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Volume 237

Pim de Voogt Editor

 Reviews of
Environmental Contamination
and Toxicology
VOLUME 237

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 Reviews of
Environmental Contamination
and Toxicology

Editor
Pim de Voogt

Editorial Board
María Fernanda Cavieres, Valparaiso, Chile
James B. Knaak, Getzville, New York, USA
Annemarie P. van Wezel, Nieuwegein/Utrecht, The Netherlands
Ronald S. Tjeerdema, Davis, California, USA
David M. Whitacre†, Summerfield, N Carolina, USA

Founding Editor
Francis A. Gunther

VOLUME 237
 Coordinating Board of Editors
DR. PIM DE VOOGT, Editor
Reviews of Environmental Contamination and Toxicology

University of Amsterdam
Amsterdam, The Netherlands
E-mail: [email protected]

DR. ERIN R. BENNETT, Editor

Bulletin of Environmental Contamination and Toxicology

Great Lakes Institute for Environmental Research

University of Windsor
Windsor, Ontario, Canada
E-mail: [email protected]

PETER S. ROSS, Editor

Archives of Environmental Contamination and Toxicology

Vancouver Aquarium Marine Science Center

Vancouver, BC, Canada
E-mail: [email protected]

ISSN 0179-5953 ISSN 2197-6554 (electronic)

Reviews of Environmental Contamination and Toxicology
ISBN 978-3-319-23572-1 ISBN 978-3-319-23573-8 (eBook)
DOI 10.1007/978-3-319-23573-8

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Foreword

International concern in scientific, industrial, and governmental communities over

traces of xenobiotics in foods and in both abiotic and biotic environments has justi-
fied the present triumvirate of specialized publications in this field: comprehensive
reviews, rapidly published research papers and progress reports, and archival docu-
mentations. These three international publications are integrated and scheduled to
provide the coherency essential for nonduplicative and current progress in a field as
dynamic and complex as environmental contamination and toxicology. This series
is reserved exclusively for the diversified literature on “toxic” chemicals in our
food, our feeds, our homes, recreational and working surroundings, our domestic
animals, our wildlife, and ourselves. Tremendous efforts worldwide have been
mobilized to evaluate the nature, presence, magnitude, fate, and toxicology of the
chemicals loosed upon the Earth. Among the sequelae of this broad new emphasis
is an undeniable need for an articulated set of authoritative publications, where one
can find the latest important world literature produced by these emerging areas of
science together with documentation of pertinent ancillary legislation.
Research directors and legislative or administrative advisers do not have the time
to scan the escalating number of technical publications that may contain articles
important to current responsibility. Rather, these individuals need the background
provided by detailed reviews and the assurance that the latest information is made
available to them, all with minimal literature searching. Similarly, the scientist
assigned or attracted to a new problem is required to glean all literature pertinent to
the task, to publish new developments or important new experimental details
quickly, to inform others of findings that might alter their own efforts, and eventu-
ally to publish all his/her supporting data and conclusions for archival purposes.
In the fields of environmental contamination and toxicology, the sum of these
concerns and responsibilities is decisively addressed by the uniform, encompassing,
and timely publication format of the Springer triumvirate:
Reviews of Environmental Contamination and Toxicology [Vol. 1 through 97
(1962–1986) as Residue Reviews] for detailed review articles concerned with

v
vi Foreword

any aspects of chemical contaminants, including pesticides, in the total environ-

ment with toxicological considerations and consequences.
Bulletin of Environmental Contamination and Toxicology (Vol. 1 in 1966) for
rapid publication of short reports of significant advances and discoveries in the
fields of air, soil, water, and food contamination and pollution as well as method-
ology and other disciplines concerned with the introduction, presence, and
effects of toxicants in the total environment.
Archives of Environmental Contamination and Toxicology (Vol. 1 in 1973) for
important complete articles emphasizing and describing original experimental or
theoretical research work pertaining to the scientific aspects of chemical con-
taminants in the environment.
The individual editors of these three publications comprise the joint Coordinating
Board of Editors with referral within the board of manuscripts submitted to one pub-
lication but deemed by major emphasis or length more suitable for one of the others.

Coordinating Board of Editors

Preface

The role of Reviews is to publish detailed scientific review articles on all aspects of
environmental contamination and associated (eco)toxicological consequences.
Such articles facilitate the often complex task of accessing and interpreting cogent
scientific data within the confines of one or more closely related research fields.
In the 50+ years since Reviews of Environmental Contamination and Toxicology
(formerly Residue Reviews) was first published, the number, scope, and complexity
of environmental pollution incidents have grown unabated. During this entire
period, the emphasis has been on publishing articles that address the presence and
toxicity of environmental contaminants. New research is published each year on a
myriad of environmental pollution issues facing people worldwide. This fact, and
the routine discovery and reporting of emerging contaminants and new environmen-
tal contamination cases, creates an increasingly important function for Reviews. The
staggering volume of scientific literature demands remedy by which data can be
synthesized and made available to readers in an abridged form. Reviews addresses
this need and provides detailed reviews worldwide to key scientists and science or
policy administrators, whether employed by government, universities, nongovern-
mental organizations, or the private sector.
There is a panoply of environmental issues and concerns on which many scien-
tists have focused their research in past years. The scope of this list is quite broad,
encompassing environmental events globally that affect marine and terrestrial eco-
systems; biotic and abiotic environments; impacts on plants, humans, and wildlife;
and pollutants, both chemical and radioactive; as well as the ravages of environmen-
tal disease in virtually all environmental media (soil, water, air). New or enhanced
safety and environmental concerns have emerged in the last decade to be added to
incidents covered by the media, studied by scientists, and addressed by governmen-
tal and private institutions. Among these are events so striking that they are creating
a paradigm shift. Two in particular are at the center of ever increasing media as well
as scientific attention: bioterrorism and global warming. Unfortunately, these very
worrisome issues are now superimposed on the already extensive list of ongoing
environmental challenges.

vii
viii Preface

The ultimate role of publishing scientific environmental research is to enhance

understanding of the environment in ways that allow the public to be better informed
or, in other words, to enable the public to have access to sufficient information.
Because the public gets most of its information on science and technology from
internet, TV news, and reports, the role for scientists as interpreters and brokers of
scientific information to the public will grow rather than diminish. Environmentalism
is an important global political force, resulting in the emergence of multinational
consortia to control pollution and the evolution of the environmental ethic. Will the
new politics of the twenty-first century involve a consortium of technologists and
environmentalists, or a progressive confrontation? These matters are of genuine
concern to governmental agencies and legislative bodies around the world.
For those who make the decisions about how our planet is managed, there is an
ongoing need for continual surveillance and intelligent controls to avoid endanger-
ing the environment, public health, and wildlife. Ensuring safety-in-use of the many
chemicals involved in our highly industrialized culture is a dynamic challenge,
because the old, established materials are continually being displaced by newly
developed molecules more acceptable to federal and state regulatory agencies, pub-
lic health officials, and environmentalists. New legislation that will deal in an appro-
priate manner with this challenge is currently in the making or has been implemented
recently, such as the REACH legislation in Europe. These regulations demand sci-
entifically sound and documented dossiers on new chemicals.
Reviews publishes synoptic articles designed to treat the presence, fate, and, if
possible, the safety of xenobiotics in any segment of the environment. These reviews
can be either general or specific, but properly lie in the domains of analytical chem-
istry and its methodology, biochemistry, human and animal medicine, legislation,
pharmacology, physiology, (eco)toxicology, and regulation. Certain affairs in food
technology concerned specifically with pesticide and other food-additive problems
may also be appropriate.
Because manuscripts are published in the order in which they are received in
final form, it may seem that some important aspects have been neglected at times.
However, these apparent omissions are recognized, and pertinent manuscripts are
likely in preparation or planned. The field is so very large and the interests in it are
so varied that the editor and the editorial board earnestly solicit authors and sugges-
tions of underrepresented topics to make this international book series yet more
useful and worthwhile.
Justification for the preparation of any review for this book series is that it deals
with some aspect of the many real problems arising from the presence of anthropo-
genic chemicals in our surroundings. Thus, manuscripts may encompass case stud-
ies from any country. Additionally, chemical contamination in any manner of air,
water, soil, or plant or animal life is within these objectives and their scope.
Manuscripts are often contributed by invitation. However, nominations for new
topics or topics in areas that are rapidly advancing are welcome. Preliminary com-
munication with the Editor-in-Chief is recommended before volunteered review
manuscripts are submitted. Reviews is registered in WebofScience™. Inclusion in the
Preface ix

Science Citation Index serves to encourage scientists in academia to contribute to the

series. The impact factor in recent years has increased from 2.5 in 2009 to almost 4 in
2013. The Editor-in-Chief and the Editorial Board strive for a further increase of the
journal impact factor by actively inviting authors to submit manuscripts.

