Fish Diseases and Disorders, Volume 2: Non-infectious Disorders, Second Edition doc

Fish Diseases and Disorders, Volume 2:
Non-infectious Disorders,
Second Edition
This page intentionally left blank
Fish Diseases and Disorders,
Volume 2: Non-infectious Disorders,
Second Edition
Edited by
John F. Leatherland
Department of Biomedical Sciences
Ontario Veterinary College
University of Guelph
Guelph
Canada
and
Patrick T.K. Woo
Department of Integrative Biology
College of Biological Science
University of Guelph
Guelph
Canada
CABI is a trading name of CAB International
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© CAB International 2010. All rights reserved. No part of this publication
may be reproduced in any form or by any means, electronically,
mechanically, by photocopying, recording or otherwise, without the
prior permission of the copyright owners.
A catalogue record for this book is available from the British Library,
London, UK.
Library of Congress Cataloging-in-Publication Data
Fish diseases and disorders.–2nd ed.
p. cm.
Includes bibliographical references and index.
ISBN-10: 0-85199-015-0 (alk. paper)
ISBN-13: 978-0-85199-015-6 (alk. paper)
1. Fishes–Diseases. 2. Fishes–Infections. I. Woo, P.T.K. II. Title.
SH171.F562 2006
639.3–dc22
2005018533
ISBN-13: 978 1 84593 553 5
Commissioning editor: Rachel Cutts
Production editor: Fiona Harrison
Typeset by AMA Dataset, Preston, UK.
Printed and bound in the UK by the MPG Books Group.
v
Contents
Contributors vii
Preface ix
1. Introduction: Diagnostic Assessment of Non-infectious Disorders 1
John F. Leatherland
2. Neoplasms and Related Disorders 19
John M. Grizzle and Andrew E. Goodwin

3. Endocrine and Reproductive Systems, Including Their Interaction with the
Immune System 85
John F. Leatherland
4. Chemically Induced Alterations to Gonadal Differentiation in Fish 144
Chris D. Metcalfe, Karen A. Kidd and John P. Sumpter
5. Disorders of Development in Fish 166
Christopher L. Brown, Deborah M. Power and José M. Núñez
6. Stress Response and the Role of Cortisol 182
Mathilakath M. Vijayan, Neelakanteswar Aluru and John F. Leatherland
7. Disorders of Nutrition and Metabolism 202
Santosh P. Lall
8. Food Intake Regulation and Disorders 238
Nicholas J. Bernier
9. Immunological Disorders Associated with Polychlorinated Biphenyls and
Related Halogenated Aromatic Hydrocarbon Compounds 267
George E. Noguchi
vi Contents
10. Disorders of the Cardiovascular and Respiratory Systems 287
Anthony P. Farrell, Paige A. Ackerman and George K. Iwama
11. Hydromineral Balance, its Regulation and Imbalances 323
William S. Marshall
12. Disorders Associated with Exposure to Excess Dissolved Gases 342
David J. Speare
13. Welfare and Farmed Fish 357
Peter Southgate
Glossary 371
Index 395
vii
Contributors
Paige A. Ackerman, Faculty of Land and Food Systems, Centre for Aquaculture and Envi-

ronmental Research (CAER), & Department of Zoology, University of British Columbia
Vancouver, BC V6T 1Z4, Canada
Neelakanteswar Aluru, Department of Biology, Woods Hole Oceanographic Institution,
Woods Hole, Massachusetts, USA
Nicholas J. Bernier, Department of Integrative Biology, University of Guelph, Guelph,
Ontario, N1G 2W1, Canada
Chris L. Brown, Marine Biology Program, Florida International University, Miami, FL
33181, USA
Anthony P. Farrell, Faculty of Land and Food Systems, Centre for Aquaculture and Envi-
ronmental Research (CAER), & Department of Zoology, University of British Columbia
Vancouver, BC V6T 1Z4, Canada
Andrew E. Goodwin, Aquaculture/Fisheries Center, University of Arkansas at Pine Bluff,
Pine Bluff, Arkansas 71601, USA
John M. Grizzle, Southeastern Cooperative Fish Disease Project, Department of Fisheries
and Allied Aquacultures, Auburn University, Auburn, Alabama 36849, USA
George K. Iwama, University of Northern British Columbia, Prince George, British Columbia,
Canada
Karen A. Kidd, University of New Brunswick, Saint John, NB, Canada
Santosh P. Lall, National Research Council of Canada, Institute for Marine Biosciences,
1411 Oxford Street, Halifax, NS B3H 3Z1, Canada
John F. Leatherland, Department of Biomedical Sciences, Ontario Veterinary College,
University of Guelph, Guelph, Ontario, N1G 2W1, Canada
William S. Marshall, Department of Biology, St. Francis Xavier University, Antigonish,
Nova Scotia, B2G 2W5, Canada
Chris D. Metcalfe, Trent University, Peterborough, ON, Canada
George E. Noguchi, US Fish and Wildlife Service, Division of Environmental Quality,
Arlington, VA, USA
José M Núñez, The Whitney Laboratory for Marine Bioscience, 9595 Ocean Shore Blvd.,
St. Augustine, FL 32080 USA
Deborah M. Power, Centro de Ciências do Mar (CCMAR), Universidade do Algarve, Campus

de Gambelas, Portugal
viii Contributors
Peter Southgate, Director, Fish Veterinary Group, Inverness, UK
David. J. Speare, Atlantic Veterinary College, University of Prince Edward Island,
Charlottetown, PEI, C1A 4P3, Canada
John P. Sumpter, Brunel University, Uxbridge, Middlesex, UK
Mathilakath M. Vijayan, Department of Biology, University of Waterloo, Waterloo, Ontario,
N2L 3G1, Canada
ix
Preface
As for the fi rst edition of this volume, the chapters comprise comprehensive discussions of
the some of the major non-infectious disorders of fi nfi sh. It is the second volume of a three-
volume series on fi sh diseases and disorders; Volume 1 deals with parasitic diseases and
Volume 3 with microbial diseases. Reviews in the three volumes are written by leading
international authorities who are actively working in the area or who have contributed
greatly to our understanding of specifi c diseases or disorders.
The present book includes non-infectious disorders of development and growth and
various aspects of the physiology of wild and captive species, including nutritional physi-
ology, feeding activity, cardiovascular physiology, ionic and osmotic regulation, stress
physiology, reproduction and endocrine physiology. In addition, chapters dealing with
issues related to the diagnosis of non-infectious disorders, tumourigenesis and problems
related to supersaturated gas issues in aquaculture practice are included. Because of the
increasing concern of the effects of ‘anthropogenic’ chemicals on aquatic organisms, par-
ticularly, but not exclusively, those that act as hormone mimics or hormone-disrupting
chemicals, several chapters address this issue from different perspectives. These chapters
review the known effects of such chemicals on the endocrine, reproductive and immune
systems, and explore the use of fi sh as sentinel organisms for the detection of such chemi-
cals and monitoring of ‘ecosystem health’. In addition, because of the increasing interest in
animal welfare issues in aquaculture practice, a chapter dealing with this topic is included
in this volume.

