v>EPA
United Statet Office of Water
Environmental Protection Agency Washington, D.C. 20400
EPA 430/09-37-004
June 1987
Guidance for Conducting Fish
Liver Histopathology Studies
During 301 (h) Monitoring
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May 1S37
GUIDANCE FOR CONDUCTING
FISH LIVER HISTOPATHOLGY
STUDIES DURING
301 (h) MONITORING
Prepared by:
Tetra Tech, Inc.
11820 Northup Way, Suite 100
Bellevue, Washington 98005
Prepared for:
Marine Operations Division: 301 (h) Program
Office of Marine and Estuarlne Protection
U.S. Environmental Protection Agency
401 M Street SW
Washington, D.C. 20460
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PREFACE
This document v«as preoared by U.S. EPA's Marine Ooerations Division
(Office of Marine and Estuanne Protection), in response to requests from
U.S. EPA regional offices and coastal municipalities for assistance on
technical issues raised during issuance of 301(h)-modified NPDES permits.
Under regulations implementing Section 301(h) of the Clean Water Act,
municipalities that discharge sewage to marine waters are required to
conduct monitoring programs to 1) evaluate the impact of their discharge on
marine biota, 2) demonstrate compliance with applicable water quality
standards, and 3) measure toxic substances in the discharge. Fish liver
'listopathology is one important biological impact that is monitored by
selected dischargers.
The purpose of this document is to provide guidance for designing and
conducting field surveys of fish liver histopathology as part of 301
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CONTENTS
Page
PREFACE 11
LIST OF FIGURES v
LIST OF TABLES vi
ACKNOWLEDGEMENTS vii
1.0 INTRODUCTION 1
1.1 BACKGROUND 1
1.2 PURPOSE AND SCOPE 2
2.0 BACKGROUND INFORMATION 4
2.1 THE LIVER OF FISHES *
2.1.1 Structure 4
2.1.2 Function 7
2.1.3 Relation to Chemical Contaminants 8
2.2 FISH LIVER HISTOPATHOLOGY 12
2.2.1 General 12
2.2.2 Cellular Alterations 15
2.2.3 Neoplasia 1?
2.2.4 Hepatocarcinogenesis Models for Fishes 22
2.3 REVIEW OF HISTORICAL DATA 29
2.3.1 Laboratory Studies 29
2.3.2 Field Studies 39
3.0 GUIDANCE FOR CONDUCTING FIELD STUDIES 57
3.1 STUDY DESIGN 57
3.1.1 Species Selection 57
3.1.2 Age Limits 60
3.1.3 Sample Size 65
3.1.4 Sampling Season 79
3.1.5 Station Location 81
ill
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3.2 FIELD SAMPLING PROCEDURES 32
3.2.1 Fish Acauismon 32
3.2.2 Holding Time ana Conditions 83
3.2.3 Labeling ana Coding 33
3.2.4 Liver SubsamoHng 34
3.2.5 Tissue Fixation 35
3.2.6 Ancillary Data 86
3.3 LABORATORY PROCEDURES 93
3.3.1 Tissue Processing 93
3.3.2 Histopathological Evaluations 97
3.3.3 Quality Assurance/Quality Control 103
3.4 DATA ANALYSIS AND INTERPRETATION 105
3.4.1 Age and Sex Effects 105
3.4.2 Growth and Condition 106
3.4.3 Comparisons Among Stations 107
3.4.4 Relationships with Ancillary Variables 111
4.0 SUMMARY 114
4.1 INTRODUCTION 114
4.2 BACKGROUND INFORMATION 114
4.3 GUIDANCE FOR CONDUCTING FIELD STUDIES 117
4.3.1 Study Design 117
4.3.2 Field Collection 119
4.3.3 Laboratory Procedures 121
4.3.4 Data Analysis and Interpretation 122
5.0 REFERENCES 125
6.0 GLOSSARY 142
APPENDIX A - SUMMARY OF HEPATIC LESIONS OBSERVED IN FISHES AFTER
LABORATORY EXPOSURE TO VARIOUS CHEMICALS A-l
IV
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FIGURES
Number Page
1 Schematic of the fish liver and associated organs 5
2 Schematic of major contaminant pathways in relation to the
fish liver 9
3 Generalized biotransformation pathway for exogenous
chemicals 11
4 Distribution of times to first neoplasm for a variety of
fishes exposed to a variety of chemicals in the laboratory 37
5 Relationship between hepatic lesions and size or age of
Atlantic hagfish. ruffe, and English sole 61
6 Length frequency distributions of various age groups of
male and female English sole from Commencement Bay, WA 64
7 Sample sizes required to detect one individual affected
with a lesion with 95 percent confidence, given various
population sizes and prevalences 67
8 Example of a 2 x 2 contingency table 70
9 Power of the G-test vs. sample size when lesion prevalence
at the reference site is 0.1 percent 75
10 Power of the G-test vs. sample size when lesion prevalence
at the reference site is 5.0 percent 75
11 Effects of sample size on the minimum detectable prevalence
at a test site relative to the prevalence at the reference
site 78
12 Seasonal variation of hepatic lesions in English sole from
the Duwamish River, WA 80
13 Results of simulation experiments showing the proportion of
Type i errors in tests of the null hypothesis that lesion
prevalence at both the reference and test sites equals
10 percent 110
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TABLES
Number
1 Chemicals that have induced hecatic lesions in
fishes following laboratory exposure 30
2 Species in which hepatic lesions have been induced
following laboratory exposure to chemicals 35
3 Summary of field studies in which elevated prevalences of
hepatic neoplasms have been found in feral fishes 40
4 Characteristics of fishes found to have elevated prevalences
of hepatic neoplasms in field studies 58
A-l Summary of hepatic lesions observed in fishes after lab-
oratory exposure to various chemicals A-l
vi
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ACKNOWLEDGMENTS
This technical guidance document was produced for the U.S. Environmental
Protection Agency under the 301(n) post-decision technical supoort contract
No. 68-01-6938, Allison J. Ouryee, Project Officer. This document was
prepared under the direction of Or. Thomas Ginn (Program Director) of Tetra
Tech, Inc. The authors of this document were Or. Scott Becker and Mr. Thomas
Grieb of Tetra Tech, Inc.
This document was reviewed by the following individuals:
Dr. Bruce Boese (U.S. Environmental Protection Agency)
Or. John Couch (U.S. Environmental Protection Agency)
Ms. Allison Ouryee (U.S. Environmental Protection Agency)
Dr. Steve Ferraro (U.S. Environmental Protection Agency)
Or. George Gardner (U.S. Environmental Protection Agency)
Dr. Stephen Goldberg (Whittier College, CA)
Mr. Kris Lindstrom (K.P. Lindstrom and Associates)
Or. Andrew Lissner (Science Applications International Corporation)
Dr. Charles Menzie (Project Consultant)
Dr. Brian Melzian (U.S. Environmental Protection Agency)
Or. Robert Murchelano (National Oceanic and Atmospheric Administration)
Mr. Mark Myers (National Oceanic and Atmospheric Administration)
Or. Thomas O'Connor (National Oceanic and Atmospheric Administration)
The comments from these reviewers improved the quality of this document and
are gratefully acknowledged.
vii
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1.0 INTRODUCTION
1.1 BACKGROUND
A wide variety of pathological conditions has been found in feral
(i.e., wild) fishes collected from marine, estuarine, and freshwater habitats
throughout the world (e.g., Amlacher 1970; Mawdesley-Thomas 1972; Reichen-
bach-Klinke 1973; Ribelin and Migaki 1975; Snieszko and Axelrod 1976; Roberts
1978; Sindermann 1979, 1983; Sindermann et al. 1980; Mix 1986). In many
cases, these pathological conditions have been associated with some form of
environmental pollution. Despite these associations, the use of fish
pathology as a quantitative tool for evaluating the consequences of environ-
mental pollution is a relatively new endeavor. Major requisites for
conducting such quantitative studies are appropriate study designs (including
species selection, size or age limits, sample sizes, station locations),
field sampling methods, and laboratory analytical techniques (Sindermann et
al. 1980). After reviewing historical field and laboratory studies of fish
pathology, Johnson and Bergman (1984) concluded that changes must be made in
many of the approaches and methods used traditionally in such studies, if
results are to be useful for addressing the objectives of aquatic toxicology.
The U.S. Environmental Protection Agency (EPA) has selected fish liver
histopathology as one of the indicators of biological impacts for selected
marine dischargers holding 301(h)-modified NPDES permits. The use of fish
liver histopathology as an environmental assessment tool by U.S. EPA is
consistent with its use by the National Oceanic and Atmospheric Adminis-
tration (NOAA) as a major indicator of long-term biological conditions in
coastal waters of the U.S. (e.g., Susani 1986; Susani et al. 1986). The
liver is an appropriate organ for evaluation for the following reasons:
• It is the organ primarily responsible for the metabolic
homeostasis of the entire fish and, as such, is associated
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intimately with the chemical contaminants tnat may enter a
fish inhabiting a oolluted environment (see Section 2.1.3)
• A variety of field studies have found idiopathic neoolasms and
other lesions in the livers of fishes inhabiting polluted
environments (see Section 2.3.2)
t The liver is the organ most often altered pathologically in
laboratory exposures of fishes to chemicals, including carcin-
ogens (Gingerich 1982)
• Various national and international workshops have recognized
the value of fish liver histopathology as an indicator of
environmental pollution (e.g., Sindermann et al. 1980;
U.S. EPA 1986).
1.2 PURPOSE AND SCOPE
This document provides guidance for conducting quantitative field
studies of fish liver histopathology as part of 301(h) monitoring programs.
At present, no comprehensive sources of such guidance are available. The
document is directed primarily at the non-pathologists involved in writing
301(h)-modified NPOES permits and in overseeing field studies of fish liver
histopathology. Although this document is directed at non-pathologists,
various sections may also be useful to pathologists.
This document addresses the following four major components of quanti-
tative field studies of fish liver histopathology:
• Study design
• Field sampling
• Laboratory analysis
• Data analysis and interpretation.
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Although the emphasis of this document is on liver histopathology, many of
the considerations addressed for each component may also pertain to a
/anety of other kinds of pathological conditions in fishes.
General recommendations for each of the four major study components are
made in Section 3.0. These recommendations were made as detailea as
possible without sacrificing their site-specific applicability. For
example, because specific objectives generally vary among different studies,
exact specifications for such considerations as sample sizes, station
locations, staining procedures, and methods of data analysis could not be
made. Instead, the various acceptable options for each feature are presented
along with their respective benefits and limitations. Literature citations
were used to support recommendations whenever possible.
Before the four study components are discussed, a major section (i.e.,
Section 2.0) is presented on the background information needed to understand
many of the recommendations made throughout the document. Section 2.0 first
describes the general structure and functions of the fish liver and the
relationship between the liver and chemical contaminants that enter the
fish. Considerations related to pathological conditions in the fish liver
are discussed next. These considerations include descriptions of the
general cellular alterations that follow cell injury, a review of the
processes involved in ne'oplasia, summaries of hepatocarcinogenesis models
for rainbow trout (Salmo gairdneri) and English sole (Parophrys vetulus),
and reviews of historical laboratory and field studies relating fish liver
histopathology to pollution or, more specifically, to chemical contamination.
A summary of the major points described throughout this document is presented
in Section 4.0.
Because many of the terms used in this document are unfamiliar to
anyone without a background in pathology or cellular biology, a glossary
(Section 6.0) is provided at the end of the document.
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2.0 BACKGROUND INFORMATION
2.1 THE LIVER OF FISHES
This section describes the general structure and functions of a fish
liver and how the organ is involved with the treatment of exogenous toxic
contaminants. This information is a prerequisite for understanding how
pathological conditions of the liver arise.
2.1.1 Structure
Although the structure of the liver generally is similar in all fishes,
some considerable interspecific differences exist (review in Gingerich
1982). Such differences might be expected for a group of animals that
includes approximately 20,000 species with a variety of evolutionary
histories and a distribution across a wide range of habitats (Moyle and Cech
1982). As in other vertebrates (e.g., mammals, birds, reptiles), the liver
of fishes arises in the embryo as a ventral evagination of the developing
intestine. The anterior portion of this tissue develops into the liver,
whereas the posterior portion develops into the gall bladder.
The liver is the largest visceral organ in fishes. In most species the
liver weighs 1-3 percent of body weight (Gingerich 1982). However, in some
sharks the liver can weigh as much as 20 percent of body weight (Lagler et
al. 1962). Liver mass can vary substantially within an individual, depending
on the rate of food consumption, time since last feeding, and reproductive
state.
The liver of all fishes is located in the anterior and ventral por-
tion of the abdominal cavity (Figure 1). It is connected with the anterior
portion of the intestine by the hepatic and bile ducts. Secretions produced
in the liver are transported to the intestine through these ducts. The gall
bladder (absent in some fishes) is a relatively small organ that is closely
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LEGEND
BO =
CD =
GB =
HA =
• Bile Duct
Cystic Duct
= Gall Bladder
> Hepatic Artery
HD = Hepatic Duct
HP = Hepatic Portal Vein
HV = Hepatic Vein
IN = Intestine
LI = Liver
*• Blood
^ Liver Secretions
Figure 1. Schematic of the fish liver and associated organs.
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associated vmn the liver. It is xonnectea *uh the oile auct oy tne cystic
auct. 3ile producea in the liver is storea in tne gall blaoder. Numerous
/anations in this general arrangement exist among different ssecies
(Gingencn 1982).
The 1 wer is closely related to the circulatory system (Figure 1), and
is one or" tne most richly vasculanzed organs in fishes tGingencn i982).
Blood containing almost all of the materials digested and absorbed in the
intestine is transported to the liver through the hepatic portal vein. The
hepatic vein transports blood from the iiver directly to the heart. In most
fishes, the hepatic vein empties directly into the sinus venosus of the
heart. Oxygen-rich blood enters the liver through the hepatic artery.
Hepatocytes (i.e., parenchymal liver cells) in most fishes are morpho-
logically similar throughout the liver. The shape of hepatocytes can vary
among different species (e.g., hexagonal, oval). Unlike the mammalian
liver, biochemically and functionally heterogeneous zones of hepatocytes are
not prominent within the liver of fishes (Gingerich 1982). Hepatocytes in
the livers of both mammals and fishes are arranged in plates or sheets.
However, in most fishes the sheets are two cells thick, whereas in mammals
they are one cell thick. A network of tiny bile canal icuii and tubules is
distributed throughout the liver. These tubules contact every hepatocyte
and gather cellular secretions (i.e., bile) for drainage into the hepatic
duct.
The internal structure of hepatocytes in fishes is similar to that
found in higher vertebrates (Gingerich 1982). Generally, there is a single
nucleus per cell. Rough endoplasmic reticulum (RER) lies adjacent to the
nucleus, and mitochondria frequently are found associated with the RER.
Smooth endoplasmic reticulum (SER) usually is found near areas of glycogen
deposition, but is less prominent than in higher vertebrates. The Golgi
apparatus generally is well developed.
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Z.I.2 Function
Althougn the f'-inct'ons of the 11 /er of .-nost f:snes are similar, some
consiceraole interspecific differences may exist (Gingench 1982). As -.n
other vertebrates, the liver of fishes has a variety of functions. ~nree
major functions include the following:
• Production of bile
0 Storage of fats and carbohydrates (primarily glycogen)
• Metabolism of food material from the intestine and toxic
chemicals from the intestine and other sources (e.g., gills,
skin).
Bile is produced in the liver as a cellular secretion. It is tnen
concentrated and stored in the gall bladder and released into the intestine,
as needed. Bile is composed primarily of bile salts and metabolic waste
products. The bile salts aid in the enzymatic digestion of fats in the
intestine. Because the waste products from the liver can include toxic
chemicals or their metabolites, the bile of fishes appears to offer a major
route of elimination (i.e., eventually through faces) for a variety of
chemical contaminants (Gingench 1982).
Although the amount of fat stored in the liver can vary dramatically
among fishes, two general groups of fishes can be distinguished (Lagler et
al. 1962). In the first group, fat is stored primarily in the liver [e.g.,
flatfishes (Pleuronectiformes) and cods (Gadidae)]. In the second group,
fat is stored primarily in muscle tissue [e.g., tunas (Scomondae) and
herrings (Clupeidae)]. Glycogen is stored as an energy reserve in the liver
and is released into the bloodstream when needed.
The liver receives all material absorbed in the intestine except certain
lipids. Within the liver, proteins can be synthesized or made into carbo-
hydrates, fats can be altered in composition or made into carbohydrates,
blood cells can be destroyed, nitrogenous wastes can be transformed into
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urea for excretion by the kianeys, and toxic cnemicals can oe aetoxinea or
oreoared for elimination, [n some cases, che toxicity of certain chemicals
is ennanced by the iwer's metabolic activities (see Section 2.1.3).
Of the major functions of the liver, tne most important is orooably its
metabolic role (Romer 1970). Because it is the first organ to receive and
process almost all materials newly arrived from tne intestine, the iiver
plays a central role in the metabolic homeostasis of the wnole organism.
The liver's major role in the treatment of exogenous toxic contaminants
renders the cells of this organ {i.e., hepatocytes) highly susceptible to
toxic injury, and thus potentially useful for monitoring the effects of
environmental pollution.
2.1.3 Relation to Chemical Contaminants
As mentioned in Section 2.1.2, a major role of the liver of fishes is
the treatment of exogenous toxic contaminants. These chemicals can enter a
fish through at least three major routes: the mouth (and then the gastro-
intestinal tract), the gills, and the skin (i.e., integument). Contaminants
can enter through the mouth in several forms. They can be incorporated into
the tissue of prey organisms, attached to sediment or organic detritus
ingested incidental to feeding (e.g., in prey gut contents, in worm tubes,
adhering to prey), or dissolved in consumed ambient water (i.e., for marine
and estuarine fishes). Dissolved contaminants can enter through the gills
by diffusing into tfie bloodstream as a fish respires. Dissolved contaminants
can also enter through the skin by being absorbed from ambient water.
Contaminants inside the body of a fish are transported to the liver through
the hepatic portal vein and the hepatic artery (Figure 2).
Cnce inside the liver, a contaminant can be processed in many different
ways, depending upon such factors as the kind of contaminant, the species of
fish, and the metabolic state of the fish. Exogenous contaminants may be
stored, directly eliminated, or metabolically altered before being elimi-
nated. Metabolic alteration of contaminants is particularly germane to fish
liver histopathology, as some metabolites are highly reactive and potentially
cytotoxic, mutagenic, or carcinogenic.
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CONTAMINANT ENTRV
ENTERO HEPATIC
CVCLING
LEGEND
BD = Bile Duct
CD = Cystic Duel
GB = Gall Bladder
HA = Hepatic Artery
HD = Hepatic Duct
HP = Hepatic Portal Vein
HV = Hepatic Vein
IN = Inleslme
LI = Liver
Blood
Liver Secretions
Figure 2. Schematic of major contaminant pathways in relation to the fish liver
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The metaoolic transformation of an exogenous cnermcal -s :er>nea
oiotransformation. The general oattern of biotransformation (Figure 3) is
to convert tne contaminant to a more oolar (i.e., water soluoie) form ana to
conjugate this derivative with a highly polar endogenous comoound, «mcn
then facilitates elimination through normal routes (Tinsley t979). 3io-
transformation of exogenous chemicals can thus be divided into t-wo major
ohases (Loomis 1978): nonsynthetic reactions (i.e., metabolite formation)
and synthetic reactions (i.e., conjugation). In some cases, however,
metabolites can be eliminated without being conjugated (Connell and Miller
1984). Reactions involved with both phases of biotransformation are
catalyzed by enzymes.
Metabolite formation from exogenous chemicals is achieved primarily by
oxidation. These reactions are catalyzed by enzymes (i.e., oxygenases) of
the mixed function oxidase (MFO) system incorporated in the smooth endo-
plasmic reticulum of the cell. Metabolites may also be formed by reduction
or hydrolysis.
After metabolites are formed by oxidation, reduction, or hydrolysis,
they may be conjugated to an endogenous compound in preparation for elimina-
tion. Conjugation is catalyzed by enzymes (i.e., transferases) located in
the cytosol, mitochondria, and endoplasmic reticulum of the cell (Connell
and Miller 1984). The three major kinds of conjugation reactions involve
glucuronic acid (a glucose derivative), glutathione (a tripeptide), and an
active sulfate (Tinsley 1979).
Although metabolite formation by oxidation, reduction, or hydrolysis
generally is an important detoxification step, highly reactive electrophilic
metabolites can be produced. Because some of these reactive metabolites
interact chemically with cellular macromolecules such as ONA and RNA, they
are considered potential carcinogens, mutagens, and cytotoxins (Connell and
Miller 1984).
As with mammals, the bile of fishes appears to be a major route through
which a variety of exogenous chemicals and their metabolites are eliminated
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Cyloloxlclty
Mulagan«»lB
Catclnoganaals
EXOGENOUS
CHEMICAL
(Enzymes)
METABOLITES
NONSYNTHETIC
REACTIONS
• OXIDATION
• REDUCTION
• HYDROLYSIS
(Enzymes)
CONJUGATES
SYNTHETIC
REACTIONS
••9
• GLUCURONIOE CONJUGATION
• GLUTATHIONE CONJUGATION
• SULPHATION
ELIMINATIION
ELIMINATION
Reference Modified ham Connull and Miller 1984
Figure 3. Generalized biolransformation pathways (or exogenous chemicals
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(Gingench 1982). Exogenous-chemicals also can be eliminated through other
routes involving such structures as kidneys and gills.
The propensity of a chemical to be eliminated in the bile of fishes is
influenced by the molecular weight and polarity of the contaminant (Gingench
L982). Chemicals having both a low molecular weight (i.e., <200) and low
polarity are eliminated through the kidneys or gills, and are not concen-
trated in bile. By contrast, bile is a common route of excretion for
chemicals that are charged or highly polar and chemicals that are noncharged
but high in molecular weight (i.e., >600). Chemicals between these two
extremes (i.e., those with molecular weights of 300-600 and intermediate
polarities) appear to be eliminated in nearly equal amounts through the
kidneys and through bile.
In some cases, chemicals eliminated in bile can become incorporated
into entero-hepatic cycling (Glngerich 1982). This process involves reab-
sorbtion from the intestine, reintroduction to the liver, and resecretion
into bile. Entero-hepatic cycling reduces the elimination rate of affected
chemicals and may be responsible for prolonging the effects of certain toxic
contaminants.
2.2 FISH LIVER HISTOPATHOLOGY
2.2.1 General
The science of pathology is concerned primarily with the study of
disease. As such, it addresses the structural and functional consequences
of injurious stimuli to the cells, tissues, and organs of the body, and
ultimately the consequences to the entire organism (Robbins et al. 1984).
In general, organisms are adapted to accommodate a variety of dynamic stimuli
and thereby maintain their oodily equilibrium (i.e., homeostasis). However,
when stimuli become more severe or the response capabilities of the organism
decline, disease may result. This is true for the whole organism as well as
for each individual cell. In general, disease involves the modification,
loss, or accentuation of existing biochemical pathways and structures rather
than the generation of new pathways or structures. Pathology therefore is
12
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concerned primarily with deviations from normal structure, onysiolocy,
biocnermstry, and cellular ana molecular biology.
Pathology is concerned with four major asoects of disease (Roboins
et al. 1984):
t
Etiology - cause of disease; can be subdivided into gene-
tically related causes and acquired (e.g., infectious, nutri-
tional, chemical, physical) causes
• Pathogenesis - sequence of events (e.g., chemical, molecular,
cellular) that occur within an organism from initial stim-
ulation to final expression of a disease
t Morphologic changes - structural changes that occur as a
result of a disease; can sometimes be used to identify the
etiology and prognosis of a disease
• Functional changes - changes that result in the activities of
structures as a result of morphologic changes; can sometimes
be used with morphologic changes to identify the etiology and
prognosis of a disease.
Most field studies of fish liver histopathology have focused primarily
on the morphologic changes that occur in response to harmful environmental
stimuli (see Section 2.3.Z). Many of these changes are relatively stable
and amenable to some form of quantification. In some cases, morphologic
changes can be observed grossly (i.e., with the unaided eye). In many
cases, morphologic changes can be observed using light microscopy. Electron
^icroscopy provides an even more detailed evaluation of these changes.
Determining the specific etiologies of pathological liver conditions in
feral fishes rarely is possible because these organisms generally are exposed
to an unknown diversity of potentially harmful stimuli (e.g., infectious,
nutritional, chemical, physical). Possible interactions among stimuli that
13
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f;/ tneir individual effects (e.g., synergism, antagonism) r'yrtner
complicate etiological determinations.
Pathogenesi s and functional cnanges in the livers of feral fisnes
generally are inferred from observed morphologic changes. Because each fisn
usually is sampled once and then sacrificed, direct observations of disease
progression and functional cnanges are not possiole for individual fisn.
However, if a variety of morphologic changes is found within the livers of
single or multiple individuals, disease progression and functional changes
often can be infe-red.
Although most field studies of fish liver histopathology are limited to
observing morphologic changes, laboratory studies frequently consider the
etiology, pathogenesis, or functional changes related to the morphologic
changes. In the majority of laboratory studies, fishes are exposed to a
single stimulus under carefully controlled conditions. The pathological
conditions that result can thus be attributed with reasonable confidence to
the effects of the test stimulus. In addition, by monitoring the test
organisms over time, the pathogenesis and functional changes involved with a
particular condition often can be observed.
Although laboratory studies of fish liver histopathology have many ad-
vantages over field studies, the validity of making direct extrapolations
from laboratory results to the more complex conditions encountered in the
field generally is uncertain. To maximize the utility of both laboratory
and field studies, it is preferable that they be closely interrelated (e.g.,
Johnson and Bergman 1984).
The following three sections describe the general cellular alterations
that may follow exposure to a harmful stimulus, the ,iajor events involved in
neoplasia, and the heptocarcenogenesis models for --inbow trout and English
sole. Unless otherwise noted, the information in Sections 2.2.2 and 2.2.3
was taken from Robbins et al. (1984).
14
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2.2.2 Cellular Alterations
Many pathological conditions in the liver of fishes arise from struc-
tural or functional alterations at the cellular or subcellular level. This
is particularly true for conditions caused by chemical stimuli. Many of the
factors related to such cellular injury are summarized in this section.
The normal cell generally is in a steady state with its microenviron-
ment, and is capable of adapting to altered steady states in response to mild
stimuli without losing its viability. However, if the adaptive capability
of a cell is exceeded, the cell may experience injury. This injury is
reversible to a point (i.e., degeneration), but if the stimulus is persistent
or strong enough, irreversible injury may occur. In many cases, irreversible
injury can lead to cell death (i.e., necrosis) or carcinogenesis.
The most common causes of cell injury are obstruction of blood supply
(i.e., ischemia), infectious agents (e.g., viruses, bacteria, fungi), and
chemical agents (e.g., toxicants, nutritional imbalances). In general,
morphologic changes in an injured cell become apparent only after a critical
biochemical system within the cell has been altered. The severity of cell
injury depends on the following variables:
t Kind of stimulus
• Duration of stimulus
• Magnitude of stimulus
t Kind of cell
t Physiological ?tate of cell.