Amsterdam, The Netherlands Pim de Voogt

January 2015
Contents

Caenorhabditis elegans, a Biological Model for Research

in Toxicology .................................................................................................... 1
Lesly Tejeda-Benitez and Jesus Olivero-Verbel
Pore Water Collection, Analysis and Evolution: The Need
for Standardization ......................................................................................... 37
Jacob G. Gruzalski, James T. Markwiese, Neil E. Carriker,
William J. Rogers, Rock J. Vitale, and David I. Thal
Environmental Fate and Toxicology of Dimethoate ..................................... 53
April Van Scoy, Ashley Pennell, and Xuyang Zhang
Exposure to Crystal Violet, Its Toxic, Genotoxic and Carcinogenic
Effects on Environment and Its Degradation and Detoxification
for Environmental Safety ............................................................................... 71
Sujata Mani and Ram Naresh Bharagava
Metabolic Pathways for Degradation of Aromatic Hydrocarbons
by Bacteria ....................................................................................................... 105
Guillermo Ladino-Orjuela, Eleni Gomes, Roberto da Silva,
Christopher Salt, and John R. Parsons
A Review and Assessment of Spent Lead Ammunition
and Its Exposure and Effects to Scavenging Birds in the United States ..... 123
Nancy H. Golden, Sarah E. Warner, and Michael J. Coffey

Index................................................................................................................... 193

xi
Caenorhabditis elegans, a Biological Model
for Research in Toxicology

Lesly Tejeda-Benitez and Jesus Olivero-Verbel

Contents
1 Introduction ......................................................................................................................... 2
2 Biological Features of C. elegans ....................................................................................... 2
3 Advantages of Using C. elegans as a Biological Model ..................................................... 3
4 Applications in Medicine .................................................................................................... 4
5 Toxicity Endpoints .............................................................................................................. 4
5.1 Lethality ................................................................................................................... 5
5.2 Growth ..................................................................................................................... 6
5.3 Reproduction ............................................................................................................ 6
5.4 Fertility..................................................................................................................... 7
5.5 Lifespan.................................................................................................................... 7
5.6 Intestinal Autofluorescence ...................................................................................... 7
5.7 Locomotion .............................................................................................................. 8
5.8 Metabolism .............................................................................................................. 8
5.9 Development ............................................................................................................ 8
5.10 Feeding Behavior ..................................................................................................... 9
5.11 Oxidative Stress ....................................................................................................... 9
5.12 Patterns of Gene Expression .................................................................................... 9
5.13 Protein Expression ................................................................................................... 10
5.14 DNA Damage ........................................................................................................... 10
5.15 GFP Reporters.......................................................................................................... 10
5.16 RNA Interference (RNAi) ........................................................................................ 11
5.17 Cell Apoptosis.......................................................................................................... 11
5.18 Cell Cycle Arrest...................................................................................................... 12
5.19 Transgenerational Effects......................................................................................... 12

L. Tejeda-Benitez • J. Olivero-Verbel (*)

Environmental and Computational Chemistry Group, School of Pharmaceutical Sciences,
Zaragocilla Campus, University of Cartagena, Cartagena 130014, Colombia
e-mail: [email protected]; [email protected]

© Springer International Publishing Switzerland 2016 1

P. de Voogt (ed.), Reviews of Environmental Contamination and Toxicology
Volume 237, Reviews of Environmental Contamination and Toxicology 237,
DOI 10.1007/978-3-319-23573-8_1
2 L. Tejeda-Benitez and J. Olivero-Verbel

6 Toxicity Assessments .......................................................................................................... 12

6.1 Environmental Samples ............................................................................................. 12
6.2 Pesticides.................................................................................................................... 13
6.3 Metals......................................................................................................................... 17
6.4 Nanoparticles ............................................................................................................. 17
6.5 Drugs .......................................................................................................................... 23
6.6 Toxins ......................................................................................................................... 25
6.7 Other Chemicals......................................................................................................... 25
7 Conclusion .......................................................................................................................... 28
8 Summary ............................................................................................................................. 28
References ................................................................................................................................. 29

1 Introduction

Caenorhabditis elegans is a non-parasitic nematode which, due to its many conve-

nient features has become an important model in biology research; for example, it
was the first animal whose genome was completely sequenced. This nematode was
proposed as a model organism by Sydney Brenner in 1965 (Garcia-Sancho 2012).
Since then, it has been used in cell biological, genetic and neurobiological studies of
higher eukaryotes. Between 1970 and 1980, the complete cell lineage of this worm,
from the fertilized egg to adult, was characterized by laser ablation and microscopy
(Sulston et al. 1983). Electron microscopy and serial sectioning allowed for the
reconstruction of the entire nervous system (White et al. 1986), together with the
genetic and genomic data generated in the 1990s (Coulson et al. 1991). This organ-
ism has become a powerful tool for the discovery and functional characterization of
eukaryotic genes (Dimitriadi and Hart 2010). Many aspects of C. elegans as a toxi-
cological model have been reviewed in an excellent paper by Leung et al. (2008). In
this state-of-the-art review, the authors present an update on that report, focusing on
toxicity end points and assessments for many types of environmental pollutants.

2 Biological Features of C. elegans

The body of an adult C. elegans is approximately 1 mm long. Its transparency

allows viewing of cell types in all stages of development. It has a simple nervous
system of 302 neurons as an adult, where each neuron has a unique position
(Dimitriadi and Hart 2010; Giles and Rankin 2009). Most organisms are hermaph-
rodites, with two ovaries, oviducts, a cavity for storing sperm called the sperma-
theca, and uterus (L’Hernault 2009). Hermaphrodites produce sperm as L4 larvae
and oocytes during early adulthood; they reproduce by self-fertilization and there-
fore cannot fertilize other hermaphrodites. The males, which appear spontaneously
with a frequency of less than 0.3 %, are able to fertilize hermaphrodites. The repro-
ductive cycle of C. elegans lasts 2.5–4 days at room temperature, and with a usual
lifespan of 12–20 days (Giles and Rankin 2009).
Caenorhabditis elegans, a Biological Model for Research in Toxicology 3

Eggs

50 mm L1
Adult

100 mm
50 mm

L2
L4

100 mm 100 mm

50 mm L3

Fig. 1 Life cycle of C. elegans. Images were acquired using a dissection microscope Nikon smz
745T with 4× magnification

Embryonic development culminates in the generation of an L1 larva of 550 cells,

after 113 cells have died by apoptosis. After four larval stages, the hermaphrodite
worm becomes an adult organism with 959 cell nuclei (some syncytial), 302 of
which are neurons. Males have 1031 cell nuclei. The mature adult is fertile for 4
days, and it can live between 10 and 15 additional days. Each adult hermaphrodite
lays between 200 and 300 eggs, at intervals of about 20 min. Furthermore, the cycle
time depends upon the temperature of incubation (Garcia-Sancho 2012). When
environmental conditions are adverse, for example, during food shortages, high
temperatures, or high population densities, successful reproduction is unlikely.
Under such conditions, C. elegans can halt its development passing into an alterna-
tive L3 stage called dauer, which can survive for months. During this stage, the
nematode does not feed and its cuticle is tougher. Nematodes can re-enter the repro-
ductive life cycle at L4 when conditions are more favorable (Wang et al. 2010b).
Figure 1 shows the complete cycle of C. elegans.

3 Advantages of Using C. elegans as a Biological Model

C. elegans is used as a model in genetic research because of its convenient features.

First, its transparency allows for transgenic proteins fused to fluorescent markers to
be visible in living animals in in vivo experiments (Giles and Rankin 2009). Its
generation time is short (4 days) and it occurs by self-fertilization, ensuring rapid
4 L. Tejeda-Benitez and J. Olivero-Verbel

reproduction in the laboratory (Zhuang et al. 2014) since each adult hermaphrodite
produces 200–300 progeny (Megalou and Tavernarakis 2009).
Its excellent performance as a model in genetics has led to the development of
many tools and resources, including thousands of characterized mutants and RNA
interference libraries, useful for silencing gene expression (Giles and Rankin 2009;
Megalou and Tavernarakis 2009). RNA interference (RNAi) with this organism is
relatively simple, and therefore gene silencing is often used to dissect signaling
pathways (Adam 2009).
C. elegans has been used in toxicological research, from the whole animal level
to the level of individual cells (Zhuang et al. 2014). It is cultured in the laboratory
in a nematode growth medium (NGM), which contains NaCl, agar, peptone, choles-
terol, K3PO4, KH2PO4, K2HPO4 and MgSO4. Another suitable culture medium is K
agar, which also contains KCl (Meyer et al. 2010). The worms are maintained in an
incubator at 20 °C and the bacteria Escherichia coli OP50 is utilized as a food
source (Giles and Rankin 2009). The K medium prepared with KCl and NaCl is the
liquid used to transfer worms to fresh dishes and to carry out bioassays (Williams
and Dusenbery 1990).

4 Applications in Medicine

C. elegans is a model organism that has been important in the studies carried out to
identify and understand the functioning of the machinery in nuclear transportation
(Adam 2009). It has helped to elucidate biochemical pathways involved in diseases,
such as obesity (Finley et al. 2013; MacNeil et al. 2013), diabetes (Estevez et al. 2014;
Shi et al. 2012), and Alzheimer’s disease (Diomede et al. 2014; Lublin and Link 2013).
C. elegans is an excellent model to investigate aging because of its short lifespan, its
susceptibility to oxidative stress and the similarities with the human aging process
(Chatterjee et al. 2013; Pang and Curran 2014). This nematode has also been employed
to identify biochemical pathways and mechanisms of action of new drugs, especially
antihelmintics (Kumarasingha et al. 2014; Lublin and Link 2013; Wu et al. 2012b).

5 Toxicity Endpoints

Bioassays to assess the effects of a toxicant on C. elegans can be carried out through
different endpoints. The normal procedure for acute exposure consists of the incuba-
tion of young adults in the K medium containing the toxicant at several concentra-
tions, usually without food. In long term exposure assays, worms in the L1 stage are
used; in this case E. coli OP50 is added as food (Zhuang et al. 2014). When worm
reproduction is not required during an experiment, since brood size may affect the
results, 5-fluorodesoxiuridine is used to inhibit DNA synthesis (Wu et al. 2012a).
Endpoints can be grouped according to their effects on biological parameters, for
Caenorhabditis elegans, a Biological Model for Research in Toxicology 5

Fig. 2 End points toxicity on C. elegans. Toxicity studies with C. elegans could be carry out through
two kinds of endpoints, those evaluate effects in nematode biology and those use molecular markers

instance, lethality, growth, locomotion, and reproduction. It is also possible to use

molecular markers to determine oxidative stress, changes in gene or protein expres-
sion, DNA damage, or green fluorescence protein (GFP) expression. A classification
of endpoints, commonly utilized in toxicity research using C. elegans as model is
shown in Fig. 2.
Some of the frequently used endpoints related to toxicity assessment using
C. elegans are presented below. These assays are usually performed employing
concentration-response curves.