The second edition attempts to address emerging areas of interest and concern in fi sh-
eries health in both wild populations and captive stock, and to refl ect changing attitudes
toward the interpretation of fi sh health issues and the affects of non-infectious disorders on
production issues in the wild and captive fi sh stocks. Several chapters are included that
were not present in the fi rst edition; new authors have contributed to some of the chapters
that were present in the fi rst edition, and some chapters have been updated from the fi rst
edition.
The principal audience of this volume, as for Volumes 1 and 3, is the fi sh and fi sher-
ies research community, in aquaculture and government fi sheries management and
researchers in academe; the community comprises environmental toxicologists, pure and
applied fi sh physiologists, fi sh health specialists, and fi sh health consultants in government
x Preface
laboratories, universities or the private sector. The volume is also relevant to graduate
students and senior undergraduate students who are involved in studies related to the
health of aquatic organisms.
J.F. Leatherland and P.T.K Woo
© CAB International 2010. Fish Diseases and Disorders Vol. 2:
Non-infectious Disorders, 2nd edition (eds J.F. Leatherland and P.T.K. Woo) 1
1 Introduction: Diagnostic Assessment
of Non-infectious Disorders
John F. Leatherland
Department of Biomedical Sciences, Ontario Veterinary College,
University of Guelph, Guelph, Canada
Introduction
The term diagnosis is generally used to
describe the recognition of a disease or con-
dition by its clinical signs and symptoms;
however, the defi nition is commonly extended
to include the second stage of the identifi ca-
tion process, namely the determination of

the underlying physiological, biochemical
or molecular factors that are related to or
responsible for the disease or condition. In
human and veterinary medicine, even when
a specifi c aetiological agent is known, a
cluster of specifi c clinical signs (together
with symptoms communicated by human
patients) is used to formulate preliminary
diagnoses. Based on the clinical signs, clini-
cal tests are then used to confi rm or refute
the preliminary diagnosis, and, where pos-
sible, treatments and disease management
strategies are developed to deal with the
condition. This general approach is used
extensively in veterinary practice related to
the management of captive fi sh stocks and,
to a lesser extent, to diagnose infectious
conditions of wild fi sh populations; how-
ever, diagnosing non-infectious disorders in
fi sh has tended to be much more problem-
atic, and it has been particularly diffi cult to
link the non-infectious conditions to a
specifi c aetiological factor. Moreover, the
follow-up evaluation of the physiological and
biochemical responses of the organism rarely
provides specifi c information about the root
cause(s) of the dysfunctional condition.
This volume of the second edition of
the fi sh diseases series comprises chapters
that focus on the description of known and

generally well-documented non-infectious
disorders. The chapters examine the nature
of the disorders, the biological implications
of those disorders and the aetiologies of the
disorders, as far as these are known. Some
chapters survey the diseases and disorders
associated with a specifi c organ system,
such as the cardiovascular system; in other
chapters the focus is on a particular aspect of
fi sh disorders related to a specifi c theme,
such as disorders associated with nutritional
factors or with tumour genesis. Regardless of
the scope of the interest, a primary chal-
lenge for investigators in this particular fi eld
is to identify when a specifi c animal, a cap-
tive stock or a wild population is exhibiting
signs of a non-infectious disease or disor-
der. As will be explored in this chapter,
most of our knowledge pertaining to non-
infectious conditions is based on follow-up
studies that have been prompted by obser-
vations of poor growth, reproductive prob-
lems or grossly evident lesions within a
particular population or stock. As will be
discussed in the following pages, for several
reasons, an a priori diagnosis (or even a
2 J.F. Leatherland
posteriori diagnosis) of a specifi c problem is
often not possible.
Issues Related to the Diagnosis of

Non-infectious Disorders
Infectious diseases are diagnosed by symp-
tomatology (the study of symptoms) and the
identifi cation of the infectious agent or the
product of that agent. For non-infectious dis-
orders, because there is no infectious agent
or the product of that infectious agent, the
identifi cation of a problem is limited to the
recognition of clinical signs and symptoms.
Moreover, non-infectious diseases may not
be associated with a primary response of the
innate or acquired immune system; hence,
even immunological assessment tools may
not be applicable. Consequently, many of
the non-infectious conditions that have been
recognized and studied in fi sh to date have
been documented without the application of
specifi c diagnostic methods. In fact, many of
these cases were discovered serendipitously
and the follow-up physiological or biochem-
ical studies were made a posteriori, and it
remains to be determined if these largely
non-specifi c responses can be used as mean-
ingful diagnostic tools. In fact, for the most
part, these compensatory physiological and
biochemical responses, albeit of value and
interest to the investigator, are of limited
diagnostic value. In contrast, in the short
term, it is commonly the ‘global’ responses
of a population, such as changes in the struc-

ture of a population or changes in the repro-
ductive success of a population, that are the
primary indicators of the existence of a
health issue in that population. There are
exceptions to the rule, such as changes in the
cardiovascular physiology and xenobiotic-
induced changes in the reproductive system
of some fi shes, which are explored in later
chapters.
Figure 1.1 summarizes the several levels
of biological organisation at which responses
to non-infectious disorders can, in theory,
be detected; however, it must be emphasized
that non-infectious disorders and diseases
that have very different root causes may
elicit similar responses (such as poor
growth) when measured at the population
or stock level. The diagnostic and analytic
problems are far more challenging for stud-
ies of disorders in wild fi sh populations,
compared with studies of issues in captive
stocks. In captive fi sh stocks, high mortality
rates, reduced feeding and reduced repro-
ductive success of the stock can be readily
identifi ed by facility managers; the cause(s)
may not be directly evident but the out-
comes are. In contrast, for wild popula-
tions, the reduction in fi sh numbers could
be associated with increased mortality or
reduced reproduction or both. Increased

