chipman et al.,
animal tissue levels may sometimes be useful. 1998). A sensitive but relatively complicated method
of detecting these DNA alterations involves HPLC
6.3.6. Biochemical indices of oxidative damage separation and electrochemical detection. Malins et
A large number of biochemical and physiological al. (1990) observed oxidized guanine bases in DNA of
effects have been associated with increased fluxes of hepatic neoplasms of English sole, which were not
oxyradicals. Some biochemical perturbations that seem detected in non-neoplastic livers. Treatment with t-
particularly promising in relation to biomarkers are butyl hydroperoxide alone or in combination with
LPOX, the total oxyradical scavenging capacity (TOSC) GSH depletion, however, did not affect levels of
assay, DNA oxidation, methemoglobinemia and redox DNA oxidation in channel catfish and brown bull-
status. head, indicating that these species are relatively
insensitive for this effect (Ploch et al., 1999). Pollu-
. Lipid peroxidation, or the oxidation of polyunsatu- tant-mediated effects on DNA integrity are discussed
rated fatty acids is a very important consequence of in more detail under the genotoxic parameters (Sec-
oxidative stress and has been investigated extensively tion 6.10).
(Stegeman et al., 1992; Hageman et al., 1992). The . Several compounds (such as aromatic hydrazines,
process of LPOX proceeds by a chain reaction and, quinones, nitrite and some transition metals) have
as in the case of redox cycling, demonstrates the been shown to enhance the formation of methemo-
ability of a single radical species to propagate a globin (MetHb), a form of hemoglobin in which the
number of deleterious biochemical reactions. The heme iron is in the oxidized state (Fe3") and which is
actual chemistry of LPOX and associated production unable to bind and transport O2 (Stegeman et al.,
of various free-radical species is extremely complex 1992; Gonzalez et al., 2000). Increases in MetHb
(Kappus, 1987) and beyond the scope of this discus- formation have been observed in channel catfish
sion. Numerous studies have demonstrated enhance- exposed to naphthoquinones (Andaya and Di Giulio,
ments of LPOX in various tissues from fish species 1987) and in feral perch inhabiting BKME-contami-
exposed in vivo to a variety of chemicals, e.g. nated waters (Andersson et al., 1988).
paraquat-exposed carp (Gabryelak and Klekot, . Oxyradical-generating compounds can influence the
1985), channel catfish and brown bulhead exposed redox status of cells by imposing a drain on
to t-butyl hydroperoxide (Ploch et al., 1999), sea bass intracellular reducing equivalents, potentially affect-
exposed to heavy metals (Romeo et al., 2000) and ing a variety of metabolic processes (Stegeman et al.,
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 109

1992). These effects were discussed previously under Varanasi, 1991; Varanasi and Stein, 1991). Some
phase II cofactors (Section 6.2.1). PAHs are excreted as polar metabolites via the gall-
bladder (in bile), but most PAHs are excreted after
6.4. Biotransformation products conjugation by phase II enzymes (Vermeulen et al.,
1992). Metabolite levels in bile can be determined either
The exposure to xenobiotic compounds which are by analyzing the total level of PAH metabolites as
easily biodegradable in fish, such as PAHs and aromatic fluorescent aromatic compounds (FAC), or by selecting
amines, generally cannot be assessed by simply measur- a single metabolite as a marker for total PAH metabo-
ing their tissue levels (Melancon et al., 1992). Depending lism. 1-Hydroxypyrene (1-OH pyrene) has been selected
on the xenobiotic, its metabolism may lead to a form for this purpose because, first of all, relatively high levels
that is more easily monitored and thus may be used as a of pyrene have been detected in most sediments;
biomarker of exposure. Biomonitoring via metabolites secondly, pyrene is biotransformed predominantly into
of xenobiotic chemicals requires knowledge of the extent a single, strongly fluorescent metabolite (1-OH pyrene);
of metabolism and the types of metabolites of a and thirdly, the bioavailability of pyrene is relatively
particular compound produced by an organism. It is, high for aquatic organisms (Ariese et al., 1993a). 1-OH
however, beyond the scope of this section to review the pyrene, accounts for a large percentage of the total PAH
metabolism of all xenobiotic chemicals. Substances like metabolites in the bile of PAH-exposed fish (Krahn et
PCBs and DDTs are very resistant to metabolism in fish al., 1987). PAH metabolites can be determined using
(Melancon et al., 1992; Van der Oost et al., 1996a). synchronous fluorescence spectrometry (SFS), fixed
PAHs, on the other hand, can be biotransformed by wavelength fluorescence (FF) or HPLC (Aas et al.,
many aquatic organisms, but most effectively in the liver 2000). The results of the 1-OH pyrene SFS determina-
of fish. PAHs are metabolized by the phase I enzymes to tion in fish bile agreed very well with HPLC/fluores-
more hydrophilic products such as phenols, dihydro-
cence data (Ariese et al., 1993b). Generally, the levels of
diols, quinones and epoxides (Lech and Vodicnik, 1985;
bile metabolites are indicative of short-term exposure (1
Bucheli and Fent, 1995). The MFO system may thus
week), and therefore, provide information on recent
efficiently detoxify a large number of xenobiotics by
exposure only. It was demonstrated that the PAH
converting the parent compounds to easily excretable
metabolite levels in bile as well as the bile volumes
products. However, not only detoxification occurs, since
were highly influenced by the feeding status of the fish
certain compounds may be biotransformed to reactive
(Collier and Varanasi, 1991; Brumley et al., 1998). In
intermediates that are highly toxic, mutagenic or
order to reduce variations in PAH metabolite bile levels
carcinogenic to the fish. The oxidative metabolism of
due to feeding status, they proposed a procedure in
PAHs, for instance, proceeds via highly electrophilic
intermediate arene oxides, some of which bind cova- which the metabolite concentrations are related to the
lently to cellular macromolecules such as DNA, RNA, biliary pigment contents.
and protein (Neff, 1985). It is generally accepted that A strong increase (!/500% of control) in the biliary
metabolic activation by the MFO system is a prerequi- levels of PAH metabolites has been observed in most of
site for PAH-induced carcinogenesis (Van Schooten, the laboratory studies in which various fish species were
1991). Pulp and paper mill effluents generally contain exposed to PAHs (Table 11). These results are con-
high concentrations of resin and fatty acids, as well as firmed by field studies, in which a significant increase of
chlorophenolics. Elevated fish bile levels of these sub- FAC levels was observed in bile of fish from polluted
stances have been used as biomarkers (Leppänen et al., environments (Table 12). A significant decrease in FAC
1998). The possible uses of metabolites of both xeno- levels was not observed in any of the laboratory or field
biotic chemicals (PAHs, chlorinated phenols, resin studies (Tables 11 and 12). The FAC responses for all
acids, etc.) and endogenous metabolites (vitellogenin fish species from 15 laboratory studies and 24 field
(VTG), GSH, porphyrins, reproductive hormones, etc.) studies are summarized in Fig. 10A. A significant
as biomarkers for exposure and/or effects have been increase in biliary FAC levels was observed in 93% of
reviewed in more detail by Melancon et al. (1992). The the laboratory studies and 79% of the field studies, while
formation of DNA adducts, together with other DNA strong increases (!/500% of control) were observed in
alterations, will be discussed under the genotoxic para- 87% and 46% of the laboratory and field studies,
meters (Section 6.10.1). respectively.
Levels of biliary PAH metabolites are certainly
6.4.1. PAH metabolites in bile sensitive biomarkers to assess recent exposure to
In order to determine the exposure to and possible PAHs. Since PAH exposure cannot be reliably deter-
effects of PAHs in fish, the fate of PAH metabolites mined by measuring fish tissue levels, this parameter is a
should be investigated so as to quantify the PAH flux valid fish biomarker for ERA processes concerning
(uptake and excretion) in the animals (Collier and PAH-contaminated sites.
110 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

6.5. Stress proteins, metallothioneins and multixenobiotic However, little is yet known about the environmental
resistance relevance of stress protein responses in fish exposed to
environmental contaminants. The baseline features of
Environmental stress, as well as a variety of physical stress proteins or their responses towards various
conditions, may sometimes induce the synthesis of stressors (such as heavy metals) have been characterized
certain proteins in fish. Some of these proteins are in a number of laboratory studies with fish and fish cell
believed to play a role in protecting the cell from the cultures (e.g. Kothary and Candido, 1982; Kothary et
damage, which may result from environmental pertur- al., 1984; Chen et al., 1988; Misra et al., 1989; Dyer et
bations, while others are involved in the regulation of al., 1993; Sanders et al., 1994; Grøsvik and Goksøyr,
various genes. The best-known representatives of this 1996; Janz et al., 1997; Rabergh et al., 2000), but thus
group are the stress proteins (also known as heat shock far very few studies have been reported in which stress
proteins, (HSPs)), metallothioneins (MTs) and multi- proteins were used as biomarkers in field situations (e.g.
xenobiotic resistance (MXR) transmembrane proteins. Sanders and Martin, 1993; Triebskorn et al., 1997). The
latter study revealed that HSP70 induction is more
6.5.1. Stress proteins (HSPs) suitable as a biomarker of exposure at low temperatures.
The stress proteins comprise a set of abundant and More research in both the laboratory and the field will
inducible proteins which are involved in the protection be required before their usefulness as biomarkers in
and repair of the cell in response to stress and harmful ERA monitoring programs can be accurately evaluated.
conditions, including high or low temperature, ultravio- The mechanisms linking tissue level stress responses and
let light, oxidative conditions, anoxia, salinity stress, impairment of function at the organismal level will also
heavy metals, and xenobiotics such as teratogens and be important for this evaluation (Stegeman et al., 1992).
hepatocarcinogens (Stegeman et al., 1992; Di Giulio et Since nutrition and water quality (pH, temperature,
al., 1995). They are part of the cell’s strategy to protect salinity and dissolved oxygen) can affect the HSP
itself from damage. The stress protein response includes response, these factors should be monitored during
two major, closely related groups of gene products: the laboratory experiments and field collections.
HSP group and the glucose-regulated protein (GRP)
group. Synthesis of HSPs is dramatically increased by 6.5.2. Metallothioneins (MTs)
exposure to heat and other physical and chemical MTs constitute a family of low-molecular-weight,
stresses, while synthesis of GRP is increased in cells by cysteine-rich proteins functioning in the regulation of
such factors as deprivation of glucose or oxygen (Stege- the essential metals Cu and Zn, and in the detoxication
man et al., 1992). Sanders (1990) proposed a third class of these and other, non-essential, metals such as Cd and
of stress proteins, the stressor-specific stress proteins. Hg (Roesijadi and Robinson, 1994). The cellular inter-
These proteins appear to participate in specific bio- actions involving MTs are expected to follow two
chemical pathways involved in the metabolism of general lines, the first being the interception and binding
chemicals, metabolites or harmful by-products that are of metal ions that are initially taken up by the cell and
the result of a particular chemical or physical condition the second being the removal of metals from non-
rather than being a part of the cell’s protective system in thionein ligands that include cellular targets of toxicity.
response to general cellular damage. Two of these The latter may represent a detoxication function for
stressor-specific stress proteins are well characterized: structures, which have been reversibly impaired by
heme oxygenase and MTs (see Section 6.5.2). Each inappropriate metal binding. The role of MTs in
stress protein comprises a multigene family in which sequestering metals is well established, while their
some proteins, often called cognates, are constitutively induction by exposure to a wide variety of metals (e.g.
expressed while others are highly inducible in response Cd, Cu, Zn, Hg, Co, Ni, Bi, and Ag) is associated with
to environmental stresses. The cognates play a role in their protective function (Stegeman et al., 1992; Viar-
basic cellular physiology, and are present in the cell engo et al., 2000). Studies on the regulation of MT gene
under normal conditions (Sanders, 1990). expression provided evidence that induction by metals is
From a mechanistic viewpoint, the strategy of select- a direct response to increases in the intracellular metal
ing potential stress protein biomarkers based on the concentration which is mediated through the action of
molecular mechanisms underlying protection against metal-binding regulatory factors (Thiele, 1992). The
environmentally induced damage is sound and offers capacity for MT induction is greatest in tissues that
promise for identifying environmentally relevant bio- are active in uptake, storage and excretion, e.g. the small
marker assays (Stegeman et al., 1992). The regulation of intestine, liver and gills of fish (Roesijadi and Robinson,
the heat shock transcriptional response has been re- 1994).
viewed by Morimoto (1998), who described the mechan- Since MTs have no known catalytic function, mea-
ism as a ‘crosstalk between a family of heat shock surements of their concentrations are based upon
factors, molecular chaperones, and negative regulators’. quantitative assays of the protein itself (Stegeman et
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 111