Upon exposure to a harmful stimulus, cells may initially escape injury
by adapting to the stimulus in one of several ways. The four most important
cellular adaptive changes are:
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t Airoony
• Hypertropny
t Hyperplasia
• Metaplasia.
Atrophy represents a reduction of the structural components (and thus
size) of a cell and may be caused by decreased workload, loss of mnervation,
diminished blood supply, inadequate nutrition, or loss of endocrine stimu-
lation. Atrophied cells can be viable, although they have diminished
function. However, atrophy can progress to the point at which cells are
injured or die.
Hypertrophy represents an increase in the structural components (and
thus size) of a cell. It may be caused by increased functional demand or
hormonal stimulation and may or may not be pathologically related.
Hyperplasia represents an increase in the number of cells in an organ.
It often occurs concurrently with hypertrophy and may or may not be path-
ologically related. Pathologic hyperplasia represents a potential source
From which cancerous cell proliferation may arise.
Metaplasia represents a reversible alteration of adult cell types. It
may be an adaptive substitution of cells more sensitive to stress by other
cell types better able to accommodate harmful stimuli.
If adaptation to a harmful stimulus cannot adequately protect a cell,
some form of cellular injury usually occurs. A variety of morphologic
changes can be observed in injured cells by using light microscopy. Two
common patterns of degeneration (or reversible injury) are cellular swelling
and fatty change.
Cellular swelling is the first manifestation of almost all forms of
cellular injury. It occurs when cells lose their ability to maintain ionic
16
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and fluid homeostasis and extracellular water moves into tne cell. Because
this condition is reversible and generally indicates only mi id injury, ir.s
onncioal value is its use as an indicator of the more severe injury cnac may
r'ollow. Cellular swelling is distinct from cellular hypertropny.
Unlike cellular spelling, fatty cnange occurs less universally, it is
found primarily in cells involved with fat metabolism, such as those of the
liver. Fatty change is any abnormal accumulation of fat within ceils. It
reflects an imbalance in the production, utilization, or moDilization of
lipid material and is often accompanied by the appearance of mtracellular
fat vacuoles. As with cellular swelling, fatty change is reversible,
generally nonlethal, and may be useful as an indicator of subsequent more
serious injury.
In addition to fatty change, intracellular accumulators of other
substances can occur in injured cells. These include proteins, carbo-
hydrates, pigments, and abnormal substances. These substances generally are
harmless, but under some circumstances can be toxic.
Cells that eventually die undergo a variety of morphological changes,
the sum of which is termed necrosis. Cells actually die some time before
necrotic changes become visible under a light microscope. Dead cells usually
exhibit increased eosinophilia. The cytoplasm may become highly vacuolated
after lysosomal enzymes have digested cytoplasmic organelles. The nucleus
may shrink to become a small, dense mass (pyknosis) and eventually dissolve
(karyolysis) or break apart (karyorrhexis).
2.2.3 Neoplasia
Neoplasms (i.e., tumors) are new growths of o'.normal tissue that grow
by cellular proliferation more rapidly than norn.a; and continue to grow
after the stimulus that initiated the new growth is withdrawn (Stedman's
Medical Dictionary 1984). Neoplasms exhibit partial or complete lack of
structural organization and functional coordination with normal cells, and
usually form a distinct mass of tissue. Neoplasms can be classified as
benign or malignant (i.e., cancerous). Benign tumors generally are thought
17
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*.o oe iess rarmfui to tieir host tnan are malignant tumors, out tr.ere are
exceptions to tms oattern.
All neoplasms nave two primary components, ^'ne first component is a
group of proliferating neoplastic cells that constitute the oarencnyma (or
main ooay) of the tumor. These cells represent the "cutting sage" of tne
neoplasm and determine its nature and progression. The second basic
component of neoplasms is the supportive stroma. This stroma is comprised
of connective tissue, blood vessels, and possibly lymphatics. The stroma
supports the main body of the tumor both physically and chemically.
The neoplasm most frequently observed in fish livers is hepatocellular
carcinoma. Although this neoplasm was formerly called hepatoma, that term
is considered inexact and its use in future studies is discouraged (Squire
and uevitt 1975; Sinnhuber et al. 1977). Hepatocellular carcinomas arise
from the parenchyma 1 cells of the liver. A second kind of neoplasm commonly
observed in fish livers is cholangioceilular carcinoma. This tumor arises
from the cells of intrahepatic bile ducts. Occasionally, tumors of mixed
origin (i.e., hepatocellular and cholangiocellular) are found.
Benign and malignant neoplasms frequently can be distinguished on the
basis of the following characteristics:
• Differentiation and anaplasia
t Rate of growth
• Encapsulation/invasion
• Metastasis.
Differentiation represents the extent to which parenchyma! cells of the
neoplasm resemble comparable normal cells, both structurally and func-
tionally. In general, cells of all benign tumors closely resemble normal
cells (i.e., they are well-differentiated). Cells of malignant tumors, by
contrast, range from being well-differentiated to being very different
18
-------
(e.g., primitive-appearing) from normal cells (i.e., they are 'j.naiffaren-
•-iated).
Anaplasia is the loss of some kind of differentiation in cells ana ;$
one of the cnaractenstics used to identify malignancy. The term anaolasia
implies a reversion from a mgh level of differentiation to a lo^er (i.e.,
more primitive) level. Anaplasia is characterized by a variety of mor-
phologic and functional changes. Cells and nuclei generally vary in size
and shape (i.e., pleomorphism). Nuclei may be disproportionately large for
the cell, with the nuclear-cytoplasmic ratio approaching 1:1 instead of the
normal ratio of 1:4 or 1:6. A large number of mitoses may be present as a
result of cellular proliferative activity. Sometimes the mitotic figures
are atypical and bizarre.
In general, the functional capacities of a neoplastic cell correlate
with its level of morphologic differentiation. Thus, wel!-differentiated
cells may function quite normally, whereas undifferentiated cells may lose
their original specialized functional characteristics.
The rate at which a neoplasm grows can assist in the determination of
benign and malignant tumors. Most benign tumors grow slowly over a number
of years, whereas most malignant tumors grow at a much more rapid, and
sometimes erratic, rate. - However, there are numerous exceptions to this
pattern.
Most benign tumors are enclosed within a fibrous capsule (i.e., they
are encapsulated). The capsule is partly derived from the fibrous stroma of
the surrounding normal tissue, and partly elaborated by the tumor. Benign
tumors may compress, but do not invade, surrounding tissue. By contrast,
malignant tumors rarely are encapsulated. In addition, most malignant
tumors invade surrounding tissue through infiltrative and erosive growth.
Next to metastasis (discussed below), invasiveness is the most reliable
indicator of malignancy.
Metastasis is the appearance of neoplasms in tissue discontinuous with
the primary tumor. It results from transport of neoplastic cells through
19
-------
u.-.e oiooastraam ana t.ie lympnanc system or from seeding of oody cavir.-ss
after :ney nave been oenetrated. Metastasis unequivocally identifies a
•saoolasm as malignant and therefore ;s the most rehaole i.-.aicator of
raai icnancy. Most malignant tumors can metastasize. However, the potential
for metastasis cannot &e determined from a oathologic examination of cne
onmary neoplasm, as many factors related to ooth tne tumor ana ere nost are
involvea.
Metastasis of hepatocellular carcinomas in feral fishes usually is not
found (e.g., Oawe et al. 1964; Falkmer et al. 1976; Brown et al. 1977).
However, McCain et al. (1982) documented the metastasis of a massive
cholangiocellular carcinoma to the spleen, kidney, small intestine muscle
wall, and ventricular myocardium of an individual English sole.
In laboratory studies of rainbow trout, Hendricks et al. (1984) noted
that although metastasis of hepatocellular carcinomas has been documented
(e.g., Ashley and Halver 1963; Yasutake and Rucker 1967), it occurs in-
frequently and usually involves fish that are 3-6 yr old. Hendricks et
al. (1984) suggest that hepatocellular carcinomas may be relatively slow to
metastasize in rainbow trout because of the low temperatures of the water in
which these poikilothermic organisms live.
The large variety of carcinogenic agents capable of inducing neoplasms
can be grouped into the following three categories:
• Chemical carcinogens
• Radiant energy
• Oncogenic viruses.
There is strong experimental evidence that neoplasm formation is a pro-
gressive process involving multiple steps and multiple exposures to stimuli.
It is therefore possible that neoplasms may be induced by simultaneous or
sequential exposure to several different carcinogens.
20
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All chemical carcinogens fall into one of t*o grouos. i"ne first crouo
is termed direct-acting (or activation-inaeoenaent) carcinogens. rnese
cnemicals do not reauire any kino of modification to exert tneir carcinogenic
effect. However, they sometimes can oe chemically or enzymatically m-
activatea. In general, these chemicals are weak carcinogens.
The second group of carcinogens is termed procarcinogens. These
chemicals require some form of metabolic conversion to produce metabolites
capable of inducing neoplasms. Procarcinogens are often called parent
compounds, whereas their carcinogenic metabolites are called ultimate
carcinogens. Many procarcinogens are activated by the hepatic MFO system
(Section 2.1.3). Although procarcinogens require metabolic activation to be
carcinogenic, they can also be metabolized to noncarcinogenic end products
(i.e., detoxified). Procarcinogens include potent carcinogens such as
polycyclic aromatic hydrocaroons (PAH), nitrosamines, and aflatoxins.
Chemical carcinogenesis involves at least two stages: initiation and
promotion. Initiation results from exposure of a cell to a threshold dose
of a carcinogenic chemical. An initiated cell is altered permanently,
making it likely to give rise to a neoplasm. Because initiation is irrever-
sible, multiple subthreshold doses are as effective as a single threshold
dose.
Initiation alone cannot induce neoplasms, but must be followed by
promotion. Promotion increases the tumorigenie response of an initiated
celJ when the cell is exposed to the promoter above a threshold level.
Because initiation is irreversible, promotion does not have to follow it
immediately. Unlike initiation, multiple subthreshold doses of a promoter
will not have the promoting effect of a single threshold dose. Most
promoters do r.-.t induce tumors by themselves. However, some chemicals can
act as both initiators and promoters, and are thus called complete
carcinogens.
There is strong evidence that chemical carcinogens induce tumors by
interacting with ONA, indicating they are mutagenic. However, tumors could
also be induced by the interaction of carcinogens with RNA and proteins.
21
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Sadiant energy in tne form or ultraviolet rays, x-rays, gamma rays, ana
ionizing particles (alpha particles, oeta oarticies, arotons, -eutrons) can
induce neoplasms. Radiant energy can damage ONA ana cellular memoranes,
alter proteins, and inactivate enzymes. However, the exact event resoonsible
for producing neoplastic cells is unknown. Much of the evidence suggests
chat raaiant energy exerts its carcinogenicity througn interactions «itn
ONA, indicating a mutagemc pathway.
Both RNA- and ONA-contaim'ng viruses can induce neoplasms. Unlike
nononcogenic viruses, oncogenic viruses generally are not infectious. RNA-
oncogenic viruses are also called retroviruses. Although the exact mech-
anisms by which RNA- and DNA-oncogemc viruses induce tumors currently are
unknown, it appears that the two kinds of viruses act in different manners.
Z.2.4 Heoatocarcinoqenesis Models for Fishes
In this section, two models of hepatocarcinogenesis are discussed. The
first is based on laboratory studies of rainbow trout, and the second is
based on field studies of English sole. These two models are the most
detailed ones available for fishes, and both were derived from extensive
amounts of empirical data.
The most complete description of and nomenclature for the sequential
cellular alterations involved in animal hepatocarcinogenesis are for rats
and mice (e.g., Squire and Levitt 1975; Frith and Ward 1980; Stewart
et al. I960). By comparison, fish hepatocarcinogenesis studies are in their
infancy (Hendricks 1982). Although many of the principles and much of the
nomenclature used in rat studies have been applied to fish studies, the
degree to which hepatocarcinogenic processes in rats are analogous to those
in fishes is unknown.
Rainbow Trout—
The species of fish most studied with respect to chemically induced
hepatic neoplasms is the rainbow trout. The chemicals used most often to
22
-------
induce hepatic neoplasms in this species are aflatoxins (primary aflatoxin
31 or AFB, ), a group of potent carcinogens oroduced by the mold Asoergiilus
fi avus. The relatively large amount of information available for tnis
species has been synthesized by Sinnnuber et al. (1977), Hendricks (1982),
and Hendricks et al. (1984). Because most studies have focused primarily on
the mere presence of hepatic neoplasms rather than their developmental
processes, the pathogenesis of liver cancer in rainbow trout is not well-
documented. However, as more information is available for this species than
for any other fish, it is instructive to review the available data.
In rainbow trout, the morphologic stages involved in hepatocarcino-
genesis are as follows:
t Pale, swollen, individual cells with enlarged pleomorphic
nuclei
• EosinophiHc foci
• Basophilic foci
• Hepatocellular carcinomas.
However, the sequential nature of these stages has not been confirmed
(Sinnhuber et al. 19-77).
The enlarged cells of the first stage undergo degeneration and
necrosis, but do not form nodules of proliferating cells. Sinnhuber et
al. (1977) suggest that the toxic influence of the carcinogen interferes
with normal cell functions and division, thereby producing a polypioid,
hypertropnlc cell that eventually dUs. The number of affected cells
increases with increasing doses of aflatcxin. Islets of regenerating cells
frequently are found in livers with degenerating cells, but their role in
hepatocarcinogenesis is unknown.
Eosinopnilic foci generally are small (i.e., <0.5 mm diameter). Cells
within these foci have relatively normal nuclei, but are distinctly eosino-
23
-------
omnc, nypertroomc, ana cevoid of glycogen. Mitotic ngures are rare, a-a
*ne cslls GO r.ot compress surrounaing tissue. The sosinoomiia resui:s
orunanly from e
-------
neoplascic transformation is complete (Sinnnuoer en al. 1977; Henancks e:
al. 1984).
Aithougn many authors distinguish adenomas from carcinomas on the basis
of degree of differentiation and presence or aosence of metastases,
Sinnhuoer et al. (1977) suggest that the potential for malignant oenavior is
present in all trout tumors, and may occur given sufficient time. They
therefore recommend that all tumors induced by aflatoxin in rainbow trout be
classified as hepatocellular carcinomas.
English Sole--
Myers et al. (1987) provide the first comprehensive documentation of
close morphological similarities between idiopathic hepatic lesions in a
feral fish and the established series of lesions induced in rodents following
laboratory exposure to hepatocarcinogens. The study was conducted on English
sole collected from Eagle Harbor, Washington. The sediments in Eagle Harbor
are contaminated with a variety of hepatocarcinogens (particularly creosote-
derived aromatic hydrocarbons), and prevalences of hepatic neoplasms and
other liver abnormalities are among the highest found in English sole from
any location in Puget Sound (Maiins et al. 1985b; see Section 2.3.2).
Myers et al. (1987) identified statistically significant associations
between a variety of lesion types based on their patterns of co-occurrence.
The authors assumed that co-occurring lesions may be caused by similar
etiological agents and that these lesions may be temporally related to each
other in terms of their development. A temporal relationship implies that
the lesions may be induced in a sequence of progression that terminates with
hepatic neoplasms. The authors also compared the lesions they observed in
feral English sole, with similar lesions found by others in rodents and
rainbow trout following controlled laboratory exposure to hepatocarcinogens.
Myers et al. (1987) caution that although their results are based on strong
circumstantial evidence, conclusive proof of the hepatocarcinogenesis model
for English sole must await carefully controlled field or laboratory
experiments.
25
-------
Myers et ai. (1987) identifrea the following major neoatic lesions cr.ac
are thougnt to be related to or associated with the histogenesis of liver
neoplasms in English sole:
t Nonsoecific necrotic lesions
Hepatocellular coagulation necrosis
Liquefactive necrosis
Hydropic degeneration
Pyknosis
Hyalinization
Cystic parenchyma! degeneration
• Specific degenerative conditions
Nuclear pleomorphism
Meqalocytic hepatosis
• Nonneoplastic proliferates conditions
Nonhyperplastic hepatocellular regeneration
• Foci of cellular alteration
Eosinophilic foci
Basophlllc foci
Clear cell or vacuolated cell foci
Hyperplastic regenerative foci
• Neoplasms
Liver cell adenomas
Hepatocellular carcinomas
Cnolangiomas
Cholangiocellular carcinomas
Mixed carcinomas.
26
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Although, nonspecific necrotic lesions are known co oe caused cy a
anety of agents, Myers en ai. (1387) excluaed tnose iesions cioseiy
associated with visible infectious agents. The necrotic iesions reoortea oy
Myers et al . (1987) generally exhibited focal or multifocal aistr-autions
ana rarely were found in a large prooortion of any tiver. These :esicns
frequently were accompanied oy nemorrnage, fibrinization , .-nononucl ear
infiltrates, fibroplasia, and increased density of meianomacrophage centers.
The two specific degenerative conditions affected only negatocytes,
were diffusely distributed in nonzonal patterns, and occurred in the absence
of cellular infiltrate. Nuclear pleomorphism was characterized by nuclei of
various size and chromatin distribution/content. Aside from chose aber-
rations, hepatocytes with nuclear pleomorphism exhibited a normal aopearance.
Megalocytic hepa'.osis was characterized primarily by enlargement of both
nuclear and cellular diameters and atypical distributions or densities of
chromatin within vesicular nuclei.
Nonhyperplastic hepatocellular regeneration was the only nonneoplastic
proliferative condition found that is thought to play a role in hepato-
carcinogenesis in English sole. Although a second nonneoplastic prolifer-
ative condition (e.g., cholangiofibrosis) was found, Myers et al. (1987)
concluded that it probably was not involved in the progression toward
neoplasia. Nonneoplastic hepatocellular regeneration ranged in appearance
from the undifferentiated morphology to the later stages of parenchyma!
replacement characterized by maturing, more differentiated hepatocytes.
Foci of cellular alteration were similar to the lesions in rats and
mice that are thought to be precursors of neoplasms. Each type exnibited a
distinct pattern of alteration, and was arranged in discrete micronodular
foci. The borders of the foci blended indistinctly into the surrounding
muralia and compression of adjacent parenchyma was minimal or absent.
Eosinophilic foci ranged from 0.1 to 0.9 mm in diameter, and were
characterized primarily by slight to dramatic cellular hypertrophy, increased
cytoplasmic eosinophilia with a granular texture, and varying degrees of
27
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nuclear oleomorpmsm. Basoomlic foci rangea from O.i co 0.3 .Tim ana .v
cnaractenzed primarily oy nyperoasopm i ic cytoplasm in normai-s i zee
'leoatocytes with oieomoramc nuclei. Clear call or vacuoiacea cell r'oci
*ere smaller than the former :*io lesions (i.a., <0.4 mm) ana were character-
izea by hepatocytes with either a vacuoiatea cytoplasm or a lacy, flocculent,
ooony stained cytoplasm. Alterations of nuclei were minimal. Hyperoiastic
regenerative foci also were relatively small (i.e., 0.05-0.3 mm). In
addition, these foci were nyperplastic and characterized oy regenerative
hepatocytes that exhibited reduced size and increased basophilia. Prevalence
of hyperpiastic regenerative foci were rare compared to prevalences of the
other three kinds of foci of cellular alteration.
Neoplasms included those of hepatocellular (i.e., liver cell adenoma,
hepatocellular carcinoma) and biliary (i.e., cholangioma, cholangiocellular
carcinoma) origin. One kind of neoplasm included both hepatocellular and
cholangiocellular elements (i.e., mixed carcinoma). Of these five kinds of
neoplasm, liver cell adenomas and cholangiomas are considered benign, whereas
the remaining three neoplasms are considered malignant.
As mentioned previously, the hepatocarcinogenesis model proposed by
Myers et al. (1987) for English sole was based primarily on statistical
associations among lesions and comparisons with similar lesions founds in
laboratory studies of rodents and rainbow trout. Myers et al. (1987) propose
the following sequence of events for the histogenesis of hepatocellular
neoplasms in English sole:
• Nonspecific necrotic lesions and specific degenerative
conditions appear as the initial, subchronic to chronic
hepatocellular lesions manifesting the cytotoxic effects of
exposure to hepatocarcinogens. These conditions provide the
proper stimulus for a compensatory, regenerative, pro-
liferative response.
0 In the above environment favoring proliferation, foci of
cellular alteration can develop. Because these foci are
28
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selectively resistant to the cytotoxic effects of carcinogens,
they nave a growth advantage over normal neoatocytes.
• Autonomous, neoolastic hepatocyies arise from some of the non-
autonomous foci of cellular alteration. This transformation
may occur oy a complex multistep process of mutation followed
by selection.
Myers et al. (1987) note that the pattern of histogenesis of biliary
neoplasms in English sole presently is unclear.
2.3 REVIEW OF HISTORICAL DATA
In this section, historical laboratory and field studies of fish liver
histopatnology are reviewed. Many of the concepts and patterns described in
these sections were used to develop the recommended protocols for field
studies of fish liver histopatnology in Section 3.0.
2.3.1 Laboratory Studies
A relatively large number of chemicals have been found to induce
hepatic lesions in various fishes following controlled laboratory exposure.
The major details of many of these studies are presented in Table A-l
(Appendix A). This table was constructed by synthesizing the information
presented in review articles by Matsusnima and Sugimura (1976), Myers and
Hendricks (1982), and Couch and Harshbarger (1985), and by reviewing the
recent literature (i.e., 1982-1986) as part of the present study. The
chemicals are grouped according to the general scheme of Meyers and Hendricks
(1982), to facilitate interpretation by environmental managers.
The 87 chemicals listed in Table A-l (Appendix A) are summarized in
Table 1. As noted previously, all of the chemicals have induced hepatic
lesions in fishes. These chemicals represent a wide variety of natural and
anthropogenic products, including pesticides, fossil-fuel related compounds,
chemotnerapeutic agents, mycotoxins, plant derivatives, nitrogenous com-
pounds, and inorganic compounds. Twenty-six (30 percent) of these chemical
29
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TABLE 1. CHEMICALS THAT HAVE INDUCED HEPATIC L£5IONS
IN FISHES FOLLOWING LABORATORY EXPOSURE*
Number of Soecies
Neoplasms0Other Lesions^
Orqanochlonne insecticides
Chlordane - 1
DOT 1 7
Oieldrin - >5
Endosulfan - I
Endnn - 6
Heptachlor - 3
Hexachlorocyclohexane ~ *•
(beta isomer, lindane byproduct)
Kepone " l
Lindane ~ 3
Methoxychlor " 3
Toxaphene " 1
Qrqanochlorine herbicides
Dichlobenil - 1
Dowicide G - L
2,4-0 - J
•
Tordpn 101 (picloram and 2,4-0 as 1
amine salts)
Tordon 22K {picloram, potassium salt) - I
Industrial organochlorine compounds
PCB-Aroclor 1248 - L
PCB-Aroclor 1254 - 4
PCB-Mlscellaneous - 3
Carbon tetrachloride 1 3
Monochlorobenzene ~ l
Organophosphate Insecticides
Abate (temphos) - J
Dlazinon (Spectracide) ~ |
Dimethoate (Cygon) ~ J
Oursban (chlorpyrifos) ~ |
Dylox (trichlorfon) ~ *
Malathion - 3
Methyl parathion ~ l
30
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TABLE 1. (Continued)
Number of Soecies Affect*
Neoplasms Other Lasioi
Carbamate insecticides
Aldicarb (Temik) - i
Carbaryl (Sevin) - 3
Propoxur (Baygon) - I
Miscellaneous herbicides
Acrolein - 1
Amitrole-T - I
Dinoseb - 1
Oiquat - 1
Hydrothol 191 - 1
Paraquat-CL - 1
Fossil-fuel related compounds
Benzo(a)pyrene (BaP) 1 1
Crude oil-whole - 3
Crude oil-water soluble fraction - 2
7-12 Dimethylbenz(a}anthracene (OHBA) 2 2
Oiled sediments - 1
Chemotherapeutic agents
Copper sulfate - 3
Oiethylstilbestrol (DES) 1 t
Sulfamethazine - 1
Thiabendazole - I
Hycotoxins
Aflatoxin B, (AFB.) 5 5
Aflatoxin G{ (AFG.1) 2 2
Aflatoxin M* (AFMf) 1 I
Aflatoxin Q* (AFQh 1 1
Aflatoxicol^AFL)1 1 1
Ochratoxin A + B 1 1
Sterigmatocystine 3 3
Versicolorin A 11
Plant derivatives
Cycad nut meal 3 3
Cycasin - 1
Cyclopropenoid fatty acids (CPFA) 1 1
Gossypol - !•
Methylazoxymethanol acetate (MAMA) 2 2
31
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TABLE 1. (Continued)
Number of Soeciss Af^'eccec
Neoplasms Other Lesions
Pyrrolizidine alkaloids - 1
Tannic acid I 1
Nitroso- compounds
N,N'-dinitrosopiperazine (DNP) I 1
N-nitrosodiethyl amine (DEN) 7 7
N-mtrosodimettiylamine (OMN) 3 3
N-methyl-N'-nitro-N-mtrosoguanidine (MNNG) 1 I
N-nitrosomorpholine (NM) 2 2
Miscellaneous nitrogenous compounds
2-Acetylaminofluorene (2-AAF) 3 3
o-Aminoazotoluene (o-AAT) 4. 4
Amrnon i a 2
Benzidine - 1
Carbazone 1 1
p-Oimethylaminoazobenzene (OAA8) 3 5
Thiourea 1 1
Urethane 1 1
Miscellaneous organic and organometal1ic compounds
Bis(tri-n-butyltin) oxide - 1
Oimethylsulfoxide (DMSO) - *
Methyl mercuric chloride - 2
4-Nitro-3-(trifluoromethylJphenol - 1
PhenoJ - I
Inorganic compounds
Cadmium chloride - 8
Cupric chloride - l
Cupric sulfate - 1
Disodium arsenate - 1
Lead nitrate - 1
Mercuric chloride - 3
Sodium arsenite - 1
32
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TABLE I. (Continued)
a The list of cnermcals is based on Taole A-l (flooenau A). Chemicals a
grouoed according to Che general scneme used by Meyers and Hendncfcs (1982).
£o facilitate interpretation by environmental managers.
b These numoers are basea on Table A-L (Aooendix A). Note thac they reorese...
the numoer of umaue soecies, not the numoer of laboratory studies conducted.
c Any kind of hepatic neoplasm.
d All kinds of hepatic lesions except neoplasms. In studies where neoplasr-
were induced, other kinds of lesions rarely were reported by the author'
For the purposes of this table, it was assumed that other kinds of lesioi
were present in all studies in which neoplasms were induced.