mortalities in wild populations may not be
recognized unless there is an acute episode
and then only if the dead fi sh are found,
which is not likely to occur, for example,
with benthic species. More commonly,
increased mortality in a wild population is
suspected when the numbers of fi sh in a
population declines; however, a reduction
in the size of the population may not neces-
sarily be related to an increase in mortality
rates, although this may be one component;
several direct and indirect factors, includ-
ing ecological factors may contribute to a
decrease in population size, as summarized
in Box 1.1.
All of the factors noted in Box 1.1 have
been linked to reductions in the size of wild
populations of diverse fi sh species, and they
will be elaborated on later in this chapter.
Because the reduction in the size of a popu-
lation is the end product of the impact of
these factors, other population indicators
need to be used to examine the dynamics of
the dysfunctional state in progress and these
may be more useful indicators. For exam-
ple, the absence of an age class in a popula-
tion may be indicative of a reproductive
problem, and skewed age/size distributions
might indicate impaired growth and associ-
ated metabolic dysfunctions, which could

possibly be attributed to several factors
(Fig. 1.1). Information related to feeding
activity source and quality of diet might
provide an insight into changes in the struc-
ture of the population. Measurements of the
relative concentration of stable isotopes in
body tissues are currently being used by a
Introduction 3
Population or stock indices
Mortality rates
Age/size distribution
Numbers of age groups in the population or stock
Reproductive success
Growth rate
Population or stock size
Organism indicators
Growth and reproductive performance
Behaviour (various, but including feeding
behaviour)
Immune system competence
Gross lesions (various, but including tumours)
Organ system indicators
Organ size and morphology
Differentiation of organ systems
Histopathology
Blood chemistry:
stress hormone
glucose
pH shifts
oxygen carrying capacity

Tissue and cellular indicators
Histopathology
Tissue and cell composition:
enzymes
receptors
phospholipids
metabolites
Cellular energetics
Expression of specific genes
Apoptosis activity
Fig. 1.1. Schematic summary of the levels
of biological organization at which indica-
tors of non-infectious diseases or disorders
can be detected; at each level examples of
key investigational methods are shown. The
population or stock indicators are most com-
monly the fi rst indicators of a non-infectious
disease or disorder, although some organism
indicators (for example, prevalence of
lesions, including tumours) have also been
the fi rst indicators of a possible problem.
For the most part, the organ system indica-
tors and tissue and cellular indicators have
not been primary indicators of a possible
problem, but have been used for follow-up
diagnostic purposes.
Box 1.1. Summary of factors that may contribute directly or indirectly to a decrease in the size
of a wild population of fi sh.
Mortalities or impaired reproduction associated with contaminated environments.
Mortalities or impaired reproduction associated with hypoxic environments.

Mortalities associated with suppressed immune system function, leading to increased susceptibility
to infectious disease.
Increased predation (including increased harvesting of natural resources by recreational and
commercial fi shing).
Reductions in the availability of suitable food resources.
number of investigators (Satterfi eld and
Finney, 2002; Høie et al., 2003; Schlechtriem
et al., 2004; Dubé et al., 2006; Hutchinson
and Trueman, 2006; Rojas et al., 2006;
Williamson et al., 2009, among others) to
determine the changing history of dietary
sources of individual fi sh and populations.
This approach offers a means of determining
4 J.F. Leatherland
dynamic aspects of population stability and
could be a valuable tool in documenting
trophic-related factors involved in popula-
tion change.
Another compounding factor is the as
yet poorly understood association between
depressed immune system function and
impaired growth and reproductive success.
It is not clear whether the growth and repro-
ductive condition bring about the depressed
immune response or vice versa, or whether
these are independently part of the rela-
tively non-specifi
c ‘stress response’ in fi sh.
However, stress responses are an important
consideration in the diagnosis of all non-

infectious conditions in fi sh.
Table 1.1 summarizes some of the major
stress responses in vertebrates. The general
non-specifi
c stress response in fi sh includes
the rapid release of stress hormones, such as
adrenal catecholamines (epinephrine and
norepinephrine), within seconds of the
onset of the stressor (the so-called ‘primary
stress response’). This is followed within
minutes by an increase in the release of the
glucocorticoid hormone cortisol from the
steroidogenic cells of the interrenal gland,
leading to an increase in circulating levels
of the hormone, which lasts for several
hours. In some literary sources this increase
in plasma cortisol concentrations is consid-
ered to be a component of the ‘primary stress
response’, but the temporal differences in the
stressor-linked profi les of plasma hormone
levels of catecholamine and glucocorticoid
hormones argues for the cortisol release and
its activation of glucocorticoid receptors to be
considered as the ‘secondary stress response’.
The increase in circulating levels of the cat-
echolamine and glucocorticoid hormones
stimulates changes in blood metabolites,
such as glucose; the catecholamines stimu-
late the release of glucose from glycogen by
several tissues, but mostly by hepatocytes;

cortisol stimulates the mobilization of lipid
reserves and the production of de novo glu-
cose by hepatic gluconeogenesis using non-
carbohydrate substrates. In addition, the
increased skeletal muscle activity that com-
monly accompanies the stress response gives
rise to an increase in plasma lactic acid and
changes in plasma pH, and there may also
be changes in plasma electrolytes caused
by increased blood fl ow through the gills
and increased ion exchange across the gill
epithelium.
The release of tissue carbohydrate reser-
ves by catecholamines and the production of
new glucose by hepatic gluconeogenesis
supplies the increased metabolic needs of
cells involved in the stress response, such as
increased muscle and central nervous sys-
tem activities; these metabolic responses
represent the ‘tertiary stress response’,
which is highly benefi cial to the organism.
However, the increased chronic secretion of
cortisol has a depressive action on the immune
system (see Chapter 6, this volume), which
may increase the susceptibility of the organ-
ism to pathogens. Cortisol-induced immuno-
suppression may be considered as an
example of the ‘quaternary stress response’,
as could the suppression of growth and
impaired reproduction. The reduction in

growth may be caused by a decrease in feed-
ing or increased activity of the fi sh, leading
to energy sources being diverted from the
support of somatic growth. Reduced repro-
ductive success may also be caused by a
decrease in availability of nutrients if the
animal ceases to feed. However, stressor-
induced changes in the activity of the
hypothalamus–pituitary gland–gonad axis
may lead to impaired gamete production, and
direct inhibitory actions of cortisol on gonadal
steroidogenesis have also been reported for
some species (Reddy et al., 1999; Leather-
land et al., 2010). These various levels of
the stress response are discussed at more
length in Chapter 6, this volume.
Whilst these global responses by a pop-
ulation (or stock) are important fi rst signs,
they usually provide little immediate infor-
mation about the cause of a specifi c disor-
der; whole organism and organ indices may
provide a second level of investigation.
These might include measurement of the
mass of specifi c organs, histopathological
examination of tissues and organs to explore
for lesions, assessments of immune response,
monitoring of blood chemistry, measure-
ment of the levels of energy reserves in key
organs and assessment of the activities of
key enzymes in intermediary metabolic