al., 1992). Several existing methods (e.g. ion-exchange concerning their normal physiological function and the
chromatography coupled with atomic adsorption spec- factors that control the levels of MT in selected species
trometry, metal substitution assays, polarographic and of fish needs to be extended.
immunochemical techniques) have been reviewed by
Engel and Roesijadi (1987). In several species of fish, 6.5.3. Multixenobiotic resistance (MXR)
MT levels have been demonstrated to increase in a dose- Many fish are able to survive in environments
responsive (e.g. George and Young, 1986; George, 1989; containing high levels of multiple anthropogenic pollu-
Hogstrand and Haux, 1991; George et al., 1992a; tants or natural product toxins. Tissue levels of certain
Castaño et al., 1998) or a time-responsive (Beyer et al., contaminants are often maintained at levels below those
1997) manner after administration of heavy metals. The observed in the environment. It has been suggested that
potency of metals to induce MT may depend upon fish this fact is mediated by the MXR phenomenon, which is
species, tissue and experimental conditions. In gill tissue similar to the multidrug resistance (MDR) phenomenon
of carp, for instance, the following order of potency for that was first observed in tumour cell lines resistant to
MT induction was determined: Hg !/Cd !/Ag !/Zn anti-cancer drugs (Juliano and King, 1976). The MXR
(Cosson, 1994). Several field studies with feral fish mechanism acts as an energy-dependent pump that
have supported the experimental data that MTs seques- removes both endogenous and xenobiotic chemicals
ter heavy metals and that MT levels correlate with tissue from the cell, thus preventing their accumulation and
levels of heavy metals (e.g. Roch et al., 1982; Roch and cytotoxic effects (Kurelec, 1992; Epel, 1998). The
McCarter, 1984; Olsson and Haux, 1986; Hylland et al., protein responsible for this transport function is the
1992; Schlenk et al., 1995; Olsvik et al., 2000). In transmembrane P -glycoprotein (PGP). PGPs are found
mammals, insects and crustaceans it was demonstrated endogenously in specialized epithelial tissues involved in
that MT is induced under many other conditions besides secretion and excretion, such as gut, liver and kidney, as
metal exposure (Stegeman et al., 1992; Muto et al., well as on endothelial cells of capillary blood vessels at
the blood #/brain barrier (Bard, 2000). The physiological
1999). Both glucocorticoid and peptide hormones have
importance of PGPs in mammals has been established
been found to induce MT synthesis, while factors like
with knockout mice, in which an increased accumula-
temperature and nutritional status affect binding of
tion of xenobiotics together with an elevated sensitivity
metals to MT. It has been suggested that MT synthesis
toward these chemicals was observed (Schinkel et al.,
may be reduced in the presence of high levels of organic
1994). PGPs are able to translocate a wide variety of
contaminants due to an increased demand for cysteine
structurally and functionally diverse substrates. These
residues for GSH synthesis. Estradiol and estrogenic
compounds tend to be moderately hydrophobic, planar,
PCBs appeared to inhibit cadmium-mediated MT in-
natural products, which are often substrates for or
duction in Arctic char (Gerpe et al., 2000).
metabolites of detoxification enzymes such as cyto-
Generally, the need to search for and validate
chromes P450 (Sharom, 1997). In addition to toxin
biomarkers for the potential deleterious effects of evasion, phospholipid and steroid transport, PGPs
(heavy) metals seems less important than for organic endogenous functions may include a role in develop-
xenobiotics, since metals are a relatively small group of ment and osmotic control (Bard, 2000). The PGP-
chemicals which can be easily detected by chemical mediated MXR in aquatic organisms was first reviewed
analyses. On the other hand, it is quite clear that by Kurelec (1992). In order to place ecotoxicological
knowledge of intracellular metal compartmentation is data in context of the larger MDR field of study, Bard
essential to understanding the mechanisms of metal- (2000) recently reviewed the MXR as a cellular defence
induced cell injury (Fowler, 1987). The feasibility of mechanism in aquatic organisms.
MTs as biomarkers for metal exposure or metal-induced Numerous studies have reported induction of MXR
stress has been discussed by Stegeman et al. (1992). transport activity and elevated PGP levels in field
Given the extensive scientific information base and populations of pollutant exposed aquatic organisms or
available methods for measuring changes in MT synth- after laboratory exposures. Both natural products and
esis and its metal composition, these proteins show a anthropogenic contaminants found in the aquatic en-
strong potential for use as biomarkers for ERA of toxic vironment, as well as biotransformation products of
metals. Since MT isoforms seem to be differentially phase I enzymes, appear to be substrates and inducers of
induced by various heavy metals, MT isoform-specific the MXR transporter in aquatic organisms (Bard, 2000).
nucleotide probes to quantify the expression of these PGP induction may be a generalized response to
isoforms need to be developed. The biological function stressful conditions such as xenobiotic exposure or
of MT is by no means fully understood. It is, therefore, cellular injury. Elevated P -glycoprotein expression
still not possible to link changes in MT levels to injury at may occur via multiple mechanisms, including gene
the cellular or organismal level. Before MTs can be used amplification, transcriptional and post-transcriptional
to assess organismal health or fitness, the knowledge controls (Roninson, 1992). The factors that regulate
112 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

PGPs are not fully understood. Recent studies suggest variability in hepatic PGP levels in feral fish suggests
that PGP expression may be regulated by protein kinase that fish may have variable abilities to respond to PGP
C (PKC) mediated phosphorylation (Chaudhary and inducers (Bard et al., 1998).
Roninson, 1992), thyroid hormones, and cystic fibrosis The discovery of the MXR mechanism in aquatic
transmembrane conductance regulator (CFTR) protein organisms may have important implications on ERA
expression (Bard, 2000). In addition to well character- studies, since it interferes with important phenomena
ized detoxification systems (phase I, II and III enzymes, such as uptake, bioavailability, bioaccumulation and
HSPs, etc.), the induction of a multixenobiotic defence toxicity of xenobiotic chemicals and their metabolites.
mechanism in organisms living in polluted environments However, it has yet to be established how the induction
may explain why contaminant spills cause more severe and inhibition of MXR activity can be applied as a
adverse affects at pristine sites than in already polluted biomarker of environmental pollution. Therefore, much
areas (Bard, 2000). work remains to be done to characterize the function of
The protective role of the MXR defence mechanism PGPs in wild fish populations, and to determine how
appears to be fragile. As opposed to MXR induction, it PGPs interact with other detoxification systems. The
was also demonstrated that many classes of chemicals, literature on this topic indicates that MXR expression in
referred to as ‘chemosensitizers’, are capable of compe- aquatic organisms other than fish, such as bivalves,
titively inhibiting the MXR function (Kurelec, 1997). worms and sponges, can be reliably used as an indicator
Since these chemicals may block one of the basic of contaminant exposure and/or resistance.
biologic defence mechanisms and cause reversion of
natural resistance to pathobiologic sensitivity, they 6.6. Haematological parameters
should be considered as environmentally hazardous
chemicals. MXR-inhibition causes increased bioaccu- Especially in relation to a reduction in the use of test
mulation of xenobiotics and, therefore, elevated internal animals, haematological components may be promising
levels of toxins that may exert cytotoxic, genotoxic or fish biomarkers. Blood samples can regularly be ob-
neurotoxic effects at environmental levels not otherwise tained from test organisms, thus allowing the use of a
considered harmful. Due to the clinical importance of non-destructive approach in effect assessment. Typi-
acquired MDR in cancer cells, many studies were cally, haematological parameters are non-specific in
performed in order to discover novel agents that inhibit their responses towards chemical stressors. Nevertheless,
P -glycoprotein-mediated efflux of cytotoxic drugs (Sar- they may provide important information in effect
kadi and Muller, 1997). A wide variety of compounds assessment studies, e.g. by providing an indication as
have been shown to reverse MDR in vitro (Kurelec, to the general physiology and health status of the
1997). Several methods have been described to test organism under investigation (Beyer, 1996).
environmental samples for the presence of MXR-
inhibiting substances (Smital and Kurelec, 1997). 6.6.1. Serum transaminases
Examples of interactions of responses on PGP and The aminotransferases, alanine transaminase (ALT or
cytochromes P450 are reported for intertidal fish GPT) and aspartate transaminase (AST or GOT),
(Anoplarchus sp. ), upon field and laboratory exposures constitute a group of enzymes that catalyze the inter-
to crude oil, containing substances that are inducers for conversion of amino acids and a-ketoacids by transfer
both CYP1A and PGP (Bard et al., 1998). After 3 weeks of amino groups. The a-ketoglutarate/L-glutamate cou-
of exposure to contaminated sediments and food PGP ple serves as an amino group acceptor and donor pair in
expression was demonstrated in bile canaliculi. PGP amino-transfer reactions (Moss et al., 1986). ALT
expression was highly correlated to hepatic CYP1A in catalyses the transfer of the amino group from alanine
these fish. In another study, in which carp were exposed to a-ketoglutarate to form glutamate and pyruvate,
to water with low concentrations of diesel-2 oil induc- while AST catalyses the transfer of the amino group
tion of CYP1A was stimulated when the PGP mechan- from aspartate to a-ketoglutarate to form glutamate and
ism was inhibited in the presence of 20 mM verapamil, a oxaloacetate (Moss et al., 1986).
well-known MXR inhibitor (Kurelec, 1995). In marine An increase of enzyme activity in the extracellular
field study it was demonstrated that the CYP1A fluid or plasma is a sensitive indicator of even minor
induction in grey mullet due to XAD-7 water concen- cellular damage since the levels of these enzymes within
trates was highly correlated with the expression of MDR the cell exceed those in the extracellular fluids by more
gene in sponges (Krasko et al., 2001). In some other than three orders of magnitude (Moss et al., 1986). The
studies with fish the relationship between CYP1A measurement of enzyme activities in the serum is,
induction and elevated PGP expression could not be therefore, frequently used as a diagnostic tool in human
demonstrated (Bard, 2000). Some mammalian studies medicine (Adolph and Lorenz, 1978; Goetz, 1980). Most
suggest that CYP3A and PGP may be co-induced in research on the use of serum transaminase activities as
some circumstances (Bard, 2000). The large individual an indicator of tissue damage has, therefore, been
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 113

performed on humans, since both ALT and AST Determinations of ALT and AST activities in blood
activities are of great clinical significance (Moss et al., plasma have incidentally been applied in fish research to
1986). Following myocardial infarction, an increased indicate bacterial, viral and parasitic infections, intox-
AST activity appears in the serum, as may be expected ications and water pollution (Bucher and Hofer, 1990).
from the relatively high AST concentration in heart However, the application of serum enzyme determina-
muscle. ALT levels are within normal ranges or are only tions in fish, as an indicator of chronic intoxication,
marginally increased in uncomplicated myocardial in- seems to be questionable. In general, necrotic cells do
farction, since the concentration of ALT in heart muscle not contribute to an increased enzyme activity in the
is only a fraction of that of AST. Although the serum serum, since the greater part of the increase originates
levels of both AST and ALT become elevated whenever from damaged but still living cells, and membrane
disease processes affect liver cell integrity, ALT is the defects causing increased permeability (Adolph and
more specific enzyme for the liver. In humans, elevations Lorenz, 1978). On the other hand, toxicants can also
of serum ALT activity are rarely observed in conditions inhibit the activity or synthesis of enzymes (Goetz,
other than parenchymal liver disease (Moss et al., 1986). 1980), resulting in decreased activities in the blood.
In viral hepatitis and other forms of liver disease Consequently, serum transaminases in fish cannot be
associated with hepatic necrosis, serum AST and ALT considered reliable biomarkers to assess the effects of
are elevated even before the clinical signs and symptoms chronic pollutant exposure in ERA. AST in fish plasma,
of disease appear. It has been suggested that serum however, may be a promising effect biomarker when
levels of GSTs (Section 6.2.2) may be used as an used in short-term caging experiments, as was demon-
alternative for AST and ALT activities to indicate tissue strated by Beyer et al. (1997), Van der Oost et al. (1998).
damage (Hayes and Pulford, 1995).
Not many of the field and laboratory studies con-
sidered reported on the activities of the serum transa- 6.6.2. Other haematological parameters
Utilization of the chemically induced alterations in
minases ALT and AST in fish blood. An increase in
the heme pathway as biomarkers of both exposure and
both serum ALT and AST activity was observed in
effect has been the subject of intense study over the past
experiments in which fish were acutely exposed to
20 years in mammalian toxicology. These studies have
domestic waste water, while after prolonged exposure
delineated a number of chemical-specific responses that
transaminase activities in blood plasma dropped to the
have proven extremely useful in the early detection of
control level (Bucher and Hofer, 1990). An increased
low-dose chemical effects in mammals (Stegeman et al.,
ALT level was also observed in PAH exposed bluegill
1992). Chemically induced disturbances in the heme
sunfish, while increased AST plasma levels were ob-
pathway, which is essential for the biosynthesis of
served in carp exposed to deltamethrin and in PAH-
haemoproteins (e.g. hemoglobin) and various cyto-
exposed flounder (Table 11). Other laboratory studies chromes (e.g. cyt P450), have been reviewed by Silber-
could not demonstrate any significant alterations in geld and Fowler (1987). In a number of fish species
plasma transaminase activities. Fish serum transaminase similar specific changes in heme pathway enzyme
activities were reported in few field experiments only. A activities have been observed following waterborne
significant increase in plasma AST activity was only exposure to lead or cadmium (reviewed by Stegeman
observed in flounder and carp, caged at polluted sites et al., 1992). Since a number of relationships between
(Table 12). A significant decrease in ALT activity was these indicators and cell injury are already known in
observed in roach exposed at a BKME-polluted site mammals, interpretation of the alterations in a given
(Table 12). The ALT responses for all fish species from heme pathway enzyme activity in relation to an injur-
five laboratory studies and three field experiments are ious process in fish will also be more reliable. Other
summarized in Fig. 10C. A significant increase in ALT haematological parameters, such as hematocrit, hemo-
activity was observed in three laboratory studies (60%) globin, corpuscular volume, corpuscular hemoglobin
and none of the field studies, while a strong increase (!/ concentration, plasma osmolality, plasma lipids, albu-
500% of control) was not observed in any of the studies min, total protein and glucose, are generally less specific
considered. The AST responses for all fish species from than the serum enzymes, and may also be influenced by
eight laboratory studies and seven field experiments are natural factors such as bacterial challenges (Iwama et
summarized in Fig. 10D. A significant increase in AST al., 1986). However, in specific situations, these para-
activity was observed in 38% of the laboratory studies meters may still be useful as biomarkers of toxicant
and in two field studies (29%), while a strong increase effects in fish (e.g. Van Vuren, 1986; Tort et al., 1987;
(!/500% of control) was only observed in one labora- Reddy et al., 1991; Boon et al., 1992; Ghazali, 1992;
tory study (13%) with PAH-exposed flounder (Beyer et Allen, 1993).
al., 1997) in and one field study (14%) with caged Particularly when measured in concert with other
flounder (Beyer et al., 1997). biomarkers with more specific response patterns, such as
114 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