33
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nave ir.aucea neoatic neoplasms in one or more soecies of nsn. ~-,Q major
•grouos of cnenncals having the nighest percentages of heoatocarcinogens
include mycotoxins (100 oercenc), nitroso- compounds (100 percent i, miscel-
laneous nitrogenous comoounas (75 percent), and plant derivatives (60 oer-
cent). Sixty-one (70 percent) of the cnenncais isted in Table L have not
mauced hepatic neooiasms in fishes. i^ajor groups having no apparent
fiepatocarc i nogens include organocnlonne neroicides, organoonosonate
insecticides, carbamate insecticides, miscellaneous neroicides, miscellaneous
organic compounds, and inorganic compounds. Although these latter 61
chemicals have not induced neoplasms, they have induced other kinds of
hepatic lesions in fishes and may be capable of inducing lesions under
different sets of test conditions (e.g., different test species, different
exposure routes, higher chemical concentrations, longer test durations).
Most of the fish species in which hepatic lesions (i.e., neoplasms and
other kinds) have been induced by laboratory exposure to chemicals are
listed in Table 2. This list represents a broad taxonomic spectrum, and
includes 39 species from 20 families. The family Salmonidae is repesented
by the largest number of species (i.e., seven). The species used most
frequently in laboratory tests have been rainbow trout, guppy, cono salmon,
and zebra fish (cf. Table A-l, Appendix A).
Hepatic neoplasms have been induced in eight of the 39 species
(20.5 percent) listed in Table 2 (each denoted by an asterisk). These
species include all three poeciliids (i.e., guppy, two topminnows), two of
three cyprinodontids (i.e., sheepshead minnow, rival us), two of seven
salmonids (i.e., sockeye salmon, rainbow trout), and one of five cyprinids
(i.e., zebra fish).
Couch and Harshbarger (1985) summarized the various amounts of time
required for initial formation of hepatic neoplasms in a variety of fishes
exposed to a variety of carcinogenic chemicals. All of those studies are
included in Table A-l (Appendix A). The times to first neoplasm for all 105
fish/chemical combinations included in Couch and Harshbarger (1985) are pre-
sented in Figure 4. Some of these times probably are overestimates, because
fish were not examined until the experiments were terminated. In 59 cases
34
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TABLE 2. SPECIES IN WHICH HEDAT!C LESIONS HAVE BEEN
INDUCED FOLLOWING LABORATORY EXPOSURE TO CHEMICALS
"amily
Scienttfie Name
Common Name
Petromyzomdae
Salmomdae
Cyprinidae
Heteropneustidae
Ictaluridae
Clanidae
Batrachoididae
Oryziidae
Cyprlnodoncidae
Poeciliidae
Atherinidae
Gasterosteidae
Petromvzon marinus
Oncorhynchus kisutch
Qncorhynchus nerka
Oncorhynchus tshawytscha
Sal mo clarki
Salmo galrdneri
Sal mo trutta
Salvelinus namaycush
Bar bus conchomus
Carassius auratus
Cypnnus carpio
Danio (Brachydanio) rerio
Rhodeus amarus
Heteropneustes fossil is
Ictalurus punctatus
Clarius batrachus
Halobatrachus didactylus
Oryzias latipes
Cyprlndon variegatus
Fundulus heteroclitus
Rivulus tnarmoratus
Peocilia (Lebistes) reticulata
Poeciliopsis lucida
Poeci1iopsis monacha
Henidia beryMina
Gasterosteus aculetus
Lamorey
Coho salmon
Sockeye salmon*
Chinook salmon
Cutthroat trout
Rainbow trout*
Brown trout
Lake trout
Goldfish
Carp
Zebra fish*
Bitter-ling
Channel catfish
Walking catfish
Sapo
Medaka
Sheepsnead
minnow*
Muiratnchog
Rivulus*
Guppy*
Topimnnow*
Topminnow*
Inland
si 1verside
Threespine
stickleback
35
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TABLE 2. (Continued)
Family
Scientific Name
Common Name
Chanmdae
Centropomidae
Centrarchidae
Channa punctatus
Qghiocephalus punctatus
Oicentrarchus labrax
Lepomus cyanellus
Asian catfish
c
Robalo
Green sunfish
Sciaenidae
Mugilidae
Anabantidae
Pleuronectidae
Soleidae
Lepomus macrochirus
Lepomus imcrolophus
Leiostomus xanthurus
Mugil auratus
Trichogaster fasclatus
Parophrys vetulus
Platlchtnys flesus
Pseudopleuronectes americanus
Trlnectes maculatus
Blueqill
Redear sunfish
Spot
Lisa
c
English sole
Flounder
Winter flounder
Hogchoker
a This list is based on the studies reported in Table A-l (Appendix A).
b Species in which some kind of hepatic neoplasm has been induced in a
laboratory study are denoted by an asterisk*).
c Common name not found.
-------
20 -
18 -
16 -
U 14-
Q
H 12-
(0
O 10 —
NUMBER
01 OD
1 1
4-
2-
0
c
I:!
-•^
^^•i
-.'.1;';:;:':',1:"::;-::;;'
.iffli
hl||
p- '•
1
2
•••«•
»*••-• •
p-
•^•i
I
4
*'-•-:•-
,,,..
i
6
::
:
iry
mm
•I
;f
1
- pi
-i ^
1 1 i 1 1 1 1 1 1
3 10 12 14 16 18 20 22 24
TIME TO FIRST NEOPLASM (months)
Reference: Modified from Couch and Harchbarger 1985.
Rgure 4. Distribution of times to first neoplasm for a variety of fishes
exposed to a variety of chemicals in the laboratory.
37
-------
(56.2 oercent), neoanc neoolasms were induced within 6 mo of sxoosure to :--°
carcinogen, in 98 cases (33.3 oercent), hepatic neoolasms «ere mcuceo
«itnin I yr of exposure.
Direct extrapolation of laboratory results to field conditions are
difficult to maxe (e.g., Johnson and Bergman 1984). in many cases, the
species used for laboratory tests are selected because they are *nown to oe
very sensitive to hepacocarcinogens. In addition, the contaminant concen-
trations to which fishes are exposed in many laboratory studies are much
higher than most observed concentrations in the environment. Finally, the
duration of contaminant exposure in laboratory studies often exceeds that
which might be expected under natural conditions. Despite these limitations,
laboratory results may be useful as estimates of the worst-case conditions
that may be encountered in the environment.
With the above caveats in mind, several patterns identified in labora-
tory studies have implications for interpreting the results of field
studies. First, controlled laboratory studies demonstrate unequivocally
that many contaminants found in the environment can induce the same kinds of
hepatic lesions as those found in feral fishes from polluted haoitats. This
demonstration is essential for supporting the hypothesis that lesions
observed in feral fishes are the result of chemical contamination. It does
not, however, discredit the alternative hypotheses that lesions are induced
by other agents (e.g., nutritional imbalances, viruses).
A second laboratory result with field implications is the fact that
similar kinds of hepatic lesions in fishes have been induced by a wide
variety of chemical contaminants. Although many of these lesions are thought
to be indicative of toxic effects, their general nonspeci ficity makes
diagnosis of a single causative agent difficult, if -iot impossible (e.g.,
Meyers and Hendricks 1982). This nonspeci ficity *-s extended to field
studies by the observation of Harshbarger (1977) that nearly every kind of
neoplasm (i.e., hepatic and others) found in fishes currently was known
prior to 1940. This lack of differences has been maintained despite the
large increase in quantity and variety of toxic chemicals to which fishes
have been exposed since 1940. It therefore is highly unlikely that specific
38
-------
cypes of hepatic neoplasms in feral fishes can oe used to identify aer'n-
itively their causative agents.
A third laboratory result *ith field implications is the fact tnat
heoatic neoolasms have been inauced in certain fisnes in time intervals
snorter than 6 mo. Thus, even if a particular fish visits a contaminated
sue once and for a relatively short period of time, there is the possi-
bility that hepatic lesions, including neoplasms, could be induced if the
fish is suitably sensitive and if the contaminant concentrations in the
environment are suitably high.
2.3.2 Field Studies
Most field studies of hepatic lesions in fishes from contaminated
environments have been conducted within the last 10 yr. However, this r'oes
not necessarily mean that these lesions were not present prior to the mid-
1970s. The occurrence of hepatic lesions in many fishes initially was
discovered inadvertently as specimens were being evaluated for other purposes
(e.g., Falkmer et al. 1976; Pierce et al. 1978; Smith et al. 1979). in
these cases, the presence of grossly visible nodules led to detailed
microscopic evaluations of the affected livers. Once a putative relationship
between environmental contamination* and hepatic lesions in fishes had been
established, many subsequent studies were designed specifically to evaluate
microscopic hepatic lesions in fishes from unsurveyed, contaminated areas.
Thus, the scarcity of data on hepatic lesions prior to the mid-1970s
probably was due largely to the lack of studies designed specifically to
evaluate these abnormalities.
This section reviews most of the field studies that have documented
elevated prevalences of hepatic neoplasms and other liver abnormalities in
feral fishes collected from chemically contaminated environments (Table 3).
These studies include nine geographic locations (seven in the U.S. and two
in Europe), freshwater (five) and saltwater (four) habitats, and 1Z species.
Most of the historical field studies (7 of 17, or 41 percent) have been
conducted in Puget Sound, Washington (Table 3). The highest prevalence of
39
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TABLE 3. SUMMARY OF FIELD STUDIES IN WHICH ELEVATED PREVALENCES
OF HEPATIC NEOPLASMS HAVE BEEN FOUND IN FERAL FISHES
Location
Puget Sound, WA
Fox River, IL
Black River, OH
Torch Lake, MI
Hudson River, NY
Boston Harbor, MA
Deep Creek
Lake, MD
Elbe Estuary,
Germany
Gull mar Fjord,
Sweden
Study3
Pierce et al. 1978
McCain et al. 1982
Mai ins et al. 1984
Mai ins et al. 1985a
Malins et al. 1985b
Tetra Tech 1985
Krahn et al. 1986
Brown et al . 1973
Brown et al. 1977
Baumann et al. L98Z
Baumann and
Harshbarger 1985
Black et al. 1982
Smith et al. 1979
Murchelano and
Wo Ike 1985
Oawe'et al . 1964
Kranz and Peters
1985
Falkmer et al . 1976
Species
Engl ish sole
English sole
Starry flounder
English sole
Rock sole
Pacific staghorn
sculpin
English sole
English sole
English sole
English sole
Brown bullhead
Brown bullhead
Brown bullhead
Brown bullhead
Sauger
Walleye
Atlantic tomcod
Winter flounder
White sucker
Ruffe
Atlantic hagfish
Samel e
Size
62
673
350
2,190
1,379
422
106
115
1,014
249
283
284
?
125
23
22
264
200
12
551
23,600
Percent
Neoolasm-
32.3
0-12.9
0-3.0
0-16.2
0-4.8
0-1.7
0-7.5
0-25.7
0-8.3
0-20.7
12.4
13.8
1.2-33.0
38.4
100.0
>27.3
25.0
8.0
25.0
8.0
0.6-5.8
a The details of all of these studies are presented in the text.
b Scientific names of species are presented in Table 4.
c Prevalence or range of prevalences found for any kind of hepatic neoplasm in the
species of interest.
40
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neoplasms found in any field stuay was 100 percent (i.e., saugers .n Tore-
Lake, Michigan). However, in ail otner cases, maximum neoplasm orsvaience
less than 40 percent.
The following reviews describe the design of each field scuay, :r,e
observed prevalences of hepatic neoplasms and putative preneoplastic lesions,
the microscopic characteristics of the observed liver abnormalities, any
relationships between lesions and other variables (e.g., age, sex, chemical
concentrations), and the major conclusions reached by the authors. Much of
the information presented in this section was used to develop the recom-
mendations made later in Section 3.0.
Puget Sound, Washington —
Study 1 — Pierce et al. (1978) collected 62 English sole from the
Ouwamish River in Puget Sound, Washington from July 1975 to January 1976.
For comparative purposes, 18 English sole were collected from Point Pully, a
Puget Sound reference area. Microscopic examination revealed that 20 fish
(32.3 percent) from the Ouwamish River had hepatic neoplasms. None of the
fish from Point Pully had neoplasms.
Most neoplasms were minimum-deviation basophilic nodules or eosino-
philic nodules. The basophilic nodules frequently compressed surrounding
tissue. In some cases, they appeared to have invaded surrounding tissue.
The eosinophilic nodules, by contrast, frequently exhibited numerous areas of
invasiveness.
A variety of nonneoplastic abnormalities were found in English sole
from the Duwamish River. These included increased size and number of melanin
macrophage centers, centrolobular fatty degeneration and necrosis, cord
disarray, increased hepatocyte basophilia, and hepatocellular hypertrophy
associated with bizarre nuclei and multiple nucleoli.
The authors conclude that chemical contaminants are the suspected cause
of the observed liver abnormalities in English sole, but that other agents
such as pathogens and nutritional deficiencies cannot be ruled out. They
41
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note tnat sediments of tie Quwannsh River are contaminated «icn DOT, 3C3s.
coooer, and lead and that trie liver aamage ooservea in Englisn sole resemoies
chat induced in otner fisnes oy PC3s and other cnlorinated hyarocaroons.
Study 2--McCain et al. (1982) collected 673 English sole ana 350 starry
fiounoer (Platichthys stellatus) from four areas of Puget Souna between
Octooer 1978 and October 1980. Three of the areas (Duwarmsh River, Snohomish
River, Lake Washington Ship Canal) are chemically contaminated to various
degrees. The fourth area (McAllister Creek) is an uncontaminated reference
area. All four areas are influenced by fresh water to some extent.
In English sole, hepatic neoplasms (i.e., minimum deviation nodules,
liver cell adenomas, hepatocellular carcinomas, cholangiocellular carcinomas,
and mixed carcinomas) ranged from 0 percent in McAllister Creek and the
Snor-omish River to 8.2 percent and 12.9 percent in the Lake Washington Ship
Canal and the Duwamish River, respectively. Prevalence of putative pre-
neoplastic lesions [i.e., hepatocellular regeneration, hepatocellular
eosinophilic hypertrophy (subsequently referred to as eosinophilic foci)]
ranged from 0 percent in McAllister Creek and the Snohomish River to 9.4
percent and 10.2 percent in the Duwamish River and the Lake Washington Ship
Canal, respectively. A variety of nonneoplastic liver abnormalities were
also found in higher prevalences in the Lake Washington Ship Canal and the
Duwamish River compared with the Snohomish River and McAllister Creek.
These included megalocyt'ic hepatosis, cholangiofibrosis, necrosis, and hemo-
siderosis.
In starry flounder, adequate sample sizes were available only for the
Ouwamish River and McAllister Creek. Prevalence of hepatic neoplasms (i.e.,
minimum deviation nodules, liver cell adenomas, and cholangiocel lular
carcinomas) was 3.0 percent in the Ouwamish River, compared to 0 percent in
McAllister Creek. Prevalence of putative preneoplastic lesions (i.e.,
hepatocellular eosinophilic hypertrophy) was 1.4 percent in the Ouwamish
River, compared to 0 percent in McAllister Creek. Nonneoplastic liver
abnormalities exhibiting elevated prevalences in the Ouwamish River compared
to McAllister Creek included megalocytfc hepatosis, fatty change, and
necrosis.
42
-------
McCain et al. (1982) found that neither sex of English sole from cr.e
Ouwarmsh River was affected disproportionately by any of the heoatic lesions
evaluated. The authors did find, however, that prevalence of total hepatic
lesions was positively related to fish length, ana therefore indirectly to
fish age.
Study 3—Mai ins et al. (1984) collected 2,190 English sole, 1,379 rock
sole (Lepidopsetta bilineata). and 422 Pacific staghorn sculpin (Lepto-
cottus armatus) from 19 urban and nonurban areas throughout Puget Sound.
Hepatic neoplasms were found in all three species and included minimum-
deviation basophilic nodules, liver cell adenomas, hepatocellular carcinomas,
cholangiocellular carcinomas, and cholangiomas. Prevalences of neoplasms in
English sole, rock sole, and Pacific staghorn sculpin exhibited the following
ranges: 0-16.2 percent, 0-4.8 percent, and 0-1.7 percent, respectively.
Mai ins et al. (1984) also found a variety of putative preneoplastic
lesions in fish livers, including nodular eosinophilic hypertrophy, hyper-
basophilic foci, clear cell foci, and hyperplastic regenerative islands.
Prevalences of preneoplastic lesions in English sole, rock sole, and Pacific
staghorn sculpin exhibited the following ranges: 0-24.3 percent, 0-9.5
percent, and 0-3.4 percent, respectively.
Mai ins et al. (1984) also found a number of nonneoplastic abnormalities
in fish livers. The most prevalent nonneoplastic abnormalities were megalo-
cytic hepatosis, cholangiofibrosis, steatosis, and hemosiderosis.
In general, highest prevalences of most liver abnormalities were found
in major urbanized areas for all three fishes. Lowest prevalences generally
were found in nonurban areas. Using multivariate and bivariate statistical
analyses, Mai ins et al. (1984) found positive associations between sediment
concentrations of aromatic hydrocarbons and certain liver lesions in English
sole and Pacific staghorn sculpin, and between sediment concentrations of
metals and certain liver lesions in English sole.
43
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Stuay d—Malms et ai. (I985a) collected 66 Enghsn soie from a concern-
mated area of Puget Sound near Mukilceo, Washington during June ana Juiv of
.983. For comparative purposes, 40 Enghsn sole were samoled from a P'jce:
Souna reference area near Presiaent Pome. Heoatic neooiasras C-.e., ,-nmimum-
aeviation noaules, liver cell adenomas, hepatoceilular carcinomas, ana
cnolangioce) iuiar carcinomas) were identified rmcroscooical ly m f--/e f'sn
(7.5 percent) from Mukiiteo and in no fish from President Point. Putative
preneoplastic lesions (i.e., eosinopnilic foci and hyperoasoom1ic foci)
were found in 11 fish (16.7 percent) from Mukilteo and m no fish from
President Point.
Most nonneop 1 astic abnormalities found in fisn livers were more
prevalent at Mukilteo than at President Point. These included degeneration,
necrosis, and regeneration. By contrast, steatosis and hemosiderosis were
more prevalent at President Point.
Chemical analyses showed that sediment concentrations of aromatic
hydrocarbons, chlorinated compounds, and carbazole were substantially higher
at Mukilteo than at President Point. By contrast, sediment concentrations
of toxic metals (except lead) were similar at both sites. In fish iwers,
PCB concentrations at Mulkilteo were 17 times as nigh as those at President
Point. Concentrations of hexachlorobenzene were also elevated m 1wers
from Mukilteo. By contrast, aromatic hydrocarbons and carbazole generally
were not detected in livers from either site. In fish bile, concentrations
of benzo(a)pyrene-like and naphthalene-like metabolites in fish from Mukilteo
were 6 times and 3 times, respectively, as high as those in fish from
President Point. In fish stomach contents, concentrations of aromatic
hydrocarbons and PCBs in fish from Mukilteo were 22 times and 3 times,
respectively, as high as those in fish from President Point.
Malins et al. (1985a) concluded that their findings support the
statistical relationships identified by Malins et al. (1984) between
sediment concentrations of aromatic hydrocarbons and hepatic lesions in
English sole. The authors note that they had documented for the first time
the bioavailability of organic chemicals through the diet of English sole.
They also note that the absence of detectable concentrations of aromatic
44
-------
Hydrocarbons in the livers ana the apoarent presence of metabolites in tf.e
bile supports the Hypothesis that biotransformation of aromatic fiyorocaroons
by English sole is both raoia and extensive.
Study 5— Mah'ns et a). (1985b) captured 75 English sole from Eagle
Harbor, v»asnington between November L983 and April 1984. Eagle Haroor is a
small embayment in Puget Sound that is contaminated by creosote. For
comparative purposes, the authors used the same 40 English sole from
President Point as described in Malins et al. (1985a). Hepatic neoplasms
(i.e., liver cell adenomas, hepatocellular carcinomas, cholangiocellular
carcinomas, and mixed carcinomas) were identified microscopically in 20 fish
(26.7 percent) from Eagle Harbor and in no fish from President Point.
Putative preneoplasms (i.e., eosinophilic foci, basophilic foci, and clear
cell foci) were found in 33 fish (44.0 percent) from Eagle Harbor and in no
fish from President Point.
Most nonneoplastic abnormalities found in fish livers were substantially
more prevalent at Eagle Harbor than at President Point. These abnormalities
included degeneration, necrosis, regeneration, steatosis, and nemosiderosis.
Chemical analyses showed that sediment concentrations of aromatic
hydrocarbons and the heterocycles carbazole and dibenzofuran were elevated
substantially compared to President Point. By contrast, sediment concen-
trations of chlorinated hydrocarbons and toxic metals were not elevated
substantially. In fish muscle and liver tissue, concentrations of aromatic
hydrocarbons, carbazole, and chlorinated hydrocarbons generally were
relatively low. Naphthalene and alkylated naphthalenes constituted the
highest proportion of aromatic hydrocarbons found in livers. Although the
concentration of PCBs was somewhat elevated (i.e., 1.1 ppm) in livers from
Eagle Harbor, it did not differ substantially from that at President Point
(i.e., 1.0 ppm). In fish bile, metabolites of aromatic hydrocarbons were
substantially elevated in Eagle Harbor compared to President Point. In fish
stomach contents, concentrations of aromatic hydrocarbons were substantially
higher at Eagle Harbor than at President Point. By contrast, concentrations
of chlorinated hydrocarbons and carbazole were similar in stomach contents
from the two study sites.
45
-------
Malms ec al. (1985o) concluded chat certain creosote comoorencs,
acting individually or synergistically, were causally linked to :he mgn
orevalence of liver aonormanties oosen/ed in tngiisn sole from Eagle
Harbor. The authors suggest that the diet is an important route of con-
taminant uotake. The authors also note that the presence of metaoolues in
01 le demonstrates that Englisn sole accumulated and actively metabolized
creosote components.
Study 6—Tetra Tech (1985) collected 896 English sole (age >3 yr) from
chemically contaminated areas of Commencement Bay during June 1984. For
comparative purposes, 118 English sole (age XJ yr) were collected from Carr
Inlet, a nonurban reference embayment. Prevalences of hepatic neoplasms
(i.e., liver cell adenomas, hepatocellular carcinomas, cholangiocellular
carcinomas, and cholangiomas) ranged from 0 to 8.3 percent in Commence-
ment Bay, and were absent from Carr Inlet. Prevalences of putative preneo-
plastic lesions (i.e., eosinophilic foci, basophilic foci, and clear cell
foci) ranged from 3.4 to 25.7 percent in Commencement Bay and was 5.1 percent
in Carr Inlet. Prevalences of megalocytic hepatosis and nuclear pleomorphism
were substantially higher in Commencement Bay than in Carr Inlet.
Tetra Tech (1985) found that prevalences of the four major kinds of
lesions evaluated did not differ (P>0.05) between the sexes of English
sole. However, prevalences of neoplasms and putative preneoplasms were both
positively correlated (P<0.05) with fish age. Prevalences of megalocytic
hepatosis and nuclear pleomorphism were not significantly correlated (PXJ.05)
with fish age.
Study 7—Krahn et al. (1986) collected 249 English sole from 11 areas
throughout Puget Sound from November 1983 to January 1984. Stations were
selected to represent a gradient of chemical contamination. Prevalence of
hepatic neoplasms ranged from 0 to 20.7 percent. Prevalence of putative
preneoplastic lesions ranged from 0 to 32.8 percent. Highest prevalences of
both kinds of lesion were found in the Duwamish River. Prevalence of
megalocytic hepatosis ranged from 0 to 86 percent, with the highest value
-------
found in Eagle Harbor. Prevalence of steacosis ranged from 0 to 41.4
oercent, with the highest prevalence found in the Duwamisn River.
In addition to fish liver lesions, Krahn et al. (1986) measured the
bile concentrations of multi-ring aromatic compounds chat fluoresce at the
benzo(a)pyrene wavelength oair. English sole from Eagle Haroor nad the
highest concentrations of biliary metabolites. Significant (P<0.05) positive
correlations were found between the relative mean concentration of biliary
metabolites at each study site and the prevalences of neoplasms, putative
preneoplasms, megalocytic hepatosis, and total lesions (i.e., one or more of
the four lesions considered). Correlations between lesion prevalences and
sediment concentrations of selected aromatic hydrocarbons were not signifi-
cant (P>0.05). The correlation between sediment concentrations of selected
aromatic hydrocarbons and relative mean concentrations of biliary metabolites
also was not significant (PXJ.05).
Krahn et al. (1986) concluded that the significant correlations between
biliary metabolites and hepatic lesions in English sole provide added
evidence of the putative relationship between aromatic compounds and liver
abnormalities.
Fox River, Illinois—
Study I—Brown et al. (1973) collected 2,121 fishes from the highly
polluted Fox River watershed near Chicago, Illinois between 1967 and 1972.
Of the over 17 species sampled, only the brown bullhead (Ictalurus nebu-
losus) exhibited unusually high prevalences of hepatic neoplasms. Of the
283 bullheads examined, 35 (12.4 percent) had hepatic neoplasms. Brown et
al. (1973) also sampled 4,639 fishes from reference sites in Canada and
found that of the 101 brown bullheads sampled in those uncontaminated areas,
2 (2.0 percent) had hepatic neoplasms.
Brown et al. (1973) conclude that increased levels of such pollutants
as mercury, lead, arsenic, toluene, crude oil, gasoline, benzanthracene,
chlorinated hydrocarbons, phosphates, sulfates, and coliform bacteria in the
Fox River system may have been responsible for the observed neoplasms.
47
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Factors such as dissolved oxygen, temperature, and nutritional /anacion
«ere considered similar in both the Fox River ana the reference area.
Study 2—3rown et al. (1977) sampled 284 additional brown bullheaas
from the Fox River watersned from 1972 to 1976 ana found that 39 [13.3 per-
cent) nad hepatic neoplasms. Of the 87 brown bullheaas samolea \n tne
Canadian reference areas from 1972 to 1976, only 1 (1.2 percent) had heoatic
neoplasms. These results were very similar to those found by Brown et
al. (1973) from L967 to 1972, suggesting that the observed patterns were
temporally stable.
Microscopic examination of the livers evaluated by Brown et al. (1977)
revealed that neoplastic cells generally were pleomorphic and frequently
multinucleate. The cytoplasm of neoplastic cells was sometimes vacuolated,
and sometimes granular and acidophilic. Some neoplasms tended to invade
surrounding tissue, but widespread metastasis rarely was observed.
Black River, Ohio—
Study 1—Baumann et al. (1982) collected brown bullheads from the
industrialized Black River near Lorain, Ohio from April to June of 1980.
For comparative purposes, 329 brown bullheads were collected from Buckeye
Lake, Ohio, a less contaminated water body, from July to August of 1980.
Hepatic neoplasms in fish from the Black River were grossly visible as small
white nodules on the surface of the liver. These neoplasms were thought to
be cholangiomas.
Microscopic examination revealed a large number of mitotic figures
throughout the neoplasms, and invasion of surrounding tissue. The central
regions of the neoplasms contained acidophilic cells and large areas of
necrosis.
The prevalence of grossly visible hepatic neoplasms in Black River fish
>3 yr old (33.0 percent) was significantly higher (P<0.01) than the preva-
lence in fish <3 yr old (1.2 percent). None of the bullheads from Buckeye
Lake had grossly visible hepatic neoplasms.