Introduction 5
pathways. The specifi city of some of these
diagnostic tests is still not well established,
but they do provide valuable information
about the nature of the animal’s physiologi-
cal condition. The third order of diagnostic
examination, which explores the organ- and
tissue-specifi c cellular and subcellular sites
of the malfunction (Fig. 1.1), has similar
limitations as regards the specifi city
of
response.
This chapter provides an overview of
this stepwise ‘diagnostic approach’; it also
outlines the strengths and weaknesses of
some of these methodologies and empha-
sizes that there is no single template that
can be applied to determine the causes of all
known or suspected environmentally related
conditions. Each outbreak of a problem needs
to be investigated using fi rst principles and
the application of the most appropriate
investigational tools.
This chapter also briefl y explores how
fi sh disorders can themselves be used as
biological indicators of environmental prob-
lems and as a measure (bioassay) of the
extent of the environmental problem. This
use of so-called sentinel organisms in the
wild as the ‘miner’s canary’ to monitor the

quality of the environment has provided an
invaluable fi rst step towards the recognition
and subsequent understanding of sometimes
broad-based problems. An excellent exam-
ple of this approach is Sonstegard’s (1977)
documentation of regional differences in
tumour prevalence in fi sh in the Great Lakes
of North America. Sonstegard used tumour
prevalence as an indicator of the extent of
contamination of different regions of the
lakes with chemicals that directly or indi-
rectly induced tumourigenesis; follow-up
studies were then used to determine the spe-
cifi c factors involved. Sonstegard’s extensive
Table 1.1. Stages of the response of fi sh to a range of stressors.
Stage of response
to stressors Biochemical and physiological changes
Period of
response
Primary Rapid upregulation of the autonomic nervous system,
increasing the adrenergic stimulation of the heart pacemaker
Rapid release of catecholamines from the interrenal chromaffi n
c
ells; increased plasma catecholamine concentration
Increased heart rate
Mobilization of carbohydrate reserves
Neural stimulation of hypothalamic corticotropin-releasing-hormone
(CRH)-secreting cells to override the negative feedback control of
plasma cortisol concentration
Within

seconds
Secondary Suppression of the negative feedback regul
ation of pituitary
adrenocorticotropic cells to allow increased adrenocorticotropin
(ACTH) secretion
Increased plasma cortisol concentrations, beginning within
minutes and progressing for several hours
Minutes to
hours
Tertiary Increased plasma glucose concentration in response to
catecholamine stimulation of hepatic glycogenolysis
Increased hepatic gluconeogenesis in response to glucocorticoid
(cortisol) stimulation, leading to increased plasma glucose
concentration
Possible increased plasma lactic acid concentrations resulting from
increased skeletal muscle activity
Hours
QuaternaryPh
ysiological responses to chronic hypercortisolism; these may
include: immunosuppression by glucocorticoids and increased
susceptibility to pathogens, impairment of growth and impairment
of reproduction
Days to
months
6 J.F. Leatherland
series of studies of the epizootiology of
tumours in Great Lakes fi sh species set the
stage for later work that used sentinel aquatic
species as markers of contaminants in vari-
ous lakes, coastal aquatic systems and rivers.

Such sentinels have been used not only to
monitor the presence of xenobiotics but
also to determine seasonal and year-to-year
changes in the level of contamination. Of
particular note is the use of sentinel species
to detect and monitor changing levels of
endocrine-modulating toxicants in the effl u-
ents of pulp mills and sewage treatment
plants; these are discussed at greater length
later in this chapter and also in Chapters 3
and 4, this volume.
During the last few decades, there has
been considerable interest in documenting
the effects of human activities on the degra-
dation and destabilization of ecosystems.
Metaphors drawn from the human health
sciences have been applied increasingly to
describe changes in ecological systems, and
terms such as ‘ecosystem health’ and ‘stressed
ecosystems’ have become commonplace in
the literature; indeed, university programmes
of similar names have been developed dur-
ing the same period. The application of the
diagnostic methods and approaches that are
currently used in human and veterinary
medicine to the diagnosis of ecological prob-
lems was proposed by Fazey et al. (2004),
and these approaches have been used to
diagnose degradation of ecosystems that are

very obviously impacted by human activi-
ties (e.g. removal of forests, draining of wet-
lands, pollution of terrestrial and aquatic
systems, global climate change, etc.). How-
ever, our level of understanding of ecosys-
tem interactions is still very limited, and
indicators have not yet been developed that
can distinguish between less severe human
impact and the ‘natural’ changes that are
characteristics of all ecosystems. Ecosystems
are very diverse and are also not static enti-
ties; their character changes with season
and with time, and each particular ecosys-
tem exhibits its own characteristic responses
to change. Ever since the emergence of life
on this planet, both short-term and long-
term climatic fl uctuations have acted as
stressors on living organisms and thus on
the interactions of those organisms within a
particular ecosystem. A change in the dynam-
ics of an ecosystem does not necessarily
mean that the system is unstable or unhealthy.
However, changes in the physiological or
clinical status of key sentinel organisms
that comprise the biotic components of a
particular ecosystem over time can be inval-
uable and sensitive monitors of ecosystem
change and signal the occurrence of change
long before there is a marked deterioration
in the ‘health’ of an ecosystem.

Human activities have had major (and
rapid) effects on the stability of ecosystems.
These include the excessive harvesting of
selected animal and plant species resulting
in reduction in species diversity, the intro-
duction of exotic organisms, the physical
disturbance of key aspects of an organism
(e.g. draining of wetlands that comprise the
breeding areas for many aquatic ecosystems),
changes in the availability of nutrients (e.g.
fertilizer or pesticide runoff from cultivated
land, the drainage of municipal sewage into
aquatic systems or the depletion of nutri-
ents following the introduction of exotic
species), the contamination of ecosystems
by toxic chemicals, and the potential effects
of climate change and associated meteoro-
logical changes. All aquatic ecosystems
have been impacted to some extent by one
or more of these activities, and although
attempts have been made to artifi cially ‘sta-
bilize’ ecosystems, once the signs of change
are evident, attempts to reverse the change
have been largely ineffective. The human-
associated escalation in the rate of environ-
mental change has accompanied the spread
of human populations. In particular the
spread of industrial activities has led some
evolutionary ecologists to conclude that the
planet is well on its way toward a third