CYP1A-mediated responses, blood parameters may be in the exposed animals. In addition, a decreased migra-
valuable biomarkers for effect assessment. tion of granulocytes was observed. Sex-related differ-
ences in the immune responses were evident in many
6.7. Immunological parameters parameters, e.g. in the number of blood ISC and splenic
ASC. The studies by Aaltonen et al. (2000a,b) suggest
The immune system comprises a network of cells that steroids may contribute to immunemodulation in
capable of rapid proliferation and differentiation, regu- fish. The causal relationships between immunotoxic
lated by a variety of soluble factors, and is closely pollutants and fish diseases as well as the ecological
integrated with other organ systems and functions. As significance of such effects in the field still remain
such, it is extremely vulnerable to insult from exogenous unclear. It is possible that immunosuppressive effects
chemicals, especially after chronic exposure or repeated of pollutants serve as causal factors in the origin of fish
short exposures (Weeks et al., 1992). In mammals it was diseases with multifactorial etiology, e.g. various
observed that both cell-mediated and humoral (anti- skin diseases such as lymphocystis, papillomas, fin
body-mediated) immunity may be depressed by pollu- erosion, fin rot and skin ulcers (Vethaak, 1993). In
tants such as PAHs, PCBs, PBBs, OCPs (e.g. dieldrin, certain situations, however, the opposite effect, i.e.
lindane, DDTs and HCB), organometals (e.g. methyl- protection of the fish against pathogens, may be
mercury and organotins) and heavy metals such as Pb observed after pollutant exposure (MacFarlane et al.,
and Cd (Vos et al., 1989). The immune system, there- 1986).
fore, was considered to be a promising field in which As with other biomarker responses, immune re-
new candidate biomarkers for environmental monitor- sponses provide an integrated measure of exposure
ing could be found. It should, however, be emphasized over time and may reflect the combined results of
that the immune system can be influenced by a large simultaneous exposure to several chemicals. It is, how-
variety of stressors, which implies that immunological ever, not possible to determine which chemical has
biomarkers may be useful and sensitive, but often non- caused the observed effect as none of the changes in
specific (reviewed by Weeks et al., 1992). immune function can be attributed to a specific com-
The immunology of fish species is less well-character- pound or class of chemicals (Wester et al., 1994). It
ized than in mammalian species, although the know should be taken into account that a number of other
ledge has increased rapidly in recent years. As in stresses, such as handling, transportation or social
mammals, the immune system biomarkers in fish are interactions among individuals, may cause immunolo-
considered to have considerable potential for applica- gical disturbances in fish as well. In addition, three main
tion in pollution biomonitoring (Wester et al., 1994). An categories of hormones (i.e. corticoids, catecholamines
increasing number of studies have demonstrated im- and opioid peptides) have direct and indirect effects on
mune effects in fish after exposure to environmental various aspects of the immune system (Weeks et al.,
pollutants (e.g. Robohm, 1986; Payne and Fancey, 1989; 1992). Although highly reliable and reproducible assays
Secombes et al., 1991; Pulsford et al., 1992; Zelikoff, of immune function exist, much research has to be
1993; Arkoosh et al., 1994; Dunier et al., 1994; Lemaire- carried out in order to establish the most responsive
Gony et al., 1995; Sanchez-Dardon et al., 1999; Aalto- immunological alterations and to validate their ecotox-
nen et al., 2000a,b). Several immunological parameters icological relevance. Nevertheless, immunological bio-
may potentially be used as biomarkers in fish, e.g. white markers have an important role to play in monitoring
blood cell (leukocyte) and lymphocyte status (measured the health of fish prior to the occurrence of devastating
as blood cell or differential counts), non-specific defence disease outbreaks and as early-warning indicators for
factors (such as lysosomal activity and levels of acute the potential harm of environmental chemicals. Due to
phase proteins in body fluids), weight and morphology the fundamental physiological role of the immune
of leukocyte producing organs (such as spleen, thymus system, any kind of impairment in the immunological
and kidney), melanomacrophage centers (number, size resistance of fish may be interpreted as an important
and histopathological examination), macrophage func- signal in ERA. Since many parameters of the immune
tion (chemotaxis, phagocytosis, pinocytosis and chemi- system are similar in different organisms, fish may serve
luminescence), increased susceptibility to bacterial as sentinels of potential environmental hazards for
infections, and others (reviews of Weeks et al., 1992; humans (Weeks et al., 1992).
Wester et al., 1994). In recent studies with roach exposed
to pulp and paper mill effluents (Aaltonen et al. 6.8. Reproductive and endocrine parameters
2000a,b), fish were immunized with bovine and
gamma-globulin (BGG) 3 weeks before sampling. Spe- Decreased reproductive capability in feral organisms
cific anti-BGG antibody secreting cells (ASC) and may be considered as one of the most damaging effects
immunoglobulin secreting cells (ISC) were suppressed of persistent pollutants released by man. A number of
and the plasma levels of anti-BGG antibody were lower xenobiotics with widespread distribution in the environ-
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 115

ment are reported to have endocrine activity which tion through hatching. These findings suggested that the
might affect reproduction and thus might threaten the responses in CYP1A activity interfered with the ability
existence of susceptible species (Colborn et al., 1993; of the general class of cyt P450 enzymes to regulate sex
Peterson et al., 1993; White et al., 1994). Animals at high steroids. Due to their broad substrate specificity, cyt
trophic levels, generally having limited reproduction P450 isoenzymes may accelerate testosterone clearance
rates, are likely to be the most vulnerable in this regard. rates and affect estradiol levels in contaminant-exposed
An example is formed by the indications that PCB fish (Spies et al., 1990). Binding of xeno-estrogenic
pollution of Swedish waters is linked to uterine occlu- xenobiotics (e.g. DDTs, HCHs and alkylphenols) to the
sions and stenoses in female ringed seal, resulting in estrogen receptor also causes estrogenic effects (Harri-
reproductive failure and, consequently, a rapid decrease son et al., 1995). A national fish survey in the US, which
of the seal population (Helle et al., 1976a,b). Another sampled fish from 25 streams in 13 states, suggests that
example is the occurrence of imposex (development of water-soluble pesticides such as atrazine and other
male sexual characteristics in female gastropods) in the herbicides in current use may be affecting the endocrine
common whelk due to organotin exposure (Mensink, systems of fish (Renner, 1997). The pesticides do not
1999). accumulate in fish, but lab studies demonstrated that
Effects on reproductive competence as a response to they are able to permanently change steroidal pathways.
pollution stress have also been demonstrated in fish. Although no cause and effect could be established, the
There is increasing evidence that low-level pollution may variation in unusual hormone levels in carp (determined
decrease the fecundity of fish populations, leading to a as the median ratio of 17b-estradiol to 11-ketotestoster-
long-term decline and eventually extinction of important one) appeared to be best explained by high concentra-
natural resources. This, together with overfishing, may tions of pesticides in the water. Reproductive steroids
account for a decline in major fisheries, such as those in were also affected in feral perch and roach exposed to
the North Sea, in the industrialized West. In an BKME effluents in Finland (Karels et al., 1998).
extensive review, Kime (1995) presented an overview The synthesis of VTG, a precursor of yolk proteins, is
of the effects of sublethal pollution, both industrial and affected by estradiol. It was demonstrated that PCB-
agricultural, on all aspects of fish reproduction, from exposed fish were less capable of producing VTG (Spies
gonadal development through to spawning, together et al., 1990). An impaired reproductive function due to
with a discussion of how some of these effects may be a decreased plasma VTG levels was reported for female
result of disturbance of the reproductive endocrine rainbow trout exposed to Cd (Haux et al., 1988). VTG
system. Evidence has been presented demonstrating synthesis can also be induced in male fish exposed to
that pollutant effects may occur at multiple sites of the endocrine disrupting chemicals such as alkylphenols,
reproductive system. They may cause lesions, hemor- thus leading to a so-called feminisation of male fish
rhage, or malformations in the gonads, pituitary, liver (Gimeno et al., 1996). The VTG response in fish may
and the brain. Production and secretion of hormones of thus be used as a sensitive biomarker of exposure to
the hypothalamus, pituitary, and gonads is usually estrogenic compounds. A pronounced increase in
inhibited and their metabolism by the liver can be plasma VTG levels in male fish was observed in many
altered. There is also a considerable literature on the laboratory studies (e.g. Sumpter and Jobling, 1995;
survival of eggs, larvae and fry, which are particularly Arukwe et al. 2000; Lindholst et al., 2000; Hemmer et
susceptible to pollutants, and may have a major impact al., 2001) and field experiments (e.g. Purdom et al.,
on population dynamics (Kime, 1995; Hugla and 1994; Mellanen et al., 1999; Lye et al., 1999; Larsson et
Thome, 1999). al., 1999). The latter field study revealed that a
Xenobiotics may reduce reproductive success of fish substantial part of the observed estrogenic effects was
by interacting directly with the germ cells (Armstrong, due to 17a-ethinyloestradiol, a synthetic oestrogen used
1990), resulting in a high rate of mitotic chromosome in contraceptives that was present in effluents of sewage
abnormalities (Crosby Longwell et al., 1992). Negative treatment plants receiving (mainly) domestic waste-
correlations between hatching success and ovarian tissue water. The occurrence of hermaphrodite fish in the sites
burdens of chlorinated hydrocarbons have been ob- receiving this type of waste water might thus be due to
served, for instance, in North Sea whiting and in the presence of (xeno-)estrogenic compounds in the
flounder from the Baltic Sea (von Wersternhagen et effluents (e.g. Sumpter, 1995; Jobling et al., 1998).
al., 1981; von Wersternhagen et al., 1989). Spies et al. However, vitellogenesis may also be affected by pollu-
(1988) investigated relationships between impairment of tants with known affinity for the estrogenic receptor,
the reproductive success of the starry flounder from the such as nonylphenol, bisphenol A, PCBs and PAHs.
Pacific coast of North America and environmental Nicolas (1998) reviewed the present understanding of
pollution. It was found that the hepatic CYP1A activity the effects of PAHs on vitellogenesis in fish. Another
was inversely related to the egg viability, the fertilization potential biomarker for estrogenic effects in male fish
success and the successful development from fertiliza- might be the induction of zona radiata proteins (ZRP),
116 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