5.1 Lethality

This assay is performed to determine the death rate derived from acute toxicity in a
concentration-response curve basis. 10 ± 1 young adults are transferred in micro-
plates which contain different concentrations of the toxicant and a negative control.
The exposure is carried out at 20 °C during 24 h in the absence of food. Then, the
number of live and dead worms is counted through visual inspection using a dissect-
ing microscope (Williams and Dusenbery 1990; Ellegaard et al. 2012; Helmcke and
Aschner 2010; Kim et al. 2012; Wu et al. 2012a; Zhuang et al. 2014). Death is
assumed when there is no movement during an observation period of 30 s (Rui et al.
2013; Shen et al. 2009; Wang et al. 2009a; Wu et al. 2013).
6 L. Tejeda-Benitez and J. Olivero-Verbel

5.2 Growth

The effect of a toxicant in the development of the nematode can be evaluated by

measuring the body length of synchronous worms before and after exposure, then
comparing them to a vehicle-control. The bodies of the worms are observed employ-
ing a light microscope with 10X magnification and with image analysis software,
such as Image-Pro® Express, ImageJ, or Fiji (Boyd et al. 2010; Cha et al. 2012; Höss
et al. 2009b; Meyer et al. 2010; Roh and Choi 2011; Shen et al. 2009; Wang et al.
2010a; Yu et al. 2013a, b). Some authors have reported the warming of the worms
to 50 °C in order to make them straight and ease the process of measuring their
length (Wang et al. 2009a). The immobilization of the worms can also be achieved
by using sodium azide (Turner et al. 2013). Growth can also be evaluated by regis-
tering the length of a curve, drawn from the tip of the head to the tip of the tail along
the dorsal-ventral half of the animal intestine, using the reference line. Width mea-
surements are taken in the vulva, drawing a line on the ventral side of the animal
between the front edge and the posterior periphery of the vulva (Rudel et al. 2013).
Other authors have proposed measuring the surface area for the flat worm (Rui et al.
2013; Wu et al. 2013; Zhuang et al. 2014). Currently, some laboratories have high-
tech equipment, such as COPAS Biosort, which measures the optical density of the
worm as an endpoint of growth (Hunt et al. 2012, 2013). The advantage is that the
COPAS Biosort can analyze hundreds of nematodes per minute, and it can also
evaluate mortality and fluorescence statistics (Hunt et al. 2012; Sprando et al. 2009).
For growth assays, some authors perform 24 h exposure periods with E. coli OP50
as food (Boyd et al. 2010; Cha et al. 2012; Roh et al. 2009), whereas in other stud-
ies, E. coli uvrA, previously killed by UVA radiation is used; in this case, the expo-
sure is carried out for 72 h and feeding is re-dosed every 24 h (Turner et al. 2013).

5.3 Reproduction

Brood size is the end point used to evaluate whether a toxic environment affects
reproduction of the nematodes, placing exposed adult or L4 worms onto fresh plates.
The number of offspring at all stages is counted and compared with a control group
(Cha et al. 2012; Gomez et al. 2009; Höss et al. 2009b; Höss et al. 2013; Kim et al.
2012; Leelaja and Rajini 2013; Menzel et al. 2009; Li et al. 2012b; Roh et al. 2009;
Rui et al. 2013; Smith et al. 2013; Wang et al. 2009a, 2010a, b). Counting is facili-
tated by heating the worms to 50 °C and staining them with Bengal red (Höss et al.
2013). In several studies the fertility rate is calculated by counting the total number
of larvae at the end of the test and dividing by the total progeny recovered to the total
parents (Rudel et al. 2013). This assay may also be carried out using the COPAS
Biosort by measuring optical density (Boyd et al. 2010). Moreover, the gonad size,
obtained by image analysis under a microscope, has been utilized to evaluate the
effects on reproductive organs (Wu et al. 2011). Toxic effects may also be seen as
Caenorhabditis elegans, a Biological Model for Research in Toxicology 7

changes in the egg-laying pattern and the number of eggs or larvae at different time
intervals (Gomez et al. 2009; Smith et al. 2013). Finally, the rate of egg laying can be
estimated by placing adult worms exposed to fresh plates and counting the number
of eggs laid in 1 h (Jadhav and Rajini 2009; Shashikumar and Rajini 2010).

5.4 Fertility

Reproductive toxicity can also be assessed by calculating the percentage of L4 larvae

that develop fertilized eggs after exposure. Gravid hermaphrodites are considered to
have at least one egg inside their bodies (Höss et al. 2009a, b; Roh and Choi 2011;
Wang et al. 2009a). To count the number of eggs in the uterus, nematodes can be trans-
ferred to a bleach solution, which dissolves the body of the worm, directly exposing
the eggs and allowing them to be counted under a light microscope (Wu et al. 2011).

5.5 Lifespan

Healthy worms at the L4 larval stage are exposed to a toxic agent, for example 24 h
and then placing them on NGM plates with E. coli OP50. To prevent the production
of offspring, 5-fluorodeoxyuridine is added. Worms are transferred to new plates
every 3 days. The number of survivors is recorded daily until all animals die. The
survival rate is calculated by dividing the number of live nematodes by the total
number of nematodes, including both live and dead worms. The lifespan is defined
as the time period between the L4 larval stage and death (Cha et al. 2012; Li et al.
2009, 2012b; Shen et al. 2009; Wang et al. 2010a; Zhuang et al. 2014).

5.6 Intestinal Autofluorescence

The intestinal lysosomal lipofuscin deposits that accumulate over time in the nema-
todes generate autofluorescence, feature used as a marker of aging. Treated nema-
todes are placed on an agar pad on a glass slide, then the fluorescent signals are
captured by a fluorescence microscope. A band filter of 525 nm is employed to detect
the endogenous intestinal fluorescence, and images are analyzed using software such
as Magnafire®. Lipofuscin levels can be measured using the software ImageJ, by
determining the mean pixel intensity in the intestine of each animal. Adults need to be
photographed on the same day to avoid the light variation related to the intensity of the
fluorescence source (Boyd et al. 2010; Helmcke and Aschner 2010; Rui et al. 2013;
Shen et al. 2009; Wang et al. 2010a; Wu et al. 2012a, c, 2013; Zhuang et al. 2014).
8 L. Tejeda-Benitez and J. Olivero-Verbel

5.7 Locomotion

Effects on the locomotion of nematodes have been linked to a deterioration of the

neural network which can be evaluated based on several criteria, such as head
thrash, body bend frequency, and basic movements (Yu et al. 2013a). Each exposed
nematode is transferred to a plate containing 60 μL of K medium on the top of the
agar. After a recovery period of 1 min, the number of head trashes is counted for
1 min. A head trash is defined as a change in the direction of bending in the body.
To test the body bend frequency, nematodes are collected in a second plate, and then
the number of times that the body bends in a period of 20 s is recorded. The bend of
the body is observed as a change in direction of the upper pharynx along the Y axis,
assuming that the nematodes are moved along the X axis. To test the basic move-
ments, the number of sinusoidal forward movements is counted at an interval of
20 s. Locomotion behavior of control and treated nematodes should be analyzed
simultaneously to avoid possible influences of the light-darkness cycle (Giles and
Rankin 2009; Li et al. 2009, 2012a, b; Matsuura et al. 2013; Roh and Choi 2011;
Rui et al. 2013; Wu et al. 2012a, 2013; Xing et al. 2009a; Yu et al. 2013a, b; Zhuang
et al. 2014). Alternatively, immobility is determined by counting the number of
immobile worms, usually registering a response when touched by platinum wire
(Jadhav and Rajini 2009; Leelaja and Rajini 2013; Roh and Choi 2011).

5.8 Metabolism

To assess the state of metabolism, the pharyngeal pumping speed and the average
cycle length of defecation can be evaluated. For testing the pumping rate, the nema-
todes are placed on NGM agar plates with food. After a few minutes, the pumping
movement of the pharynx is counted for a minute under a microscope (Jadhav and
Rajini 2009). To test the average cycle length of defecation, every nematode is
observed individually for a fixed number of cycles. A cycle is defined as the interval
between initiation of two successive steps of muscle contraction (Liu et al. 2013;
Wu et al. 2012a; Zhao et al. 2014a, b).

5.9 Development

The effects of toxicants on the development of nematodes can be investigated by

counting the number of individuals in each stage of their life cycle: egg, L1, L2, L3,
L4 and adults, at regular time intervals up to 96 h after treatment (Roh and Choi
2011). The development through the larval stages can be estimated using the fol-
lowing criteria: L1 if they have four or fewer gonadal cells; L2 if they possess over
four gonadal cells which have begun to spread along the length of the animal; L3 if
there is a further extension of the gonad, and vulval morphogenesis has started; L4
if there is a dorsal rotation of the gonad; and adults if they have observable eggs
(Helmcke et al. 2009). Entrance into the dauer state can be used to analyze toxicity,
Caenorhabditis elegans, a Biological Model for Research in Toxicology 9

since dauer formation is induced by causing starvation in nematodes. Usually,

treated nematodes in state of gravidity are placed on agar plates until laying eggs at
20 °C. This progeny is changed to 27 °C, and 72 h later. The organisms in the dauer
stage are counted (Wang et al. 2010b).

5.10 Feeding Behavior

Some toxics can affect the feeding and foraging behavior in C. elegans. Jones and
Candido (1999) described a procedure to assess feeding behavior by monitoring the
decline in the density of the bacterial food in liquid cultures of nematodes by mea-
suring absorbance at 550 nm. Another method consists of the use of agar with round
holes located equidistant from the center of the dish. Each hole is filled with bacte-
rial suspension in K medium. Toxic solutions are placed in different holes, and
nematodes are inoculated in the center of the plate. The number of nematodes in the
interior of each hole is counted at various intervals of time. This test shows whether
the test nematodes try to avoid contaminated food (Monteiro et al. 2014).