48
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Saumann et al. (1982) noted that the Black River is contaminated ay a
range of organic contaminants, but that the oasic difference oetween
that waterway and Buckeye Lake is the presence of industrial effluents
containing PAH. Chemical analyses conducted in conjunction with tne
pathology study documented high levels of PAH in Black River oottom sedi-
ments and elevated levels (relative to Buckeye Lake) in tissue of Black
River bullheads. The authors concluded that PAH were the most likely cause
of the hepatic neoplasms observed in the Black River bullheads.
Study 2—Baumann and Harshbarger (1985) collected 125 brown bullheads
from the Black River in 198Z. Microscopic examination revealed that 48 fish
(38.4 percent) had hepatic neoplasms. Cholangiocellular carcinomas (28.8
percent) were more common than hepatocellular carcinomas (19.2 percent).
Neoplasms were equally common in 3- and 4-yr-old fish. Chemical analyses
showed that sediment concentrations of PAH in the Black River were 1,000
times greater than those in Buckeye Lake. In addition, tissue concentrations
in Black River bullhead were elevated relative to those of Buckeye Lake
fish. Dioxins, dibenzofurans, DOT, PCBs, arsenic, and cadmium were not
unusually elevated in Black River bullheads relative to Buckeye Lake fish.
The authors concluded that the elevated prevalence of hepatic neoplasms in
Black River bullheads was chemically induced and the result of exposure to
PAH.
Torch Lake, Michigan—
Black et al. (1982) collected 23 saugers (Stizostedion canadense) and
22 walleye (Stizostedion vitreum) from Torch Lake, Michigan in September 1979
and July 1980. Hepatic neoplasms (diagnosed microscopically as hepato-
cel lular carcinomas) were grossly visible as nodules in all (100 percent) of
the saugers and in at least six (27.3 percenc, of the walleyes. Visible
nodules ranged from 2 to 20 mm in diameter. Microscopically, neoplastic
cells exhibited increased basophilia and moderate anaplasia. Cells had
large nuclei and nucleoli, but only mild pleomorphism. Fibrosis was not
common. Few mitoses were evident and neoplasm growth appeared to be slow.
The neoplasms compressed and sometimes evoked atrophy in surrounding hepato-
49
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eyres. Parasitic trsmatoce cysts and melanin macropnage centers *ere oreser.c
in most I r/er sections.
Black et al. (1982) noted that the saugers they evaluated were very oia
(i.e., probably >IZ yr old). They also notea that gonads frequently aooearea
atroonic in the saugers and less frequently so in the walleyes, suggesting
tnat the copulations of these species in Torch Lake may tnereoy oe negatively
affected (i.e., in terms of reproductive capacity).
Black et al. (1982) suggest that the copper mining wastes discnarged to
Torch Lake may be directly or indirectly responsible for the high preva-
lences of hepatic neoplasms in resident saugers and walleyes. Since 1900,
over 20 percent of the lake has been filled with copper tailings. In
addition, mine water pumpage and untreated municipal sewage were also
discharged to the lake for many years. The authors suggest that some
chemical component(s) of the mine wastes (e.g., copper, selenium, arsenic)
may be carcinogenic. Alternatively, the mine wastes may be interacting with
the sewage wastes to produce carcinogens (e.g., metal-catalyzed nitro-
samines). The authors suggest there is no relationship between the para-
sitic trematodes and hepatic neoplasms.
Hudson River, New York-
Smith et al. (1979) evaluated hepatic neoplasms in 254 adult Atlantic
tomcod (Microgadus tomcod) collected from the Hudson River from December
1977 to February 1978. The presence of these neoplasms was noted inci-
dentally as fish were being processed in the laboratory for growth, mor-
tality, and reproduction studies. Fish were divided into three .categories
according to the gross characteristics of the liver abnormalities. Only
four livers were examined microscopically: two from one group and one from
each of the other two groups. Based solely on gross characteristics, Smith
et al. (1979) estimated that approximately 25 percent of the 264 livers
contained hepatic neoplasms. However, that figure may underestimate the
true prevalence, as. microscopic examination may have revealed neoplasms in
livers that lacked grossly visible neoplasms. Based on gross examinations,
none of the neoplasms exhibited metastasis.
SO
-------
Microscooic examination of the liver of the one fish from cne grouo
-.aving the fewest numoer of gross abnormal i ties snowea excessi/e -/acuolation
suggestive of fat deposition. Also oosen/ed were focal areas of suocaosuiar
congestion and mild hemorrnage.
Microscopic examination of the single liver from the group cnarac-
terized by small (1-3 mm) light grey pustule-like lesions revealed several
small neoplasms. The neoplasms were not encapsulated and appeared to be
invading surrounding normal tissue. Neoplastic cells generally were poorly
differentiated and enlarged. Nuclei of neoplastic cells were pleomorphic,
swollen, and vesicular. Nucleoli were also swollen and mitoses were
uncommon. The cytoplasm of all neoplastic cells exhibited increased
basophilia. Necrotic cells were scattered diffusely throughout the neo-
plasms.
Microscopic examination of two livers from the groups characterized by
dark red or purple lesions of various sizes revealed numerous small neo-
plasms that were histologically similar to those described for the liver
with light gray lesions. However, in one liver from the third group, a
single neoplasm involved approximately 60 percent of the liver. Focal areas
of sinusoidal congestion and subcapsular hemorrhage were also found in one
liver from the third group. In the more advanced neoplasms from the third
group, cells were greatly enlarged (i.e., 5-6 times normal), highly pleo-
morphic, and often binucleate or multinucleate. The nuclearrcytoplasmic
ratio appeared to be reduced, nucleoli were often swollen, and the cytoplasm
was frequently vacuolated.
Smith et al. (1979) noted that livers of some of the Atlantic tomcod
contained relatively high levels (i.e., 10.9-98.2 ppm) of PCBs (Aroclor 1016
and 1254), and suggested that those chemicals may have caused the observed
neoplasms.
51
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3oston Harbor, Massachusetts—
Murcnelano ana Wolke (L985) collected 200 ^nter flounaer
pleuronectes amencanus) from Boston Harbor, Massacnusetts in April and June
1984. Microscopic examination revealed that 15 fish (3.0 percent) naa
neoatic neoolasms and 20 fish (10.0 oercent) had euner neoplasms or outatv/e
preneoolastic lesions. Neoplasms included hepatocellular (2.5 percent) ana
cholangiocellular (7.0 percent) carcinomas, cholangiomas (0.5 percent), and
adenomas (0.5 percent). Preneopiastic lesions included basophilic (3.5 per-
cent) and vacuolar (4.5 percent) foci. The authors note that prevalences of
preneopiastic lesions may have been higher had more liver sections been
examined for each fish.
The most common nonneoplastic abnormalities observed in the Boston
Harbor fishes were increased numbers of melanin macrophage centers (68 per-
cent) and hepatocyte vacuolation (68 percent). Other nonneoplastic lesions
included pericholangitis, vasculitis, focal necrosis, biliary hyperplasia,
and cholangioflbrosls.
Murchelano and Wolke (1985) noted that only fish collected off Oeer
Island had grossly visible hepatic lesions. Deer Island is the discharge
point for much of Boston's primary-treated municipal sewage. The authors
also noted that the high incidence of vacuolated cells and increased numbers
of melanin macrophage centers were consistent with the action of a hepa-
totoxin. However, they do not speculate as to what kind of hepatotoxin may
have been responsible for the observed liver abnormalities.
Deep Creek Lake, Maryland—
Study l--0awe et al. (1964) performed gross neocropsies on six fishes
from Deep Creek Lake, Maryland during September 1963. Of 12 white suckers
(Catostomus commersoni) evaluated, 3 (25 percent) had intrahepatic bile-duct
neoplasms, none of which was detectable by external inspection or palpation.
All of the fish with tumors were relatively old (i.e., 5-15 yr).
52
-------
Microscopic evaluation of the three livers with neoplasms revealea cnat
none of the neoplasms had metastasized. !n all cases, parasitic orotozoans
(i.e., probably a haolosoonciium soecies) were present within tne neoolastic
eDitrtelium. However, similar protozoans *ere also found in the livers of
fish without tumors.
Oawe et al. (1964) caution that the low sample size and relatively old
age of many of the fish may bias the apparently high prevalence of neo-
plasms. The authors suggest that the neoplasms may have been caused by the
parasitic protozoans, carcinogenic hydrocarbons (i.e., from boating ac-
tivity), pesticides used to eradicate mosquitos, or rotenone used to sample
fish in the lake at regular intervals.
Study 2—Oawe et al. (1976) collected 74 white suckers from Deep Creek
Lake between 1964 and 1974. Sixty-six of those fish were similar in length
to those sampled by Oawe et al. (1964) in 1963. None of the 74 fish
collected after 1963 had liver neoplasms. Oawe et al. (1976) also collected
3,134 white suckers from a wide variety of aquatic habitats throughout the
U.S. and Canada and found only one fish with a liver neoplasm. That
individual was taken from Pleasant Valley Lake, Maryland. Thus, the high
prevalence of hepatic neoplasms found in 1963 may have represented an
isolated case, rather than a general trend.
Elbe Estuary, Germany—
Kranz and Peters (1985) collected 551 ruffe (Gvmnocephalus cernua) from
the Elbe Estuary near Hamburg, Germany from 1980 to 1982. Nodules suspected
of being neoplastic were grossly visible in 8 percent of the livers. Micro-
scopically, the initial stages of the nodules were seen as small groups of
greatly enlarged basophilic cells. In the larger nodules, signs of necrosis
were evident. The trabecular arrangement of the cells disintegrated and
cells became increasingly pleomorphic. Vascular congestion sometimes
occurred. Nuclear pleomorphism was slight. Melanin macrophage centers were
large and numerous in the surrounding parenchyma.
S3
-------
D3rtiai discoloration* or r,-,e Mver ^ere grossly e>/iaent •.n 39
of tne fish. Microscopic examination revealeo cnat tnese aiscoioracions
«ere onmarily areas of fatty vacuoiation mat resulted from excess storage
of lioias. Glycogen also appeared co oe accumulated in some of tnese areas.
7ne authors noted that the ooserved excessive accumulation of iipia was
orooaoly pathological and similar to the kind of liver iipoia degeneration
that results from improper nutrition ana reaction to certain ooilutants.
Liver nodules were absent in small ruffe (i.e., <17 cm in lengtn).
However, prevalence of nodules showed a positive association with size for
large ruffe. Because size often correlates with age, nodule prevalence may
have been a function of fish age. Condition (i.e., weight x 100/length ) was
significantly lower for fish with nodules than for fish without gross liver
abnormalities.
Kranz and Peters (1985) noted that the Elbe Estuary is affected by a
variety of pollutants. They also noted that similar abnormalities were
found in fishes following exposure to pesticides, PCBs, crude oil, and heavy
metals. Finally, they suggested that fat-soluble hydrocarbons were possible
causes of the observed abnormalities in the Elbe Estuary.
Gull mar Fjord, Sweden—
Study I—Falkmer et al. {1976) sampled Z3.600 hagfish (Myxme gluti-
nosa) from Gullmar Fjord, Sweden from 1972 to 1975. For comparative
purposes, 1,183 hagfish were collected from the nearby open sea during 1972
and 1974. Many of the observed hepatic neoplasms were grossly visible as
small white spots on the surface or within the parenchyma of the liver.
Although liver color varied considerably among individuals, there was no
association with neoplasms. No gross evidence of metastasis was observed.
Microscopic evaluation revealed two major kinds of neoplasms: hepato-
cellular and cholangiocellular. Both kinds of neoplasm frequently occurred
in the same liver, but hepatocellular neoplasms generally were more common
(i.e., 2-3 times) than cholangiocellular neoplasms. Hepatocellular neo-
plasms exhibited a range of characteristics. Some of these neoplasms were
54
-------
nodular hyperplasias of questionaole neoolastic nature. They aid nor
comoress the adjacent liver parenchyma ana formea bounaanes that oftan were
difficult to discern. A second croup consisted of slightly larger nodules
that were comoosed of highly differentiated heoatocytes tnat comoressed or
evoked atroohy in surrounding tissue. No cellular or nuclear Dleomoronisms
were exmbued in these neoplasms and the number of muotic figures *as low
or absent. Falkmer et al. (1976} classified this second group as benign
liver cell adenomas. A third group consisted of the largest hepatocellular
neoplasms and was classified as carcinomas. Areas of necrosis and hemorrhage
occurred frequently and invasive growth was evident. However, both the
degree of cellular atypia and the number of mitotic figures were relatively
low.
As with hepatocellular neoplasms, the characteristics of cholangio-
cellular neoplasms covered a wide range. The larger neoplasms were definite
carcinomas, being either highly or poorly differentiated.
In 1972, prevalence of neoplasms in the Gullmar Fjord was 5.8 percent
compared to 2.8 percent in the open sea. In 1974, prevalence of neoplasms
in the fjord was 0.6 percent, compared to 0.9 percent in the ooen sea.
Between 1972 and 1975, prevalence of neoplasms declined from 5.8 percent
(1972) to 2.9 percent (1973) to 0.6 percent (1974 and 1975).
Falkmer et al. (1976) compared the body weight of hagfish with neoplasm
prevalence and found a positive relationship. Because body weight generally
correlated with age, these results suggest that neoplasm prevalence exhibits
a positive association with age. Falkmer et al. (1976) also noted that
neoplasms were absent in small hagfish (i.e., <25 g). Falkmer et al. (1976)
concluded that because hagfish from the open sea generally were smaller than
those from Gullmar Fjord, observed differences in neoplasm prevalence between
the two areas may have been biased.
Study 2—Falkmer et al. (1977) collected 3,700 hagfish from Gullmar
Fjord in 1976 and found a neoplasm prevalence (i.e., 0.6 percent) identical
to that found in 1974 and 1975. Preliminary chemical analyses showed that
composites of livers (with and without neoplasms) from hagfish captured in
55
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the fjora contamea PC3s an a concentration of 5 pom (-vet *eignt),
the concentration in comoosited rivers from the ooen sea was aoproximateiy
0.2 oom. Given tnat use of PCSs was pronibited in Sweden in 1971-72 and tnat
neoplasm prevalence in hagnsn from che Gullmar Fjord declmea from 5.3
oercent in 1972 to 2.9 percent in 1973 and to 0.6 oercent in 1974-76, Falkmer
et at. (1977) suggest tfiat ?CBs may have been the orimary cause of tr.e
observed neoplasms.
56
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3.0 GUIDANCE FOR CONDUCTING FIELD STUDIES
This section presents recommenced procedures for conducting fiela
studies of fish liver histopathology during 301(h) monitoring studies.
Included are recommendations regarding study design, field samohng proce-
dures, laboratory methods, and data analysis and interpretation. Recom-
mendations were made as specific as possible without sacrificing their
general applicability. Many of the recommendations are based on the
information presented in Section 2.0 of this report.
3.1 STUDY DESIGN
3.1.1 Species Selection
Different fish species can exhibit markedly different sensitivities to
toxic contaminants in the environment based on such factors as habitat, prey
type, life span, migratory behavior, and genetic constitution. Many of
these factors for the 12 species in which elevated prevalences of hepatic
neoplasms were found in field studies are summarized in Table 4.
All of the species listed in Table 4 spend most of their time near the
seafloor, in close proximity to any contaminants that may be present in
bottom sediments. Seven of the species are known to sometimes bury them-
selves in sediment and thus further enhance possible contact with sediment
contaminants. Ten of the species prey primarily upon benthic invertebrates,
many of which are relatively stationary. In contaminated areas, there is a
high probability that those invertebrates will also be contaminated and
thereby transfer contaminants to the.r piscine predators. At least four of
the fishes exhibit some degree of homing ability. This implies that although
these species may migrate (e.g., seasonally), they may also have the ability
to relocate contaminated areas and thereby be exposed repeatedly to con-
taminants. Finally, individuals from most of the 12 species commonly reach
57
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I ABLE 4. OIARACIERISMCS OF FISHES FOUND 10 HAVE ELEVAJED
PREVALENCES OF HEPAIiC NEOPLASMS IN FIELD STUDIES9
Family
Kyuinidae
(hagfisnes)
Catostomidae
(suckers)
Ictaluridae
(bullhead catfishes)
Gadidae
(codfishes)
£ Percidae
(perches)
Cottidae
(sculpins)
Pleuronectidae
(right eye flounders)
Common Name
Atlantic hagfish
White sucker
Brown bullhead
Atlantic tomcod
Ruffe
Sauger
Walleye
Pacific staghorn
sculpin
Rock sole
English sole
Starry flounder
Winter flounder
Scientific Name
Myjiine glutinosa
Catostomus commersoni
Ictalurus nebulosus
Hicrogadus tomcod
Gymnucephalus cernua
SUJQStedion canadense
ltli9Jtedion vltreuro
Leptocottus armatus
Lepidopsetta bilineata
Parophrys vetulus
Platichthys steflatus
Pseudopleuronectes americanus
Primary
Habitat
Bottom
Bottom
Bottom
Bottom
Bottom
Bottom
Bottom
Bottom
Bottom
Bottom
Bottom
Bottom
Bury?b
Yes
Yes
Yes
Yes
Ves
Yes
Yes
Primary llomuuj
Prey Ability.'-
r
Bl Yes
Bl.l'
Bl.r
in
ui.r
F Yes
Bl
Ul
Bl Yes
Bl
Bl Yes
. .
References: Bigelow and Schroeder (19S3), Hart (1973), Scott and Grossman
(1973), Day (1976).
b Partially bury themselves in sediment as part or normal behavior.
c Bl » benlhic invertebrates, F = fish, P - plant material.
^ Evidence exists that fish can intentionally return to specific locations.
-------
ages >_3 yr. The potential therefore exists t~at some of these fisnes may oe
exoosed to contaminants for many years.
3ased on Table 4, it appears that bottom-awelI ing, oottorn-feeaing
species in contaminated areas have a high ootential for oemg affected oy
liver aononna) i ties. This is consistent witn conclusions reacned oy Dawe ec
al. (1964) and Harshbarger (1977). However, a numoer of other species witn
characteristics similar to those of the fishes listed in Table 4 were sampled
in contaminated areas and did not exhibit liver abnormalities (e.g., 3rown
et al. 1973, 1977; Falkmer et al. 1976; Kurelec et al. 1981; Sloof 1983).
Interspecific differences in the presence or absence of liver lesions
may largely be the result of interspecific differences in sensitivity to
toxic chemicals. For example, such differences are evident in the sensi-
tivities of various salmonids to aflatoxins (Hendricks 1982). Rainbow trout
is very sensitive to aflatoxin carcinogen!city, but brown trout (Sal-
mo trutta) and brook trout (Salvelinus fontinalis) are much less sensitive.
In addition, coho salmon (Oncorhynchus kisutch) and sockeye salmon (0. nerka)
are relatively insensitive to aflatoxins.
Based on the previous discussion, the most important requ^ite for a
monitoring species is sensitivity to toxic chemicals. That is, che species
should have a high probability of developing hepatic lesions following
exposure to chemical contaminants. It is likely that this species will be a
bottom-dwelling, bottom-feeding fish, but all fishes having these charac-
teristics cannot be expected to be sensitive. When selecting a target
species for a fish liver nistopathology study, historical information
regarding the sensitivities of the species likely to be encountered in a
contaminated area should be reviewed. In the absence of such information,
preliminary field surveys or laboratory tests may be required to evaluate
this characteristic. Preliminary field studies should evaluate candidate
species at the most contaminated study sites. Laboratory tests should
expose candidate species to chemical concentrations high enough to induce
lesions in at least one species.
59
-------
Once sensitive soecies nave oeen identified, at least two otrer criteria
should be met. First, the soecies must be oresent m both contaminated ana
•jncontarmnated areas so that statistical conpansons *'th reference condi-
tions can be made. Second, the species snould not be mghly migratory, so
that residence time in the contaminated area would be too short to mauce
liver lesions or that migration between contaminated ana uncontaminated areas
rtOulo aestroy gradients in the orevaiences of liver lesions ana chereoy
confound interpretation of prevalence data from multiple sampling sues.
Other desirable characteristics of a monitoring species are that it can
be caotured easily to provide desired sample sizes at reasonable cost and
that it be either commercially or recreationally valuable.
Most of the recommended criteria for a monitoring species require that
considerable information be available regarding the characteristics of the
species. Unfortunately, this kind of information is incomplete for many
species. Based on the results of historical studies, the knowledge of which
species are sensitive to chemical contamination is probably the most
important information to have when designing a fish liver histopathology
study.
3.1.2 Age Limits
Several field studies have found a positive relationship between
prevalence of hepatic neoplasms or putative preneoplasms and age, length, or
weight of fish (Figure 5). Because length and weight generally increase with
increasing age, it is presumed that age is the primary factor in all cases.
In all of these studies, hepatic neoplasms were absent in the youngest
fish. Elevated prevalence of hepatic neoplasms in older fish relative to
younger individuals has also been noted by Baumann et al. (1982), Mai ins et
al. (1982), and M-.fain et al. (1982).
The patterns in Figure 5 suggest that age may confound interpretation
of the results of certain fish liver histopathology studies. For example,
prevalence of hepatic lesions in fish from a contaminated area could be
higher than prevalence in a reference area partly because fish in the former
60
-------
30
H«oatle Ntoolumi In
Atlantic Hagllsh
I
20
40 60 30
BODY WEIGHT (g)
I
100
Hepatic Moduli*
In Ruff*
g,
01
o
LU
QC
a.
12 14 16 18 20 22 24
10 -I
5 -
LENQTH (em)
Hipatle Neoplasm*
In English Sol*
3 4 S 6
AGE (ytart)
20 -|
10 -
Htpatlc Prtnaoplism*
In English Sol*
n
345 S >7
AGE (y«srt)
Rgure 5. Relationship between hepatic lesions and size or age of
Atlantic hagfish (Falkmer et al. 1976), ruffe (Kranz and Peters
1985) and English sole (Tetra Tech 1985).
61
-------
area may be oider than fivi in tne iatter area. ~o estimate me elevation
in lesion orevalence chat may oe tne result solely of chemical contaminat'on,
age Differences between fish from different areas must be minimized.
Ideally, fish should be comoared only within age classes. However,
oecause this kind of stratification frequently reduces samole sizes oelow
oesiraole levels, it may not always oe practical. An alternative to making
comparisons based on age classes is making comparisons based on samples
having similar age frequency distributions.
In making comparisons based on age frequency distributions, strategies
can vary from evaluating as broad an age range as possible to evaluating a
specific component of the total population. If the objective is to evaluate
lesion prevalence in the overall population of a species, the entire age
spectrum should be considered. However, if the objective is to evaluate
lesion prevalence in that component of the population most likely to be
affected by lesions, age limits may be imposed on the comparisons. For
example, because hepatic neoplasms were not found in the youngest hagfish
(Falkmer et al. 1976), ruffe (Kranz and Peters 1985), and English sole
(Tetra Tech 1985) from contaminated areas (Figure 5), future studies may
elect to exclude fish younger than the age at which neoplasms begin to
appear.
It generally is not practical to determine fish age in the field.
Instead, some hard structure (e.g., otoliths, spines, scales, opercular
bones, vertebrae) of each fish is retained and later analyzed for annual
markings in the laboratory. If the study design calls for comparisons to be
stratified by age, fish collected in the field can be stratified by an
easily measured index of age (e.g., length), pending subsequent confirmation
of actual age. For example, if only fish older than a certain age are to be
evaluated histopathologically, a lower size limit corresponding to the
minimum age can be imposed on the sample collected in the field. Because
indirect measures of age are not totally accurate, the number of fish
collected in the field should exceed the sample size desired for histo-
pathological analysis.
62
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The use of an indirect estimate of age (e.g., length, ^eight) for
evaluating age differences among study areas is not recommended, Because
".Hey generally are not suitably accurate, especially for older fish. The
indirect measure of age usea most commonly is length. However, the lengtn
frequency metnod of age estimation is useful only for young fish from
populations in which spawning occurs during a single, snort period ana
individuals grow at nearly the same rate (Royce 1972). Many species do not
meet these criteria. Spawning may be protracted over a relatively long
period, or individuals may grow at different rates depending on endogenous
and exogenous factors. As fishes grow older, differential growth rates
generally increase the observed range of lengths within an age group.
Several other factors may influence length/age relationships. Because
fish from contaminated and reference areas may represent different popu-
lations with different growth rates (potentially due, in part, to contami-
nation), length/age relationships may vary between these areas. Because
some species exhibit sexual differences in growth rates (Royce 1972),
failure to stratify length/age relationships by sex may confound length/age
relationships.
Several of the problems associated with the length frequency method for
estimating age of English sole are illustrated in Figure 6. All of the fish
shown in Figure 5 were collected from a single embayment (i.e., Commencement
Bay, Washington) and age was determined from otolith (sagitta) analysis.
For both males and females, the observed length range increased as fish grew
older. For example, the length range for females at age 3 was 5 cm (i.e.,
22.5-27.5 cm), whereas the range at age 7 was 12 cm. (25.5-37.5 cm). Thus,
the ability to accurately estimate age from length declines with increasing
age. As demonstrated in Figure 6, the median size of females was larger
than that for males at ages greater than 3. Furthermore, this disparity
between the sexes increased with increasing age.
In addition to stratifying samples prior to comparisons, age can be
used to evaluate the growth of fish using a length-at-age analysis (see
Section 3.4.2). This kind of analysis is valuable for determining whether
hepatic lesions are associated with reduced growth.
63
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AGE 3
0 J // . . T* . ' —. —r
u //I I I I I I I I ^^ | T
AGE 7
20
TOTAL LENGTH (cm)
MALES -*-Q FEMALES
Note: Median age denoted by arrow
Reference: Modified from Telra Tech 1985.
Figure 6. Length frequency distributions of various age groups of male
and female English sole from Commencement Bay. WA.
64
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Based on the orevious discussion, it is recommeicea en at age ne
cetermined directly for all fish evaluated hi stooacnoioqica 1 I y. The
oreferrea method for direct age determination in fishes is the annual ring
method, using some kind of hara bofly part. Many of these techniques are
reviewed in Chilton and Beamish (1982) and Jearld (1983).
3.1.3 Sample Size
Most fish liver histopathology data collected in the field are expressed
in the form of a proportion or percentage. The numbers represent the
prevalence of a pathological condition in the sample evaluated. For
example, if 10 of 50 fish were found to have hepatic neoplasms, the pre-
valence of hepatic neoplasms in that sample would be 0.20 (10/50) or 20 per-
cent [(10/50)xlOO)]. In an epidemiological context, prevalence is defined
as the number of cases of a disease in a given population at a given time
(Klontz 1984). Prevalence is distinct from incidence, another commonly used
epidemiological measure, which is defined as the number of new cases of a
disease in a population over a period of time (Klontz 1984). Prevalence
therefore represents a static "snapshot" of the level of a disease in a
population, whereas incidence is a dynamic property concerning the rate of
introduction of a disease into a population.