also known as vitelline envelope proteins (Hyllner et al., neuromuscular systems (e.g. Murphy, 1986). It is,
1991). Recent studies with juvenile salmon indicated therefore, assumed that this enzyme is more important
that the ZRP response was more sensitive to various than the non-specific esterases. This assumption, how-
environmental pollutants than the VTG response (Ar- ever, can only be confirmed when the physiological
ukwe and Goksøyr, 1997; Arukwe et al., 2000), thus functions of non-specific esterases are elucidated more
providing a sensitive means of detecting exposure to fully.
environmental estrogens. Both VTG and ZRP can be Many organophosphate (OP) and carbamate pesti-
analyzed by measuring mRNA expression or plasma cides are reported to be effective ACHE inhibitors. The
protein levels. ACHE inhibition has, therefore, been used to assess the
After years of exposure to low levels of environmental nature and extent of the exposure of wildlife to
contaminants, the sexual competence of both male and agricultural and forest sprays (e.g. Greig-Smith, 1991;
female fish may be impaired, which might eventually Zinkl et al., 1991). Evidence for variation of ACHE in
lead to a decrease in fitness or even extinction of the the muscle tissues of dab and flounder was demon-
population. There is, surprisingly, little difference be- strated along a pollution gradient in the North Sea
tween the effects of different classes of pollutants (e.g. (Galgani et al., 1992). A significant depression of
heavy metals, organophosphorous pesticides, organo- ACHE-activity in fish from OP-polluted sites was also
chlorine pollutants and [poly-]aromatic compounds). observed in muscle tissues of brown trout and flounder
The literature covered in a review of Kime (1995) leaves in Newfoundland, Canada (Payne et al., 1996), in
no doubt that all types of pollutants have a serious muscle tissue of the three-spined stickleback from
inhibitory effect on fish reproduction, even when in parathion polluted streams Germany (Sturm et al.,
minute quantities. Taylor and Harrison (1999) presented 1999, 2000), and in brain tissue of menhaden and
an overview of the main evidence for endocrine disrup- mummichog from polluted rivers in South Carolina
tion in wildlife, focussing on reproduction effects. They (Fulton and Key, 2001). It was unknown whether
concluded that in most cases a causal link between the inhibition observed in field studies was due to pesticides,
observed abnormalities and chemical exposure has not other factors, or a combination of both. Payne et al.
been established. In addition they described priority (1996) suggested that complex mixtures of contami-
research projects for the UK, ultimately aimed at nants, other than pesticides, could be important sources
determining the population-level significance of endo- of ACHE-inhibiting compounds in the aquatic environ-
crine disruption. It is assumed that fish make excellent ment; they provided preliminary evidence for ACHE-
bioindicators of the harmful effects that might be inhibiting activity present in extracts of used engine oil
expected in mammals in general, and human popula- and wood leachate. Since the in vitro sensitivity of
tions in particular (Kime, 1995). It is, therefore, of ACHE and BCHE and their respective in vivo responses
paramount importance for a reliable ERA, that sensitive in the field differed significantly, these enzymes should
reproductive biomarkers have to be developed and be considered separately in studies with fish. In a study
validated for their ecotoxicological significance. An with juvenile rainbow trout it was demonstrated that
overview of the methods and strategies to monitor the malathion induced CHE inhibition correlated signifi-
impact of endocrine-disrupting chemicals is given by cantly with behavioral measures, such as deviations in
Sadik and Witt (1999). In vitro VTG assays may also be swimming speed and turning rate (Beauvais et al., 2000).
used to assess the estrogenic potency of newly developed Although a number of field studies have successfully
chemicals or existing environmental pollutants (Pellisero used CHE inhibition in fish as a biomarker of pesticide
et al., 1993; Folmar et al., 2000). exposure, more research is required before this para-
meter can be used in ERA programs. Additional
6.9. Neurotoxic parameters research is needed to better explain the species-specific
differences in the relationship between ACHE inhibition
With respect to neural functions, enzymes of interest and mortality and to investigate other physiological
are cholinesterases (CHE) (Payne et al., 1996). Two perturbations associated with ACHE inhibition (Fulton
types of CHE are recognized; firstly, those with a high and Key, 2001).
affinity for acetylcholin (ACHE), and secondly, those
with affinity for butyrylcholin (BCHE), also known as 6.10. Genotoxic parameters
non-specific esterases or pseudocholinesterases (Walker
and Thompson, 1991; Sturm et al., 2000). Fish brain The exposure of an organism to genotoxic chemicals
contains ACHE, but no BCHE, while muscle tissues may induce a cascade of events (Shugart et al., 1992).
contain both ACHE and BCHE (Sturm et al., 2000). Genetic ecotoxicology can be defined as the study of
ACHE is involved in the deactivation of acetylcholin at pollutant-induced changes in the genetic material of
nerve endings, preventing continuous nerve firings, biota in nature and has two aspects: (1) initially, the
which is vital for normal functioning of sensory and genotoxicity of pollutants, such as structural alterations
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 117

of the DNA, and (2) consequently, the procession and DNA adducts are generally determined in the liver,
expression of DNA damage in mutant gene products, since this is the key organ for biotransformation of
resulting in long-term heritable effects, such as changes xenobiotics. Levels of hepatic DNA adducts may be
in gene frequency within exposed populations, muta- indicative of cumulative exposure of fish to genotoxic
tional events, etc. (Shugart, 1996). The detection and compounds over a longer period of time (several
quantification of various events in this sequence may be months), as opposed to bile metabolites, which are
employed as biomarkers of exposure and effects in indicative of recent exposure (several days) (Varanasi et
organisms environmentally exposed to genotoxic sub- al., 1989b). In a recent study by Malmström et al. (2000)
stances. Within the discipline of genetic toxicology, it was demonstrated that DNA adducts could be
research has taken place at three levels (Maccubbin, detected in both liver tissue and leucocytes. However,
1994): the adduct levels in liver were higher and the adduct
patters were different in leucocytes. Further investiga-
#/ identifying the kind and determining the frequency of tions are needed to convincingly elucidate whether the
genetic diseases; use of non-invasive sampling methods will be appro-
#/ studying the mechanisms of how chemical and priate for environmental biomonitoring of DNA ad-
physical agents cause genetic disorders; ducts in fish. Currently, methods of varying sensitivity
#/ evaluating agents for their potential to cause genetic exist for the measurement of DNA adducts, including
damage. 32
P-postlabeling, HPLC/fluorescence spectrometry and
immunoassays using adduct-specific antibodies (Shugart
The study of DNA adducts in human and animal
et al., 1992). With the most sensitive method, the 32P-
models has been an important part of the latter two
postlabeling assay, it is possible to determine levels as
levels of research. A more general approach involves the
low as one adduct per 109 nucleotides. Even in wild
detection of DNA strand breaks that are produced,
brook trout from remote areas in Newfoundland (with-
either directly by the toxic chemical (or its metabolite) or
out a known input of pollutants) elevated DNA adduct
by the processing of structural damage (Shugart et al.,
levels were detected in liver and brain tissues (Ray et al.,
1992). DNA base composition, oncogene activation,
1995).
cytogenetic effects and tumorigenesis also have the
Since the formation of PAH #/DNA adducts is
potential to be used as biomarkers. The study of genetic
thought to be a necessary step in the carcinogenic action
toxicology in aquatic systems is mainly focused on
of PAHs, it is possible that there is a relationship
carcinogenesis in fish and shellfish.
between the amount of DNA adducts and cancer risk
(Dunn, 1991). There is a good (but not perfect)
6.10.1. DNA adducts correlation between the level of chemical binding to
Paradoxically, the same metabolic processes that are DNA and carcinogenic potency and organ specificity
responsible for the efficient elimination of biodegrad- (Maccubbin, 1994). In general, however, it is difficult to
able substances such as PAHs from the organism are demonstrate consistent quantitative relationships be-
also able to activate environmental carcinogens to tween DNA adduct levels in organisms, and formation
DNA-reactive forms (Dunn, 1991). In environments of tumours or histologically related lesions and environ-
with high PAH levels, greater P450 induction could mental contamination. Adduct formation is likely to be
contribute to higher steady-state levels of activated a complex function of various factors that may influence
carcinogens and, consequently, to a greater formation carcinogen metabolism (Dunn, 1991). Apart from
of DNA adducts or to enhanced oxidative DNA pollutant levels, these factors may be related to the age
damage (Stegeman and Hahn, 1994). Watson et al. and sex of the organisms, as well as to season, water
(1998) demonstrated that the induction of CYP1A (due temperature and food availability. Kurelec et al. (1989)
to BNF pre-treatment) significantly increased the in vivo indicated that the overwhelming majority of DNA
DNA adduct formation in channel catfish after expo- modifications in various freshwater fish species are
sure to 2-amino-anthracene (AA), indicating the invol- caused by natural factors rather than by man-made
vement of CYP1A in the bioactivation of AA. Similar chemicals, since they did not observe any statistically
observations were reported for CYP1A induced zebra- significant differences between the DNA adduct levels of
fish exposed to aflatoxin B1 (Troxel et al., 1997). fish from polluted and unpolluted sites. However, since
Greater P450 induction, however, is not necessarily statistically significant differences in hepatic DNA
associated with a greater risk of carcinogenesis, since adduct levels have been unambiguously demonstrated
the formation and persistence of critical genetic lesions in other field studies (Table 12), this theory is evidently
is also influenced by phase II defence and repair not valid for all fish species. Maccubbin (1994) reviewed
processes. It is, nevertheless, most likely that liver DNA adduct formation in fish, both in vitro and in
PAH-DNA levels in fish reflect the extent of exposure vivo, and concluded that fish have been shown to
to carcinogenic or mutagenic PAHs. activate carcinogens to products that form DNA
118 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

adducts, which are the same ones as described in rodent tive but much easier to perform, like the ELISA assay of
studies. Since both phase I and II enzyme activities may Van Schooten et al. (1992) which uses polyclonal
influence DNA adduct formation, a thorough charac- antibodies against benzo[a]pyrene-7,8-diol-9,10-epox-
terization of the metabolic pathways of the carcinogen is ide-DNA.
necessary to evaluate adduct formation relative to
species sensitivity (Maccubbin, 1994). In addition, the
6.10.2. Secondary DNA modifications
route and duration of the exposure are important
Exposure to toxic chemicals may cause secondary
aspects to be considered when attempting to extrapolate
concomitant types of DNA alterations. Besides direct
results from laboratory studies to real-world situations,
adduct formation, damage may include strand breaks in
in order to predict the ultimate biological effect of a
the DNA polymer, changes in the DNA’s minor base
chemical. With respect to field studies, more work is
composition or an increase in the level of DNA repair
needed in identifying adducts observed in wild fish.
(Shugart et al., 1992). Apoptosis, also known as
Both laboratory and field studies on DNA adduct
programmed cell death, is a physiological and irrever-
formation in fish have been reviewed by Pfau (1997). A
sible process in tissue homeostasis that leads to DNA
strong increase (!/500% of control) in the hepatic levels
fragmentation of multiples of 180 #/200 basepairs. Apop-
of DNA adducts was observed in most laboratory
tosis could be demonstrated by an increased number of
studies in which various species of fish were exposed
small DNA fragments in liver of dab exposed to PCBs
to PAHs (Table 11). These results are confirmed by a lot
and cadmium (Piechotta et al., 1999) and in ovarian
of field studies, in which a significant increase of DNA
follicular cells from prespawning white sucker exposed
adduct levels was observed in the liver of fish from
to BKME (Janz et al., 1997), thus indicating its
polluted environments (Table 12). A significant decrease
possibility to be used as a biomarker of effects
in DNA adduct levels was not observed in any of the
Many toxic chemicals cause strand breaks in DNA,
laboratory or field studies (Tables 11 and 12). The DNA
either directly or indirectly. Alkaline unwinding and
adduct responses for all fish species from 17 laboratory
COMET assays are able to estimate the increase in the
studies and 30 field studies are summarized in Fig. 10B.
level of breaks above background resulting from ex-
A significant increase in hepatic DNA adduct levels was
posure to these chemicals (Shugart, 1990a). This method
observed in 100% of the laboratory studies and 70% of
is well suited for routine, in situ monitoring of feral fish
the field studies, while strong increases (!/500% of
because of its ease, speed and low cost. In laboratory
control) were observed in 65% and 30% of the labora-
studies, an increase in strand breaks was detected in the
tory and field studies, respectively.
fathead minnow and the bluegill sunfish, which were
Due to the strong and consequent responses of
chronically exposed to waterborne benzo[a]pyrene (Shu-
hepatic DNA adduct levels to PAHs exposure, this
gart, 1988). The feasibility of utilizing this technique on
parameter is considered to be an excellent biomarker for
environmental species as a general biomarker for pollu-
the assessment of PAH exposure as well as a sensitive
tion-related genotoxicity is being evaluated (Everaarts et
biomarker for the assessment of potentially genotoxic
al., 1994).
effects. It is important to combine the measurements of
Chemical carcinogens have been shown to produce
DNA adduct formation as a molecular dosimeter with
hypomethylation of DNA as a result of their effect on
analysis of carcinogen metabolism and determination of
certain maintenance enzymes (Shugart et al., 1992). This
tumour formation to provide insights in the mechanisms
loss of DNA integrity can be easily measured using ion-
involved in chemical carcinogenesis (Maccubbin, 1994).
exchange chromatographic techniques. Hypomethyla-
This type of integrated study may be used in ERA to
tion, as measured by the loss of 5-methyl-deoxycytidine,
provide information about potential exposure and risk
has been demonstrated in fish exposed to benzo[a]pyr-
of environmental carcinogenesis that may be found in
ene (Shugart, 1990b). The onset and persistence of this
contaminated waterways. In addition to their use as a
phenomenon was found to be correlated with other
biomarker for exposure and effects of genotoxins, DNA
types of DNA-damaging events, such as strand breaks
adducts may provide information about the biological
and adduct formation.
effect and potential risk of a chemical, since it has been
suggested that any chemical that forms DNA adducts,
even at very low levels, should be considered to have 6.10.3. Irreversible genotoxic events
carcinogenic and mutagenic potential (Maccubbin, The consequence of structural perturbations to the
1994). The first criterion for a valid biomarker, i.e. the DNA molecule, such as adducts and secondary mod-
relative ease with which it can be measured, is not met ifications, may result in lesions that become permanent.
when DNA adducts are determined using the 32P- Affected cells often exhibit altered function indicative of
postlabeling assay, which is expensive and time con- a subclinical manifestation of genotoxic disease. These
suming. In this connection it might be interesting to irreversible genotoxic events have been reviewed by
improve immunological methods, which are less sensi- Shugart et al. (1992):
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 119