5.11 Oxidative Stress

Several markers of oxidative stress can be determined in worms after exposure to the
examined agent, both within the organism and the supernatant (Helmcke and
Aschner 2010; Leelaja and Rajini 2012; Shashikumar and Rajini 2010). The produc-
tion of reactive oxygen species (ROS) and oxidative damage may be determined by
fluorescence measurements, usually labeling the nematodes with 5-(y-6)-
chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (Eom et al. 2013; Leelaja
and Rajini 2013; Li et al. 2012a; Rui et al. 2013; Wu et al. 2012a, 2013; Zhuang et al.
2014), and reading with a laser scanning confocal microscope (Eom et al. 2013;
Helmcke and Aschner 2010; Rudgalvyte et al. 2013; Liu et al. 2012; Wang et al.
2010a; Wu et al. 2012a). Oxidative damage on macromolecules has been analyzed
by detecting carbonylated proteins (Wang et al. 2010a, b; Wu et al. 2011). Moreover,
oxidative stress can be evaluated by quantifying changes in gene expression of oxi-
dative stress-related genes by Real Time PCR or GFP reporters, such as sod-1, sod-
2, sod-3, sod-4, sod-5, gst-4, gst-5, gst-8, gst-24 and gst-42 (Rui et al. 2013).

5.12 Patterns of Gene Expression

One method used to investigate the change in expression of genes in C. elegans

exposed to environmental pollutants is the use of DNA microarrays or Real Time
PCR (Eom et al. 2013; Menzel et al. 2009; Li et al. 2012a; Roh et al. 2009; Roh and
Choi 2011; Rudgalvyte et al. 2013). As reference genes, act-1 (Zhuang et al. 2014;
Wang et al. 2014a) and ubq-1 (Wu et al. 2011) are commonly used. This method
has been applied to evaluate the effect on C. elegans gene expression for various
10 L. Tejeda-Benitez and J. Olivero-Verbel

environmental toxicants, such as river sediments (Menzel et al. 2009), veterinary

drugs (Zhuang et al. 2014), sodium fluoride (Li et al. 2012b), nanoparticles
(Eom et al. 2013), and metals (Roh et al. 2009; Wang et al. 2014b; Rudgalvyte et al.
2013) among others.

5.13 Protein Expression

Induction of proteins by exposure to pollutants can be evaluated by traditional tech-

niques such as ELISA and Western Blot. The ELISA technique was used to deter-
mine HSP90 protein expression in wild type C. elegans exposure to zinc at different
temperatures (Wang and Ezemaduka 2014). Western blots were used to evaluate the
effect of lead on HSP90 expression (Wang et al. 2014b).

5.14 DNA Damage

C. elegans has been used to evaluate DNA damage through various techniques. One
approach is the use of the qPCR technique to detect damage and DNA repair. This
test works on the principle that DNA damage inhibits the progression of the poly-
merase used in qPCR (Roh et al. 2009; Li et al. 2012b). The amount of long PCR
product provides a measure of the frequency of the injury (Leung et al. 2010).
Another alternative is the comet assay, which was used to evaluate the genotoxic
profile of river sediment (Menzel et al. 2009). More recently, the pathway of base
excision repair has been proposed as a mechanism to assess the damage to DNA by
specific qPCR (Hunter et al. 2012). Transgenic strains can also be used to assess
DNA damage. The strain xpa-1 is deficient in the mechanism of nucleotide excision
repair, and its growth is significantly affected when there is damage to DNA.
Therefore, the growth assay on this strain is an indicator of genotoxicity (Leung
et al. 2010). The transgenic strain hus-1::GFP is utilized to assess DNA damage.
HUS-1::GFP foci represents DNA double-strand breaks, allowing quantification by
counting the number of bright foci per 20 pachytene gonadal germ cells (Hofmann
et al. 2002) which can be observed and counted under a fluorescence microscope
(Wang et al. 2014a).

5.15 GFP Reporters

Transgenic nematodes carrying the GFP gene fused to various stress-inducible gene
promoters have been developed for the study of various biochemical pathways. GFP
strains are placed in wells containing the sample solutions and suitable controls. The
plates are incubated at 20 °C, performing fluorescence readings within 4–6, 8–20,
Caenorhabditis elegans, a Biological Model for Research in Toxicology 11

and 24–40 h for short, moderate and long exposures, respectively. GFP expression is
quantified using a fluorometer with a wavelength of 485 nm excitation and 525 nm
emission (Anbalagan et al. 2012; Anbalagan et al. 2013; De Pomerai et al. 2010;
Roh et al. 2010; Roh and Choi 2011). Alternatively, the observation of fluorescence
can be achieved under a light microscope, capturing images that are then analyzed
by specialized software (Li et al. 2009; Shen et al. 2009; Wang et al. 2010a; Polak
et al. 2014). The COPAS Biosort system has also been employed to measure fluores-
cence (Hunt et al. 2012; Turner et al. 2013). The transgenic strain F25B3.3::GFP
with fluorescence expression in neurons has been utilized to study heavy metal
(Du and Wang 2009; Helmcke et al. 2009) and pesticide toxicity (Negga et al. 2011).

5.16 RNA Interference (RNAi)

This technology has been widely used to study gene function. Bacterial RNAi is
introduced for 48 h at room temperature for the expression of dsRNA. Double
stranded (ds) RNA expression is induced in HT115 bacteria containing the gene-
sequence of interest inserted in the L4440 vector, or else the empty vector as control
(Kamath and Ahringer 2003). Approximately ten nematodes in stages L1–L3 are
placed on the plate seeded with induced RNAi or vector-control bacteria and incu-
bated at 20 °C. After 36–40 h, the worms are transferred to another plate seeded
with the same bacteria and grown to adulthood, at which point cultures are synchro-
nised by egg isolation, and the eggs transferred onto new plates with RNAi bacteria.
To evaluate the efficiency of dsRNA feeding over 1000 worms are evaluated by
using semiquantitative PCR (Cheng et al. 2014; Kumar et al. 2010; Roh and Choi
2011). RNAi can be used to evaluate genetic pathways involved in toxicant
responses. For instance, RNAi has been involved in the transcription of the DAF-16
factor in an unpredicted upregulation of the cyp-34A9 reporter gene by exposure to
high levels of cadmium (De Pomerai et al. 2008). In another case, gene knockdown
by RNAi was used to determine the effects on reproduction due to PCB52 exposure;
several genes were identified as having a crucial role, being the most remarkable the
cytochrome P450s group (Menzel et al. 2007).

5.17 Cell Apoptosis

To assess apoptosis in the cells of the nematode, acridine orange is used. After expo-
sure to the toxicant for 24 h, the nematodes are immersed in mixed medium with
acridine orange at 20 °C for 2 h. Then they are placed on top of agar allowing them to
recover for 10 min. Finally, they are examined under an inverted fluorescence
microscope with an excitation wavelength of 515 and 488 nm absorption. Apoptotic
cells appear yellow or yellow-orange showing increased DNA fragmentation,
whereas intact cells are uniformly green (Li et al. 2012b; Wang et al. 2009b, 2014a, c).
12 L. Tejeda-Benitez and J. Olivero-Verbel

Another technique involves staining with SYTO 12 for 4 h at room temperature,

followed by seeding with food for 30 min, washing with M9 buffer, and final observa-
tion under a fluorescence microscope with a red filter (Cha et al. 2012). Alternatively,
the transgenic strain ced-1::GFP is used for visualization of apoptotic bodies in a
fluorescence microscope (Cheng et al. 2014; Kumar et al. 2010).

5.18 Cell Cycle Arrest

To investigate whether the exposure to a toxicant causes cell cycle arrest in the
germline, the number of cores of mitotic cells is determined by staining with
4′,6-diamidino-2-phenylindole. The number of mitotic nuclei present at the distal
end of the germline is counted under a fluorescence microscope (Cheng et al. 2014;
Kumar et al. 2010; Wang et al. 2014a).

5.19 Transgenerational Effects

Sublethal endpoints such as locomotion and growth can be evaluated in the off-
spring of exposed parents. Wild-type N2 nematodes at the L3 larval stage are
exposed during the time when sperm, ova and eggs begin to form, providing a win-
dow of prenatal exposure (Yu et al. 2013b). Exposed worms are placed on several
plates. Some of them are used for measuring the parents after 24 h of exposure, and
the others, for obtaining the generations. This assay has been used to assess the
effects of antibiotics (Yu et al. 2011) and heavy metals on the growth and locomo-
tion of exposed parents and their first generation (Yu et al. 2013b).

6 Toxicity Assessments

Most currently known toxicants can be assessed using C. elegans as a model. The
following are some research studies related to toxicity of environmental matrices,
metals, pesticides, nanoparticles, and other chemicals.

6.1 Environmental Samples

C. elegans has been used as a model to assess the toxicity of environmental samples
such as soils, sludges, and river sediment. The sediment of the Danube, the Rhine
and the Elbe Rivers in Germany were studied by analyzing the changes in gene
Caenorhabditis elegans, a Biological Model for Research in Toxicology 13

expression profiling using DNA microarrays of the entire genome. At the same
time, the reproduction and DNA damage were evaluated using the comet assay
technique (Menzel et al. 2009). In a study of the toxicity of contaminated soils from
Germany, fertility, growth, and reproduction were evaluated using the wild type
Bristol N2 strain (Höss et al. 2009b). Organic extracts of contaminated soil from
Spain were evaluated using transgenic strains of C. elegans carrying GFP reporter
genes driven by promoters sequences from five stress-related genes, hsp-16.2, gpx-
6, hsp-6, gst-1, and cyp34A9; allowing the identification of different mechanisms of
toxicity (Anbalagan et al. 2012). Aqueous extracts of the same soils were evaluated
using 24 similar GFP transgenic reporter strains, correlating this data with the con-
centrations of metals present in the soil (Anbalagan et al. 2013). A summary of the
results generated from these investigations is shown in Table 1.