One of the major considerations wren designing a fish liver histo-
pathology study is the sample size required to meet the objectives of the
study. As objectives may vary widely among studies, it is not possible to
make a single set of recommendations in the present report. Instead, two of
the more common objectives that may be encountered during fish liver
histopathology studies are evaluated. The principles identified as part of
these evaluations apply to most kinds of objectives and can therefor-, be
used to guide sample size determinations for specific studies.
Objective 1—
One possible objective of a fish liver histopathology study is to
determine whether a pathological condition (e.g., hepatic neoplasms) is
65
-------
oresent in a oopulation of fish' This oojectr/e mignt be encountered during
a reconnaissance study in an unsurveyed area or during a monitoring study or"
cemooral cnanges of fisn neaith in a oreviously uncontarmnated area, "he
emphasis of these studies would be to collect a single individual having the
pathological condition of interest.
A critical consideration in achieving Objective 1 is the minimum samole
size required to detect a single occurrence of the pathological condition in
the test population of fish. This minimum sample size is dependent primar-
ily upon the following variables:
• Population size
t Prevalence of the condition within the population
• Level of desired confidence.
Simon and Schill (1984) present tables of required sample sizes in relation
to a variety of specifications for the three variables listed above. Those
tables are based largely on earlier work conducted by Ossiander and Wedemeyer
(1973) and McOamel (1979).
For the present study, the data presented by Simon and Schill (1984) are
displayed graphically (Figure 7) for a variety of conditions that may be
encountered during field surveys for a relatively rare (i.e., UO percent
prevalence) pathological condition in a fish population. Prevalences of
that magnitude might be expected for hepatic neoplasms in most environments.
The desired confidence level was set at 95 percent; population prevalences
were set at 1, 2, 3, 4, 5, and 10 percent; and population size ranged from
100 to 10,000 fish.
Above a population size of approximately 1,000 fish, the required sample
size stabilizes for all population prevalences except 1 percent (Figure 7).
For a population prevalence of 1 percent, the required sample size begins
to stabilize substantially at population sizes greater than 3,000 fish.
Because the fish populations surveyed by most field studies probably exceed
66
-------
UJ
N
cn
UJ
-j
Q.
2
<
CO
Q
UJ
E
2
DC
300 -
280 -
260-
240-
220-
200-
180-
160-
140-
120-
100-
80-
60-
40-
20-
T
2
T
3
45678
POPULATION SIZE ( x 1,000)
Prevalence
In
Population
10%
10
Rgure 7. Sample size required to detect one individual affected with a
lesion with 95% confidence, given various population sizes
and prevalences.
67
-------
1,000 individuals, peculation size should have a negligible er"fect on
reauired sample sizes when population prevalence is >2 oercent.
At population sizes greater tnan 1,000 fish, che population orevalence
nas a substantial influence on the sample size required to detect a single
affected fish, "or example, approximate sample sizes of 30, 60, and 150
fish are required for population prevalences of 10, 5, and 2 percent, respec-
tively. A sample size of between 260 and 300 fish is required for a
population prevalence of 1 percent.
The results of Figure 7 can be used to determine the sample size
required for a reconnaissance or monitoring study by specifying the minimum
population prevalence that is desired to be detectable, based on the capture
of a single fish having the pathological condition of interest. This
assumes a confidence level of 95 percent and a population size greater than
1,000. For example, if 5 percent is the desired minimum detectable popu-
lation prevalence, a sample size of 60 must be collected to be 95 percent
confident that the survey would collect at least one affected individual.
With a sample size of 60 fish, one could not be 95 percent confident that an
affected individual would be collected if population prevalences were less
than 5 percent. Thus, prevalences less than 5 percent would be considered
undetectable at 95 percent confidence if 60 fish were sampled. If a sample
size of 30 fish is used, population prevalences as high as 9 percent would
not be detectable with 95 percent confidence. To be 95 percent confident of
detecting a pathological condition at its earliest stages (i.e., prevalence
<1 percent), sample sizes greater than 300 fish must be collected. Because
sample sizes of that magnitude often are unaffordable, most researchers win
have to accept the fact that very low prevalences of a pathological condition
will not be detectable with 95 percent confidence.
Objective 2—
A second possible objective that may be specified for a fish liver
histopatnology study is to determine whether prevalence of a particular
lesion at a test site differs significantly from that at a reference site.
This objective may be encountered in a study designed to test whether
68
-------
prevalence in a contaminated area is elevated above the level tnat *ouia oe
expected in the absence of contamination.
A common method of comparing prevalences between two areas is the test
of independence using 2 x 2 (i.e., two-way) contingency tables (cf. Sokal
and Rohlf 1981). The significance of these comparisons can be maae using
either the chi-square statistic or G-statistic, the latter of which is
recommended by Sokal and Rohlf (1981).
As part of the present study, the G-test of independence was evaluated
at various sample sizes using the 2x2 case. The goal was to determine the
statistical power of this test at the various sample sizes that may be used
during most fish liver histopathology studies (i.e., 0-300 fish). The power
of a statistical test is the probability of correctly rejecting the null
hypothesis when, in fact, it is false. Power analyses were conducted over
the range of prevalences that might be expected for most hepatic lesions in
contaminated and reference areas (i.e., 0-25 percent). Results are presented
graphically to provide quick reference to the approximate levels of statis-
tical power that can be achieved for various study designs and various
environmental conditions.
The general layout of a 2 x 2 contingency table is presented in
Figure 8. The table is divided into two classes based on the kind of study
area (i.e., rows) and two classes based upon the presence or absence of
hepatic lesions in sampled fish (i.e., columns). Multiway contingency
tables with more than two classes can also be used to summarize pathology
results from more than two study areas.
In Figure 8, the expected prevalence (i.e., that at the reference site)
and the observed prevalence (i.e., that at the test site) can be computed
and compared to provide a statistical test of the null hypothesis of
independence between study site and lesion prevalence. In most fish liver
histopathology studies, a fixed number of fish are collected at each study
site. Thus, the totals (i.e., the marginal sums N^ * N^ and N21 + N22) In
the third column are fixed in each analysis. The test of independence
therefore consists of computing the probability of obtaining the observed
69
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REFERENCE
SITE
TEST SITE
MARGINAL
SUMS
NUMBER OF
FISH WITH
LESIONS
Nn
N21
N0
NUMBER OF
FISH wir Hour
LESIONS
Nt2
N22
N-2
MARGINAL
SUMS
Nt.
N2-
TOTAL SAMPLE
SIZE - N
NOTE. SUBSCRIPTS ARE DEFINED IN EQUATION 1
Figure 8. Example of a 2 x 2 contingency table.
70
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(or greater) departures from indeoenaence of lesion prevalences (i.e., :ne
numbers that can vary), out of ail oossible tuo-way caoles *ntn tne same
.Tiarginal totals for study sites.
The G-test of indeoendence is a likelihood ratio tast (Neyman and
Pearson 1928; Neyman 1950). The likelihood ratio criterion (exoressed as G)
for testing the null hypothesis of Independence is:
r s
•• n n •„
s . . '*' ^1 in
where:
N a total number of samples collected
N.. = number of observations in the i, jth cell of the r x s contin-
gency table
N., - marginal -sum of observations in the i row of the r x s
contingency table
N.. - marginal sum of the observations in the j column of the
table
r - number of rows in the r x s contingency table (r-2 in a 2 x 2
table)
s - number of columns in the r x s contingency table (s=2 in a 2
x 2 table).
Under the null hypothesis of independence (HQ), the distribution of 2
ln(G) tends to a x distribution as n-»«, where f is the degrees of
71
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freeaom (f=l for a 2 x 2 test). For small samole sizes, ic cannoc 5e
assumed that this approximation is close. As a result of deviations from
the asymtotic distribution of the test statistic, the actual Type I error of
the G-test tends to be higher than the nominal level. The aporoximation is
also poorest when r and s are small and when p. = N^/N and p.j = N.j/N are
near 0 or 1. Tnerefore, in applying the G-test in the analysis of 2 x 2
contingency tables with small sample sizes (i.e., N <. 200), the use of
correction factors has been recommended (e.g., Sokal and Rohlf 1981). This
subject is treated in detail in Section 3.4.3. Because different studies
may use different correction factors, the power analyses conducted in the
present section did not employ correction factors. They therefore represent
a more generalized evaluation of the G-test.
Two kinds of power analyses were conducted. In the first set of
analyses, the probability of detecting selected differences in lesion
prevalences between reference and test sites was calculated. In the second
set of analyses, the minimum detectable difference in prevalence at the test
site (i.e., compared to the reference site) was evaluated for different
levels of prevalence at the reference site and at a fixed level of power.
Determination of the power of the G-test involves the calculation of the
area under the curve in the critical region on the noncentral chi-square
probability density (C*). Thus, the power of the test can be found by
evaluating the integral:-
«— 19 * /" 2
P(C*|f,X) » V^e" - ". r / x * " e" * dx (2)
k! 2 rlj * k) ,
k«o / V* (a)
where:
A = the noncentrality parameter
f • degrees of freedom.
72
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The value of the noncentral ity parameter (X) may tie obtained from tne
following general rule, if under the null hypothesis (H0), the test statistic,
T(X., X....Xn) is asymptotically distributed as centra! v f, then for n
finite, the approximating noncentralUy parameter ( \) under an alternative
hypothesis (K*) is simply the value of the test statistic, "(X^, ;<2...;
-------
The resuics of the first set of oower analyses are summarized -.
Figures 9 ana 10. These figures snow the cower of the G-cest in relation co
une numoer of samoies collected at eacn location for selected crevaience
avels at both tne reference ana test sues. These analyses were conauctea
:"or eaual sample sizes at each study site, and the samole sues (i.e.,
marginal sums) in Figures 9 and 10 represent the number of samoles collected
at eacn site.
Several patterns are apparent in Figures 9 and 10. First, at a fixed
power, larger sample sizes are required to detect smaller elevations in
lesion prevalence. For example, if lesion prevalence in the reference area
is 0.1 percent (Figure 9) and power is fixed at 0.9, the approximate sample
sizes required to detect elevations in lesion prevalences at the test site
of 20, 15, 10, and 5 percent are 35, 50, 75, and 160 fish, respectively.
A second pattern identified by tne power curves is that at a fixed
sample size, power increases as the elevation in lesion prevalence at the
test site increases. For example, if lesion prevalence in the reference
site is 0.1 percent (Figure 9) and sample size is fixed at 40 fish, the
approximate values of power to detect elevations in lesion prevalences at
the test site of 5, 10, 15, and 20 percent are 0.35, 0.65. 0.85, and 0.95,
respectively.
A third pattern identified by the power curves is that at a fixed sample
size and elevation of lesion prevalence above reference levels, power
declines as reference prevalence increases. For example, at a sample size
of 40 and elevation in prevalence of 10 percent, the approximate values of
power to detect the elevated prevalence when reference prevalences are 0.1
percent (Figure 9) and 5 percent (Figure 10) are 0.65 and 0.30, respec-
tively. This suggests that every effort should be made during a fish liver
histopathology study to locate reference stations in as uncontaminated an
area as possible to enhance the probability that prevalence of chemically
induced hepatic lesions will be very low (i.e., as close to 0 percent as
possible).
74
-------
ion
I
o
a
z
o
o
01
HI
a
0.6-
0.4-
m 0.2-
m
o
DC
a
0.0
LESION
PREVALENCES (%)
REFERENCE TEST
SITE
-O-
0.1
01
0.1
01
SITE
5
10
15
20
I
80
120
160
200
I
24C
280
SAMPLE SIZE (MARGINAL SUM)
Note: Signilicance level ^ 0 Ob
Figure 9. Power of the G-lest vs. sample size when lesion prevalence at the reference site is 0.1%
-------
en
m
<
m
O
DC
Q-
1.0-1
0.8-
0.6-
DC
01
o
z
O
I-
o
UJ
LU
O
O 0.4 -
0.2 -
0.0
LESION
PREVALENCES (%)
REFERENCE TEST
SITE SITE
-O- 5 10
-•-5 15
•*• 5
5
20
2b
40 80 120 160 200 240
SAMPLE SIZE (MARGINAL SUM)
280
Note: Signihcance level - 0 OS
Figure 10. Power of the G-test vs. sample size when lesion prevalence at the reference site is 50%
-------
The oower curves oresentea in Figures 9 ana 10 can ae usea to guiae :-e
selection of samoie sizes for oiannea studies, if preliminary information
exists regaraing lesion prevalences in reference ana test areas, erese
/alues can be aoolied to Figures 9 and 10 to determine the samoie sizes
needed to detect specific elevations in lesion prevalence with various
degrees of statistical power. The power curves can also oe usea in an a
posteriori analysis in which the focus is on the evaluation and inter-
pretation of statistical analyses. For example, if lesion prevalence in the
reference area was known (or assumed) to be close to 0 percent, and the
study objective was to have an 80 percent probability of detecting a lesion
prevalence of 10 percent at a test site, Figure 9 indicates that ap-
proximately 60 fish should be collected at each site. In instances where
the null hypothesis has been accepted, the information provided in these
plots also can be used to evaluate the probability of th1; corresponding type
II error (i.e., the probability of accepting a null hypothesis when it is
false).
A second set of power analyses was conducted to provide a different
view of the power of the G-test in specific applications. These analyses
provide information concerning the relative benefits in terms of increased
test sensitivity that can be obtained for corresponding increases in sample
size. These analyses were conducted at a fixed power of 0.80. The minimum
detectable prevalence at a test site that could be discriminated statis-
tically (P<0.05) from that at the reference site was calculated for reference
site prevalences between 0.1 and 20 percent. The analyses were conducted by
fixing the noncentrality parameter (A) In Equation 3 for a power of 0.80 and
solving the resulting equation for the number of lesions at the test site
{N21, see Figure 8). This is possible because the total numbers of samples
at both the reference and test sites are equal in these evaluations, and the
marginal sums for the reference site corresponding to the selected prevalence
levels are fixed. The values of N2- were obtained by setting the resulting
equation equal to zero and using the Newton-Raphson method to solve the
single unknown (N-,).
Results of the second set of power analyses are presented in Figure 11.
They demonstrate that if prevalence at the reference site is constant, the
77
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70 T SAMPLE SIZE
CD
20.0
00 2.5 5.0 7.5 100 125 150 17.5
PREVALENCE (%) AT REFERENCE SITE
Note: Significance level - 0 05
Statistical power - 0 80
Figure 11. Effects of sample size on the minimum detectable prevalence at a test site relative
to the prevalence at the reference site.
-------
minimum detectable prevalence at the test site decreases «icn -ncreasing
ssrnole size. However, the rate of decrease is not r.near. "or examoie, .^nen
=sion orevalences at the reference site are near 0 oercent, :he aooroximate
-11 mmum aetectable prevalences at the test sue at sample sizes of 20, 40.
50, 100. and 200 fish are 26, 14, 10, 6, and 3 percent, resoecti /ely. "hus.
by increasing sample size by 20 fish from N=20 to N=40, the minimum aetec-
table prevalence declines by 12 percentage points. 3y adding another
20 fish from N=40 to N=60, the minimum detectable prevalence declines by
only 4 percentage points. To realize an additional decline of 4 percentage
points, 40 fish must be added from N=60 to N«100. Finally, the addition of
100 fish from N=100 to N=200 reduces the minimum detectable prevalence by
only 3 percentage points. Thus, the value of adding additional replicate
samples declines as sample size increases.
Results of the secjnd set of power analyses (Figure 11) also aemonstrate
that as prevalence at the reference site increases, the margin (or dif-
ference) between that value and the minimum detectable prevalence at the
test site also increases. For example, if N=60 and reference site pre-
valences are 0, 5, and 10 percent, the differences between those prevalences
and the corresponding minimum detectable prevalences at the test site are
approximately 10, 15, and 20 percent, respectively. Thus, as prevalence at
the reference site increased within this range, the minimum detectable
elevation in prevalence above reference levels doubled. These results
support the recommendation made earlier in this section that every effort
should be made to ensure that prevalences at the reference site are as low
as possible.
3.1.4 Sampling Season
Litt'2 information is available regarding seasonal variation in
prevalenc:: of hepatic lesions in fishes. McCain et al. (198Z) evaluated
seasonal variation in the prevalences of neoplasms and putative preneo-
pI asms in livers of 551 English sole from the Ouwamish River, Washington
(Figure 12). No significant difference among seasons (P>0.05; G-test of
heterogeneity) was found for either neoplasms or preneoplasms.
79
-------
30-
20 -
«-. 10-
01
o
z
01
_1
s
oc
Q.
10-
HEPATIC NEOPLASMS
P>0.05, G-tast
HE
P>
PATIC
0.05,
..•• '
PHE
G-tes
NEOPLASMS
t
JANUARY APRIL JULY OCTOBER
(182) (95) (112) (162)
MONTH
(Sample Size)
Reference: Modified from McCain tt al 1982.
Figure 12. Seasonal variation of hepatic lesions in English sole from
the Duwamish River, WA.
80
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Seasonal variation in tne orevaience of neoatic neooiasnis couia resu't
rrom the seasonal .-nigrations etmoitea oy many fisnes if fish witn 'esions
senavs differently tnan fisn without lesions. For examoie, . f f;sn *icn
lesions do not migrate, lesion prevalence would be at a minimum *nen nsn
without lesions migrate into a contaminated area and *oula peak «nen fish
without lesions leave the area. Seasonal variation in cne prevalence of
rapidly induced lesions also may vary if fish are more sensitive to lesion
induction during particular times of the year.
Ideally, fish liver histopathology surveys should be conducted during
the times of year when lesion prevalences are expected to peak (Sindermann
et al. 1980). This strategy allows the worst-case conditions to be evalu-
ated. It also increases the likelihood that the observed prevalences can be
discriminated statistically from reference conditions. In the absence of
information on seasonal variation in lesion prevalences, interannual
comparisons should be made only between surveys conducted during the same
season.
3.1.5 Station Location
Appropriate locations of sampling stations depend upon the oojectives
of different studies. To evaluate the elevation of lesion prevalences above
an expected level as a possible consequence of chemical contamination,
stations frequently are located in contaminated and uncontaminated (i.e.,
reference) areas. This pair-wise approach allows the observed prevalence in
the contaminated area to be compared statistically with the prevalence that
would be expected in the absence of contamination (i.e., the observed
prevalence in a reference area). An additional case can be made for the
association between lesion prevalences and contamination if stations are
located along a gradient of contamination (i.e., from highly contaminated to
moderately contaminated to uncontaminated).
In all of the above circumstances, it is recommended that chemical
analyses of sediments be conducted in conjunction with fish histopathology
to confirm the degrees of sediment contamination. It is also recommended
that stations be located in areas where the spatial extent of contamination
81
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is large enougn to reasonaoly exoect that the sampled fish may nave soent a
consideraole amount of time within the influence of trie measured concami-
3.2 FIELD SAMPLING PROCEDURES
3.2.1 Field Acquisition
One concern when determining prevalences of hepatic lesions in fishes
is that the collection technique does not bias the results. Bias will occur
if fish with lesions are sampled differently than fish without lesions
(Sindermann et al. 1980). For example, & passive collection technique
(cf. Hubert 1983) that relies on fish feeding (e.g., hook-and-line, long-
line) or fish movement (e.g., gill nets, traps) may undersample fish with
lesions if their desire or ability to feed or move is reduced. By contrast,
an active capture technique (cf. Hayes 1983) such as otter trawling (e.g.,
Tetra Tech 1987) may oversample fish with lesions if their swimming ability
is reduced to the point that they would be less likely to escape the
oncoming net than would fish without lesions.
At least one potential instance of sampling bias has been reported in
the literature. Dawe et al. (1976) found high prevalences of hepatic
neoplasms in white suckers from Deep Creek Lake, Maryland, using rotenone
poisoning, but failed to find similar lesions in suckers from other local-
ities by gill-netting the fish during spawning runs. The authors suggest
that the suckers with neoplasms may not have taken part in spawning runs and
therefore could not be sampled by the gill-netting technique.
Given the possible influence of collection technique on observed lesion
prevalences, it is recommended that the technique used in each study of fish
liver histopathology be selected to account for any known behavioral
differences between fish with and without lesions. Unfortunately, little
information is available regarding this topic. However, if some behavioral
information exists, or if reasonable speculations can be made, this in-
formation should be used to evaluate the collection technique.
82
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A second 'on
-------
Each specimen should oe given a unique coae numoer. The coae numoer
snoula de used co laoei aii samoies tnat vviii be analyzed in cr.e laooratory
(e.g., iiver samoies, otohths). The coding system can oe simDie, out roust
orevent the laooracory personnel from knowing any of the cnaracteristies of
the fish from wmch each samole *as taken, including age, sex, nealtn, ana
location of capture. This lack of knowledge will ensure that tne analysis
is conducted objectively. The process of ensuring sample anonymity at the
time of laboratory analysis is called a "blind" system.
3.2.4 Liver Subsamplinq
Before being necropsied, each fish should be weighed (nearest g, wet
weight) and measured (nearest mm, total length). The fish should then be
scanned for grossly visible external abnormalities by a person trained to
recognize those conditions. The fish should then be sacrificed by severing
the spinal cord at the brain stem in a manner that poses no risk of damage
to the liver OP to the body parts used for aging.
Following severance of the spinal cord, the abdominal cavity should be
opened, ensuring that the liver is not damaged in the process. Following
primary incision, the entire liver should be removed gently from the
abdominal cavity to provide a full view of the organ. When removing the
liver, extreme care should be taken to avoid puncturing the gall bladder, as
the bile stored within that organ is extremely caustic to liver tissue
(Hendricks et al. 1975). If a liver is damaged by contact with bile, it
should not be used for histological analysis.
Following liver removal, the fish should be scanned for grossly visible
internal abnormalities. The sex of each fish and its reproductive state
should also be noted at this time.
Each liver should be scanned for grossly visible abnormalities. The
color and texture of the organ should also be noted. Color charts can be
used to help standardize color descriptions. Particular attention should be
paid to describing any abnormal foci or nodules. It may be useful to weigh
84
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each liver, pnotograph each anomaly, and identify on a-agrams «nere suo-
samoles were removed.
The process of tissue collection should be guided by the oresence or
aosence of grossly visible abnormalities, in the absence of aDnormaluies,
a tissue subsample (i.e., section) should be resected from tne entire aeptn
of the liver along its longest axis. When visible abnormalities are present,
the tissue section should be taken so that the entire depth of the anomaly
is sampled. The section should contain both normal and abnormal tissue, so
that the pathologist can see the border between the two kinds of tissue. If
more than one kind of abnormality is visible within a liver, each kind
should be described and subsampled. Multiple sections within a single liver
should be coded separately, so that histological preparations can be related
to gross observations. To ensure proper fixation, each tissue section
should not exceed 4 ir.n in thickness (Luna 1968).
3.2.5 Tissue Fixation
Adequate fixation is essential for accurate histological determinations
(Luna 1968; Yevich and Barszcz 1981). The goals of fixation are to:
t Preserve cells and their constituents in as lifelike a state
as possible
• Prevent postmortem changes such as autolysis
t Protect and harden soft tissues to allow for easy manipulation
during subsequent processing
« Convert the normal semi-fluid consistency of ceils to an
irreversible semi-solid consistency
• Aid visual differentiation of tissue structure when using
stains.
85
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To acmeve tnese goals, fixation snouid be performeo immediately after
tissue removal, ana as soon as oossiole after ceath of tne organism. .1
addition, the tmcxness of the resectea tissue snouia oe
-------
The following kinds of data snould be included in most -"ISM i •./er -nsco-
oachology surveys:
• Fish age
• Fish sex
f Fish length
• Fish weight
• Gross pathological observations.
Age-
As described in Section 3.1.2, certain hepatic lesions in fishes are
associated positively with increasing fish age. It is therefore critical
that age dependence be evaluated for all lesions considered in a study. If
age dependence is found, age differences among samples must be removed
before statistical comparisons can be made. As recommended in Section 3.1.2,
age should be determined directly using the annual ring method applied to an
appropriate hard body structure.
A variety of" hard body structures have been used for aging fish,
including otoliths (primarily the sagittae), fin rays, scales, spines, and
vertebrae (Jearld 1983). The method used for each kind of structure is
different, but all require that they be performed by a well-trained and
experienced individual. Also, different methods may be optimal for different
species. Methods of fish aging are reviewed by ChiUon and Beamish (1982)
and Jearld (1983).
Sex-
Few field studies have examined whether hepatic lesions are found
disproportionately in one sex. None of the studies evaluating sex dependence
of hepatic lesions of English sole from Puget Sound found statistically
87
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significant relationsnios between lesion orevalence and fisn sax (McCain e:
al. 1977, 1982; Malms et ai. 1982; Tetra Teen 1985;
-------
or not t-at individual *ill oe usea for histopathological analysis,
snould be measured prior to necropsy for chose individuals selectea for
mstooachoiogical analysis. Total length is Che length from t-.e ancenor-
•nost oart of the fish to tne tip of the longest caudal fin rays. Two kinds
of coca! length can be measured (Anderson and Gutreuter 1983). Maximum TL
is aetermined *hen the lobes of the caudal fin are compressed dorso-ven-
trally, whereas natural TL is measured when the caudal fin is in us natural
state. To be consistent with the convention used by most fishery investi-
gations in the U.S., maximum TL should be measured (Anderson and Gutreuter
1983).
In some cases, erosion of the caudal fin in a substantial segment of a
population may require that a measurement other than total length be used
for affected individuals. If this occurs, it is recommended that maximum
standard length (SL) be used as a substitute. Standard length is the length
from the anterior-most part of the fish to the posterior end of the hypural
bone. Anderson and Gutreuter (1983) state that in practice, SL may be
measured to some external feature such as the last lateral line scale, the
end of the fleshy caudal peduncle, or the midline of a crease that forms
when the tail is bent sharply. Standard length can be related to total
length by developing a regression relationship between these two measures
for a sample that covers the complete length range observed in the popu-
lation.
Weight--
Weight generally is used in conjunction with length to evaluate fish
condition (see Section 3.4.2). It is recommended that weight be determined
individually for each fish selected for histopathological analysis. Weight
should be measured to the nearest gram (wet weight) of the whole body prior
to necropsy.
Gross Pathological Observations—
Gross observations of external abnormalities in all fishes sampled (both
target and nontarget species) are relatively inexpensive and should be
89
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oerformea routinely wnen Conducting fish 11/er mstooatnology surveys.
Gross ooservations of internal aonormaluies of all individuals selectao for
"iistaoatnological analysis also is recommended. Althougn gross ooser/ations
ceneraily are not definuv/e evaluations of fish health, they may be very
useful for uncovering oreviously unknown oathological conditions in fisnes
from oolluted areas. For examoie, liver abnormalities in Atlantic :omcoo
from the Hudson River, New York (Smith et al. 1979) and in Englisn sole from
the Duwamish River, Washington (Pierce et al. 1978) were discovered inci-
dentally, as fishes were being evaluated for other purposes. In addition to
uncovering previously unknown pathological conditions, gross observations
can also be related to microscopic observations of the liver to investigate
possible associations between different kinds of pathological conditions.