. Cytogenetic assays , like standard chromosome ana- As an overall conclusion, it can be stated that the
lysis, sister-chromatid exchange (SCE) and erythro- detection of structural DNA changes and ensuing events
citic nuclear abnormalities (ENA), are sensitive non- has been demonstrated and documented as a viable
specific indicators for mutagenic damage, which have scientific tool for in situ BM of the genotoxicity of
shown promising, results in laboratory experiments. chemicals in ERA.
An increase in ENA frequency was observed in eel
exposed to certain PAHs and resin acids (Pacheco
6.11. Physiological and morphological parameters
and Santos, 1997) and cyclophosphamide (Pacheco
and Santos, 1998). The micronucleus assay is less
As opposed to most of the biochemical parameters
sensitive than the former assays, but it reveals the
that have been discussed in this review, physiological
consequences of both spindle anomalies and chro-
and morphological parameters are higher-level re-
mosomal breakage and has been applied to feral fish.
sponses following chemical and cellular interaction,
Most of these techniques are labor-intensive, so they
which are generally indicative of irreversible damage
have limited utility as routine screening assays.
(Hinton et al., 1992). The main objective of this review is
. Flow cytometric measurement (FCM ) is a technique
to discuss the feasibility of biological parameters in
that measures several cellular variables, including
ERA, so that most emphasis is placed on the use of
DNA, RNA, protein, specific chemicals and numer-
biomarkers as an early-warning system for potential
ous morphological attributes in suspended cells. The
hazards due to environmental contaminants. Therefore,
efficiency of measuring the effects of chronic mutagen
the use of physiological and morphological parameters
exposure with this technique has been demonstrated
as biomarkers will be discussed only briefly.
in both laboratory and field studies. Due to its low
cost and high speed and sensitivity this method has a
tremendous potential for use as an initial screening 6.11.1. Histopathology
procedure. The main advantages and limitations of The feasibility of using histopathological parameters
flow cytometry for biochemical studies are reviewed in fish as a biomarker for aquatic pollution has been
by O’Connor et al., 2001. reviewed by Hinton et al. (1992), Hinton (1994).
. Oncogene activation and mutation rates are two Epidemiological studies on the occurrence of fish disease
parameters, which may be used in the future to assess in relation to their usefulness in monitoring marine
the impact of DNA mutations, although much pollution have been reviewed by Vethaak and ap
research is still needed to validate the assays for their Rheinallt (1992). They concluded that, on a worldwide
use as potential biomarkers. Oncogene activation is a scale, the most convincing examples of a causal relation-
DNA-based assay to measure specific nucleotide ship between fish disease and pollution was provided by
changes in oncogenes and other appropriate genes intensive and detailed studies carried out in North
of chemically exposed animals. The use of DNA America, particularly on liver pathology. Mix (1986)
sequencing and restriction fragment length poly- critically reviewed the neoplastic and cancerous diseases
morphism data in the study of environmental muta- in aquatic organisms and their relationship to environ-
gens has yet to be investigated. The p53 gene has been mental pollution. Only a small number of studies could
sequenced for several fish species with a view to the be identified in which the data were considered to
possible use of mutations in the highly conserved support an association between pollution and neoplasia
domains of p53 to identify genotoxins in the aquatic (Brown et al., 1977; Kimura et al., 1984; Malins et al.,
environment (Cachot et al., 1998; Bhaskaran et al., 1985; Myers et al., 1994). Triebskorn et al. (1997),
1999). Schramm et al. (1998) described methods to study the
liver ultrastructure using quantitative and semi-quanti-
The involvement of xenobiotic-metabolizing enzymes tative electron microscopy. The biomarker responses
in carcinogenesis has been demonstrated in some observed in these studies were correlated with the results
mammalian studies, which reported altered levels and obtained by behavioral, limnological and analytical
activities of these enzymes in preneoplastic and neoplas- investigations, and reflected the levels of pollution at
tic lesions (Stegeman et al., 1992). The decrease in phase each site. The species specific sensitivity for histopatho-
I enzymes involved in electrophile production from logical lesions were demonstrated in a study with
chemical carcinogens, combined with the increases in rubberlip surfperch and rainbow surfperch exposed to
phase II enzymes involved in the detoxification and a natural petroleum seep (Spies et al., 1996). Total lesion
elimination of electrophiles, suggests an adaptive re- scores were not different between two groups of
sponse to a toxic environment. In the few reported rubberlip surfperch, while pronounced differences in
studies with fish, however, GST levels in liver neoplasms gill lesions were observed between exposed and reference
were either depressed or unaffected (Stegeman et al., rainbow surfperch. It was suggested that certain lesions
1992). (e.g. abnormal branching of gill filaments) are biological
120 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

markers for xenobiotics that induce CYP1A (Spies et al., Several laboratory studies considered for this review
1996). reported a significantly increased LSI value in fish
It is generally assumed that histopathological bio- exposed to PCBs, OCPs, BKME, PCDDs and PAHs
markers are valuable as indicators of the general health (Table 11). Increased LSI values were also reported in
of fish and mirror the effects of exposure to a variety of field studies with bream, brown bullhead, chub, cod,
anthropogenic pollutants (Hinton et al., 1992). No dab, English sole, perch, plaice, shorthorn sculpin and
geographic or ecosystem limitations are apparent. Acute white sucker from contaminated sites (Table 12).
changes are seen when contaminant levels are suffi- Significantly decreased LSI values were observed in
ciently high, while chronic duration is required to laboratory studies with fish exposed to OCPs, PCBs and
determine sublethal aspects of change. Many alterations PAHs (BNF) and in field studies with redbreast sunfish
persist even after exposure to a toxicant has ceased so and tilapia at polluted sites (Tables 11 and 12). The LSI
that host responses to prior toxicity can also be used to responses for all fish species from 24 laboratory studies
determine effects. Responses are relatively easily recog- and 28 field studies are summarized in Fig. 10E. A
nized, provided that proper reference and control data significant increase in LSI values was observed in 38% of
are available. Sufficient information is at hand to the laboratory studies and 43% of the field studies, while
assemble cellular or histopathological biomarker ap- strong increases ( !/500% of control) were not observed.
proaches and to apply them in integrated field studies Bagenal and Tesch (1978) proposed a CF for fish,
(Hinton, 1994). based upon the ratio between body weight and length:
100 &/body weight (g)/(length (cm))3. This factor may be
6.11.2. Gross indices affected if the availability of food is limited or if the food
Gross indices are sometimes indicative of toxicant consumption of the fish is impaired due to stress factors.
effects (Mayer et al., 1992). Plant or animal condition as None of the laboratory studies considered for this
determined by morphology, appearance and other gross review reported a CF value that was significantly
characteristics should not be overlooked in assessing different from the controls (Table 11). Increased CF
contaminant impact. A first-level screen to identify values were reported in field studies with brown bull-
potential pollutant exposure and effect can be accom- head, dab, English sole and perch from contaminated
plished on the basis of overt and relatively simple sites (Table 12). Significantly decreased CF values were
measures of condition. Such measures may serve to only observed in a field study with white sucker
identify the most sensitive members of a fish population. (Hodson et al., 1992). The CF responses for all fish
In addition, they may provide information on energy species from 7 laboratory studies and 23 field studies are
reserves and possibly the ability of animals to tolerate summarized in Fig. 10F. A significant increase in CF
toxicant challenges or other environmental stresses values was observed in none of the laboratory studies
(Mayer et al., 1992). Morphological parameters that and 17% of the field studies, while strong increases (!/
are often determined in field research are the liver 500% of control) were not observed.
somatic index (LSI), to identify possible liver diseases, The condition of the liver and of the whole body, as
and the condition factor (CF), to assess the general measured with the LSI and CF values, can provide
condition of fish. information on potential pollution impacts. Although
The LSI is the ratio between the weight of the liver these parameters are not very sensitive and may be
and the total body weight of the fish: 100 &/liver weight affected by non-pollutant factors (e.g. season, disease,
(g)/body weight (g) (Slooff et al., 1983). Slooff et al. nutritional level), they may serve as an initial screening
(1983) summarized a number of studies, which sup- biomarker to indicate exposure and effects or to provide
ported the hypothesis that there was a causal relation- information on energy reserves (Mayer et al., 1992). The
ship between liver enlargement and exposure to condition indices are quite general and non-specific, but
chemical pollutants. Based on both biochemical ana- their low cost, ease and rapidity still make them valuable
lyses and histological observations it was concluded that ERA tools.
liver enlargement in bream from polluted sites was
mainly caused by hypertrophy (increase in cell size). In 6.12. Toxicological significance of fish biomarkers
contrast to this, Poels et al. (1980) found the liver
enlargement in young rainbow trout that were experi- A major challenge of biomarker development with
mentally exposed to polluted Rhine water to be the respect to ERA is to define the significance of biomarker
result of hyperplasia (increase in cell number). It was responses in terms of ecological effects of the pollutants.
suggested that this discrepancy might be explained by For ERA it is not sufficient to show that biomarker
age differences of the fish, since the fast growing liver levels differ among sites or even that a biomarker level is
tissue of juvenile fish will respond more readily by a abnormally high at a site (Suter, 1990). The relation-
hyperplasic reaction than the liver tissue of full-grown ships between responses at different levels of biological
fish. organization as well as the relevance and time scales of
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 121