6.2 Pesticides

In the environment, C. elegans as a free-living nematode, is exposed to various pes-

ticides used in agriculture as well as to persistent organic waste that can contaminate
soil for long periods of time (Anbalagan et al. 2013). Some of the most recent stud-
ies relating to the toxicity of pesticides in C. elegans are summarized in Table 2. As
many pesticides are neurotoxic, the well-defined nervous system of C. elegans is a
suitable tool to assess the neurotoxicity induced by these chemicals (Gomez et al.
2009; Leelaja and Rajini 2012, 2013; Lewis et al. 2013; Negga et al. 2011; Roh and
Choi 2008, 2011; Shashikumar and Rajini 2010; Meyer and Williams 2014).
Fluorescence expression by GFP reporter genes has been employed to study the
toxicity of pesticides such as Glyphosate, Paraquat, 2,4-days, Endosulfan,
Cypermethrin, Carbendazim, Chlorpyrifos, Diuron, Rotenone, DDT, Deltamethrin,
and Dichlorvos (Anbalagan et al. 2013). In another report, Chlorpyrifos was studied,
and although it did not cause severe DNA damage, it inhibited growth of xpa-1 defi-
cient strain, whose mechanism of nucleotide excision repair is deficient (Leung et al.
2010). The herbicide Glyphosate and the fungicide dithiocarbamate have been stud-
ied to assess mortality and neurological damage in C. elegans. Neuronal damage by
exposure to these pesticides was verified by using the transgenic strain F25B3.3::GFP
(Negga et al. 2011). The effect of Paraquat, Diquat, and Parathion on brood size was
evaluated with COPAS Biosort, with Paraquat showing the highest toxicity (Boyd
et al. 2010). Acetylcholinesterase activity of pesticides has also been assessed in
nematodes exposed to Fenitrothion and Monocrotophos (Leelaja and Rajini 2013;
Roh and Choi 2011). Studies with tributyltin reported that this biocide caused cell
apoptosis in C. elegans via DNA double-strand breaks (DSBs) (Wang et al. 2014a).
Furthermore, tributyltin chloride caused increased sterility and embryonic lethality
by DSBs and checkpoint activation in the germline (Cheng et al. 2014). Insecticidal
proteins such as Cry, used in transgenic corn, were studied and showed dose-depen-
dent inhibitory effects on C. elegans reproduction (Höss et al. 2013).
Table 1 Evaluation of environmental samples using C. elegans as a model
Environmental sample Strain End point Result Reference
Sediments from three N2 Reproduction, DNA damage, Disaccharide and glycogen Menzel
rivers in Germany: changes in gene expression metabolism and functional pathways et al. (2009)
Danube, Rhine and Elbe were affected
Water and sediment basins N2, gpdh-1, gpdh-2, mtl-2, mtl-2::GFP, pcs-1, smf-2, sod-3 Growth and GFP expression Inhibition of growth. Turner
affected by coal mining in et al. (2013)
Virginia (United States)
Contaminated soils N2 Fertility, growth and reproduction Toxicity was correlated with Höss et al.
from Germany the organic fraction (2009a)
Organic extracts of hsp-16.2::GFP, gpx-6::GFP, hsp-6::GFP, gst-1::GFP, GFP expression Induction of expression Anbalagan
contaminated soils in cyp34A9::GFP of transgenes et al. (2013)
Southeast Spain
Aqueous extracts of soil hsp-16.1::GFP:lacZ, cep-1::GFP, hsp-16.2::GFP, GFP expression Correlation between metals Anbalagan
sod-3::GFP, cyp-35a2::GFP, daf-16::GFP, gpx-6::GFP, and expressed transgenes et al. (2012)
hsp-6::GFP, gpx-4::GFP, cyp34A9::GFP, hsp-3::GFP,
mtl-1::GFP, elt-2::GFP, gst-1::GFP, skn-1::GFP, gst-4::GFP,
sod-4::GFP, cyp-29A2::GFP, ctl-2::GFP, hsf-1::GFP,
mtl-2::GFP, hsp-60::GFP, hsp-70::GFP y sod-1::GFP
Wastewater from N2, daf-2 Life span, dauer formation Life span decreased Wang et al.
recycled paper plant (2010b)
Particulate matter N2, unc-47::GFP Lethality, lifespan, growth, Effects on lifespan, reproduction, Zhao et al.
reproduction, locomotion, intestinal locomotion behavior, and intestinal (2014a,b)
autofluorescence, oxidative stress, development exposed and their
defecation behavior, gene expression progeny
Surface water containing N2 Locomotion, pharyngeal pumping, The surface water was Ju et al.
brominated organic defecation, mechanical sensory neurostimulatory (2014)
compounds stimulus, chemotaxis, thermotaxis
Dispersed oil crude N2, (bcIs39 [(lim-7)ced-1p::GFP + lin-15(+)]), Cell apoptosis, gene expression Increasing of germ cell apoptosis Polli et al.
and TJ1(cep-1(gk138) I) following a CEP-1-dependent pathway (2014)
Soil-derived Fe oxide N2, sod-2(ok1030) Uptake of Fe, growth, reproduction The toxicity of ferrihydrite, goethite Höss et al.
colloids and akaganeite depends on aggregate (2015)
size and specific surface area
Table 2 Evaluation of pesticide toxicity
Pesticide Strain End point Result Reference
Phosphine N2 Oxidative stress Delayed development and oxidative stress Leelaja and
Rajini (2012)
Fenitrothion N2, cyp35a2 Paralysis, growth, fertility, Cyp35a2 involved in toxicity Roh and Choi
development, AC activity (2011)
Monocrotophos N2 Fertility, oxidative stress, Decreased oxidative stress, paralysis and brood Leelaja and
AC activity, paralysis size. AC inhibition Rajini (2013)
Cypermethrin N2 Expression of hsp-16, rate Brood size, egg laying and life span decrease in Shashikumar
of egg laying, life span, a dose-dependent fashion and Rajini
brood size, oxidative stress Increased ROS and carbonylated proteins (2010)
Dithiocarbamate and N2, F25B3.3::GFP Lethality and neuronal Neurotoxicity was verified by fluorescent Negga et al.
glyphosate damage expression at neuronal level (2011)
Cry proteins N2, bre-5 Reproduction, gene Reproduction inhibition Höss et al.
expression MAPK pathway upregulated (2013)
Gliphosate, Paraquat, 2,4-D, hsp-16.1::GFP:lacZ, cep-1, GFP expression Dichlorvos and Rotenone induced several stress Anbalagan
Endosulfan, Cypermethrin, hsp-16.2, sod-3, cyp-35a2, daf-16, response genes in a dose- dependent manner et al. (2013)
Carbendazim, Chlorpyrifos, gpx-6, hsp-6, gpx-4::GFP, cyp34A9, Endosulfan, DDT, Carbendazim, Deltamethrin,
Diuron, Rotenone, DDT, hsp-3, mtl-1, elt-2, gst-1, skn-1, Cypermethrin, 2,4-D and Chlorpyrifos induced
Deltamethrin, Dichlorvos gst-4, sod-4, cyp-29A2, ctl-2, hsf-1, few genes
mtl-2, hsp-60, hsp-70, and sod-1
Chlorpyrifos N2, emb-8, glp-1, xpa-1 Growth and DNA damage No DNA damage was detected, but the xpa-1 Leung et al.
Caenorhabditis elegans, a Biological Model for Research in Toxicology

mutant was more sensitive than the wild type (2010)

(continued)
15
 16

Table 2 (continued)
Pesticide Strain End point Result Reference
Chlorpyrifos N2 Gene expression, changes The free concentration of chlorpyrifos quickly Roh et al.
of free concentration of diminished and the expression of cyp genes (2014)
chlorpyrifos in the medium varied with the volume of exposure medium
and the test duration
Diquat, Paraquat, Parathion N2, myo-2::GFP Body length, optical Parathion > Diquat > Paraquat Boyd et al.
density, GFP expression, (2010)
reproduction
Tributyltin N2, ced-3, ced-4, egl-1, ced-9, cep-1, Cell apoptosis, Cell cycle Induction of apoptosis in germline via DNA Wang et al.
cep-1, clk-2, hus-1, lin-45,mek-2, arrest, gene expression, damage (2014a)
ksr-1, mpk-1, nsy-1, mek-1, jkk-1, DNA damage
mkk-4, jnk-1, sek-1, pmk-1
Imidacloprid and Tiacloprid N2 Reproduction Synergistic effect after exposure to the mixture Gomez et al.
(2009)
Dichlorvos N2 Pharynx pumping, Correlation between the biochemical effects Jadhav and
contraction of the nose, and behavior parameters Rajini (2009)
paralysis, egg laying rate
Monocrotophos N2 Locomotion, lifespan, egg Decreased locomotion, lifespan, egg laying, and Salim and
laying, growth, AC activity brood size Rajini (2014)
L. Tejeda-Benitez and J. Olivero-Verbel
Caenorhabditis elegans, a Biological Model for Research in Toxicology 17

6.3 Metals

Metals, in particular those named as heavy metals, constitute one of the most impor-
tant groups of environmental toxicants, and reach the ecosystems from sources such
as oil refineries, mining, and industrial effluents, causing severe toxic effects on
living systems. This group has been one of the most studied using C. elegans as a
biological model. Several of the reports related to the effects of heavy metals on C.
elegans are presented in Table 3. The effects of different metals such as Ag, As, Cr,
Cd, Cu, Hg, Mn, Pb, Ni and Zn have been studied for several end points such as
lethality (Williams and Dusenbery 1990), lifespan, fertility, growth, intestinal auto-
fluorescence, GFP expression, morphology changes (Shen et al. 2009; Rudel et al.
2013; Hunt et al. 2012), neuronal damage, neurodegeneration, neuronal loss, and
axonal degradation (Du and Wang 2009; Xing et al. 2009a, b). On the other hand,
exposure to Zn, Cd, Hg, Cu, Fe, Cr, and As has also been monitored using GFP
transgenic reporter strains (De Pomerai et al. 2010).