major extinction, comparable in many ways
to the mass extinctions that categorized the
end of the Palaeozoic and Mesozoic eras
(Ward, 1994). Therefore, although sentinel or
indicator organisms have played a central
role in monitoring both changes in environ-
mental conditions and the rate of environ-
mental change, reversing these changes has
proved to be a challenge that is currently
beyond the limits of our ability.
Introduction 7
Fish as Sentinel Organisms
Non-infectious disorders of particular wild
species have been used effectively to signal
detrimental changes at a particular site or
within an ecosystem. In some cases, fi sh
that are susceptible to particular contami-
nants have been placed in cages in aquatic
systems that are thought to be contaminated.
Two examples of the use of sentinel fi sh
species illustrate their value. One series of
studies (summarized in Chapter 3, this vol-
ume) examined the effects of sewage treat-
ment effl uent on vitellogenin synthesis in
fi sh held downstream of the effl uent. Vitello-
genin is a phospholipoprotein that is trans-
ferred to the oocytes during gonadal growth
and maturation, a process referred to as
vitellogenesis. Vitellogenin is synthesized
by the liver under the infl uence of oestro-

gen, and therefore it is normally only syn-
thesized by sexually mature females. The
presence of vitellogenin in juvenile fi sh and
adult males is indicative of the presence of
environmental oestrogens (xeno-oestrogens).
Sentinel fi sh held in cages downstream of
sewage treatment plants in several countries
were found to have elevated plasma vitello-
genin levels, suggesting that the sewage
treatment microfl ora were not able to fully
metabolize the oestrogens (including contra-
ceptive oestrogens) excreted by the human
population from which the effl uent is received.
A second example of the application of sen-
tinel fi sh species has been the examination
of the effects of bleach kraft mill effl uent
(BKME) on the reproductive biology of fi sh
in river and lake systems and of the disper-
sal of the effl uent within the ecosystem
(summarized in Chapter 3, this volume).
The physiological responses of the sentinel
animals have provided evidence of the pres-
ence of a contaminant or mixture of con-
taminants and, to some extent, the level of
the contaminant.
For both freshwater and marine aquatic
systems, teleost fi shes have proved to be par-
ticularly valuable as sentinels as they occupy
various trophic levels in an ecosystem; they
accumulate xenobiotic chemicals both via

the food chain and directly from the water
column via the gills; and they ‘biomagnify’
many xenobiotic factors in specifi c tissues to
a level that can be measured using currently
available chemical analysis.
The value of such sentinels as bioassay
systems is that they can be used as indica-
tors without necessarily having a priori
knowledge of the nature of the environmen-
tal insult (physical or chemical). This is par-
ticularly important in assessing the effects
of man-made chemicals on the environment,
because the total number of newly synthe-
sized chemicals continues to increase at a
rate that exceeds our capacity to undertake
meaningful toxicology screening, and our
knowledge of the interactions of chemicals
in biological systems is still rudimentary.
Moreover, the method is especially valuable
in situations in which there is a mixture of
chemicals being introduced into the envi-
ronment, as is the case for BKME.
An additional value of the sentinel
approach over the direct chemical measure-
ment approach is the high level of sensitiv-
ity of the former for some classes of toxicants.
Many environmental chemicals exert their
effect by interacting with receptor proteins
on the plasma membrane of cells. A low
level of receptor–ligand (toxicant) interac-

tion brings about changes in cellular activ-
ity, and the cellular response is biomagnifi ed
to the point that the physiology of the senti-
nel organism is changed to a degree that can
be measured.
Each category of toxicant in a mixture
of toxicants in a given ecosystem will have
its own unique mode of action at the cellu-
lar or subcellular level; therefore, there is no
single protocol to test for all toxicants, or
even for all toxicants in a particular class of
chemicals. For example, heavy metals exert
their effects via different pathways. Some
factors, such as organic phosphate, exert
effects directly on an organ system; for
example, the organic phosphates act on the
central nervous system (Katzung, 2001).
Members of the aromatic halogenated hydro-
carbon group of chemicals, which includes
the dioxins and polychlorinated biphenyl
(PCB) families, exert a range of biological
effects (Bruckner-Davis, 1998; Rolland,
2000a,b). In the case of the PCB family, the
toxicity of different PCB congeners is
8 J.F. Leatherland
dependent on the structure of the congener.
Some congeners act on the nucleus of cells,
where they interact with the aryl hydrocar-
bon receptor (AhR). This leads to the
increased expression of some genes, includ-

ing those that code for the synthesis of cyto-
chrome P450 (CYP) enzymes, which are
mixed-function oxidases involved in detoxi-
fying an animal of a range of compounds.
The xenobiotic is a ligand for the AhR pro-
tein; ligand activation of the AhR causes it
to form a heterodimer with a nuclear trans-
locator protein, such as ARNT; the het-
erodimer acts as a transcription factor for the
genes that encode for specifi
c CYP enzymes.
Other PCB congeners do not elicit a CYP

response but can affect thyroid hormone
metabolism (Brouwer et al., 1998; Porterfi eld
and Hendry, 1998; Naz, 2004). Other cellular
sites of action of xenobiotics include actions
on metabolic events, either by affecting cel-
lular enzyme gene expression or by acting
directly on the interaction of an enzyme
with its substrate via multiple routes of
action, membrane transport processes, and
hormone and growth factor receptors in the
plasma membrane or nucleus of target cells
(Naz, 2004). Toxicants that act as ligands for
several families of hormone or growth factor
receptors may either activate the receptor
(i.e. act as an agonist) or prevent the receptor
binding to its native ligand (i.e. act as antag-
onists). These xenobiotic–receptor relation-

ships may be transient or persistent. Persistent
toxicants have a relatively long biological
half-life, usually because the toxicants can-
not be readily metabolized. Persistent ago-
nistic compounds may have a relatively low
affi nity for a specifi c receptor relative to the
native ligand, but their long half-life gives
them an increased biological potency; this
is the case for weak xeno-oestrogenic chemi-
cals such as bisphenol A, which have a long
biological half-life (Bjerregaard et al., 2007;
Crain et al., 2007). This is particularly evi-
dent in fi sh because these compounds induce
the synthesis of vitellogenin by the livers of
fi sh exposed to environmental compounds
that are weak oestrogens (Harries et al.,
1996); vitellogenin is a phospholipoprotein
that is normally only found in female fi sh
that are undergoing gonadal maturation; the
presence of vitellogenin in immature female
fi sh and male fi sh is commonly used as an
indicator for the presence of environmental
xeno-oestrogens (Crain et al., 2007). Alterna-
tively, persistent antagonistic toxicants bind
to receptors without activating the receptors;
the occupation of the binding site on the
receptor may prevent the normal interac-
tion between the receptor and its natural
ligand, a hormone or other form of cytokine
or growth factor; an example is the anti-