these biomarker responses are illustrated in Fig. 11 . Induction of cyt P450 isozymes can alter the rates of
(Adams et al., 1989). Responses at each level provide endogenous substrate metabolism, either directly as
information that helps to understand and interpret the catalysts or indirectly by competition for reducing
relationship between exposure and adverse effects. It is equivalents from P450 RED. Since cyt P450 isozymes
generally accepted that ecological relevance is inversely are involved in the metabolism of steroid hormones,
related to criteria like sensitivity and specificity (De such effects may eventually result in reproductive
Zwart, 1995). Effects at a higher level of biological failure.
organization (population, community, etc.) have a high . Direct or indirect inhibition or inactivation of P450
biological and toxicological relevance, but may be by substrates or non-substrates can affect the capa-
insensitive due to the presence of alternative pathways city for xenobiotic metabolism or for endogenous
in an ecosystem. functions, depending on the P450 form inhibited.
The question whether or not biochemical alterations, . Failure to complete a catalytic cycle following sub-
like hepatic EROD induction, are indicative of the strate binding, electron transfer and O2 binding can
development of irreversible toxic effects after prolonged result in the formation and release of oxygen radicals,
or elevated exposure to the causative agents, is not easy which are toxic and mutagenic themselves.
. Receptor mechanisms involved in the regulation of
to answer. It is hard to predict, therefore, to what extent
some P450 genes can act in toxic mechanisms
the biochemical alterations in a certain population may
independent of the catalytic role of P450.
eventually influence the health of this population or the
entire ecosystem. Several comprehensive studies, how- Establishing the network of interactions between
ever, have demonstrated clear relationships between P450 enzyme function and/or regulation and other
environmental pollution and fish disease (Section molecular processes will reveal the full significance of
6.11.1). In a study with English sole from the Puget these enzymes in toxic mechanisms (Stegeman and
Sound area on the Pacific coast of the USA, for Hahn, 1994). Linkages exist between the P450 system
instance, cause-and-effect relationships could be demon- and other biochemical systems, including those invol-
strated between pollution levels (PAHs), exposure ving heme synthesis and degradation, HSPs, steroid
(biliary FACs), biomarker responses (increased receptors, antioxidant enzymes, MTs, oncogenes, tu-
CYP1A activities) and biological consequences, such mour suppressor genes, etc. (Fig. 12). These complex
as preneoplastic hepatic lesions and hepatic neoplasms interactions illustrate the difficulty of evaluating and
(Myers et al., 1994). Similar relationships were reported assessing the toxicological consequences of impaired
by Vethaak et al. (1996), who observed increased biliary biotransformation processes due to exposure to envir-
FAC levels, EROD induction and skin and liver diseases onmental pollutants. Chronically intoxicated fish can
in flounder after long-term exposure to PAH- and PCB- reduce the inflow of toxicants by building up morpho-
contaminated sediments in a mesocosm study. logical barriers and they may activate mechanisms of
One effect that has been clearly demonstrated to be a detoxification (Lindström-Seppä and Pesonen, 1986;
consequence of enzyme induction is an increased Andersson et al., 1988), which make the fish more
clearance of endogenous substrates (Stegeman and resistant to toxicants (Bucher and Hofer, 1990). More-
Hahn, 1994). There are, however, numerous possible over, many variables without any association to pollu-
toxic mechanisms involving altered levels and activities tion may have an additional impact on the various
of biotransformation enzymes and receptors. An exam- enzyme systems (see Section 6.13).
ple is the link between routes I and II of Fig. 6, i.e. There are indications that some phase II enzymes may
increased phase I biotransformation due to the induc- be induced by the same AhR-mediated mechanism as
tion of cyt P450 isozymes. Although primarily a path- the phase I enzymes (Owens, 1977; Pickett and Lu,
way for detoxification, there are various ways whereby 1989). The ultimate toxicity of any specific xenobiotic
cyt P450 activity or regulation can elicit toxic effects chemical is related to the status of induction of phase I
(Stegeman and Hahn, 1994): as well as phase II enzymes and to the balance between
bioactivation and detoxification reactions (i.e. the
. Many protoxicants, promutagens, and procarcino- balance between formation of toxic and non-toxic
gens are converted to reactive, toxic products by cyt metabolites) (Vermeulen, 1996). Since the extent of
P450. In some cases, if the capacity of the phase II phase II enzyme induction is usually lower than that
enzymes (e.g. GSTs and UDPGTs) is insufficient to of cyt P450, the rate of removal of toxic metabolites by
conjugate these products, electrophilic metabolites conjugation may be reduced relative to the rate of their
can bind to nucleophilic centers in cellular macro- formation by oxidative reactions (Hodgson, 1994).
molecules (DNA, RNA, proteins) leading to mem- Formation and persistence of critical genetic lesions
brane impairment, cellular toxicity, mutations or may be influenced as much by detoxification and repair
even carcinogenesis. processes as by the oxidative metabolism creating the
122 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

activated carcinogenic derivative (Stegeman and Hahn, ecological factors that control and modulate exposure
1994). This balance between bioactivation and detox- to, as well as effects of, environmental contaminants.
ification is crucial for the overall assessment of potential However, these same factors may also complicate
hazards due to the exposure to toxic substances, and interpretation of the significance of the biomarker
thus for the ERA using fish biomarkers. We would, responses in ways that may not always be anticipated
therefore, like to emphasize the importance of using (McCarthy, 1990). Many non-pollution-related vari-
combinations of existing fish biomarkers, both for the ables may have an additional impact on the various
classification of the environmental quality and for the enzyme systems, and may thus interfere with biomarker
identification of potential environmental risks. An responses when experimental conditions are not thor-
example of an integrated biomarker approach, indicative oughly analyzed or controlled. Examples of such ‘con-
of the balance between bioactivation and detoxification, founding’ or ‘modifying’ factors are the organisms’
was recently presented as the biotransformation index health, condition, sex, age, nutritional status, metabolic
(BTI ), expressing the ratio between phase I and II activity, migratory behavior, reproductive and develop-
activities as EROD:UDPGT or EROD:GST ratios (Van mental status, and population density, as well as factors
der Oost et al., 1998). A suggestion for an integrated like season, ambient temperature, heterogeneity of the
biomarker for oxidative stress might be to express the environmental pollution, etc. Unfortunately, most avail-
ratio between SOD or CAT activities and LPOX, as able toxicity data rarely quantify the potency that
proposed by Peters et al. (1994). However, these indices, confounding factors are likely to exhibit in natural
together with other potential integrated biomarkers, environments (De Kruijf, 1991). Moreover, estimates of
should first be validated in both laboratory and field confounding factor interactions are scarce, as evidenced
research before they can be incorporated in ERA by the extensive use of uncertainty factors in risk
programs. assessment to address unknowns (Power and McCarty,
More research is required before the toxicological 1997).
significance of changes in levels and activities of stress Several examples of the impact of confounding
proteins, MTs and hematological parameters can be factors on CYP1A indices have been reported in recent
clearly evaluated, validated and qualified. The toxico- years. Highly elevated CYP1A levels were observed in
logical significance of other biomarkers, such as im- mature male dab collected from off-shore stations with
munological, reproductive, genotoxic, physiological and low water temperature due to stratification, while
morphological parameters, is more evident (Fig. 11). considerably lower CYP1A levels were observed at
Despite indications that certain biomarker responses stations with higher water temperatures in vertically
are an early warning for adverse effects on the health or mixed areas, including polluted coastal stations (Slei-
fitness of individual organisms, it will be hard to derink et al., 1995a). The effect of the water tempera-
correlate these responses with effects on population, ture, which was inversely related to the CYP1A levels,
community or ecosystem levels. The sensitivity of appeared to dominate over the effect of PCB contam-
population and community responses to naturally vary- ination, which showed a positive correlation with
ing environmental factors implies that observed field CYP1A levels. Elevated water temperatures caused
responses to stress, even in the most carefully selected changes in antioxidant defenses by increasing SOD
cases to compare references and polluted sites, cannot be activity and decreasing GSH levels and GPOX activity
attributed solely to the action of the stressor in question in gills of the freshwater catfish (Parihar et al., 1997).
(see Section 6.13). These difficulties have led to the Eggens et al. (1996) demonstrated significant seasonal
suggestion that generalized risk assessment may be fluctuations in CYP1A indices in flounder. Sex-related
impossible, because each population and community is differences in EROD activity were observed during the
so tightly integrated into its own particular ecosystem spawning period (January #/March), while sex-related
that it is unique (Power and McCarty, 1997). differences in CYP1A protein levels were observed
during the post-spawning period (March #/June). The
6.13. Limitations of biomarkers activity of endogenous antioxidant systems (GPOX,
CAT, GST) in rainbow trout and black bullhead are
A successful implementation of biomarkers in envir- influenced by age and maturation (Otto and Moon,
onmental monitoring programs requires a good under- 1996a). A 7 weeks food deprivation also affected
standing of the mechanisms underlying the responses. detoxification enzyme activities (notably GST, UDPGT
The same attributes that make the biomarker approach and GRED) in the liver of rainbow trout (Blom et al.,
a powerful tool for biological (effect) monitoring also 2000). Moreover, nutritional status (long-term food
caution against its rapid and indiscriminate application deprivation) influenced PCB tissue levels and biomarker
without the benefit of carefully accumulated experience. responses (EROD and CYP1A) in arctic charr (Jørgen-
Biomarker responses are powerful because they inte- sen et al., 1999). It has been demonstrated that bacterial
grate a wide array of environmental, toxicological and infections seriously affect the activity of biotransforma-
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 123

tion enzymes in carp. Levels of cyt P450 and activities of themselves constitute a hazard to indigenous organisms.
EROD, UDPGT and GST as well as enzyme induction Once exposure has occurred and substances are bioa-
of these enzymes after 3MC exposure were significantly vailable, a sequence of biological responses may take
decreased after infection with Listeria monocytogenes place. Whether the well-being of the organism is
(Chambras et al., 1999) or the bacterial endotoxin eventually affected will depend upon many factors,
lipopolysaccharide (Marionnet et al., 1998). Similar some intrinsic (e.g. age, sex, health and nutritional
effects have been observed when Mediterranean scorpio status of the organism) and others extrinsic (e.g. dose,
fish were maintained under laboratory conditions in the duration, route of exposure to the contaminant and the
presence of a tropical alga (Caulerpa taxifolia ), which is presence of other chemicals). These intrinsic and ex-
known to produce repulsive toxic compounds, such as trinsic factors represent barriers to the assessment of
caulerpenyne derivatives (Uchimura et al., 1999). exposure and subsequent risk from that exposure
A further limitation of using biomarkers for ERA is (Shugart et al., 1992; Heugens et al., 2001). However,
the fact that relationships between biomarker responses biomarker responses can help to circumvent these
and field population-level effects are (presently) not well problems to a large extent by focusing on relevant
defined. Ecosystems respond in aggregate to the anthro- molecular events that occur after exposure and metabo-
pogenic and natural influences acting on them, as is lism. It is, therefore, of major importance to incorporate
illustrated in Fig. 13. In addition, since various sub- the use of biomarkers in assessing potential hazards and
stances may affect the same biomarkers, most biomar- risks due to the presence of toxic environmental
ker responses are not specific for individual compounds. pollutants.
Their dose #/response behavior is often not predictable
due to inadequate basic research. For mobile species 7.1. Conclusions on fish bioaccumulation markers
such as fish, the actual duration of exposure is un-
certain. It is, therefore, important to carefully define In order to elucidate the aquatic behavior of environ-
reference conditions. In order to avoid potential arte- mental contaminants and to assess exposure of aquatic
facts, particular care in sampling and handling of organisms, fish bioaccumulation markers may be ap-
samples is required. plied. It is virtually impossible to predict the fate of
xenobiotic substances with simple partitioning models.
When performing risk assessment on potentially hazar-
7. Summary and conclusions dous substances the mere consideration of the BCF is
insufficient and may be completely misleading (Franke,
In the preceding chapters a wide array of bioaccu- 1996). Instead, the complexity of bioaccumulation
mulation markers and biomarkers, which can be or are should be considered, which means that data on the
being used to demonstrate exposure to and effects of overall bioaccumulation process, including toxicoki-
environmental contaminants, have been discussed. netics, metabolism, BSAFs, organ-specific bioaccumu-
Based upon the data presented in this review, there is lation and bound residues, are of greater significance
little doubt that measurement of bioaccumulation and and should be related to critical body burden concen-
biomarker responses in organisms from contaminated trations for ecotoxicological endpoints. Predictive mod-
sites offers great promises for providing information els may eventually become powerful tools for realistic
that can contribute to environmental monitoring pro- simulation of pollutant behavior and may be used in the
grams designed for surveillance, hazard assessment, future to protect ecosystems and human health (Far-
regulatory compliance or documenting remediation. rington, 1991). However, caution must be exercised that
The biomarker approach clearly permits acquisition of the elegance and complexity of a series of coupled
information that cannot be obtained from the measure- mathematical equations does not evoke a false sense
ment of chemical residues in environmental and biolo- that accurate predictive capabilities of wide-ranging
gical media. However, in some cases it still has to be applicability are a proven reality. Most models have
demonstrated that biomarkers respond in a regular and been tested on relatively few chemicals, biota and
predictable manner to increasing exposure and that ecosystems, and for relatively short periods of time
higher-level effects are predictable from biomarker levels only. Whereas more complex sets of equations provide
and activities (Suter, 1990). At present, most routine accurate output at the cost of much input, simple
programs on environmental or ecological risk assess- functions are parameter-scarce but yield less precise
ment are still based upon measurement of a selected outcomes (Hendriks, 1995b). The complex of site-
group of chemicals in various environmental compart- specific factors which may influence the bioconcentra-
ments, and on comparing the results with legislative tion, biomagnification, biotransformation and bioavail-
threshold values or safety standards (Suter, 1993; De ability of chemicals (e.g. sediment organic carbon,
Zwart, 1995). However, xenobiotic chemicals, in the particulate OM, presence of other chemicals), as dis-
forms found in the environment, often do not by cussed in Section 5, make it almost inevitable that
124 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