6.4 Nanoparticles

The toxicological potential of nanoparticles (NPs) is receiving increased attention

because of their massive release into the environment. Although a number of manu-
factured NPs are employed for medical and clinical purposes, the interaction
between nanomaterials and biological systems remains unknown. For this reason,
the NPs have joined the group of Emerging Contaminants and every year more stud-
ies on the subject are performed with C. elegans (Table 4). It has been considered
that the main mechanism of nanotoxicity is oxidative stress (Zhao et al. 2014a).
Toxicity of hydroxylated fullerene nanoparticles was studied using C. elegans, and
it was demonstrated that water-soluble fullerol NPs have a potential for inducing
apoptotic cell death (Cha et al. 2012). The study of TiO2 NPs was carried out by
analyzing different toxicity endpoints such as lethality, reproduction, growth, loco-
motion, intestinal autofluorescence, and oxidative stress. TiO2 NPs caused severe
deficits in gut development, defecation behavior, and changes in gene expression
(Rui et al. 2013; Zhao et al. 2014a). The toxicity of TiO2, ZnO, and SiO2 NPs has
been compared using endpoints including lethality, locomotion, growth, reproduc-
tion, and production of ROS. The order of toxicity was ZnO > TiO2 > SiO2 (Wu et al.
2013). In a study of the intake of silver NPs by image analysis, there was absorption
of silver NPs in the body, transgenerational transfer, and inhibition of growth (Meyer
et al. 2010). The lethal effects of AgNPs on C. elegans are increased if the exposure
is through E. coli OP50 (Ellegaard et al. 2012). In other research, reduction was
observed in survival and reproduction and there was interaction of Ag NPs with
biological surfaces of C. elegans, causing severe edema (Kim et al. 2012).
Other documents randomly have
different content
 1946-1971 223 likes Gorky and Shakespeare, and it is to
this that we owe two of his finest fikns. The Lower Depths (Donzoko,
1957) is an apparently literal film version of the Gorky play; though
the time is the eighteenth century and the place, Edo, now Tokyo.
But Kurosawa has illuminated this rather disagreeable drama with a
visual magnificence and a really unexpected beauty that makes one
quite forget that the original was a play. Not a trace of the stage is
left— it is all pure ciaema. The fihn— hke all of Kurosawa's best films
— has a very definite and completely individual style. A consistent
set of rules governs characters, camera movement, formal
composition, and editing. Hiese rules all unify the fihn, making it a
bit more consistent than life itself, and gives that higher realism
which we usually call art. Particularly interesting were the new
interpretations: the Baron was presented as a real Japanese
tonosarm, a completely lordly lord; the harlot was shown as utterly
hysterical; and Luka, the old pilgrim-played by Bokuzen
Hidaribecame a figure of fun, but at the same time he is the only
one who knows what life is about. The Castle of the Spider's Web
(Kumonosu-Jo, 1957), also known as The Throne of Blood, was
another adaptation, one might almost say an illumination, of a well-
known story: that of Shakespeare's Macbeth. In transplanting
Shakespeare to the Middle Ages of Japan, Kurosawa did little to
change the original. Though the characters all had Japanese names,
and though the locale was obviously Japan, the story of vaulting
ambition and deep
 224 JAPANESE CINEMA desire is both timeless and
international in its appeal and it is this quality which Kurosawa both
preserved and amplified. Kurosawa saw in the picture a continuation
of a major theme: "I keep saying the same thing in different ways: if
I look at the pictures I've made, I think they say, Why is it that
human beings aren't happy?' Both Ikim and Record of a Living Being
are such pictures-T/ie Throne of Blood on the other hand shows why
they must be unhappy." The reasons are the most elemental rules of
the human comedy-Kurosawa and Shakespeare both agree on just
what they are. So, in this fihn Kurosawa continued his thesis that
power invariably corrupts. Since the story was a legend and the
theme timeless, however, he discarded the extreme realism of other
historical fihns, and created a film style which so completely suited
the plot that form and content became visually one. Using only a
handful of components-drifting fog and smoke, rainy forests, the
shining surface of armor, the sheen of natxiral wood, the
translucence of cloudy skies, the dead white of human skin-
Kurosawa, with his cameraman, Azakazu Nakai, created a film with a
definite texture. It was as though you could feel it with the hand.
More, it was a nearly perfect fusion of fihn and sound-and one
studded with examples of the director's talent. Just a few were: a
marvelous scene— not in Shakespeare-where hundreds of birds,
disturbed by the destruction of their forest, fly into the banquet haU
and circle around the distraught hero; the sequence where
 1946-1971 225 tiny nocturnal soimds form an ostinato to
the murder of Duncan; the appearance of the ghost of Banquo
where Kurosawa, disdaining trick photography, used the actor
himself, made-up in pure white, lights creating a halo arovmd him;
the march of the forest, photographed using slow motion and
telescopic lens, the trees swaying like gigantic seaweed as wave
after wave descends upon the castle, and the superb climax with
Macbeth— beautifully played by Toshiro Mifune— immobilized by
arrows, the shafts falling in formation on either side of him, piercing
his armor, his body, the final arrow transfixing his neck. The
restrained prodigality of this fihn is but an indication of the multiple
worlds that Kurosawa has created. Equally fitting to its subject was
the world of The Lower Depths, one in which the poverty of the
characters was perfectly matched by a photographic finish, which
was the precise opposite of the luxurious patina of the Macbeth film.
It is carefully threadbare, directorial effects being hidden, or
appearing only in negative form. The only method of punctuation
Kurosawa allowed himself was the simple cut. He confined himself
and his actors to just two main sets. He allowed his film no stars as
such. There was little music, its substitute being the rhythmic sound
of the hyosMgi, the clappers which call the audience's attention at
the beginning of the traditional theater. These limitations were
combined in the brilliant final scene: after the death of the
prostitute, the cynic takes a drink, turns to the camera and says:
"What a shame! Just when the party was beginning." At the same
time there is a single clap from the hyosMgi and instantly the
 2,2.6 JAPANESE CINEMA end title glows for a second, then
disappears, wresting, almost jerking us away from what is indeed a
floating world, but one which is also ours. Just as different was the
double world that Kurosawa created for Ikiru and Record of a Living
Being— two films which share a common milieu, one which is seen
in Drunken Angel and Stray Dog, as well as the slight but charming
Wonderful Sunday (Subarashiki Nichiyobi, 1947 )> but now the ruins
have been rebuilt, sumps have been drained, all is prosperity again.
The ruin, the devastation, is in the hearts of men. In order to
emphasize this, Kurosawa in these two films, this double story of
two men realizing themselves, one in death, the other in madness,
used the most flamboyant, the most prodigal, the most attention
getting of styles. The restraint of The Lower Depths and of
Rashomon is completely missing. Here Kurosawa shouts for
attention. Record of a Living Being, wonderfully photographed by
Azakazu Nakai, as was IJdru, showed us a disordered world. The
camera, Uke the hero, peers myopically at objects such as
telephones and typewriters and they become huge and menacing.
The family is habitually seen as a unit, perfectly composed, the
staring wide-angle lens holding every member. There is always a
distracting element, catching and irritating the eye, as in the scene
where the distraught hero goes to visit his young mistress —the jets
fly constantly overhead, obscuring the dialogue; an electric fan
blows the pages of a magazine at the lower
 1946-1971 227 comer of the picture frame; and all of this
incessant and meaningless motion confuses and fatigues the eye.
Ikiru presents the same disordered environment, but intensifies it.
The fihn is long and varied; it winds and imwinds; it shifts from
mood to mood, from present to past, from silence to a deafening
roar-all in the most unabashed and absorbing fashion. One can
compare it only to Citizen Kane, a film Japan did not see until its
1961 television showings, in which, for much the same reason, the
resources of the camera are strained to the limit: the marvelous
"night town" sequences in which camera motion is consimimately
used to suggest the teeming city; the scenes in the oflSce, so
hushed and imderplayed that they all but shout for attention; the
final funeral sequence (the last third of the film) where the life of the
dead official is reviewed over and over again, each repetition getting
a bit nearer the truth. In all of Kiu-osawa's best films the style fits
the story so closely that the resulting emotional experience is literally
imforgettable. This is seen even in his debut fihn, Sanshiro Sugata,
and in its 1945 sequel, where the judo story was reflected in athletic
cutting, and in the bounce and pace of the tempo. It is seen fully
matured in two of the director's very finest pictiures: Rashomon and
Seven Samurai. The former is extremely well known, probably the
most famous Japanese fihn ever made. Its theme, Hke that of Ikiru,
is basically existential: a man is what he does, not what he intends
nor what he beheves; truth is just as relative as anything else and
reahty is a matter of in 
 228 JAPANESE CINEMA terpretation. Kurosawa, who
certainly does not consider himself an existentiahst, based the film
on two stories of Ryunosuke Akutagawa, and was assisted by
Shinobu Hashimoto, whose first produced scenario this was. The
script as it originally stood-the work of three individuals standing
outside and even against the traditional Japanese current—
questioned all values. A lord, his bride, and a bandit meet in a wood.
The result is the death of the lord and the probable violation of the
bride. There was one other witness, a woodcutter who claims to
have witnessed the entire incident, and who compares the various
conflicting versions with a servant and a monk, both of whom, like