androgenic action of some organochlorine
compounds such as the DDT metabolite
DDE (Kime, 1998; Rolland, 2000b; Norris
and Carr, 2006). Yet other xenobiotics inter-
act with proteins that are not receptors; for
example, nonylphenol impairs gonadal
steroidogenesis by inhibiting the movement
of cholesterol into the mitochondria of ster-
oidogenic cells, thus reducing the synthesis
of the precursor steroid, pregnenolone (Kort-
ner and Arukwe, 2006). Cholesterol fl ux into
the mitochondria requires the presence of
activated steroidogenic acute regulatory
(StAR) protein; nonylphenol may prevent the
activation of StAR or prevent its insertion
into the outer mitochondrial membrane.
Epizootiological Measures of Disorders
Widespread disruptions of population sta-
bility caused by a disease outbreak, habitat
destruction, depletion of food sources or the
application of other environmental stres-
sors may be accompanied by gross epizootic
indications of distress. This is the case for
both captive and wild fi sh, and the most
common ‘population indicators’ include
high mortality, skewed age/size distribu-
tions, impaired growth performance, low
body metabolite reserves and impaired
reproductive success (Fig. 1.1). In addition,
as indicated earlier in the chapter, epizoot-

ics of gross lesions, particularly neoplasms,
have been used as population indices, usu-
ally as indicators of the presence of contam-
inants (e.g. Sonstegard, 1977). The major
limitation in the use of population indices
as a diagnostic tool is their lack of specifi -
city; few population indices are disease-,
disorder- or condition-specifi c.
Introduction 9
Mortality or reduction in population size
Each species of fi sh can tolerate environ-
mental changes to which they are continu-
ally exposed; these may include temperature,
pH and salinity of its aquatic environment;
the availability of oxygen (and presence of
carbon dioxide); and the availability of food
(Fig. 1.2). The major organ systems undergo
adaptive responses that adjust the homeo-
static processes within this ‘tolerance range’.

At the upper and lower ends of the tolerance
range, the fi sh will physiologically resist
further physiological changes, but these so-
called ‘resistance ranges’ are small and home-
ostatic balance is disturbed. If the homeostatic
balance is not recovered rapidly, the animal
reaches the extreme upper or lower end of
the resistance range, at which point it dies;
these are the upper and lower lethal points
for a particular variable (Fig. 1.3). Death

occurs as the end result of the breakdown of
homeostatic processes, which can result
from a myriad of events, including the pres-
ence of infectious agents or changes in the
abiotic environment that exceed the upper
or lower limits of the animal’s tolerance
range, as well as metabolic disorders and
contamination of the environment by natu-
ral or man-made toxicants or infectious dis-
ease (Fig. 1.3). As such, although it is the
most dramatic indicator of acute or chronic
problems, the death of a signifi cant percent-
age of a population (or captive stock), unless
there is a diagnosable infectious aetiology,
provides little direct information about the
nature of the problem.
As indicated in an earlier section of this
chapter, the disappearance of wild fi sh stocks
cannot, per se, be directly attributed to increa-
sed mortality. Mortality caused by contami-
nated environments or infectious disease
could be part of the problem, but, equally,
changes in predator–prey relationships,
ABIOTIC
FACTORS
pH
Salinity
Oxygen availability
Ambient temperature
Food availability

HOMEOSTASIS
Organ systems involved:
Integument
Gills
Kidneys
Liver
Gastrointestinal tract
Cardiovascular system
Nervous and endocrine systems
Musculoskeletal system
Blood/tissue factors
regulated:
Osmotic and ionic balance
pH
Oxygen tension
Carbon dioxide tension
Nutrient levels
Fig. 1.2. Schematic summary of the relationship between abiotic factors and homeostasis, the
ph
ysiological factors that are regulated and the main organ systems involved in homeostatic regulation.
Abiotic factors impose a persistent adaptive stress on the organism, which can be accommodated within the
normal homeostatic (physiological) range. The various organ systems that are involved are shown – it should
be noted that these encompass virtually all of the body organ systems; only the reproductive system is not
included. Some, but not all, of the blood and tissue factors that are regulated are also shown.
10 J.F. Leatherland
excess harvesting of fi sh stocks (or of the
primary prey species of a particular fi sh
stock), and factors such as contaminants,
loss of spawning habitats or changes in
water condition, such as hypoxia, resulting

in reduced reproductive success, could be,
and probably are, also involved.
Examples of the effects of such cumula-
tive events on fi
sh populations abound, but
the catastrophic declines in the Atlantic
cod (Gadus morhua), lake trout (Salvelinus
namaycush) in the Great Lakes of North
America, and sockeye salmon (Oncorhyn-
chus nerka) stocks along the Pacifi c coast of
North America bear testimony to the problem
faced by a particular species, as does the
drastic decline of the commercial fi shing
base in the Mediterranean Sea. It should be
emphasized that although these examples
represent recent events (most within the last
60 years), archaeological evidence attests to
the long-term effect of human activities on
animal and plant populations. Even in the
absence of human activity, the fossil record
provides similar evidence of the ‘constancy
of change’ in population and community
structures.
Thus, in captive or wild populations,
high mortalities may provide an immediate
indication of an acute or chronic problem
(including infectious diseases) that exceeds
the animal’s tolerance and resistance
ranges, but the mortalities may also be
indicative of environmental issues related

to the availability of reproductive resources.
Even if the mortalities are related to fac-
tors exceeding the resistance limits of the
fi sh, the specifi c cause of death can only be
Disturbed
homeostasis
DISRUPTING FACTORS:
Changing biotic factors
Toxicants
Infectious agents
Genetic disorders
Compensatory
responses
Compensatory
responses
Homeostasis
re-established,
possibly with new
set points
Cellular dysfunction
Death of organism
Changes within tolerance range
Changes beyond tolerance range
Fig. 1.3. Schematic representation of the processes which cause the organism’s normal physiological range
to be pushed beyond the tolerance range; physiological variations within the tolerance range can be ac-
commodated, possibly with some adjustment to the homeostasis set points. Variations beyond the tolerance
range cause the animal to resist further physiological change for short periods of time, but the process can-
not be reversed; the animal will succumb when it reaches the upper or lower limits of the range – the upper
and lower lethal points.
Introduction 11

established by the application of other diag-
nostic methods.
Changes in age/size distributions
Changes in the age/size distribution may be
useful indicators, particularly of problems
faced at specifi c stages in the life cycle. For
example, the loss of early year classes may
be indicative of an impaired recruitment of
the population into brood stock or, equally,
this may be caused by reproductive prob-
lems. Further, if a specifi
c age group within
a population is small, this may be an indica-
tion of impaired growth effi ciency or increased
size-specifi c mortality. A major limitation of
this approach is that it requires a long-term
study and necessitates the removal of a sig-
nifi cant number of a resident population.
Random sampling methods usually use
lethal techniques, and the most accurate
ageing techniques rely on the examination
of the annual growth rings of the otoliths of
the inner ear and are therefore only possible
post-mortem. Furthermore, all of the limita-
tions as regards the interpretation of the
results of such studies that applied to the
use of mortality rates as indicators of prob-
lems within a population are equally true in
the evaluations of age/size data.
Impaired growth performance