serious discrepancies will exist between model-estimated dated for their ecotoxicological relevance before they
and actual body burdens. Predictive models will, there- can be used in standardized biomonitoring programs. In
fore, remain of limited value until they have been reverse, for the biomarkers with a clear ecological
applied and validated in ‘real world’ situations. Rather relevance, research should be focused on increasing
than relying on models to estimate exposure, BAM (in their sensitivity and selectivity. Since it seems unlikely
fish, SPMDs or invertebrates) may allow actual mea- that biomarkers will replace all chemical analyses,
surement of exposure and a more accurate assessment of biomonitoring should always be performed in concert
adverse health outcomes to the aquatic community with CM. In addition, it is of paramount importance
(Valberg et al., 1996). that the potential impact of confounding factors (as
The ideal bioaccumulation marker of exposure is one discussed in Section 6.13) has to be taken into account
that is chemical-specific, accurately measurable in trace
when interpreting biomarker data.
quantities, measurable in easily sampled biological In order to assess exposure to or effects of environ-
media or by non-invasive techniques, and is well
mental pollutants on aquatic ecosystems, the following
correlated with previous exposure (Valberg et al.,
suite of biomarkers (biological and biochemical para-
1996). Contaminant levels in fish may be used as
meters) may be examined in fish:
indicators of exposure, although confounding factors
like age, sex, season and mobility may have an . biotransformation enzymes (phase I and II);
additional impact on bioaccumulation. Since pollutant . oxidative stress parameters;
levels in fish blood are hard to detect, the most . biotransformation products;
promising fish bioaccumulation markers for exposure . stress proteins, MTs and MXR proteins;
assessment are body burdens (generally muscle, liver . hematological parameters;
tissue or whole body) of persistent organic pollutants, . immunological parameters;
like PCBs and OCPs (e.g. HCB, DDTs, drins and . reproductive and endocrine parameters;
HCHs). Since PCDD ad PCDF levels in fish tissues . genotoxic parameters;
are very low as compared with the sediment levels, their . neuromuscular parameters;
value as bioaccumulation markers remains question- . physiological, histological and morphological para-
able. Easily biodegradable compounds, such as PAHs meters.
and chlorinated phenols, do not tend to accumulate in
fish tissues in quantities that reflect the exposure. All fish biomarkers discussed in this review are
Exposure to these compounds may be assessed by evaluated for their potential use in ERA programs,
measurement of biliary metabolite levels. In order to based upon the six criteria that were proposed in Section
make the results of different studies more comparable, 2 of the present paper. The evaluation is summarized in
future bioaccumulation research should preferably be Table 13 and elaborated in the following paragraphs.
carried out using standardized procedures. BSAFs
should, therefore, be determined as the ratio between . The phase I biotransformation enzymes, notably
LW-based tissue levels and OM-based sediment levels. CYP1A, definitely belong to the most sensitive fish
A study combining bioaccumulation and ecotoxicity biomarkers known at present. The value and feasi-
testing, including measurement of biota tissue levels, is bility of these biomarkers have been demonstrated in
considered a promising approach which should be numerous laboratory and field studies with various
pursued in future research (Franke, 1996). In other fish species. Consequently, there is growing interest in
words, a reliable ERA procedure preferably encom- using CYP1A induction in fish as a biomarker to
passes both bioaccumulation markers and biomarkers. indicate the exposure of aquatic organisms to
CYP1A-inducing compounds and to evaluate the
7.2. Conclusions on fish biomarkers degree and possible risk of environmental contam-
ination. CYP1A protein levels and EROD activity
It would be ideal for ERA purposes to have a limited can be incorporated in ERA programs, provided that
set of specific biomarkers indicating the exposure and the experimental design considers all intrinsic and
assessing the hazards of all major classes of pollutants as extrinsic variables that may potentially influence this
well as non-specific biomarkers that assess accurately parameter, as well as xenobiotics that inhibit CYP1A
and completely the health condition of the organism and activity (e.g. organotins). No sensitive pollution-
the ecosystem (Peakall and Walker, 1994). In the ‘real related responses have been observed in fish for other
world’ we have some promising biomarkers to assess P450 isozymes (e.g. CYP3A) thus far. At present,
exposure and effects of toxic substances, but much more CYP1A levels and activities have been validated for
research is required before we approach the ideal use in various areas of (environmental) toxicological
situation. It should be emphasized that the most research, such as:
sensitive or selective biomarkers should also be vali- #/ research on toxic mechanisms of xenobiotics;
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 125

Fig. 11. A theoretical visualization of the relationships between ecological relevance and time-scales of pollutant-induced biomarker responses.
Adapted from Adams et al. (1989).

#/ toxicity screening; tors, linkage between enzyme activities and higher

#/ identification of exposure to specific compounds; levels of organization (e.g. organ or whole animal)
#/ quantification of impact and exposure of a suite of and the basic chemistry of purified forms. Current
organic trace pollutants; research on the latter topic may reveal specific
#/ identifying subtle early effects (‘early-warning’); isoforms of GST and UDPGT that are more sensitive
#/ health and ecosystem monitoring; indicators of exposure or effects than the measure-
#/ triggering of regulatory action. ment of total activity.
. Due to the important role of phase II enzymes in . Since many environmental contaminants exert toxic
detoxification they should be considered in future effects related to oxidative stress, this phenomenon
biomonitoring programs as well, although their may be another important feature for biomarker
sensitivity towards pollutant exposure is limited. An development. However, antioxidant enzymes are
integrated biomarker approach, using combinations generally less responsive to pollutants than phase I
of biomarkers such as the biotransformation index and II enzymes and the relationships between re-
(BTI, reflecting the ratio between phase I and II sponse and contaminant exposure are still less well
activities), may be more useful for monitoring since established. Their function in detoxification pro-
this index reflects the balance between bioactivation cesses, however, motivates continued research on
and detoxification (Van der Oost et al., 1998). The the potential use of SOD, CAT and GRED in ERA
BTI may also be indicative of the susceptibility of programs. Other promising parameters that may be
organisms to toxic xenobiotics with carcinogenic useful to indicate oxidative stress are the GSH:GSSG
properties. Further studies are required, however, to ratio and LPOX products such as aldehydes.
improve the usefulness of phase II enzymes as . Biliary levels of (conjugated) metabolites of easily
biomarkers in ERA procedures; e.g. with regard to biodegradable xenobiotics, such as PAHs, chlori-
baseline activities, non-pollution confounding fac- nated phenols and resin acids, have been validated
126 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

as fish biomarkers of exposure and may now be used considered as too expensive and time-consuming to
in ERA monitoring programs. Additional research be applied in routine ERA programs. Other DNA
has to be performed to establish the impact of modifications, such as strand breaks, may also be
confounding factors and to define clear relationships feasible as biomarkers for genotoxic chemicals (e.g.
between biomarker responses and the organisms’ COMET assay and DNA unwinding assay).
health. . When physiological, histological and morphological
. The feasibility of stress proteins (HSPs), MTs and parameters in fish are affected, this is generally
multi xenobiotic resistance (MXR) as fish biomarkers indicative of irreversible damage or disease. These
for ERA monitoring needs to be further evaluated parameters are, therefore, not suitable as early-
with additional research, especially as to baseline warning signals in ERA programs. Histopathological
data on their normal physiological function and the parameters are, however, relatively easy determined
influences of non-environmental factors in the field. and valuable to evaluate the ecotoxicological signifi-
. Increased enzymatic activity, such as that of transa- cance of other biomarkers. Gross indices, such as the
minases, in the blood of fish may be indicative of LSI and the CF, can be easily measured and may be
impaired membranes or cell damage in specific used to assess the condition of the liver and the
organs, but the initial effects tend to decrease with general health of fish, respectively. Immunhistochem-
time. In short-term caging studies, however, the AST ical detection of cellular and tissue related abnorm-
activity may be a feasible biomarker for ERA alities offer ‘earlier’ warning signals than are
purposes. More research is needed, however, to provided by normal pathological and histopatholo-
establish clear relationships between transaminase gical examination of tissue samples.
responses and pollutant exposure. Other hematologi-
cal parameters may be valuable for effect assessment A visual overview of the biomarker evaluation (Fig.
when measured in concert with more sensitive and 14) demonstrates that phase I enzymes (e.g. hepatic
selective biomarkers. EROD and CYP1A), biotransformation products (e.g.
. Assays to determine adverse effects of xenobiotics on biliary PAH metabolites), reproductive parameters (e.g.
the immune system (e.g. leukocyte and lymphocyte plasma VTG) and genotoxic parameters (e.g. hepatic
status) may become important non-specific effect DNA adducts) are currently the most valuable fish
biomarkers because of their high ecological signifi- biomarkers for ERA purposes. It has to be emphasized,
cance. More research on cause-and-effect relation- however, that the value for other biomarkers will be
ships and the influences of confounding non- elevated when additional research on certain topics has
pollution-related stressors is required, however, be- been performed successfully. Moreover, certain biomar-
fore these parameters can be used in ERA programs. kers will be considered more valuable for specific
. Many environmental contaminants are known to situations, other that determining the overall environ-
have endocrine-disrupting properties. Effects of pol- mental quality in ERA programs. The overview is
lutants on the endocrine system may be of major simplified, since all criteria are given the same value
importance in ERA programs, since impairments in (good ["/], 1; fair [9/], 0.5 and poor [%/], 0) and no
reproductive capability may have a serious impact on weight factors are included to indicate the importance of
fish populations. Sensitive assays, such as levels of the different criteria. It is not possible to propose weight
VTG and ZRPs are promising, but they have to be factors that are generally applicable, since these will
further validated for their ecological significance. depend upon the purpose of the investigations the
. The inhibition of ACHE in fish tissues may be a biomarkers are applied for. It is, for instance, not
promising biomarker for the assessment of exposure advisable to use the 32P-postlabeling assay for DNA
and effects of complex mixtures of contaminants. adduct determinations in routine monitoring programs
Compounds such as OP and carbamate pesticides, with large amounts of samples because of the expensive
have been demonstrated to depress ACHE activity, and time-consuming assay. If, on the other hand, only a
but various other organic trace pollutants may cause small amount of samples has to be investigated, e.g. to
the same effects. More research on the impact of confirm the presence of potentially carcinogenic sub-
confounding factors and the identification of the stances in the environment, this assay will be quite
(classes of) compounds responsible for observed suitable since the first criterion is less important.
effects in field trials is required, before this parameter The main role of biomarkers in environmental
can be used in ERA programs. assessment is to determine whether or not, in a specific
. The formation of hepatic DNA adducts in fish is environment, organisms are physiologically normal
considered to be a valid biomarker for exposure to (Peakall and Walker, 1994). The use of biomonitoring
PAHs and for the assessment of potential genotoxic methods in the control strategies for chemical pollution
effects. 32P-postlabeling is a very sensitive assay for has several advantages over chemical monitoring (De
measuring DNA adduct levels, but it may be Zwart, 1995). Firstly, these methods measure effects in
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 127