Gross external observations are relatively inexpensive because they do
not require specialized equipment or preparation techniques and thus can be
made as individuals are sorted from the catch. In addition, gross external
observations generally do not require that a trained pathologist be aboard
the sampling vessel. However, it is extremely important that at least one
individual on board be trained by a qualified pathologist to identify the
various kinds of pathological conditions that may be encountered. Sindermann
et al. (1980) stress that pathological observations made by untrained
personnel are usually useless and often misleading. For example, at least
two pathological conditions (fin erosion and skin ulcers) can easily be
confused with the external damage that fishes may suffer as they are dragged
along the seafloor in an otter trawl.
Given the potential usefulness of gross observations and the need for
accurate and verifiable determinations, it is recommended that represen-
tative fishes having each kind of pathological condition be archived for
each major survey, and that the conditions be confirmed by a qualified
pathologist. This verification step is especially important if different
personnel make the gross observations during different surveys. For all
suspected pathological conditions that cannot be identified in the field,
representative specimens should be archived for later evaluation by a
qualified pathologist.
90
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Sinaermann et al. (1980) reviewed the literature on the r2'anonsn]D or
fish pathology to aollution in marine ana estuanne environments, ana
ioentined the r'ol lowing four grossly visible conaitions as acceotaole for
immediate use in monitoring programs:
t Fin erosion
• Skin ulcers
• Skeletal anomalies
t Neoplasms (i.e., tumors).
Fin erosion is found in a variety of fishes from polluted habitats. It
probably is the most frequently observed gross abnormality in polluted areas
(Sindermann 1983). In demersal fishes, the dorsal and anal fins are the
ones most frequently affected whereas in pelagic fishes, the caudal fin is
the one primarily affected. The causes of fin erosion are unknown and
likely complex. They may include chemical contaminants, low dissolved
oxygen, and pathogens. Fin erosion has been induced in fishes after
laboratory exposure to petroleum and PCBs (Couch and Nimmo 1974; Minchew and
Yarbrough 1977).
Skin ulcers have been found in a variety of fishes from polluted
habitats. Next to fin erosion, they are the most frequently reported gross
abnormalities in polluted areas (Sindermann 1983). Prevalence of ulcers
generally varies with season, and is often associated with organic en-
richment. The primary cause of skin ulcers may be pathogenic organisms
(e.g., Vibrio spp.) associated with pollution.
Skeletal anomalies frequently are more prevalent in fishes from polluted
areas than in fishes from uncontaminated areas. Most observed skeletal
anomalies involve the spinal column and include fusions, flexures, and
vertebral compressions. Skeletal anomalies also include abnormalities of
the head, fins, and gills. Skeletal anomalies have been induced in fishes
91
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after laboratory exoosure to kepone and heavy metals (Sinaermann 5t oi.
i960).
Neoplasms or tumors have been found in eievacea prevalences in a variety
of ooiluted areas througnout the world. The most freauentiy reoorted grossly
/isible tumors include liver tumors, skin tumors (i.e., epidermal aapi Hamas
and/or carcinomas), and neun lemmomas. Liver tumors nave been induced in
fishes after laboratory exposure to a variety of chemicals (see Section
2.3.1). Two kinds of growths nave been described as epidermal "papiilomas"
and pseudobranchial "tumors" in the literature (Sindermann et al. L980).
The predominant and pathognomonic cell type in these growths is the presently
unidentified X-cell. Available evidence suggests that this cell probably is
a protozoan parasite, possibly an amoeba of the family Harmanellidae (Oawe
1981; Myers 1981). No relationship between the prevalence of these skin
anomalies and pollution has been demonstrated conclusively.
It is recommended that any survey of fish liver histopathology examine
fishes for fin erosion, skin ulcers, skeletal anomalies, and neoplasms, at a
minimum. The occurrence of parasites should also be recorded. In addition
to the five conditions listed above, any additional grossly visible patno-
logical conditions that are suspected of occurring in a specific locality
should be monitored.
Other Ancillary Data—
In addition to the kinds of ancillary data recommended for all fish
liver histopathology studies (i.e., those discussed previously), several
other kinds of data may prove useful when interpreting observed patterns of
lesion prevalences, including:
• Contaminants in sediment
• Contaminants in tissue
a Contaminants in stomach contents
92
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• Contaminant metaoolites in bile
t Stomach contents
• Sediment toxicity
• Benthic infaunal assemblages
• Identities and abundances of nontarget species.
Each of these kinds of data is discussed in Section 3.4.
3.3 LABORATORY PROCEDURES
3.3.1 Tissue Processing
Embedding—
Before a fixed tissue can be sectioned (i.e., sliced into very thin
sections for microscopic analysis), it must be embedded in a firm medium
(Luna 1968). The medium ensures that thin, uniform sections can be cut.
The most common embedding medium used for fish tissue being prepared for
light microscopy is paraffin. Other media considered suitable for light
microscopy include celloidin and carbowax, as well as the relatively new
plastic materials (e.g., metnacrylate, epoxies) developed for high-resolution
light microscopy and electron microscopy (Johnson and Bergman 1984).
It is recommended that paraffin be used to embed tissues being prepared
for routine histopathological evaluation of liver abnormalities in fish.
Paraffin is readily available in commercial laboratories and is relatively
inexpensive. It allows examination of much larger tissue sections than do
many of the more specialized techniques (e.g., methacrylate embedment).
However, other media may be used if the objectives of the study go beyond
routine histopathological examination using light microscopy.
93
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The oaraffins commonly used to emoea fish tissue inciuae Paraolasc,
Paraolast Plus, and Paraolast Extra. Of these mecna, Paraaiast ;<:,-a
generally oroviaes trie oest results in terms of ease of sectioning ana
Gegree of resolution.
it is recommended that embedding be conducted using an automated tissue
embedding center. Automated methods usually are better at proviaing high
quality, uniform, and reproducible results than are manual methods. The
automated center should provide a guaranteed uniform temperature during
embedment. The use of vacuum infiltration during embedment is recommended.
Tissues generally are embedded in plastic cassettes (marked with unique
specimen numbers) for ease of sectioning and subsequent storage and re-
trieval.
When paraffin is used as an embedding medium, tissues must first be
dehydrated and cleared in solutions miscible with paraffin. Dehydration
entails removing all extractable water from the tissue by having a dehydrant
diffuse through the tissue. This generally is accomplished by immersing the
tissue in a graded series of increasing concentrations of the dehydrant.
The dehydrant used most frequently is alcohol (e.g., ethanol).
Following dehydration, the tissue must be cleared using a reagent that
is miscible with paraffin and the dehydrant. Clearing renders the tissue
amenable to paraffin infiltration by removing the dehydrant. As the
dehydrant is removed, the tissue clears. When the tissue becomes trans-
parent, the clearing process is considered complete. Commonly used clearing
agents include xylene, toluene, and chloroform.
Following clearing, the tissue is impregnated by paraffin. Impregnation
is the complete removal of the clearing reagent by substitution with
paraffin. Impregnation usually requires two or three baths in paraffin
under a controlled temperature that keeps the paraffin above its melting
point. The temperature of the bath should never rise more than 5° C above
the melting point of the paraffin, as excessive shrinking and hardening of
the tissue may result. When a vacuum is applied during impregnation, it
helps remove air, gases, and any remaining clearing agent. The vacuum also
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araws the oaraffin into ail areas of the tissue, esoeciaiiy tnose areas .er>
-oid by the evacuation of air.
Following impregnation, emoedding of the tissue is completed oy orooerly
orienting it in melted paraffin, ^hen the paraffin solidifies, •.; arovices
a firm medium for keeping intact all parts of the tissue when sections are
cut.
Sectioning—
Following embedment, tissues are sectioned (i.e., cut) into very thin
slices from the paraffin block using a microtome equipped with a very sharp
stainless steel blade (Luna 1968). High quality sectioning facilitates the
pathologist's task of accurately identifying tissue and cellular abnor-
malities.
The quality of sectioning depends greatly on the ability of the
sectioning technician and the quality and condition of the sectioning
equipment. The technician must have adequate manual dexterity and must be
well-trained. Quality of sectioning should be preferred over oerformance
rate. The most critical component of the microtome is the knife. The knife
should always be maintained at its highest degree of sharpness, so that
sections ribbon off the paraffin block in a flat, unwrinkled manner. The
knife should be cleaned after each use by removing accumulated paraffin with
a piece of gauze saturated with xylene.
The ideal section should be of uniform thickness and free from compres-
sion, wrinkles, and knife marks. Unsatisfactory sections should always be
discarded and new ones taken. For histopathological analysis of fish liver
tissue, it is recommended that sections be 4-5 urn in thickness. Sections of
this thickness can bJ produced readily by most commercial laboratories.
Mounting—
Following sectioning, tissues are mounted onto glass microscope slides
(Luna 1968). This procedure involves floating tissue sections in a warm-
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water oath (50 C) to fully exoand tr.e section, ana then transferring :-e
section onto a glass slide. The slide may be precoatea «ith albumin -.3
facilitate adhesion. Tne section must he flat on cne slide xitn 10
wnnkles, tears, or ouboles present. Slides sometimes are heated to ensure
the firm aahesion of the section to the glass.
Staimng--
After tissue sections are mounted on microscopic slides, they can be
stained using dyes to differentiate various tissue and cellular elements
(Luna 1968). Staining enhances the pathologist's ability to recognize
individual tissues and cell types, and to detect pathological alterations.
• A wide variety of stains and staining procedures are available, both for
routine and specialized purposes. The most common staining procedure used
for fish liver tissue is initial staining with hematoxylin, followed by
counterstaining with eosin. The hematoxylin and eosin procedure is often
abbreviated as H&E staining. Hematoxylin imparts a blue or purple tint to
alkaline (basic) cellular elements. Eosin, by contrast, imparts a pink or
red tint to acidic elements. Cellular elements stained by hematoxylin are
termed basophilic, whereas those stained by eosin are termed eosinophilic.
Because numerous methods of H&E staining are available, it is recommended
that several be evaluated before a fish liver histopathology study begins,
and that the one providing the best results for the species of interest be
selected for use in the study.
Although H&E staining is suitable for most diagnostic purposes, it may
be necessary to use more specialized staining techniques to identify
accurately certain tissue and cellular elements. Some adjunct staining
techniques used in fish pathology include Periodic Acid-Schiff (PAS),
Masson's trichrome, Prussian blue reaction for hemosiderin, and Best's
carmine for glycogen. The choice of suitable special stains will depend
upon the kinds of conditions detected. The need for special stains should
be determined by the pathologist who examines the tissues.
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Following any staining procedure, the tissue sections must oe c
rfitn class covers! IDS. "he covers! IDS are atcacnea to cue slide oy using
mounting meaium. Several mounting media are commercially availaole. "he
one tnat is cnosen should oroviae good optical clarity and snould arotect
the tissue for long-term storage. A commonly used mounting medium is Protex.
Slide Coding—
In general, slides should be given the same code number as that given to
each specimen in the field. However, in some cases the pathologist may be
capable of discerning the site of capture from this code numoer. For
example, the same pathologist may have been involved with the field col-
lection of tissue sections. In such cases, it is recommended that a second
code number be substituted for the original code number on each slide to
ensure complete objectivity of histopathotogical evaluations.
3.3.2 Histopathologicai Evaluations
Qualifications of the Pathologist—
Probably the most important factors for ensuring accurate histo-
pathological evaluations are the qualifications of the pathologist making
those evaluations. Pathology is a science that relies considerably on
training and experience. It is therefore recommended that, at a minimum,
the pathologist be formally trained in the fields of human, veterinary, or
comparative pathology. In addition, it Is recommended that the pathologist
have demonstrated experience in the histologic examination of fish tissue.
This second requirement is necessary because pathological conditions in fish
tissue may not directly resemble similar conditions in other groups of
organisms (e.g., mammals). Ideally, the pathologist should have experience
with the species of interest, because interspecific differences exist in the
appearance and structure of fish livers. If a pathologist who meets all of
the above criteria is not available for a particular study, it is recommended
that the pathologist chosen for the study work closely with an experienced
fish pathologist, until adequate experience has been gained to work indepen-
dently.
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Equipment—
TO adeauately perform tne tasks required of a diagnostic pathologist,
it is essential that high quality optical equipment be employee. The
microscooe should be a modern instrument eauiooed with multiple oojectv/es
and the capability of magnifications up to a minimum of 500 X. Ideally, the
microscope should also be equipped with a camera system, so that observed
abnormalities can be documented photographically.
Examination of Sections—
For each fish, at least one section should be examined microscopically.
During this examination it is imperative that the entire tissue area be
evaluated at a minimum magnification of 100-200 X. The investigator should
begin by scanning the entire section at 50-X power to obtain an overall
impression of the section. Subsequently the pathologist should examine each
field in the section at a magnification of 100-200 X, and increase magnifi-
cation to 400-500 X when necessary to verify the presence and characteristics
of subtle abnormalities.
Descriptions of Lesions—
The field of fish histopathology does not have the long history enjoyed
by the fields of human and veterinary pathology. As a consequence, the
level of knowledge concerning the clinical effects of many lesions in fishes
is incomplete. It 1s possible that future field studies will evaluate
species for which prior histopathological data or even data on normal
histology are not available. To avoid assignment of unwarranted prognostic
connotations, it 1s recommended that descriptive, rather than diagnostic,
terms be employed when evaluating the new species. For species that have
been studied extensively, the use of diagnostic terms may be appropriate.
The nomenclature used in descriptive histopathology is contained in most
basic pathology texts (e.g., Robbins et al. 1984; Smith et al. 1972).
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Coding and Recoraing Abnormal!cies —
As eacn tissue section is examined, individual aonormal ities snouic se
aescrioed on a pathology record sheet. In studies for whicn chere are
multioie examiners (pathologist*), all cases bearing significant aonor-
mahties should be set aside for confirmation oy the cmef pathologist.
After confirmation, the abnormalities may then be entered in an appropriate
computer format for storage and analysis.
Presently, the only available coding system specifically designed for
use in fish histopathology studies is that maintained by the National Ocean
Data Center (NOOC) in Washington, DC. This system is the one used by the
U.S. EPA Ocean Data Evaluation System (ODES). All fish liver histopathology
data collected during 301(h) monitoring studies will be entered into ODES,
and therefore will be coded in NODC format.
The NOOC Fish Histopathology Code {i.e., File Type 13) was developed
for use in descriptive and diagnostic fish histopatholgy studies. The code
was developed by L.D. Rhodes and M.S. Myers of the Northwest and Alaska
Fisheries Center (National Marine Fisheries Service, NOAA) in Seattle,
Washington. This coding system serves the following basic purposes:
• Permit the recording of unique histopathologically evinced
disease entities (i.e., lesions), infectious conditions,
parasitic conditions, and cellular alterations onto computer
formats for convenience in later entry, storage, and analysis
• Provide a standardized nomenclature for lesions detected in
tissue sections
• Permit an assessment of the distribution and relative sever icy
of any lesions detected, including any host response to
infectious or parasitic agents.
The basic organization of this coding system was adopted from the
Systematized Nomenclature of Pathology (SNOP) system which has been used in
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•;anous forms by nospuals and animal research institutes for over .0 yr.
However, the NODC code is designed specifically for jse in fisn n:sto-
pachoiogy studies and does not proviae for entry of the kind of ciimcal
data that the SNOP system allows. The organizational scheme of the NOOC
Fish Histopathology Code allows for specific identification ana descnation
of the following features:
• The organ affected
t The suborgan or tissue involved
• The lesion itself
t The distribution of the lesion within the organ (e.g., focal,
multifocal, or diffuse)
t The relative severity of the lesion
t Any host response resulting from reaction to an infectious or
parasitic agent.
The NOOC code also is designed to be interfaced, via the unique specimen
identification (accession) number, to other data formats within File Type 13
that are capable of documenting other essential information such as site,
method, time and date of fish capture, bottom and surface water temper-
ature (station header record), sex, sexual maturity, age, weight, length,
and gross pathology data (gross pathology record). This kind of information
facilitates the epizootiological analysis of the histopathology data and
intersite comparison of lesion prevalences.
Specifically relating to lesion descriptions, the NOOC Fish Histopath-
ology Code is organized into repeating units of 12 digits that describe a
specific lesion according to organ affected (3 digit code), suborgan or
tissue type (3 digits), lesion description (3 digits), distribution
(1 digit), severity (1 digit), and degree of host response in the case of
parasitic/infectious agents (1 digit). On a typical 80-column data format,
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:ms permits the description of five lesions. However, a mucn larger numoer
of lesions can oe tiescnoea for a particular soecimen as a result of ere
seauence number in Column 80 tfiat permits entry of additional descriptions
in suoseauent rows.
The organ coae permits entry of up to 999 different organ types for a
particular specimen, and therefore is quite flexible. This code therefore
permits expansion beyond the 97 organ types used currently. It generally is
organized into broad anatomical groupings, such as elements of the gastro-
intestinal tract, other digestive organs (liver and exocnne pancreas), and
excretory, circulatory, reproductive, endocrine, skeletal, immune, and
nervous systems, along with specific identification of skin and fin anatom-
ical entities (e.g., caudal fin).
The suborgan/tissue code is also highly flexible and permits expansion
of the current code, because it permits up to 999 different identifiers. It
also is generally organized into broad groupings of tissue types, including
epithelial subtypes (e.g., hepatocellular epithelium); connective tissue and
the cells and other elements composing connective and supportive tissues;
hematopoietic (blood forming) tissues and blood cell types; elements of the
cardiac and circulatory system; elements of the central and peripheral
nervous system; and elements of the skin, excretory, and reproductive
systems. Currently, 353-identifiers are available within this subcode.
The lesion code itself generally is organized according to broad
categories characteristic of different pathological processes. Within the
3-digit format fop this code, the first digit (001) is reserved for identifi-
cation of normal tissue. Generally, codes up to 099 are reserved for
protozoal infectious agents; 100-199 for metazoan parasites and bacterial,
viral, and rickettsial infections, 200-299 for inflammatory disorders;
300-399 for degenerative and necroUc conditions; 400-499 for cellular
organelle changes (i.e., generally applicable to observations made at the
electron microscope level); 500-699 for miscellaneous cellular and extra-
cellular alterations; 700-799 for growth disorders such as tissue atrophy,
proliferation, regeneration, and hyperplasia; 800-899 for preneoplastic and
neoplastic conditions; and 900-999 for vascular disorders such as thrombosis
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and congestion. Within chese categories, there exist numerous avaiiaoie
ooen codes snould otner descriptors be neeaea.
The distnoution code (L digit) assesses the involvement of a lesion
within an organ or suborgan according to its aistnoution. It uses a scale
of 1 to 5 to descnoe focal, focal to multifocal, multifocal, .-nutti focal to
diffuse, or diffuse distributions, respectively.
The seventy code (1 digit) uses a scale of 1 to 7. It describes the
relative severity of a condition from minimal (1) to severe (7).
The final subcode in the NOOC Fish Histopathology Code is the host
response code (1 digit). It is used exclusively to describe the severity of
host reaction to an infectious/parasitic agent. This inflammatory response
is coded on a scale of 1 to 8, describing no observable response (1) to a
severe response (8).
The NODC Fish Histopathology Code utilizes a nomenclature for patho-
logical description derived from several sources to properly and specifically
describe any observed lesions. Most terms are derived from the pathology
text of Bobbins et al. (1984), which is a standard reference for human
pathology, including morphologic descriptions of histologic lesions.
However, because this text deals strictly with human pathology, specialized
texts for fish pathology" (e.g., Ribelin and Migaki 1975; Roberts 1978) and
for veterinary pathology (e.g., Smith et al. 1972) have been used for
specialized terms applicable to fishes. Identification of parasites in
tissue sections follows the criteria set forth in the monograph of Chitwood
and Lichtenfels (1972). The nomenclature for specific degenerative,
proliferative, preneoplastic, and neoplastic conditions in the liver of
fishes has been adopted from terms used to describe similar lesions in mice
(Frith and Ward 1980), rats (Stewart et al. 1980), and rainbow trout
(Hendricks et al. 1984).
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3.3.3 Quality Assurance/Quaiity Control
ImersDecific Consiaerations--
Some fisn liver histopathology studies may involve a ar/erse array or
species from numerous geographic locations. Compared with mammals, fish are
a relatively primitive group of animals with a long period of phylogenetic
development. Because of this relatively long evolutionary history, the
anatomical and histoiogical differences that exist between different species
of fish (even closely related ones) are much more profound than are those
that exist between different species of mammals. This diversity is il-
lustrated by the fact that an experienced pathologist can readily distinguish
three sympatric species of flatfish (pleuronectidae) from Puget Sound,
Washington simply on the basis of liver architecture. The hepatic tissues
of these three firnes are so distinct in terms of distribution of hepato-
pancreas and melanin macrophage centers, and nepatocellular morphology that
pathologists can readily sort slides by species without having to refer to
data sheets. Such interspecific differences make it necessary for pathol-
ogists to become intimately familiar with the target species before beginning
a field study, so as to accurately recognize anatomical features and to
correctly distinguish seasonal or maturationat changes from pathological
alterations. Such interspecific differences also make it almost impossible
for a pathologist unfamiliar with a given species to interpret accurately
verification samples received under the auspices of a QA/QC program.
Internal Verification of Identification—
For studies in which multiple pathologists in the same laboratory are
used to read slides, all cases bearing significant lesions should be
examirjd and verified by the senior pathologist. In addition, at least 5
percent of the slides read by one pathologist should be selected randomly
and read by a second pathologist without knowledge of the diagnoses made by
the initial reader.
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External Verification or" Identification—
At least 5 oercent of the slides reaa ^itmn a laooratory snouia oe
suomuted for independent diagnosis to a pathologist not involved with tne
laboratory. These slides should be chosen to represent the range of
onthological conditions founa during a study, and the external oatnologist
should not be aware of the diagnoses made by laboratory personnel. The
external pathologist should have experience with fishes and, ideally, with
the species of interest.
Reference Collection-
Each laboratory should build a reference collection of slides that
represents every kind of pathological condition found in various studies
conducted by laboratory personnel. Each of these slides should be verified
by an external pathologist having experience with the species of interest.
These slides can then be used to verify the diagnoses made in future studies
to ensure intralaboratory consistency among studies. The slides also can be
compared with those of other laboratories to ensure interlaboratory con-
sistency. A reference collection of photographs also can be made, out
should not be substituted for a slide collection.
Photographic Record—
The chief pathologist should develop a photographic record that
documents the significant classes of lesions encountered during the course
of each study. The photographs should be of sufficient quality to illustrate
clearly the diagnostic features of each lesion. Where necessary, multiple
photographs taken at increasing levels of magnification should be included.
The photographs should bear a label that indicates the degree of magnifi-
cation and the code number of the tissue photographed.
Slide Set—
The chief pathologist should prepare a set of microscope slides that
bear representative examples of major lesions encountered during each
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study. The slide set sr.oula also contain representative normal si ides :r.at
•llustrate tne range of physiological variation encounterea over tne course
of tne investigation. The s'nae set snoula be accomoamea by «mten
aescnptions of eacn slide including the coae numoer, critical diagnostic
features, and final diagnosis.
3.4 DATA ANALYSIS AND INTERPRETATION
Some of the general considerations for analyzing data generated during
fish liver histopathology surveys are described in this section. The details
of data analysis may vary widely among studies, depending upon the kind of
data collected and the study objectives. Although all of those details are
not specified in this section, the general directions that detailed analyses
should follow are recommended.
For 301(h) monitoring, two major kinds of analysis generally will be
made. The first kind of analysis involves comparisons among stations during
single time periods. The objective of this kind of analysis is to evaluate
gradients in lesion prevalence away from a discharge point or to compare
prevalences at stations close to a discharge point with prevalences in a
reference area. The second kind of analysis involves comparisons among
different time periods at single stations. The objective at this second
kind of analysis is to evaluate temporal changes in lesion prevalences.
Both kinds of analysis can be conducted using the G-test tool in ODES.
3.4.1 Age and Sex Effects
As recommended in Section 3.2.6, the sex and age of each fish selected
for histopathological analysis should be determined. When data on lesion
prevalences are ready to be analyzed, they should first be tested for statis-
tically significant relationships with sex or age.
If the prevalence of a particular lesion is related to either sex or
age, the sex ratio or age distribution at all stations that will be compared
should be tested for significant differences among stations. If such
differences are found, individuals should be removed from stations until the
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adjusted sex ratios or age distnoutions do not differ significantly among
stations. Once these adjustments nave oeen made, lasion orevaiences of t-.e
remaining fisn can be compared without interference from the effects of se.x
or age.
An alternative to adjusting samples wnen relationshios between lesion
prevalence and sex or age are found is to stratify comparisons among stations
by sex or age class. In doing so, however, sample sizes may be reduced
substantially and the statistical power to detect significant differences
among stations also would decline.
IF no relationships are found between lesion prevalence and sex or age,
it is not necessary to evaluate sex and age differences among stations.
Instead, comparisons of lesion prevalences among stations can be made
directly.
3.^.2 Growth and Condition
In many fish liver histopathology studies, the question arises as to how
contamination or the presence of hepatic lesions is affecting the overall
health of each fish. Two general indices of fish health that are measured
frequently in studies of fishes are growth and condition. To evaluate these
indices, the weight (nearest gram), length (nearest millimeter), and age of
each individual for histopathological analysis should be measured (see
Section 3.2.6).
Growth can be estimated as the length of an individual fish at a given
age. Use of growth as an index of fish health assumes that unhealthy fish
grow less rapidly than their healthy counterparts. Growth might be con-
sidered a relatively long-term indicator of fish health, as it may require
many months for differences in length between healthy and unhealthy fish to
be large enough for statistical discrimination. Potential effects of
pollution OP hepatic lesions on the growth of fish can be evaluated by
comparing the lengths of each age class between fish from contaminated and
reference areas or between fish with and without hepatic lesions.
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Condition is a measure of the "fatness" of a fish and can oe estimated
as the weight of an individual relative to that individual's length. Use of
condition as an index of fish nealth assumes that the condition of unnealtny
fisn will be reduced relative to their healthy counterparts. Condition
migm be considered a relatively short-term index of fish health, as it may
only require several weeks for differences between healthy and unneaithy
fish to be large enough for statistical discrimination.
Condition can be expressed as a weight-length regression relationship
(Ricker 1975), and then compared among stations or between fish with and
without hepatic lesions by using analysis of covariance. Condition of each
fish may also be expressed in the form of an index that incorporates the
weight and length of each individual. Index values can then be compared
statistically among stations or between fishes with and without Ipsions.
Three indices of fish condition used freouently are Fulton's condition
factor (the most common), the relative condition factor, and Relative Weight
(Anderson and Gutreuter 1983).
3.4.3 Comparisons Among Stations
[n many fish liver histopathology studies, the prevalences of hepatic
lesions are compared statistically among stations having various degrees of
contamination. The simplest case is a pair-wise comparison between a
contaminated site and a reference site. As noted in Section 3.1.3, the
statistical test recommended for this kind of comparison is the G-test of
independence, using a 2 x 2 contingency formulation. This test also can be
used with multiway contingency tables to compare lesion prevalences among
more than two stations.