In its simplest terms, growth is a measure of
the change in the total energy content of an
animal over time (Brett and Groves, 1979).
It is the net difference between the acquisi-
tion and assimilation of nutrients and the
metabolism of those nutrients to generate
metabolic energy and heat (Fig. 1.4). Growth
performance is affected by the quantity,
quality, palatability and digestibility of the
available nutrients, the rate of metabolism
and activity, and factors that alter energy par-
titioning needs (e.g. gonadal development).
Consequently, in real terms, growth of fi sh, as
with that of all animals, is an extremely
complex process and still surprisingly poorly
understood. Recent excellent reviews by
Katsanevakis and Maravelias (2008) and
Kuparinen et al. (2008) illustrate the com-
plex nature of modelling and understanding
fi sh growth at a population level. In part,
the limitations of our understanding of
growth physiology are related to the imper-
fect methods currently available for measur-
ing growth rates and growth performance of
fi sh, particularly animals in the wild. Of
these, changes in body length and mass (and
condition factor) with time are widely used
and have limited value for measures of wild
populations, unless used in combination
with valid age data (see above). More recently,

measurement of the RNA:DNA ratios or of
ornithine decarboxylase activity (the rate-
limiting enzyme for nucleic acid produc-
tion) in specifi c tissues have been used as
indirect measures (Houlihan et al., 1993;
Arndt et al., 1994; Mercaldo-Allen et al.,
2008), as have measurement of the isotope
signature or stable isotope composition of
otolyth and scale rings (Satterfi eld and
Finney, 2002; Høie et al., 2003; Gao et al.,
2004; Hutchinson and Trueman, 2006) and
amino acid uptake by scales in vitro (Gool-
ish and Adelman, 1983; Farbridge and
Leatherland, 1987). In addition, changes in
the activity of key metabolic enzymes in
specifi c tissues have been used as measures
of growth by some authors (Mathers et al.,
1992, 1993; Pelletier et al., 1993, 1994; Gud-
erley et al., 1994). All of these approaches
have strengths and weaknesses, and, with
some exceptions, they are all a posteriori
measures of growth. The problem of meas-
uring growth in the long term is further
compounded by the uneven nature of
growth in fi sh. Fish inhabiting temperate
regions do not exhibit a constant rate of
growth; there are daily variations in growth
rate, which overlay seasonal differences
that are correlated with annual and semi-
lunar rhythms (Leatherland et al., 1992).

Moreover, depending on the gender and
phase of the life cycle (early ontogeny, sexu-
ally immature, sexually maturing, etc.),
growth rate stanzas (Brett, 1979), expressed
as changes in body weight over time, vary
markedly (Ricker, 1979).
For any given set of conditions, the
daily rate at which food is consumed is the
12 J.F. Leatherland
prime determinant of growth rate in fi sh
(Brett, 1979). However, annual seasonal
cycles exert a major infl
uence on the growth
performance of wild ectothermic animals
such as fi shes, particularly for species that
inhabit temperate climates. Annual rhythms
of photoperiod, light intensity and water
temperature often determine the amount of
available food, the length of time that an ani-
mal can feed and the metabolic rate (Brett
and Groves, 1979). Although the infl uence
of these abiotic factors on growth perform-
ance of fi shes is well established, there is
no comprehensive understanding of how
they exert their infl uence. Furthermore, the
multiple interactions between abiotic and
biotic factors in a complex ecosystem (and
particularly disturbed ecosystems) are
poorly understood. Consequently, the use
of growth performance of wild fi sh species

as a measure of environmental impact has
limited value, unless it is combined with
other investigational approaches; growth rates
of individuals in a population are diffi cult
to determine, and even if growth rates can
be determined, the association of altered
growth rate with a particular cause is usu-
ally very diffi cult to discern.
The established growth performance
measures outlined above are considerably
Skeletal and soft tissue
growth
Energy partitioning:
nutrient storage and
mobilization
Activity
level
Feeding
behaviour
and food
intake
Reproduction
Photoperiod
Photointensity
Oxygen levels
pH
Temperature
Environmental
stressors
Genetics

Food quality and
quantity
Fig. 1.4. Schematic representation of the interactive nature of metabolism and energy partitioning pro-
cesses in fi shes. The bold arrows indicate sites of action of environmental factors, such as photoperiod and
temperature and environmental stressors ([e.g. toxicants, high population density, food deprivation, etc.) on
the interactive net. The dashed arrows represent the energy partitioning interactions that occur as a result of
life history events and activities.
Introduction 13
easier to apply to evaluate captive stocks.
‘Optimal’ growth performance for a given
species reared under established conditions
on a particular diet is easy to measure, and
thus any reduction in growth rate can be
readily identifi
ed. However, even for these
well-controlled situations, the value of

impaired growth as a diagnostic tool is lim-
ited because it is only a preliminary indica-
tor of a problem. Under controlled conditions,
such as those found in many fi sh-farming
situations, the quality and quantity of die-
tary sources probably exert the most signifi -
cant infl uence on growth performance. A
reduction in growth rate, under these condi-
tions, is indicative of reduced food intake,
impaired digestion and/or assimilation, or
altered metabolism resulting in a reduced
effi ciency of nutrient assimilation. Specifi c
identifi cation of the cause is not possible

and other diagnostic methodologies are
required to determine the aetiology.
Impaired reproductive success
and early ontogeny defects
This topic area is explored extensively in
Chapters 3 and 4 of this book. In brief, repro-
ductive problems and embryo development
problems related to environmental contami-
nants have been reported in many wild fi sh
populations (Kime, 1995, 1998; Monosson,
1997; Rolland 2000b; Norris and Carr, 2006),
and there are likely to be issues in many
species that have not yet been identifi ed.
These studies have shown that virtually all
aspects of reproduction and early ontogeny
may be affected, but the fi rm evidence of
cause–effect linkages between exposure of
the organism to contaminants and the
observed reproductive and developmental
effects has proved to be diffi cult. Moreover,
in some instances, reproductive or develop-
ment issues were attributed incorrectly to a
contaminant aetiology. For example, M74
Syndrome in Baltic Sea Atlantic salmon
(called Early Mortality Syndrome in the
Great Lakes) is characterized by the sudden
mortality of late yolk-sac-stage embryos.
The condition was subsequently shown to
be a vitamin B defi ciency caused by over-
fi shing of the primary prey species of the