Fig. 12. Linkage between P450 and other biochemical systems. This figure illustrates the complex interactions that are known to occur between
biochemical systems involved in responses to pollutant exposure. Further linkages remain to be discovered. AhR, Ah receptor; ALAS, d-amino-
levulinic acid synthase; ARE, antioxidant responsive element (electrophilic response element); ARNT, Ah receptor nuclear translocator; BR,
bilirubin; BV, biliverdin; CO, carbon monoxide; DRE, dioxin responsive element; EH, epoxide hydrolase; GSH, glutathione; GST, glutathione S -
transferase; HAH, halogenated aromatic hydrocarbon; HO, heme oxygenase; HQ, hydroquinone; HSF, heat shock factor; HSP90, 90 kDa heat
shock protein; HSRE, heat shock response element; M, metal; MRE, metal responsive element; MRF, metal response factor; MT, metallothionein;
NO, nitric oxide; NOS, nitric oxide synthase; cyt P450, cytochrome P450; PP, protoporphyrin; Q, quinone; QR, quinone reductase (a.k.a. DT-
diaphorase); SOD, superoxide dismutase; SQ, semiquinone radical; XRE, xenobiotic response element. Adapted from Stegeman and Hahn (1994)

which the bioavailability of the compound(s) of interest implement a biomarker-based environmental monitor-
is integrated with the concentration of the compounds ing program, consisting of five major tasks:
and their intrinsic toxicity. Secondly, most biological
measurements form the only way of integrating the . Task I: preliminary survey: proof of the principle. The
effects on a large number of individual and interactive primary objective of this research is to compare the
processes. A disadvantage of most of the biological qualitative pattern and quantitative responses of a
effect measurements is that it may be very difficult to suite of biomarkers in sentinel species from sites
relate the observed effects to specific aspects of pollution polluted with specific types of contaminants, com-
or to effects on the level of populations, communities or pared with the responses of organisms from pristine
ecosystems. In view of the present chemically oriented reference sites.
pollution abatement policies and the need to reveal . Task II: development, standardization and validation
specific chemical problems, it is most probable that of key biomarkers. The main objective of this research
is to standardize protocols for existing biomarker
biological effect analysis will never totally replace
measurements, develop and modify new biomarkers
chemical analyses. The biomarker approach, therefore,
as needed and acquire the fundamental understand-
should not be considered as a replacement for conven-
ing of the relationship between exposure, biomarker
tional assessment techniques, but as an important
responses and adverse effects in sentinel species at the
supplementary approach of great ecological relevance
cellular, organismal or population level.
(Depledge and Fossi, 1994).
. Task III: environmental monitoring: biomarkers of
exposure. The objective of this research is to increase
understanding of the qualitative pattern and quanti-
tative responses of a suite of biomarkers in sentinel
8. Perspectives species until a capability is developed to identify the
extent of exposure and the nature of the contaminant
McCarthy (1990) proposed a research plan for focus- to which the organisms are being exposed. The
ing and coordinating resources necessary to develop and emphasis of this task is on developing a capability
128 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

to assess exposure rather than to evaluate the adverse

effects or long-term biological significance of that
exposure.
. Task IV: linking biomarker responses to community
level effects. The objective of this task is to establish
and verify the relationships between exposure,
changes in rapidly-responding suborganismal bio-
markers and long-term adverse effects at the popula-
tion or community level. The successful completion
of this objective will validate the use of molecular,
biochemical and physiological level biomarkers as
short-term predictors of long-term adverse effects.
. Task V: linking biomarker responses in sentinel species
to human epidemiology. The objective of this research
is to determine if there are correlations between Fig. 13. The complexity of stress #/response relationships. The dose #/
patterns in the extent, nature and geographic dis- response paradigm, although necessarily simple for experimental
practice, does not adequately account for the multiple, simultaneous
tribution of biomarker responses in sentinel species
stressors to which all species are subjected in natural environments.
and patterns of epidemiological evidence of increased Adapted from Power and McCarty (1997).
risk to human health.
purposes of environmental monitoring programs, such
The environmental monitoring program that emerges
as first carrying out cost-effective measurements in a
from the research plan proposed by McCarthy (1990)
stepwise approach, obtaining insights into the cause of
should be the final development of the field monitoring
observed effects in the field, studying trends in time or
in Task III, illuminated by the laboratory research and
spatial variation, or using biomarker responses as
development described in Task II, with ecological
insights provided by Task IV. In terms of budget and signals of negative effects on the ecosystem. The
effort McCarthy (1990) anticipated three phases: the characteristics and specific research needs for the
initial phase (i.e. the implementation of the preliminary application of biomarkers to perform screening, diag-
survey of Task I), the learning phase (i.e. the laboratory nosis, trend monitoring (both in time and space) or risk
studies and field sampling of Tasks II and III, and assessment are outlined in Table 14 (adapted from Den
accumulation of field data for Tasks IV and V) and the Besten, 1998). The use of biomarkers for risk assessment
routine monitoring phase (i.e. the mature phase of the at the community and ecosystem level is still rather
environmental monitoring program). At present, the ambitious. In the assessment of site-specific risks,
research on most of the biomarkers described in this
review is either in the initial phase (novel biomarkers) or
in the learning phase. Peakall and Walker (1994) stated
that the biomarkers are in the same stage of develop-
ment as the analyses of environmental chemicals were in
the 1960s, that is, that reasonably precise measurements
were made on local samples. For chemical analyses this
eventually led to national and international programs,
supported by QA and quality control, being set-up. At
present, several international programs (e.g. BEEP,
CITY FISH and BEQUALM) are being carried out in
order to standardize methods for a suite of (fish)
biomarkers.
Den Besten (1998) discussed the concepts for imple-
mentation of biomarkers in environmental monitoring.
Although it has been demonstrated that (fish) biomar- Fig. 14. Visualization of the evaluation of 12 groups of fish
kers are useful monitoring tools, it is clear that more biomarkers by adding up the results of a judgment (good, 1; fair, 0.5
information is needed about the relationships between and poor, 0) using six biomarker criteria. phase I, phase I biotrans-
biomarker responses and the health and fitness of formation enzymes; phase II, phase II biotransformation enzymes;
antiox, antioxidant enzymes; biotrans, biotransformation products;
organisms, and even more so between biomarker proteins, stress proteins, metallothioneins & MXR-proteins; blood,
responses and the risks for the ecosystem. With respect serum transaminases; immune, immunological parameters; repro,
to future biomarker research it is important to realize reproductive parameters; genotox, genotoxic parameters; histopath,
that different concepts are needed for the specific histopathological parameters; morpho, morphological parameters.
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149
Table 13
Fish biomarker evaluation, using six biomarker criteria

Criteria Biomarkers!

Phase 1 Phase 2 Antiox Biotrans Proteins Blood Immune Repro Neurotox Genotox Histopath Morpho

EROD GST UDPGT SOD CAT bile FAC HSP MT AST ALT Leuko lympho VTG ZRP ACHE DNA add Tissue LSI CF
CYP1A MXR DNA dam

Reliable and " " " " " " 9 " " % 9 "
easy assay
Sensitive to " 9 % " 9 9 9 " 9 " % %
pollution
Known base- " 9 9 " % 9 9 9 9 " " "
line data
Confounding 9 9 9 9 % 9 9 9 % 9 9 9
factors
Response me- " 9 % " 9 % % " 9 " 9 9
chanism
Toxicological 9 " 9 9 9 " " 9 9 " " 9
significance
Overall 5.0" 4.0" 2.5" 5.0" 2.5" 3.5" 3.0" 4.5" 3.0" 4.5" 3.5" 3.5"

Symbols: ", good; 9, fair; %, poor. Abbriviations: phase I, phase I biotransformation enzymes; phase II, phase II biotransformation enzymes; antiox, antioxidant enzymes; biotrans,
biotransformation products; proteins, stress proteins, metallothioneins & MXR-proteins; blood, serum transaminases; immune, immunological parameters; repro, reproductive parameters; genotox,
gemotoxic parameters; histopath, histopathological parameters; morpho, morphological parameters (individual biomarker abbriviations in text of Section 6).

129
130 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149

Table 14
Concepts for the implementation of biomarkers in environmental monitoring (adapted from Den Besten, 1998)

Concept Purpose Use of biomarkers Specific needs for further im-

plementation of this concept

Screening Cost-effective use of biomarkers as a Simple biomarker measurements in a Sensitive biomarkers with
first screening step tiered approach (together with che- specificity for certain types of
mical analyses and bioassays) contaminants or certain types
of effect mechanism
Signalling effects (early-warning sys- Biomarkers of exposure and of toxic
tem) effect
Biomarkers incorporated in bioassays
Diagnosis To signal the possible cause for Large batteries of biomarkers (effect Development of a suite of
observed adverse effects in popula- indicators, in part non-invasive) are biomarkers
tions of a certain species applied in studies on animals from
affected populations; responses are
compared with those in reference
populations
Insight in the relationship
between change in homeosta-
sis (healthy state), biomarker
response and fitness or per-
formance of the organism
Trend monitoring Study biomarker responses in time Repeated sampling and biomarker Knowledge of the influence of
measurements in time confounding factors
To compare changes of biomarker Translation of existing quality
responses in time with quality objec- objectives in biomarker re-
tives; check improvement after reme- sponse criteria
dial action
Monitoring of spatial variation Assessment of site-specific biomarker Comparison of biomarker responses Biomarkers must be applic-
responses (gradients) between sites able in common species (for a
region considered)
Knowledge on confounding
factors; reference values
Site-specific risk assessment Study of contaminant bioavailability Biomarkers as indicators of exposure Biomarkers must be applic-
and related risks at polluted sites (hot able in local species or in situ
spots) bioassays
Signalling effects at higher levels of Use of selected biomarkers as early- Responses of predictive bio-
organisation warning signals of adverse effects on markers must be related to
the individual, population or com- effects on higher organisation
munity level levels
Risk assessment: ecosystem structure Predicting effects on ecosystem level Application of selected biomarkers in Biomarkers must be applic-
and ecosystem function a range of species able in different species (in-
cluding key organisms)
Estimation of the fraction potentially Sensitivity comparisons be-
affected species (PAF) tween species
Application of selected biomarkers in
key organisms

information from biomarkers should be used in combi- Due to the interdisciplinary nature of biomarker
nation with other biological data (e.g. species abun- studies and the need for integration of numerous
dance) and chemical data (Den Besten, 1998). Ellis research specialties, long-term progress will be acceler-
(2000) discussed the advantages and limitations of four ated by general agreement on a common research
different risk assessment approaches (chemical specific strategy. Future research should be focused on the
limits, biological assessment, direct toxicity assessment possible implementation of biomarkers in environmen-
[DTA] and biomarker techniques) in urban receiving tal monitoring programs. However, since monitoring
waters. The inability of DTA procedures to satisfacto- information requirements and monitoring objectives are
rily evaluate chronic, sub-lethal risks increased the very situation-specific and are strongly dependent on
interest in using in situ biomarkers for the fingerprinting national water management policies, it is unlikely that
of stress-response properties as a means of diagnosing the near future will show a global trend towards
risk assessment for integrated urban runoff management unification of standard biomonitoring protocols (De
(Ellis, 2000). Zwart, 1995). The ultimate objective for applied envir-
 R. van der Oost et al. / Environmental Toxicology and Pharmacology 13 (2003) 57 #/149 131

onmental research should be to make biomarkers more tribution of different routes of exposure (McCarthy
usable in ERA. We, therefore, want to propose some and Shugart, 1990).
guidelines for ecotoxicological research programs, in . In order to reduce the amount of fish that has to be
order to actually incorporate fish biomarkers in ERA killed for biomarker research, emphasis has to be put
monitoring: on the development of non-destructive and non-
invasive biomarkers. As an alternative to fish bio-
. Efforts have to be made to design a set of fish markers, artificial devices, such as passive sampling
biomarkers, covering the exposure and/or early (e.g. SPMD) combined with cell-line bioassays, may
effects of the entire spectrum of potentially toxic be used to monitor the water quality. Pilot experi-
substances which may be present in the aquatic ments in this field have to be compared with studies
environment. on fish, in order to correlate the results to data from
. All biological and biochemical parameters that may the ‘real world’.
be used to assess exposure to and effects of environ- . New and promising developments in the biomarker
mental pollutants have to be objectively evaluated field are the so-called ‘genomics’ and ‘proteomics’.
according to the six biomarker criteria, as proposed Genomics is based upon the application of DNA
in Section 2. microarrays that allow the expression of hundreds to
. It is essential that more research should be carried out many thousands of genes to be monitored simulta-
to demonstrate relationships between biomarker neously, thus providing a broad and integrated
responses and effects on pathology, survival, growth picture of the way an organism responds to a
or reproduction at the level of individual organisms changing environment (Gracey et al., 2001). The
(Den Besten, 1998). The knowledge on these relation- entire protein complement of the genome, the ‘pro-
ships should then be used to design numerical teome’, can now be analyzed for changes associated
standards for biomarkers. with specific treatments, using ‘peptide mass profil-
. Standard procedures should be developed with re- ing’, a combination of two-dimensional gel electro-
gard to sampling, sample treatment, assay conditions, phoresis and mass spectrometry (Shepard et al.,
etc. preferably in international programs such as 2000). Proteomics research provided certain protein
BEEP, CITY FISH and BEQUALM. expression signatures (PESs), which are specific sets
. The selection of indicator species is going to be, at of proteins, present or absent, indicating specific
least to some extent, site-specific. Instead of investi- toxicity profiles.
gating the same responses in thousands of fish In conclusion, it can be stated that fish biomarkers are
species, however, national as well as international promising tools for ERA, as supplements to existing
agreements should be made on the selection of a few chemical measures. Much work has to be done, how-
sentinel species. In these indicator species a suite of ever, in order to test and interpret biomarker responses
candidate biomarkers must be thoroughly investi- and to develop acceptable QA procedures. Only when
gated and validated in both laboratory and field both scientific and legal credibility of this information is
experiments. established, the biomarker techniques can be fully
. Interpretation of results obtained by research on feral applied in routine monitoring programs. It seems
fish will remain complicated since the impact of the obvious that CM alone is insufficient for a reliable
various factors that are possibly affecting biomarker classification of water quality. Therefore, the efforts to
responses cannot always be established due to the incorporate biological compounds to the ERA research
unknown life history of the fish specimens. In this will eventually be worthwhile.
respect the feasibility of caging or mesocosm experi-
ments with cultured fish should be examined further.
. In order to facilitate a reliable comparison of results
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