As noted in Section 3.1.3, values of G should be adjusted when sample
sizes are small (N < 200). At least two correction factors have been recom-
mended in the literature: Yates' correction for continuity and Williams'
correction (Sokal and Rohlf 1981). Yates' correction requires that observed
values in the 2 x 2 table be adjusted by adding or subtracting a value of
0.5. Williams' correction for a 2 x 2 table requires that the calculated
value of G be divided by q, where:
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,\ /_* __ _" __ A
V Kl-N21 N12'N22 V
6(N)
( }
Based on the results of simulation exoenments, Grizzle (1967) snowed
that the application of Vates' correction to the chi-square test statistic
(*2) produces a test that is unduly conservative. Grizzle (1967) also
reported that the likelihood ratio test statistic (i.e., G-test statistic]
behaved almost exactly like x2. Similar sampling experiments to evaluate
the performance of the Williams' correction have not been published.
However, Sokal and Rohl f (1981) indicate a preference for the application of
the Williams' correction factor to the G-test statistic for small sample
sizes.
To evaluate the effect of Yates' and Williams' corrections on the
performance of the G-test, a series of simulations was conducted as part of
the present study. These simulations were conducted in the following
sequential manner:
• Equal sample sizes (i.e., N • 20-100) were specified for each
site, and a true null hypothesis was assumed for a lesion
prevalence (p) of 10 percent at the reference and test sites.
• For individual sampling conditions, random samples were
generated from binomial distributions, with parameters n and
p corresponding to the selected sample sizes and prevalences,
respectively. The method used to generate the binomial
variables employed the fact that a binomial random variable
is the sum of n independent Bernoulli random variables.
• The procedure of sample generation and analysis was repeated
10,000 times for each set of sampling conditions. All
calculated values of the G-test statistic were saved and
subsequently analyzed to determine the proportion of values
greater than or equal to the critical value corresponding to
a significance (I.e., Type I error) level of 0.05. The
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observed level of Type I errors in eacn simulation exoerimenc
was used to evaluate trie effect of the correction factors on
test oerformance.
Each of the simulation experiments representing the selected
conditions was repeated three times: once with the Yates1 correction aopliea
in the calculation of the G-test statistic, once using the Williams'
correction, and once with no correction applied to the value of 2 ln(G).
Three sets of experiments and a total of 24 individual simulation experiments
were performed.
Results of the simulation experiments are summarized in Figure 13. This
figure also shows the performance of the G-test with and without the
application of the selected correction factors. The test results based on
the use of the Yates' corrrection factor, for example, indicate that the
proportion of tests in which the null hypothesis was falsely rejected (i.e.,
probability of Type I error) is substantially less than the nominal level
(i.e., 0.05) over the range of sample sizes evaluated. The test statistic
resulting from the application of Yates' correction is classified as
conservative, because the frequency of rejecting a true null hypothesis
(i.e., incorrectly concluding that differences in the prevalence of lesions
exist) is decreased over the nominal level of the test.
The use of unconnected values of the G-test statistic will lead to
errors in the direction opposite to that described for use of Yates'
correction (Figure 13). That is, the frequency of rejecting a true null
hypothesis will be increased over the nominal level of the test when sample
sizes are small. For example, when simulated sample sizes at each sampling
location were less than 30, the actual probabilities of the Type I error
obtained at the nom1n«, 0.05 significance level were greater than 0.076.
The actual probabilities of the Type I error obtained at the 0.05 signifi-
cance level in the simulation experiments were greater than the nominal
level for all sample sizes less than 80.
When the Williams' correction factor was applied to the value of the
test statistic, the G-test performed very close to its expected chi-square
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o.io n
m
oc
O 0.08
OC
DC
111
0.06-
u.
O
Z
O
O
Q.
O
EC
Q.
0.04 H
0.02 -
000
UNCORRECTED VALUES
NOMINAL TYPE I ERROR
YATES-CORRECTION
WILLIAMS'CORRECTION
20
40
60
i
80
100
I
120
SAMPLE SIZE
Figure 13. Results of simulation experiments showing the proportion of Type I errors in tests cf
the null hypothesis that lesion prevalence at both the reference and test site equals 100%
-------
•nstnbuiton (Figure 13). Over tr.e range of samole sizes evaluatea M.e.,
20-100), :he actual orooaoilicy of a Type ! error corresooncnng co r.~e
nominal 0.05 significance 'evel ranged between 0.041 ana 0.061. "he .
efficacy of the Williams' correction factor was esoecially evident at the
smaller samole sizes. .ror examole, at sample sizes of 20, :ne actual
probability of a Type I error ootained at the nominal 0.05 significance
level was 0.081 for unconnected values of the test statistic, 0.011 for
values corrected with the Williams' factor, and 0.013 using the Yates1
correction.
Based on the simulation experiments conducted as part of this study, it
is recommended that the Williams' correction be applied to the G-test for
independence when lesion prevalences are compared among study sites and
sample sizes at each site are small (i.e., N<_80). The Williams' correction
shouid also be applied when multiway contingency tables are used and sample
sizes are small. The formula for Williams' correction for multiway tables
is more complex than that used for 2 x 2 tables and is presented in Sokal
and Rohlf (1981).
3.a.A Relationships with Ancillary Variables
Relationships between prevalences of hepatic lesions and a variety of
ancillary variables carr and have been evaluated in an attempt to determine
potential causes of the observed lesions. A pair-wise approach to evaluating
potential causes is useful. FOP example, if lesion prevalences and the
values of a variable are both high in a contaminated area and low in a
reference area, a case can be made that the variable may be causally related
to the hepatic lesions. However, a correlational approach is much more
convincing than a pairwise approach. In such an instance, a gradient in
lesion prevalence is related directly to a similar gradient (positive or
negative) in the values of a variable.
The most common ancillary variable that has been related to prevalence
of hepatic lesions in fishes has been chemical concentrations in bottom
sediments. In most cases, a pairwise approach has been used. However,
Mai ins et al. (1984) used a correlational approach.
Ill
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3ecause a wide variety of cnemicais generally is founa -n contaminates
seaiments, ana because many of these cnetmcals covary across stations, :t
•-areiy is possiole to test the effects of single chemicals, «nile holding
oners constant. The most common manner in which to analyze sucn data is to
conduct a multivanate analysis that generates factors composed of covarying
chemicals (e.g., Mai ins et al. 1984). The chemicals that load most strongly
on each factor can then be considered the major characteristics of the
factor. Factors can then be correlated with lesion prevalence. When a
statistically significant positive correlation is found, the major charac-
teristics of the factor are considered the putative causes of the lesions.
A second variable that commonly is measured in conjunction with lesion
prevalence is chemical contamination of fish tissue (e.g., Tetra Tech
1986). The tissues examined most frequently are muscle and liver tissue.
The goal of these analyses is to relate tissue concentrations to lesion
prevalences. The inference usually made is that the chemicals found in
tissue may have been causally related to the observed hepatic lesions.
However, this inference must be made with considerable caution, as many
organic compounds (including potent carcinogens) are rapidly metabolized in
the liver of fishes (see Section 2.1.2), and thus rarely are found in muscle
or liver tissue (e.g., Malins et al. 1985a,b). Krahn et al. (1986) demon-
strated that measuring metabolites in bile, rather than parent compounds in
tissue, may be a more meaningful way of relating lesion prevalence to those
compounds that are metabolized rapidly.
Several studies have measured chemical concentrations in the stomach
contents of fish from contaminated and uncontaminated areas (e.g., Malins et
al. 1985a,b). In general, stomach contents from polluted areas contain
substantially higher concentrations of chemical contaminants than do stomach
contents from uncontaminated areas. The inference is that diet is a major
route by which contaminants may enter the fish. Although this inference is
correct, no quantitative measure of importance can be made because other
potential routes (i.e., gills, skin) are not measured, and the fraction of
chemicals that actually is absorbed from the stomach contents is unknown.
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The stomacn contents (; .e., 2rey comoosifon) of f;sn from contannna:ea
areas .-night oe ccmoarea to tne stomacn contents of fisn from reference areas
to determine *netner tne diet in contaimnateo areas is reduced in Quantity
or auahty relative to that in the reference area. The inference is that
dietary deficiencies may facilitate or even cause lesion induction in fisn
livers, .-or example, a variety of studies have found that nutritional
imbalances can induce hepatic abnormalities in fishes (e.g., Smeszko 1972)
or enhance the toxicity of chemicals to fishes (e.g., Mehrle et al. 1977).
In addition, outright starvation can induce such abnormalities (e.g., Segner
and Holler 1984).
In addition to variables that may relate directly to induction of
hepatic lesions in fishes, a variety of relatively independent biological
indicators measured in conjunction with fish liver histopathology may assist
the interpretation of observed patterns of lesion prevalence. Several kinds
of parallel indicators measured in past studies of fish liver histopathology
include sediment toxicity (i.e., using bioassays), alterations of benthic
invertebrate assemblages, and diversity and abundance of nontarget fish
species (e.g., Tetra Tech 1985).
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d.O SUMMARY
4.1 INTRODUCTION
The U.S. EPA has selected fish liver histooathology as one of the
indicators of biological impacts for selected marine dischargers Holding
301(h)-modifled NPDES permits. This document provides guidance for conduc-
ting quantitative studies of fish liver histooathology as part of 301(h)
monitoring programs. At present, no comprehensive sources of such guidance
are available. The document is directed primarily at the non-pathol-
ogists involved in writing 301(h)-modified NPDES permits and in overseeing
field studies of fish liver histopathology. Although this document is
directed at non-pathologists, various sections may also oe useful to
pathologists. The following four major components of quantitative field
studies of fish liver histopathology are addressed:
t Study design
• Field sampling
• Laboratory analysis
• Data analysis and interpretation.
4.2 BACKGROUND INFORMATION
The liver is the organ primarily responsible for the metabolic horaeo-
stasis of the whole fish and, as such, is associated intimately with the
contaminants that may enter a fish living in a polluted environment. The
liver's central role in the treatment of exogenous toxic contaminants
renders the cells of that organ highly susceptible to toxic injury. Within
the liver, exogenous contaminants can be stored, directly eliminated, or
metabolically altered before being eliminated. Metabolic alteration of
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contaminants may oroauce T, gniy reactive metaooiues :~at ^oteitiaiiy
c/totoxic, nutagemc, or carcinogenic.
riepatocarcinogenesis models nave oeen oroposea for t>o f-.snes: -amoow
:rout and Enghsn sole, "he moaei for rainbow trout is oasea on laooratory
exoenmenis ana includes tne following morpnologic stages:
• Pale, swollen, individual cells with enlarged pleomoromc
nuclei
• Eosinophilic foci
t Basophilic foci
• Hepatocel lular carcinomas.
The sequential nature of these stages has not been confirmed.
The nepatocarcinogenesis model for English sole is based on field data
from a feral copulation, and includes the following morphologic stages:
• Nonspecific necrotic lesions
t Specific degenerative conditions
Nuclear pleomorphism
Megalocytic hepatosis
• Nonneoplastic proliferative conditions
Nonhyperplastic hepatocel lular regeneration
US
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• roci of ceiluiar alteration
Eosinoom 1 ic foci
Basoom I ic foci
Clear ceil or vacuolatea ceil foci
Hyoeroiastic regenerative foci
• Neoplasms
Liver eel 1 adenomas
Hepatocellular carcinomas
Cholangiomas
Cholangiocellular carcinomas
Mixed carcinomas.
The sequential nature of these stages lacks laboratory confirmation.
However, the similarities of these stages to the documented sequence of
changes in the livers of rats and mice suggest that the four stages observed
in English sole are sequentially related.
Laboratory exposures of fishes to chemicals have been conducted for
over 39 species and 87 chemicals. The major groups of chemicals that have
induced hepatic neoplasms in test fishes include mycotoxins, mtroso-
compounds, miscellaneous nitrogenous compounds, and plant derivatives.
Laboratory results have at least three major implications for field studies.
First, they demonstrate under controlled conditions that many chemicals
found in the environment can induce the same kinds of hepatic lesions as
those found in fishes from polluted habitats. Second, they demonstrate that
hepatic neoplasms can be induced in some fishes in as short a period as
6 mo. Third, laboratory results show that many chemicals induce similar
kinds of hepatic lesions in fishes, and thereby indicate that specific
lesions generally cannot be used as indicators of the effects of specific
chemicals in complex field situations.
At least 17 field studies have documented elevated prevalences of
hepatic neoplasms in fishes from polluted environments. These studies have
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oeen conaucted in marine, estuanne, and freshwater habitats ~n tne U.S. ana
Eurooe. "ne hignest prevalence of neoacic neooiasms ooserveo in a Dooulacion
of feral nsn *as LOO aercent for saugers from Torch Lake, Micmcan. In all
other studies, .-naximum neoplasm prevalence «as
-------
as a ootential conrounoing influence on ooservea iesion orevaiences. if a
is founa, :~e stuay aesign s.ioura oe moaifisa accorai.-.giy.
•then tne oojecti.'e of a fisn liver m stooatnoloay study is to aetect a
single occurrence of a oatnological condition in a fish oooulation, Figure 7
can oe usea to guiae :.u.e aetermi nation of reauirea samoles sizes for a
connaence level of 95 percent. For examole, assuming that :,uie target
population comprises more than 1,000 individuals, approximate sample sues
of 30, 60, and 150 fish would be required if the prevalence of the patho-
logical condition in the population was 10, 5, and 2 percent, respectively.
Results of power analyses for tne G-test of independence are presented
in Figures 9, 10 and 11, and should be used to determine the samote sues
required for comparing lesion prevalences among stations or among sampling
periods. Two general principles can be derived from those analyses:
• At a fixed power and a fixed lesion prevalence in a reference
area, smaller elevations in prevalences in a test area can be
discriminated statistically by increasing sample sizes.
• At a fixed sample size and a fixed elevation in lesion
prevalence at a test site, power decreases as the lesion
prevalence in the reference area increases above 0 percent.
Sample size and reference prevalence are therefore two critical aspects of
the study design that influence the magnitude of test-site prevalence that
will be considered significantly different from tne reference prevalence.
During the design of a fish liver histopathology study, every effort should
therefore be made to maximize sample sizes (within cost constraints) and to
minimize reference prevalence (i.e., by appropriate location of the reference
station).
Little information is available regarding seasonal variation in
prevalences of hepatic lesions in fishes. One study of seasonal variation
in prevalence of hepatic neoplasms and preneoplasms in English sole found no
substantial differences among seasons. However, in the absence of infor-
118
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•nation on seasonal /anation in lesion orevalences, interannuai comparisons
snould be maae oniy oetween studies conducted during tne same season.
Approonace locations r'or sampling stations deoend uoon tne oojectv/es
of different studies, "o evaluate tne elevation of lesion orevaiences aoove
an expected level as a possible conseauence of chemical contamination,
stations frequently are located in contaminated and uncontaminated (i.e.,
reference) areas, "his pairwise approach allows the observed orevalence in
the contaminated area to be compared statistically with the prevalence that
would be expected in the absence of contamination (i.e., the observed
prevalence in a reference area). An additional case can be made for the
association between lesion prevalences and contamination if stations are
located along a gradient of contamination (i.e., from highly contaminated to
moderately contaminated to uncontaminated). Regardless of most study
objectives, stations generally should be located in areas where the spatial
extent of contamination is large enough to reasonably expect that the
sampled fish may have spent a considerable amount of time within the
influence of the measured contamination.
4.3.2 Field Collection
Gross observations of external abnormalities in all fishes sampled
(both target and nontarget species) are relatively inexpensive and should be
performed routinely when conducting fish liver histopathology surveys.
Although gross observations generally are not definitive evaluations of fish
health, they may be very useful for uncovering previously unknown patho-
logical conditions in fishes from polluted areas. To ensure that abnormal-
ities are Identified accurately, at least one person in the field should be
trained by a qualified pathologist to recognize the various kinds of
abnormal conditions that may be encountered. If an abnormality cannot be
identified in the field, representative specimens should be archived for
later evaluation by a qualified pathologist. At a minimum, fishes should be
examined for the following grossly visible external abnormalities:
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• .-m erosion
• Skin uicers
t Skeletal anomalies
• Neoplasms (i.e., tumors)
t Parasites.
The target soecies should be collected in an unbiased manner to
evaluate the true prevalence of hepatic lesions in the target population.
Because some kinds of cellular alterations may begin immediately after fish
are collected, sampling duration should be relatively short (e.g., 5-10 mm
hauls when trawling) and fish should be necropsied as soon as possible after
collection (i.e., preferably within 15 min). If fish cannot be necropsied
immediately, they should be held alive in a flow-through seawater tank.
Before being necropsied, each fish should be weighed, measured, and
examined for grossly visible external abnormalities. The aodommal cavity
of each fish should then be opened, and the liver should be removed. The
gall bladder should not be punctured at this stage, as the bile within it
Mill damage liver tissue upon contact. The fish should be scanned for
grossly visible internal abnormalities and the sex and reproductive state
should be noted.
The process of tissue collection should be guided by the presence or
absence of grossly visible abnormalities. In the absence of abnormalities,
a tissue subsample (i.e., section) should be resected from the entire depth
of the liver along its longest axis. When visible abnormalities are present,
the tissue section should be taken so that the entire depth of the anomaly
is sampled. The section should contain both normal and abnormal tissue, so
that the pathologist can see the border between the two kinds of tissue.
Liver subsamples should be fixed immediately after resection. The
volume of fixative should be at least 10 times that of the tissue subsample.
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In general, ::ssues snouid rgmain in the fixative for at lease ^8 h. ,-resnty
areoared fixati/e should be used at ail tines. Althougn no singie fixative
is icea) ror ail situations, ;tte most common fixatives usea for i-ght-
Tiicroscooy stuaies of fisn tissue are Bouin's fluid and 10 percent neutral
ouffe^eo formaiin.
1.3.3 Laboratory Procedures
Before being sectioned, each liver subsample should be embedded in
paraffin (preferably Paraptast Extra). It is recommended that embedding be
conducted using an automated tissue embedding center to provide high quality,
uniform, and reproducible results. Before being embedded, tissue should be
dehydrated and cleared in solutions miscible with paraffin.
Pol lowing embedment, tissue should be sectioned using a microtome
equipped with a very sharp stainless-steel blade. Sections should be of
uniform thickness (i.e., 4-5 urn) and free from compression, wrinkles, and
knife marks.
Following sectioning, tissues should be mounted on glass slides, and
should be flat on the slides with no wrinkles, tears, or bubbles present.
Tissues can then be stained using different dyes for different purposes.
The most common staining procedure used for fish liver tissue is initial
staining with hematoxylin, fallowed by counterstaining with eosin. Following
staining, tissues should be covered with glass covers lips. Each slide
should be coded to ensure complete objectivity of histopathological evalu-
ations.
To ensure accurate histopathological evaluations, the pathologist
making those evaluations should be formally trained in the fields of human,
veterinary, or comparative pathology. It is also recommended that the
pathologist have demonstrated experience with the histologic examination of
fish tissue in general and, ideally, with the target species of each study.
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The 'aentity of aii iwer lesions snouid be coaea using cne National
Ocean Data Center (MOOC) Fish Histooatnolocy Coae. "iiat coding system -3
:l:e one usea oy U.S. EPA's Ocean Data Evaluation System (GOES).
Proceaures ~'or quality assurance/aual uy control (QA/QC) of iesion
identifications snouid include the following:
• Within a laboratory, all cases oeanng significant lesions
should be examined and verified by the senior pathologist
t At least 5 percent of the slides read by one pathologist
should be read by a second pathologist within the laboratory
• At least 5 percent of the slides read within a laboratory
should be submitted for independent diagnosis by a pathologist
outside the laboratory
• Each laboratory should build a reference collection of slides
that have been verified by a pathologist outside the labor-
atory
• A set of photographs and slides should be prepared and
archived for all major lesions observed in each study.
4.3.4 Data Analysis and Interpretation
The details of data analysis may vary widely among studies, depending
upon the kind of data collected and the study objectives. For 3Ql(h)
monitoring, two major kinds of analysis generally will be made. The first
kind of analysis involves comparisons among stations during single time
periods. The objective of this kind of analysis is to evaluate gradients in
lesion prevalence away from a discharge point or to compare prevalences at
stations close to a discharge point with prevalences in a reference area.
The second kind of analysis involves comparisons among different time
periods at single stations. The objective of this second kind of analysis
is to evaluate temporal changes in lesion prevalences. Both kinds of
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analysis can oe conducted using the G-test tool in GOES. .c is recommences
:nat Williams' correction factor oe aoohed to the G-statistic «nen samoles
3-:es are smai1 (i .e.. <30).
3efore any comoansons are made among stations, trie ootential relation-
snios oetween lesion orevaience and both age ana sex of fish snould be
tested. If significant relationships are found, the age or sex distributions
at selected stations may require adjustment so that comparisons among
stations are made using equivalent age or sex distributions. !n this
manner, the confounding influence of age or sex will be removed from the
comparisons.
Length and weight of fish can be used to develop indices of growth
(e.g., length-at-age) and condition (e.g., various weight/length relation-
ships). Comparisons can then be made between fish with end w'thout hepatic
lesions to determine whether the presence of lesions is related to reductions
in growth or condition.
Relationships between prevalence of hepatic lesions and a variety of
ancillary variables can and have been evaluated in an attempt to determine
potential causes of the observed lesions. A pairwise approach to evaluating
potential causes is useful. FOP example, if lesion prevalences and the
values of a variable, are both high in a contaminated area and low in a
reference area, a case can be made that the variable may be causally related
to the hepatic lesions. However, a correlational approach is much more
convincing than a pairwise approach. In such an instance, a gradient in
lesion prevalence is related directly to a similar gradient (positive or
negative) in the values of a variable.
The most common ancillary variable that has 'ieen related to prevalence
of hepatic lesions in fishes has been chemical concentrations in bottom
sediments. Additional variables that may be useful, either directly or
indirectly, when interpreting patterns of lesion prevalence include the
following:
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• lontaminants in tissue
• Contaminants :n stomacn contents
• Contaminant metabolites in bile
• Stomacn contents
• Sediment toxicity
• Benthic infaunal assemblages
• Identities and abundances of nontarget species.
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-------
-------
-------
_owe-Jmae, L. and A.J. ^nmi. .934. Short-term ana 'ong-ter/n ejects or"
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-------
Sinnnuoer, 3.O., J.Q. Henanc'
-------
icanton, M.F. .365. -eoat'c leooiasms of aauanum fisn s
-------
rfales. J.H., 3.0. Sinnnuber, J.O. Henanc:
-------
S.O GLOSSARY
1 ic
Adenoma
Adenomatous
Anaplasia
Anterior
Atrophy
Atypia
Basophilic
Benign
Biliary
Cancer
Carcinogen
Carcinoma
Cholangio-
cellular
Cholangip-
Hbrosis
Cholangioma
Congestion
Cystic
having an affinity for acid dyes sucn as eosin.
an ordinarily benign neoolasm, usual Iy well circumscribed
and tenaing to compress rather than invaae adjacent tissue.
relating to adenoma.
loss of structural differentiation and reversion to an
embryonic cell form, especially as seen in most, but not
all, malignant neoplasms.
m the front of a structure.
shrinkage of a tissue or cell as a result of structural
losses.
state of being not typical
increased affinity for basic dyes sucn as hematoxyiin.
nonmalignant character of a neoplasm.
relating to bile
general term to denote any of the various kinds of
malignant neoplasms.
any cancer-producing substance.
any of the various malignant neoplasms derived from
epithelial tissue.
related to bile duct epithelial tissue.
fibrosis of the bile ducts.
a neoplasm of bile duct epithelial origin that appears
benign.
the presence of an abnormal amount of fluid (e.g. blood)
in the vessels or passages of a part or organ.
relating to the gall bladder.
142
-------
Cytoplasm
Cytotoxic
Degeneration
Differen-
tiation
Dystrophy
Edema
Encapsulation
Endogenous
Endoplasmic
reticulum (ER)
Eosinopni1ia
Epidermis
Erythema
Etiology
Evagination
Exogenous
Fibrosis
Flexure
Focus
Fusion
the substance of a cell exclusive of the nucleus. :t
contains various organeMes and inclusions within a
orotoolasm.
detrimental or destructive to cells.
a retrogressive pathological change in cells or fssues
that may impair function. Degeneration is reversible at
some stages, but it usually leaas to necrosis.
the acquiring of a character or function different
from that of the original kind; often used to describe
the morpnologic maturation of a tissue or cell type.
defective nutrition.
an accumulation of an excessive amount of watery fluid
in cells, tissues, or serous cavities.
enclosure in a capsule or sheath.
originating or produced within the organism.
the intracellular network of tubules or flattened sacs
with (rough ER) or without (smooth ER) nbosomes on
the surface of their membranes.
staining readily with eosin dyes.
the outer layer of integument of various organs.
inflammatory redness.
the. science and study of the causes of disease and their
mode of operation.
the protrusion of some part or organ from its normal
position.
originating or produced outside the organism.
the formation of fibrous tissue as a reparative or
reactive process.
a bend or curve.
the center or starting point of a disease process.
the growth together or union of two elements.
143
-------
Golgi
apparatus
Hemorrhage
Hemosidenn
Hemosiderosis -
Hepatic
Heoatocarcin- -
ogenesis
Hepatocellular -
Hepatocyte
Homeostasis
Hyalin
Hyalinization -
Hydropic
Hyperemia
Hyperplasia
Hypertrophy
Idlopathlc
Integument
Karyolysis
a comole* of parallel, flattenea saccules, /esicies,
ana vacuoies that lies adjacent :o ine nucleus or a
cell. It is concerned with intraceilular formation
of secretory oroaucts.
bleeaing; a flow of blooa.
an insoluble form of storage iron in wmcn the micelles
of feme hydroxide are so arranged as to be visible
microscopically both with and without the use of specific
staining methods.
the accumulation of excessive amounts of hemosidenn in
tissue.
relating to the 1iver.
the process of induction of cancer in the liver.
pertaining to hepatocytes.
a parenchyma) liver cell.
the state of equilibrium (balance between opposing
pressures) in the body with respect to various functions
and to the chemical compositions of the fluids and tissues.
a clear, eosinophilic, homogeneous substance that occurs
during degeneration.
the formation of hyalin.
generally used to describe intracellular.
the presence of an increased amount of blood in a part
or organ.
the abnormal increase in the number of normal ceils in
normal arrangement in a tissue or organ, excluding tumor
formation.
increase in size of cells, tissues or organs, exclusive
of tumor formation.
denoting a disease of unknown cause.
the covering of any body or part.
apparent destruction of the nucleus of a cell by swelling
and the loss of affinity of its cnromatin for basic dyes.