himself, are sheltering themselves from the rain under the great
ruined gate which gives the film its name. It is said that the bandit
claims he tied up the lord, violated his bride before his eyes, then
killed the husband in the course of a duel; that the wife said the
bandit attacked her, then ran away, that the husband hated her for
this, that she fainted, and when she revived the knife was in his
breast; the dead lord, speaking through a mediiun— an occurrence
related by the priest— said that the violation gave the woman much
pleasure and that she tried to make the bandit kiU her husband.
When both had gone the husband kiUed himself. Then the
woodcutter admits that he actually vtdtnessed the incident. After the
attack the bandit said he would marry the woman, that he and the
lord should fight for her; the husband, however, refused to risk his
life for the sake of such a woman; this infuriated her and it was she
who
 1946-1971 229 incited them into the duel which killed her
husband. All stories are equally plausible, all are probably true— this
is what the original script intended to convey, and here it ended. It
was Kurosawa himself who added the epilogue. He has said that the
film was otherwise too short and so it probably was. At the same
time, however, the addition is so typical of the director that one
cannot well imagine the film without it. After the various stories the
cry of an infant is heard. The three find an abandoned baby and the
servant steals some of its clothing just as the woodcutter had stolen,
we find, the woman's dagger. The priest remonstrates, thinking the
woodcutter intends to strip the infant, but he merely picks it up,
saying that though he has six others at home, one more will not
matter. The priest, moved, says: "You have, I think, restored my
faith in humanity." This addition is completely typical of the director.
It is compassion which, in a way, rights all wrongs; amid a chaos of
relative values it is the only absolute. It is also typical of Kurosawa
that he should have chosen for the film the particular style that he
did. Of it, he says: "I wanted to return to the simple pictorial values
of the silent picture— it is an attempt to bring back the lost beauty
of the film." Certainly, so far as pictorial beauty goes, this is a
ravishing motion picture. Even Mizoguchi rarely equaled the beauty
of the opening scenes, with lord and wife wandering through the
forest. The shots of sunlight filtering through foliage may have been
inspired by similar scenes in the films of
 230 JAPANESE CINEMA Dovzhenko and Lang, but they
surpass them. The final sequence, with its enormously slow
dissolves, each shot farther from the three under the gate, is of the
greatest pictorial beauty. The careful composition of each shot, too,
is indicative of Kurosawa's concern for the balanced, the beautiful.
His placing of three in the forest scenes, a visible and ever-changing
triangle, is a visual tour da force. One thinks of other Kurosawa
compositions: the controlled diagonals of The Lower Depths; the
long soliloquy in The Bad Sleep Well where Mifune, alone at one side
of the screen, is balanced by a rain-flecked window and the diagonal
of the half-ghmpsed roof beyond; the exciting off-center
compositions of The Throne of Blood; the father-son scenes in Ikiru
where the father occupies the bottom half of the screen; the careful
asymmetry of the compositions in Record of a Living Being.
Kurosawa is one of the few directors in the world today who uses
pictorial composition to comment upon action. The acting technique,
always interesting in a Kurosawa film, is particularly strong in
Rashomon. Though foreign critics have found traces of the
traditional Japanese theater in the acting, there is httle indication
that Kurosawa intended this. The Rashomon acting style, actually,
came from Shingeki and, oddly, from a jungle picture, featuring lions
and tigers, which Kurosawa happened to see just before shooting
began. He asked his cast— Toshiro Mifune, Machiko Kyo, MasayuM
Mori, Takashi Shimura— to attend and take notes. After all, the fihn
was about amorality, one of the strongest animal characteristics.
What confused foreign critics was perhaps
 1946-1971 231 the grand style which Kurosawa so often
uses, personified for most people by the acting of Toshiro Mifune:
the grand gestures of Mifune and Isuzu Yamada in the Macbeth film;
the drunken dances and tantrums of the Gorky picture; the
restrained but "big" acting of Seven Samurai, larger than anything
the West has in either its fihns, in which acting is habitually the most
underplayed element, or in its theater. In Rashomon one also sees
the attention to detail which is so typical of the director. When
Machiko Kyo drops the dagger in the final version of the story it falls
point first into the earth as though into a human breast. The
extremely powerful scene where the dead husband speaks through
the mouth of the medium appears to be simplicity itself but is
actually made up of a vast number of elements, all carefully
controlled: the winds, the lightning, the banners in the background,
the very sand upon which she kneels. One thinks of teUing details in
other Kurosawa fihns: the detailed soundtrack of The Throne of
Blood with its nocturnal insects, its distant shouts, the constant
sound of footsteps; the prodigality of detail, almost a texture, in The
Lower Depths— rotten cloth, weathered wood, wrinkled skin; the
tiny naturahstic actions of the family in Record of a Living Being,
each one irritating, apparently aimless, ostensibly gratuitous; the
pond in Drunken Angel upon which float, as though in allegory, the
objects of a vanished innocence: lost letters and abandoned dolls.
The fusion of all the elements which make up the completely
individual style of Kurosawa occurs in Seven
 232 JAPANESE CINEMA Samurai (Shichinin no Samurai,
1954)— originally shown abroad, before the American remake
(1961), as The Magnificent Set5en— Kurosawa's best film and one
which, were it necessary to make the choice, I should call the finest
Japanese film ever made. In a way, it is the summation of everything
which is most Japanese about the Japanese film. It is concerned
with the present, though its story is laid in the past. It criticizes
contemporary values but insists that they are, after all, human
values. It faithfully and honestly creates the context of Japanese
Ufe, man and his surroundings. At the same time it is concerned
with timeless values and universal attitudes. It uses a controlled
realism as vehicle and presents a surface of superlative physical
beauty which serves to accentuate the beauty beneath. In many
ways Seven Samurai is both the opposite and the continuation of
Rashomon. The earlier film represents the limitations of the intellect:
four stories, each completely intellectualized, all mutually
incompatible, and all, in their way, "true." Seven Samurai on the
other hand steps beyond intellectualization. It says that only those
acts which spring from emotion are vahd acts; that action thus
motivated is itself truth. This truth is one which remains, though
universally applicable, particularly Japanese. It is one which is
shared vidth Zen and with the haiku, as well as the films of Ozu and
Kurosawa— the emotions comprehend where the intellect falters.
The basic dichotomy is one recognized and insisted upon in Japan
just as much as in the West, and Kurosawa's hu 
 1946-1971 233 manism, his Dostoevsky-like compassion,
remains his final and his strongest statement. In Seven Samurai,
however, compassion is not sentimentality. It has been tempered
with action; theory has become practice. Violent physical action,
things done, things accomplished, things completed, things
irrevocable: these are what this film is made of. The end result of all
this action (and there has rarely been so violent an "action film" as
Seven Samurai) is the distillation of a single bittersweet and ahnost
unpalatable truth, but one the emotional acceptance of which is
impossible to avoid. The story is simple. A small village, completely
defenseless, is harassed by bandits. They ask the aid of a group of
masterless samurai, men as homeless, as outside society as the
robbers they are asked to fight. They agree, and the body of the film
shows their defense, during which a number of them die. The
villagers are grateful, but it is spring planting season. They are busy,
they have things to do. The remaining samurai leave the town they
have defended, the only home they know. They stop in front of the
grave of those they left behind, then they move on. The film is an
impassioned call for cooperation among men and, at the same time,
suggests why this has always been and will always be impossible.
Like La Grande Illusion— which it resembles in no other way— Seven
Samurai demonstrates the essential sympathy binding men together,
and shows how war perverts it. But, more, the Kurosawa film goes
on to demonstrate that peace too
 234 JAPANESE CINEMA has its perversions. The samurai
fight side by side with the villagers; yet, at the end, though the
villagers have not become enemies in their turn, at the same time
they cannot be called friends. Thus the film constitutes a valid,
moving, and unnerving interpretation of the motives of men. That it
is not also depressing, even tragic, is due entirely to the sustained
conclusion, the scene by the grave, and by Kurosawa's completely
atypical but essentially satisfying use of mono no aware, that
resignation in face of the inevitable which creates the quiet and
grave beauty which, in Seven Samurai, we recognize as being very
close to what we know as catharsis. Another reason that the tragic
statement remains only implicit is that the original script— the work
of Kurosawa, Hideo Oguni, and Shinobu Hashimoto— has been
realized in terms of visual magnificence. One is so overcome by the
splendid animality, by the sheer vitality of the visuals that it is only
later that one reahzes how near tears one has been. The film is so
alive that to remember it is to remember a hundred meaningful
images: the opening sword fight in which the swordmaster shows
more about the way of the samiu-ai than have all the books ever
written about bushido; the funny scene where the head samurai—
played by Takashi Shimura— devises a plan to test his men; the
character of the last samurai— magnificently acted by Toshiro Mifune
— and his death; the love scene in the flowering hiUs; the first
attack of the bandits; their image against the sky, their slow, slow