juvenile and adult fi sh (Börjeson and Nor-
rgren, 1997). Smelt (Osmerus sp.) are the pre-
ferred prey species, but overfi shing of smelt
in the Baltic Sea and Great Lakes led to sig-
nifi cant reductions in the availability of that
species, and the Atlantic salmon increased
predation of their secondary prey species,
the alewife (Alosa pseudoharengus); alewife
contain a vitamin B inhibitor, which reduced
the ability of the adult salmon to acquire
vitamin B. As a consequence, delivery of
vitamin B from the maternal circulation into
the developing oocytes was reduced, lead-
ing to vitamin B defi ciency in the late-stage
embryos when the yolk sac reserves were
close to their fi nal stages of absorption. The
condition can be prevented by a single
immersion of the embryos in a solution of
vitamin B.
A second example of a reproductive
problem that is brought about by ‘natural’
causes is the reproductive neuroendocrine
functional changes in esturarine fi sh brought
about by seasonal hypoxia (Thomas et al.,
2007). Hypoxia has been of increasing focus
and has been related to specifi c gene expres-
sion (Rahman and Thomas, 2007) and com-
promised immunoresponse (Choi et al., 2007),
in addition to oxidative stress (Lushchak
and Bagnyukova, 2007); this may be a factor

that needs to be considered more promi-
nently in future studies of non-infectious
disorders in fi sh.
Laboratory studies, largely based on
studies of exposure of fi sh to a single chem-
ical, have provided some information about
the mechanistic basis of reported reproduc-
tive problems. The list of suspect chemicals
is long and includes polycyclic aromatic
hydrocarbons (PAHs), PCBs, dioxins, organo-
chlorine insecticides, metals (including cad-
mium, lead and selenium), phyto-oestrogens
and synthetic oestrogens (Kavlock et al.
,
1
99
6; Rolland, 2000b). However, in the cases
where effects have been seen over wide geo-
graphic regions or due to complex indus-
trial effl uents from pulp mill or sewage
treatment facilities, the causative chemicals
have often not been fully identifi ed; this
makes replication in the laboratory setting
14 J.F. Leatherland
diffi cult. Furthermore, the broad range of
chemicals on this list illustrates that repro-
ductive and development effects are infl u-
enced by multiple mechanistic pathways.
Broad generalizations of how these will
affect different species of fi

sh should be
viewed with caution, given the diversity of
reproductive strategies, reproductive life
histories and spawning strategies.
Also, the processes that are sensitive to
the impact of environmental chemicals are
diverse; thus, it should come as no surprise
that there is no simple prescription for eval-
uating reproductive and developmental fi t-
ness in fi sh. Although standardized whole
animal tests have been developed for exam-
ining the effects of anthropogenic chemicals
on reproductive processes in fi sh (summa-
rized by Leatherland et al., 1998), these tests
have been developed primarily for toxicity
testing rather than a means of diagnosing
de novo dysfunctional conditions; the tests
were not intended to be diagnostic meth-
ods, and for the most part they are not suited
to the diagnosis of emerging conditions that
are of unknown aetiology. One possible
exception is the prevalence of the yolk
phospholipoprotein vitellogenin in sexu-
ally immature fi sh of both sexes or in males
of all developmental stages; elevated plasma
vitellogenin levels in male fi sh is a reason-
ably well-established diagnostic indicator
of exposure of the fi sh to a xeno-oestrogen.
Organ, tissue and molecular indicators
Measures of tissue, organ or organism con-

tent of metabolites and calories have been
used, together with growth per se, to assess
the effi cacy of specifi c diets or feeding pro-
tocols; the most common form of proximate
analysis includes total carbohydrate, lipid
and protein levels, as well as total caloric
content. These are valuable indicators in
the confi rmation of pathologic emaciation
that is linked to infectious disease, reduced
food availability, diets that cannot be
digested and absorbed, or diets that cause
intestinal lesions that prevent the absorp-
tion of digesta. But, as with so many of the
other indicators considered in the above
sections of this chapter, the values are not
diagnostic of a specifi c condition but merely
indicative of impaired assimilation and par-
titioning of energy. In other words, they are
gross estimates of the overall ‘condition’ of
the fi sh. Most blood parameters, whether it
be haematocrit, plasma metabolite levels,
plasma enzyme activities or blood hormone
levels (summarized in Leatherland et al.,
1998), are a posteriori indicators and not
cause-specifi c; this is also true for most cel-
lular or tissue indicators. There are some
possible exceptions to this general state-
ment. One example is the group of genes
that is expressed in response to specifi c
environmental changes, such as temperature

changes and episodes of hypoxia (Lushchak
and Bagnyukova, 2007); however, even these
may be of limited value given daily and sea-
sonal changes in environmental parameters.
A second example is the group of enzymes
that is associated with detoxifi cation proc-
esses. The increased synthesis of these
enzymes or the increased expression of the
genes that encode for these enzymes is used
as an indicator of the response of the animal
to the presence of contaminants in its envi-
ronment. A list of the key enzymes in this
group is given in Leatherland et al. (1998).
Of these, induction in the hepatic activity of
mixed-function oxidases, including cyto-
chrome P4501A activity, ethoxyresorufi n-
O-deethylase (EROD) and benzo(a)pyrene
monooxygenase (B(a)PMO) (Addison et al.,
1979; Focardi et al., 1992; Arinc et al., 2000;
Corsi et al., 2004), has been used as an indi-
cator of hepatotoxic responses to environ-
mental chemicals. In addition, the induction
of the glutathione-S-transferase (GST) fam-
ily of enzymes has been used in some fi sh
species as a marker of the level of toxic chal-
lenges faced by a population or stock of ani-
mals. The GST family of enzymes in fi sh
closely resembles similar enzymes in mam-
mals (Dominey et al., 1991; Henson et al.,
2000); they contribute to the biotransforma-

tion of a wide range of compounds, includ-
ing xenobiotics and endogenous compounds.
GST enzyme levels based on functional

activity or immunohistochemical evaluation
in blood, gill, liver, kidney and intestine