144
-------
Karyorrnexis
Lesion
Liquefaction
Lymph
Lymphatic
Lymphocyte
Lysosome
Malignant
Megalocytic
hepatosis
Melanin
macrophage
center
Metabolism
Metaplasia
Metastasis
Mitochondria
Mitosis
fragmentation of the nucleus whereoy its cnromatin •=
distributee irregularly tnrougnout the cytoplasm. A
stage of necrosis usually followed by
-------
MitotK figure -
^utagemc
Mutation
Necroosy
Necrosis
Neoplasia
Neoplasm
Neunlemmoma
Nodule
Nucleolus
Nucleus
Oncogenic
Papilloma
Parenchyma
Pathogenesis
Pathology
the microscopic aooearance of a eel 1 undergoing mitosis; a
eel 1 whose cnromosomes are vi sible wi th a i ight microscooe.
navtng cne oower to cause mutations.
a change in the character of a gene that is peroetratea
in subsequent divisions of the cell in which it occurs.
postmortem examination; autoosy.
the pathologic death of one or more cells or oortion of
a tissue or organ resulting from irreversible damage.
the pathologic process that results in the formation and
growth of a neoplasm.
an abnormal tissue that grows autonomously by uncontrolled
cellular proliferation more rapidly than normal and
continues to grow after the stimuli that initiated the
new growth cease.
a benign, encapsulated neoplasm arising from the peripheral
nerve sheath (neunlemma).
a small circumscribed swelling or circumscribed mass of
differentiated tissue.
a small, rounded mass within the cell nucleus where
ribonucleoprotein is produced.
a mass of protoplasm within the cytoplasm of a cell that
is surrounded by a nuclear envelope, which encloses
euchromatin, heterochromatin and one or more nucleoli.
causing, inducing, or being suitable for the formation
and development of a neoplasm.
a branching or lobulated benign neoplasm derived from
epithelium.
the distinguishing OP specific cells of a gland or
organ, contained in and supported by the connective
tissue framework.
the mode of origin or development of any disease or
morbid process.
the science concerned with the essential nature, causes,
and development of abnormal conditions, as well as the
structural and functional changes that result from the
disease processes.
146
-------
Pencho-
lanqms
Petecmae
Pleomorphi sm
i lothermic
Preneoplastic
Prognosis
Proliferate
Pyknosis
Regeneration
Steatosls
Stroma
Trabecular
Tumor
Vacuolatiort
Vacuole
Vascular
VascuHtis
Ventral
Vesicle
Vesicular
Viscera
inflammation of the r.ssues around t^e oile aucts.
minute hemorrnagic soots.
occurrence in more Chan one form.
coid-blooaed; varying in temperature according to the
temperature of the surrrounoing medium.
preceding the formation of any kind of neoplasm.
a forecast of the outcome of a disease.
to grow and increase in number by means of reproduction
of similar forms.
a condensation and reduction in size of the cell or us
nucleus.
reproduction or reconstitution of lost or injured cells,
tissues, or body parr,.
fatty degeneration or fatty change.
the framework, usually of connective tissue, of an
organ, gland or other structure; distinguished from the
parenchyma.
containing bundles of fibers traversing the substance of
a structure.
neoplasm.
the condition of having vacuoles.
a clear space in the substance of a cell, sometimes
degenerative in character.
relating to or containing blood vessels.
inflammation of a blood or lymphatic vessel; angiitis.
the undersurface of a structure.
a small 'ac containing fluid.
characterized by or containing vesicles.
organs of the digestive, respiratory, urogenital, and
endocrine systems as well as the spleen, heart, and
great vessels.
147
-------
APPENDIX A
SUMMARY OF HEPATIC LESIONS
OBSERVED IN FISHES AFTER
LABORATORY EXPOSURE TO
VARIOUS CHEMICALS
-------
TABLE A-l. SUMMARY OF HEPATIC LESIONS OBSERVED IN FISHES
AFTER LABORATORY EXPOSURE TO VARIOUS CHEMICALS
Contaminant
QRGANOCHLORINE
Chlordane
30T
Etoosure
Soute
INSECTICIDES
W
W
W
o,w
0
0
W
W
•»
W
Soeciesd
Lake trout
Coho salmon
Lake trout
Rainbow trout
Rainbow trout
Rainbow trout
Brown trout
Guppy
Guppy
Asian fish sp.
(unspecified)
Lesions6
Focal heoatocyte vacuoiation
Focal heoatocyte degeneration
Hyperenna
Petecmae
Fatty change
Pen portal necrosis
Disorganized architecture
Hyperenna
Petechiae
Fatty change
Hepatocelluar cell carcinoma
Hepatic neoplasm
Cholangiocel lular neoplasm
Hepatic neoplasm
Nuclear hypertrophy
Hepatocyte vacuolation
Severe necrosis
Hepatocyte hypertrophy
Hepatocyte degeneration
^eferenca
Eller, unouol
.n Coucn 1975
Walsh and Ribel \
1975
Wai sh and Ribi
1975
Halver ct al .
Hendricks 193Z
Halver 1967
King 1962
King 1962
Weis 1974
Mathur 1962
Oieldrfn
? Zebra fish
W Various fishes
W Lake trout
Hepatocyte necrosis
Decreased hepatocyte size
Glycogen loss
Hepatocyte pleomorphism
Cytoplasmic vacuoles
Hyperemia
Petechiae
Fatty change
Congestion of sinusoids and
hepatic veins
Weis 1974
Mathur 1965
Walsh and Ribe
1975
A-l
-------
TABLE A-l. (Continued)
Concanunanc
Endosulfan
Endrin
Heptachlor
Route1*
W
W
W
W
W
o,w
W
W
W"
IP
W
W
Exoosure
Soecies
Coho salmon
Ophiocepnatus
punctatus
Trichoqaster
fascmus
Lake trout
Rainbow trout
Cutthroat
trout
Spot
Guppy
Goldfish
Asian catfish
Rainbow trout
Cutthroat
trout
Lesions6
Hyperemia
Petecmae
Fatty cnange
Congestion of sinusoids ana
hepatic /ems
Hepatocyte hypertrophy
Vacuotar degeneration of
cytoplasm
Necrosis
Vacuolar degeneration of
nepatocytes
Localized necrosis
Petechiae
Fatty change
Hepatocyte degeneration
Suggestive preneopiascic
changes
Focal necrosis of nepatocytes
Hepatocyte inflammation
Glycogen loss
Lipid loss
Fatty change
Reduced cytoplasmic vacuol-
atlon
Hepatocyte hypertrophy
Hypertrophy of hepatocyte
nuclei
Centrolobular necrosis
Perilobular vacuolation
Flbrosis
Heavy bile pigment deposits
Hepatocyte degeneration
Deposition of bile pigments
Reference
«aisn and Ribei m
1975
Mathur 1975
Mathur 1975
Walsh and Ribelm
1975
Wood, unpubl . ,
in Couch 1975
E1ler 1971
Lowe 1965
Mount 1962
Grant and MehrU
1970
Sastry and
Sharma 1978
wood, unpubl..
in Couch 1975
Andrews et
al. 1965
A-2
-------
TABLE A-l. (Continuea)
Contaminant
Soute
Eioosure
Soecies
Lesions
Deference
Hexachlorocyclo- W
hexane (beta isomer)
Kepone
lindane
Methoxychlor
Toxaphene
W
W
3Iueqill Hepatocyte snnnkage
Glycogen loss
Lipid loss
Loss of normal architecture
Guppy Proliferation of RER
Hepatocyte basophilia
Hepatocyte vacuolation
Sneepsnead Fatty degeneration
minnow Hepatocyte vacuolation
Small necrotic foci
Rainbow trout Focal necrosis
et
Qphiocephalus
ounctatus
Trichogaster
faseiatus
Vacuolar degeneration of
hepatocytes
Hepatocyte necrosis
Hepatocyte atrophy
Loss of normal cord pattern
Cytoplasmlc alterations
Margination of nuclear
chromatlon
Hypertrophy
Rainbow trout Nonspecific degeneration
Rainbow trout Nonspecific degeneration
81uegll1 Hepatocyte shrinkage
Hepatocyte granulation
Loss of normal cord pattern
EostnophlHc globules in
capillary lumina
Carp Vascular congestion
Hepatocyte degeneration
Rainbow trout Hepatocyte necrosis
Disorganization
of cord archi-
tecture
ai. L966
Wester et al.
1985
Goodman et a).
1982
Wood, unouoI..
in Couch 1975;
Walsh and Ribe
1975
Matnur 1975
Mathur 1975
Walsh and Ribe
1975
Cope 1966
Kennedy et
al. 1970
lakota et al. I97f
wood, unpubl.,
in Couch 1975;
Walsh and Ribel
1975
A-3
-------
TABLE A-l. (Continued)
h -.
-------
TABLE A-l. (Continued)
. EiDOSurg
Contaminant Route Species
INDUSTRIAL CRGANOCHLOR WE COMPOUNDS
PCB-Aroclor 1248 V Lake trout
Lesions6
Focal neoatocyte degeneration
Cytoplasmic vacuolation
Pleomorpnism
Reference
Eller, unouol . ,
in Couch 1975
PCB-Aroclor 1254 W
IP
PCB-Miscellaneous 0
Spot
Rainbow trout
Rainbow trout
English sole
Rainbow trout
Carp
Channel
catfish
Chinook salmon
Fatty change
Focal neoatocyte necrosis
Sinusoidal congestion
Ceroid-Hke inclusion bodies
in parenchyma] cytoplasm
Vacuolation and necrosis of
pancreatic acinar tissue
around oortal tracts with
infiltration of lymphocytes
Variable degree of vacuolation
Hepatocyte density of ques-
tlonable significance
Hepatocyte vacuolation
Enlargement of RER no
longer adjacent to
nuclei or mitochondria
Hepatocyte necrosis
Hepatocyte regeneration
Irregular nuclei
Increased lysosomes
Vacuolation
Lipid accumulation
Glycogen loss
Enlargeaent of RER
Lipid increase
Inconsistent hepatomegaly
Foci of proliferative SER
Vesiculated RER
Circular arrays of smooth
surface membranes and
myelin-Hke bodies in
hepatocyte cytoplasm
Couch 1975
Nestel and
Budd 1975
Sivarajah et
ai. 1978
Rhodes et al.
1985
Hacking et al ,
1978
Sivarajah et
al. 1978
Llpsky et al.
1978
Hawkes 1580
-------
"ABLE A-l. (Continuea;
b r
Contaminant 3oute
GI
Carbon
tetracnlonde IP
IP
Scecies
Channel
catfish
Rainbow trout
Rainbow trout
Lesions8
Prol i feration of ER
Bizarre whorls of RER
ana SER
Hepatocyte vacuolation
Compression of sinusoids
Hepatocyte necrosis
Eosinopmlic degeneration
Hydropic degeneration of
Deference
Hmton et al.
1978;
Oaunig et al .
Racicot et
al. 1975
Gingencn et
al. L978
Rainbow trout
nepatocytes
Pyknosis and coagulative
necrosis in subcapsular areas
Liquefactive necrosis and
karyolysis in centrilooular
regions
Vacuolation
Focal and laminar necrosis
D
•>
Rainbow trout Hepatic neoplasm
Heteropneustes
fossitis
Monochlorobenzene IP.U Rainbow trout
ORGANOPHQSPHATE INSECTICIDES
Abate (temeohos) w Bluegill
Ofazinon (Spec- W
tracide)
Olmethoate (Cygon) w
Asian catfish
Asian catfish
Pericentral necrosis
Fatty infiltration
Pencentral necrosis
Hydropic degeneration
Atrophy
Distortion of muralia
Variability of stain of
nepatocytes
Large foci of edema
Congestion
Hepatocyte necrosis
Granular dystrophy
Cytoplasmic vacuolation
Granular dystrophy
Cytoplasmic vacuolation
Statnam et al.
1978
Halver 1967
Sastry and Agraw«
1975
Gingench and
Oahch 1978;
Oalich et al.
1982
Eller. unpubl., i
Couch 197S
Anees 1976
Anees 1976
A-6
-------
"ABLE A-l. (Continued)
•rcntamnant
3oute
Eoosure
Soecies
Lasions
Deference
Dursoan fcnlor- W
jyn fosi
Oylox |Fri- w
cnlorfon)
Ma lathi on
W
Methyl paratnicn W
W
Sheeosneao.
minnow
Congestion
fatty cnanqe
Rainbow trout Hepatocyte cytoplasmic
vacuolation
Rainbow trout Nonspecific degeneration
Lake trout
Fatty change
Cono salmon Fatty change
Glycogen deposits
Rainbow trout Hepatocyte swelling
Sinusoid congestion
Asian catfish Granular dystrophy
Cytoplasmic vacuolation
Lowe.
;n Coucn 1975
Batten ana
LaHam 1969
«'ood, unouol.,
Walsh ana Ribe
1975
Walsh ara
1975
Wal sh ana Ribe! in
1975
wood, unoub). , .<
Wal sh ana Ribel in
L975
Anees 1976
CAR6AMATE INSECTICIDES
Aldicarb (Teimk)
Carbaryl
(Sevin)
W
W
w
w
Barbus
conchomus
Spot
Lake trout
Cono salmon
Intense vasodilation
EosinophiHc cytoplasm
Cytoplasmic vacuolation
Fatty change
Fatty change
Propoxur (Baygon) W
Carp
Hepatocyte degeneration
Kumar and
Pant 1984
Coucn 1975
Walsh and Ribelin
1975
Walsh and Ribeli
1975
Lakota et at. 19
MISCELLANEOUS HERBICIDES
Acrolein W Cono salmon
Separation of hepatocytes
within muralia
Necrosis
Hendricks 1979
A-7
-------
TABLE A-l. (Continuea)
Contaminant
noosure
Soecies
Lesions
Deference
Oinoseb
Oiquat
Hydrothol 191
Paraquat-CL
Cono salmon Hydropic degeneration of
Meoatocytes
Diffuse coaculatwe necrosis
of hepatocytes
Coho salmon Diffuse necrosis of paren-
chyma) eel Is
Cono salmon Foci of degenerate parenchyma)
cells
Foci of necrotic parenchyma1
cells
Redear sunfish Inflammation
Pigmented neoatocytes
Swollen hepatocytes
Bizarre cells
Distorted cords
Coho salmon Hydropic degeneration
particularly in centn-
lobular areas
Hendnc'ts 1979
Hendncks 1979
Hendncks 1979
Eller L969
Hendncks 1979
FOSSIL-FUEL RELATED COMPOUNDS
3eruo(a)pyrene
(BaP)
Crude oil-whole
Crude oil-water w
soluble fraction
Rainbow trout Hepatic neoplasm
Rainbow trout Glycogen loss in hepatocytes
Proliferation of ER
Presence of cochlear nbosomes
Fibrosis around sinusoids
Hogchoker Focal necrosis
Inland Hepatocyte vacuolation
sllverside Focal necrosis
Nuclear pyknosis
Hogchoker Focal necrosis
Inland Hepatocyte vacuolation
sllverside Focal necrosis
Hendncks et
al. 198Z
Hawkes 1977
Solangi and
Overstreet 1982
Solangi and
Overstreet 1982
Solangi and
Overstreet 1982
Solangi and
Overstreet 1982
A-8
-------
TABLE A-l. (Continued)
b r
'.ontaminant 3oute
7-t2 Oimetnyl- w
oenziaiantnracene
iDMBA)
W
W
Oiled sediments w
CHEMOTHERAPEUTIC AGENTS
Copper sul fate w
Oiethylstil- 0
besterol (OES)
Sul famethazine 0
Thiabendazole W or 0
MYCOTOXINS
Aflatoxin BI 0
0,E
0,E
0,E
D,E
O.E
Soecies
"oominnow
Toominnow
Topminnow
English sole
Carp
Trout
Gudgeon
Rainbow trout
Chinook salmon
Carp
Guppy
Rainbow trout
Rainbow trout
Rainbow trout
Rainbow trout
Rainbow trout
Lesions2
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Increase in 1 ipid volume
of hepatocytes
Hepatocyte lipid increase
Hepatic neoplasm
Degenerative changes in
parenchyma! cells
Hepatocyte hypertrophy
Swelling of intercellular
spaces
Vascular congestion
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Hepa*. c neoplasm
Hepatic neoplasm
Deference
Schultz ana
Schultz 1381
Schultz and
Schultz 1982b
Schultz and
Shultz 1984
McCain et al . 1'
Seichenbach-
Klinke 1975
Halver 1967
Wood et al. 195
Reichenbach-
Klinke 1975
Sato et al. 1973
Halver 1967
Lee et al. 1968
Lee et al. 1971
Sinnhuber et
al. 19 68 a
Sinnhuber et
al. 1968b
A-9
-------
A-i. (Continued)
Contaminant
toute Soectes
Lesions
Deference
Aflatoxin
Aflatoxin
Aflatoxin
Aflatoxlcol
(AFL)
0,£ Saincow trout Heoanc neoplasm
0,£ Rainbow trout Hepatic neoplasm
D,E Rainbow trout Hepatic neoplasm
O.E Rainbow trout Hepatic neoplasm
0,£ Rainbow trout Hepatic neoplasm
O.E
O.E
O.E
Medaka
Hepatic neoplasm
0 Brook trout Hepatic neoplasm
0 Sockeye salmon Hepatic neoplasm
0,E Rainbow trout Hepatic neoplasm
O.E Rainbow trout Hepatic neoplasm
Medaka
Hepatic neoplasm
Rainbow trout Hepatic neoplasm
Rainbow trout Hepatic neoplasm
Rainbow trout Hepatic neoplasm
Rainbow trout Hepatic neoplasm
D,C Rainbow trout Hepatic neoplasm
Rainbow trout Hepatic neoplasm
Sinnnuoer et
al. 1977
iaies et al . 1378
Henanc^s et
al. I380a
Henartcics et
al. 1980c
et
al. 1980f
Hatanaka et a).
1982
Wol f and Jackson
1967
wales and Sinnhui
1972
Ayres et al. 197
Hendncks et al
I980f
Hatanaka et al .
1982
Hendncks et al
1980f
Slnnhuber et a
1974
Hendricks et i
1980d
Hendrlcks et
1978
Hendricks et
1980f
Scnoenhard
et al. 1981
A-10
-------
rABLE A-i. (Contmueo)
Contaminant 3outeC
Ocnratoxm A-8
Stengmatocystine
•/ersi colorin A
PLANT DERIVATIVES
Cycad nut meal
Cycasin
Cyclopropenoid
Fatty acids
(CPFA)
[P
£
E
0
0
E
E
0
W
W
W
0
ExDOSurg
Scecies
Sainoow trout
Rainbow trout
Rainbow trout
Guppy
Medaka
Rainbow trout
Rainbow trout
Zebra fish
Guppy
Rainbow trout
Guppy
Rainbow trout
Lesions6
Nuclear swel I ing
Cytoolasmic l;oid /ac'jolation
of hepatic parencnyma
Hepatic neoplasm
Hepatic neoplasm
Cholangiocel lular neoplasm
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Acute degenerative changes
Hepatic neoplasm
Deference
Ooster et al .
Hendncfcs et a
1980e
HendMcks et a
I980f
Matsusnima
et al. 1975
Hatanaka et al .
1982
Hendricks et al
1980e
Hendncks et al
I980f
Stanton 1966
Hawkins et al .
1983
Hendncks et al
1983
Stanton 1966
Sinnhuber et al
1976; Hendncks
et al. 1980c;
Scnoennaru et
al. 1981
A-ll
-------
TABLE A-l. (Continued)
loncammant
Soecies
Lesions
Deference
jossypol
Methylazoxy-
ntetnanoi acetate
(MAMA)
Pyrrolizidine
alkaloids
Tannic acid
W
trout
Medaka
Medaka
Guppy
Rainbow trout
Foci of fatty cnanqe
Bizarre nuclei
Hepatocellular regeneration
Heoatocyte necrosis arouna
bile ducts
Inflammation of penductal
tissue
Generalized deposition of
ceroid pigment
Hepatic neoolasm
Hepatic neoplasm
Cholangiocellular neoplasm
Hepatic neoplasm
Megalocytosis
Intense eosinophilia
Nuclear aberrations
Mlcrodroplet fatty change
Hepatocyte necrosis
Focal hepatocyte regeneration
Fibrosis in hepatic parencnyma
Veno-occlusive disease in the
centrolobular and hepatic
veins
Herman 1970
Aoki ana Matsuoai
1977; Aoki ana
Matsudaira 1980:
Hatanafca et a I.
1982; Hawkins
et al. 1933
Aoki and Matsuaair
1984
Hawkins et al.
1983
Hendncks et al.
1981
Rainbow trout Hepatic neoplasm
Halver 1967
N1TROSO- COMPOUNDS
N.M'-dlnitroso-
piperazine
(DNP)
N-nitrosodl-
ethylamine (OCN)
Guppy
Guppy
Hepatic neoplasm
Hepatic neoplasm
Simon and Lapis
19B4
Khudoley 1971. 197:
Pliss and Khudoley
1975
A-12
-------
TABLE A-L. (Cantinueal
Contaminant Souts Species
Lesions
6
Deference
N-nitrosodi-
metnylamire
(OMN)
w Guooy
W Topminnow
W Medaka
0+W
Zebra fish
Heoatic neoolasm
Cholangiocellular neolasm
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Zebra fish Hepatic neoplasm
Cholangiocellular neoplasm
Rivulus Hepatic neoplasm
Cholanqiocellular neoplasm
Rivulus Hepatic neoplasm
Sheeoshead Hepatic neoplasm
minnow Cholangiocellular neoplasm
Topminnow Hepatic neoplasm
Cholangiocellular neoplasm
Guppy Hepatic neoplasm
Guppy
Rainbow trout Hepatic neoplasm
Hepatic neoplasm
Cholangiocellular neoplasm
-------
TABLE A-L. (Continuea)
Contaminant
N-methyl-T-
nuro-N-mtroso-
guantdine (MNNG)
N-nitroso-
moronoline (NM)
qoutec
w
«
e
w
w
w
w
MISCELLANEOUS NITROGENOUS
2-Acetylamino-
fluorine
(2-AAF)
o-Amlno-
azotoluene
(o-AAT)
0
0
0
w+o
V*0
0
0
Excosuro
Soecies
Zebra fisn
Zebra fisn
Rainbow trout
Guppy
Guppy
Zebra fish
Zebra fish
COMPOUNDS
Guppy
Rainbow trout
Zebra fish
Guppy
Guppy
Rainbow trout
Medaka
Lesions6
Hepatic neoplasm
Heoacic neoplasm
Cholangtoceiluiar neoolasm
Hepatic neoplasm
Hepatic neoplasm
Heoatic neoplasm
Cholangjocel lular neoplasm
Hepatic neoplasm
Hepatic neoplasm
Cholangiocel lular neoplasm
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Adenomatous hyperplasia
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Reference
31 i ss ana
-------
TABLE A-l. (Continued)
Contaminant
3outec
0
Soecies
Zebra fish
Lesions6
Heoatic neoplasm
Peferance
3l ' ss ana
1975
Halver 1967
Ermer 1970
Pllss and Khudoie)
1975
Thiourea
Urethane
0 Rainbow trout Hepatic neoplasm
D Rainbow trout Hepatic neoplasm
Halver 1967
Halver 1967
A-15
-------
TABLE A-l. (Continued)
Contaminant
Routc
Soecies
Lesions
Seference
•MISCELLANEOUS ORGANIC AND QRGANOMETALL 1C COMPOUNDS
tn-n-
butyi
DimethylsuIfoxide
(OMSO) IP
Methylmercunc IP
chlonde
(CH3HgC12)
Rainbow trout
Rainbow trout
Chinook salmon
Coho salmon
Sockeye salmon
Channel
catfish
Lisa
Sinusoid congestion Chi'
Necrosis and Kuhn 1977
Thinning and separation of
biliary epithelium from
basement metnorane
Subcapsular necrosis Benvilie et
Portal necrosis al. 1968
Periportal necrosis of exo- Kendall 1977
crine pancreas and sur-
rounding nepatocytes
Desquamation of biliary
epithelium into duct lumina
Inflammatory exudate on
surface of liver capsule
Heoatocyte vacuolation Establier et
Proliferation and dilation of al. 1978a
capillaries
Disorganization of muralia
4-Mitro-3-
(trifluoromethylJ
phenol
Phenol
Lamprey
Walking
catfish
Erythema
Focal necrosis
Vacuolation
Christie and
Battle 1963
Mukherjie and
Bhattacharya
1975
METALS
Cadmium chloride 0 Carp
(CdCl2)
IP Goldfish
Enlarged lysosomes
Glycogen loss
Formaclan of macrophage
granutomas
Koyama et al.
1979
Tafane 111 and
Summerfelt 1975
A-16
-------
rABLE A-l. (Continued)
Contaminant 3outeC
Soecies
Lesions
Deference
w
Saoo
increase n connecti-e cissue
Increase in numoers or" neoaco-
cyte nuclei
Sambow trout Glycogen loss
'.utierrez a.f.
al. 1978
An I lo et al .
1982; Larsson
and Haux 1982
Lowe-Jmae am
Niimi
Flounder
Increased glycogen levels
W
w
Cupnc chloride w.IP
(CuClz)
Cupnc sulfate W
(CuS04)
01 sodium arsenate w
(NajHAsOd)
Asian catfish Glycogen loss
Wa Ik ing
catfish
English sole
Mummichog
Winter
flounder
Green sunfish
Lipid gain
Cholesterol gain
Hepatocyte necrosis
Hepatocyte regeneration
Hepatocyte karyomegaly
Focal necrosis
Larsson ana
1982
Oubale and Shan
-------
TABLE A-l. (Continued)
Contaminant
Route
Species
Lesions
e
Deference
Lead nitrate
Mercuric chloride w
Sodium arsenlte
(NaA$02)
Sastry and
Guota I978a
Asian catfish Disorganization of muralia Sastry and
focal Heoacocyte necrosis. Quota 19786
esoecially in centnlooular
areas
Portal and cenlobular infil-
tration of inflammatory cells
Perivascular fibrosis
Dilation of intranepatocyte
spaces
Deposition of lipofuscin
granules in hepatocyce
cytoplasm
Asian catfish Perilobular necrosis
Centnlobular necrosis
Hepacocyte glycogen loss
Disarray of muralia
Cirrhosis
Lipid deposition with infil-
tration of phagocytic in-
flammatory cells in vascu-
lature and intercellular
spaces
Lisa Proliferation of dilated Estaolier et
vascular elements al. L978a
Vacuolar degeneration of
hepatocytes
Disorganization of muralia
Robalo Hepatocyte vacuolation EstaDHer et
Hepatocyte degeneration al. 1978b
Congestion of capillaries
Bluegill Fatty infiltration Gilderhaus
Focal necrosis 1966
A-18
-------
*JBL£ A-l. (Continued)
a "ms caoie is aasea on review articles oy Matsusnima and Sugiaiura (1976). .*eyers ana Her"
(1982), ana Coucn ana Harsnoarger (1985), 'as well as a seoarate re-new conducted for Me -
i?82-36 as oart of tne oresent study. All studies identified in the tnree review artic!e •
•ioc seen as cart of tne present study.
Contaminants are grouped according to cfie general scneme of Meyers and Henancks (19 •
facilitate tneir interpretaoility by environmental managers.
c W - water, 0 - diet, IP • mtraperuoneal injection, GI • mtragastnc mtucation.
Scientific names of soecies are presented in Table 2.
e Lesions generally are described using the nomenclature of the original autnors. and n"
/ague or amoiguous in some cases.
A-19
------- |