descent through the waving grasses; the last rout and the
 1946-1971 235 superb rice-planting sequence, the essence
of spring; and the final scene— so profound that its impact is felt
only when one realizes how much it is a part of the beginning, how
inevitable it is to the whole. In making the picture— it took over a
year to filmKurosawa created a style the Hkes of which was never
seen in Japan before, or since. For the first time he used
superpowered telephoto lenses to capture a feeling of intimacy. The
attack of the bandits was thus photographed: men and horses piling
up, as it were, to create sohd visual images, appearing much closer
than they really were. Later Kurosawa made this device a part of his
style: the moving forest in the Macbeth fihn; the fight on the
mountain top in The Bad Sleep Well; the train sequence in High and
Low; and the fight in Red Beard. He also used multiple cameras,
each taking the scene from a difEerent angle. Thus he was able to
cut directly, without stopping the action, from one perspective to
another. The battle in the rain is shot in this manner, and so are the
many attacks of the bandits (most of which the West has rarely
seen, since this three-and-a-half-hour film was initially shown in
most countries with one third of its length missing), as well as the
rice-planting sequence. Though this technique uses a vast quantity
of film, Kurosawa—who almost invariably shoots at the ratio of ten
to one-much prefers it. He used it even in the intimate and much-
rehearsed conversation scenes of The Lower Depths. Again, in
Yojimho, Kurosawa beautifully fitted tech 
 236 JAPANESE CINEMA nique (including extensive use of
telephoto lenses for intimacy, shooting even the most violent of
sword fights in this manner) to content, with superb results. The
story, which concerns rival gangs in a small town at the end of the
Edo period, is composed of the very stuff of the ordinary period film.
Toshiro Mifune is the masterless samurai, amused and
contemptuous, who sets everything right. But Kurosawa, like
Yamanaka before him, infused this common material with new life,
returning to even the most notorious chch^s their original value.
Cliches, fihn or otherwise, are cliches only because they happen to
be true, and in Yojimho Kurosawa's validating of material which the
common Japanese cinema had deprived of all life was particularly
striking. To take but one example: one of the staple cliches of the
period fihn is a falling fighters crying oka (from okaasan, meaning
mother) as he dies. Nowadays it is used in a most perfunctory
manner, and Kurosawa, seeing it as a challenge, deliberately
revitalized it. First in a short scene vdth a mother and her small son,
where it was used with all of the human emotion it retains in
ordinary Japanese speech; next in a very funny scene where a weak
young man, released by the rival gang, rushes to his mother, and
barely gets it out of his mouth before she viciously slaps him; and
finally in a fighting scene where a dying gang member gives back to
it all the horror and despair and longing for fife which rightfully
belongs to it. The very fact that the material is so ordinary makes
 1946-1971 237 one realize, after seeing the film, that
Kurosawa has worked a minor miracle, one in which a perfectly
controlled style gives new life to even the most hackneyed material.
Perhaps one of the reasons is that in Yojimbo and Seven Samurai,
film epics both, one broadly hiunan and comic, the other concerned
vidth idealism and its toll, with the profoimd capabilities of man,
Kurosawa insists upon the intimate. There is a great dependence
upon closeups, a constant use of deep focus, low-key photography,
and a realistic intricacy of detail which is so compelling, so real, so
very believable, that the result is immediacy— it becomes actual. In
Kurosawa, then, realism, the peculiarly Japanese realism, seen in
films as early as Souk on the Road, reaches its fullest expression. At
the same time this realism—which can be compared to the
completely difiFerent realism of the films of Ozu— is controlled and
tempered by style, a style which, though both personal and
contemporary, is part of the Japanese aesthetic. Just as in ink-
painting, in haiku, or in the woodblock print, the camera, in the
hands of a Kurosawa, a Mizoguchi, an Ozu, becomes an instrument
of evocation, calling forth the image of the reality of Japan. In this
respect all of the major directors of Japan, no matter how
traditional, how individualistic, are alike: they share with the great
painters, the great poets, the great printmakers, the great craftsmen
of their country, the ability to draw the essence from the world about
them, and to present it from an angle of vision, often
 238 JAPANESE CINEMA oblique, which is uniquely theirs,
uniquely that of Japan. It is this vision, immaculately honest in the
greatest, firmly rooted to a longed-for reality in even the least, which
is the aesthetic of Japan-and which has created some of the most
beautiful and truthful fihns ever made.
 APPENDIX Japanese Films Circulated in 16 mm in the
United States Guide to Abbreviations: AB Audio Film Center and
Brandon Films, Inc., aflSliated with CroweU, Collier and Macmillan,
Inc., 34 MacQuesten Parkway, South, Mount Vernon, N.Y. 10550 CA
Cavalcade Pictures, Inc., 959 North Fairfax Avenue, Hollywood, Calif.
90046 CO Contemporary Films, a£SHated with McGraw-Hill, Inc., 330
West 42 Street, New York, N.Y. 10036 CV Cinema Ventmes, 133
West 14 Street, New York, N.Y. 10012 FI Film Images, a division of
Radim Films, Inc., 17 West 60 Street, New York, N.Y. 10023 GR
Grove Press, Inc., Film Division, 53 East 11 Street, New York, N.Y.
10003
 240 APPENDIX JA Janus Films, 745 Fifth Avenue, New York,
N.Y. 10003 NL New Line Cinema, 121 University Place, New York,
N.Y. 10003 NY New Yorker Films, 2409 Broadway, New York, N.Y.
10024 SH Shochiku Films of America, Inc., 4417 West Adams
Boulevard, Los Angeles, Calif. 90016 SW Swank Motion Pictures,
2151 Marion Place, Baldwin, N.Y. 11510 UN Universal 16/ Universal
Education and Visual Arts, 221 Park Avenue, South, New York, N.Y.
10003 WR Walter Reade 16, Walter Reade Organization, Inc., 241
East 34 Street, New York, N.Y. 10016
 Yukio Aoshima Yasuke Chiba Heinosuke Gosho Susumu
Hani The Bell (Kane), 1967, 61 AB min. Downtown (Shitamachi), AB
1957, 59 min. The Neighhoi's Wife and SH Mine (Madamu to Nyobo),
1931, 64 min. The Izu Dancer (Izu no SH Odoriko), 1935, 94 min.
Yellow Crow ( Behold Thy SH Son) (Kiiroi Karasu), 1957, 109 min.
Children Who Draw (E o AB Kaku Kodomotachi), 1956, 44 min. Bad
Boys (Fviryo Sho- AB nen), i960, 90 min. A Full Life (Mitasareta NY
Seikatsu), 1962, 100 min. AB She and He (Kanajo to Kare), 1963,
110 min.
 APPENDIX Kon Ichikawa Tadashi Imai Hiroshi Inagald
Bwana Toshi (Buana To- AB shi no Uta), 1965, 115 min. Bride of the
Andes (An- FI des no Hanayome), 1966, 102 min. The Harp of
Burma (Bi- AB ruma no Tategoto), 1956, 116 min. Fires on the Plain
(Nobi), JA 1959= 105 minTokyo Olympiad, 1965, 93 AB min.
(American Version). Muddy Waters (Nigorie), AB 1953. 104 min. Rice
(Kome), 1957, 118 CA min. The Adulteress (Night SH Drum) (Yoru
no Tsuzumi), 1958, 95 min. Samurai (Miyamoto Mu- AB sashi), 1954,
92 min. The Rikisha Man (Mu- UN homatsu no Issho), 1958, 106
min.
 Keisuke Kinoshita Teinosuke Kinugasa Masaki Kobayashi
243 The Japanese Army (Ri- SH kugun), 1944, 88 min. The Yotsuya
Ghost Story SH ( Yotsuya Kaidan ) , 1949, Part I, 86 min.. Part II, 73
min. Carmen Comes Home AB ( Karumen Kokyo ni Kaeru), 1951, 75
min. The Tragedy of Japan ( Ni- SH hon no Higeki), 1953, 116 min.
Twenty-four Eyes CNiiushi SH no Hitomi), 1954, 155 min. The Ballad
of Narayama SH ( Narayamabushi-ko ) , 1958, 97 min. Gate of Hell {
Jigokumon ) , SW 1954, 86 min. Black River (Kuroi SH Kawa), 1957,
114 min. The Human Condition SH ( Ningen no Joken ) , Part 1,
1958, 212 min.. Part II, 1959. 181 min.. Part III, 1961, 190 min.
 244 APPENBIX Kazuo Kuroki Akira Kurosawa Harakiri
(Seppuku), 1962, SH 135 min. Ktvaidan (Kaidan), 1965, WR 160
min. Silence Has No Wings UN ( Tobenai Chimoku ) , 1967, 106 min.
Tlie Men Who Tread on AB the Tiger's Tail (Tora no O o Fumu
Otokotachi), 1945, 60 min. Drunken Angel (Yoidore AB Tenshi),
1948, 102 min. Stray Dog (Nora Inu), AB 1949, 122 min. Scandal
(Shubun), 1950, SH 105 min. Rashomon, 1950, 84 min. JA The Idiot
(Hakuchi), NY 1951, the 166 min. version. Ikiru, 1952, 140 min. AB
Seven Samurai (Shichinin AB no Samurai), 1954, 141 min. version. I
Live in Fear ( Record of AB a Living Being) (Ikimono no Kiroku),
1955, 105 min.
 245 The Throne of Blood (The AB Castle of the Spider's
Web ) ( Kumonosu-Jo ) , 1957. 105 min. The Lower Depths (Don- AB
zoko), 1957, 125 min. The Hidden Fortress FI (Three Bad Men in a
Hidden Fortress) (Kakushitoride no SanAkunin), 1958, 126 min. The
Bad Sleep Well AB (Warui Yatsu Hodo Yoku Nemmu), i960, 135 min.
Yojimbo, 1961, 110 min. AB/JA High and Low (Tengoku WR to
Jigoku), 1963, 143 min. Red Beard (Akahige), AB 1965. 185 min.
Yasuzo Masumura The Hoodlum Soldier AB (Heitai Yakuza), 1965,
103 min. Yukio Mishima Rite of Love and Death GR (Yukoku), 1965,
21 min.
 246 Kenji Mizoguchi APPE^fDIX Sisters of the Gion (Gion
SH Shimai), 1935, 95 min. Utatnaro and His Five NY Women (
Utamaro o Meguru Gonin no Onna), 1946, 95 min. Women of the
Night ( Yoru AB no Onnatachi), 1948, 75 min. The Life of Oharu
(The NY Life of a Woman by Saikaku) (Saikaku Ichidai Onna), 1952,
148 min. Vgetsu (Ugetsu Monoga- JA tari), 1953, 96 min. A Story
from Chikamatsu NL ( Chikamatsu Monogatari), 1954, 100 min.
Sansho the Bailiff ( Sansho AB Dayu), 1954, 125 min. The Princess
Yang Ktvei CV Fei (Yokihi), 1955, ca. 120 min. New Tales of the Taira
NL Clan (Shin Heike Monogatari), 1955, 120 min. Street of Shame
(Red- AB Light District) (Akasen Chitai), X956, 85 min.
 Noboru Nakamura Yoshitaro Nomura Nagisa Oshima
Yasujiro 02na 247 Twin Sisters of Kyoto SH (Koto), 1963, 105 min.
The Three Faces of Love SH (Sekishun), 1967, gx min. Portrait of
Chieko (Chi- SH eko-sho), 1967, 125 min. Tokyo Bay (Tokyo Wan),
SH 1963. 83 min. The Scarlet Camellia SH (Akai Tsubaki), 1964, 117
min. Death by Hanging (Ko- GR shikei), 1968, 117 min. Dairy of a
Shinjuku Bur- GR glar (Shinjuku Dorobo Nikki), 1968, 94 min. Boy
(Shonen), 1969, 97 GR min. I Was Born, But ... AB (Umarete wa
Mita Keredo), 1932, 89 min. The Story of Floating SH Weeds (The
Duckweed Story) (Ukigusa Monogatari), 1934, 89 min. The Only Son
( Hitori Mu- SH suko), 1936, 87 min.
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