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

May 1S37
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

     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


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

    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

             LABORATORY EXPOSURE TO VARIOUS CHEMICALS                   A-l


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



   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

     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.

                              1.0   INTRODUCTION

      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


          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).


     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.


 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.

                        2.0  BACKGROUND INFORMATION

     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

     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

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.

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

     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.

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,

     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

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.


                            ENTERO HEPATIC
                                 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
Liver Secretions
          Figure 2.  Schematic of major contaminant pathways in relation to the fish liver

      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


                   • OXIDATION

                   • REDUCTION

                   • HYDROLYSIS

                                   • GLUCURONIOE  CONJUGATION

                                   • GLUTATHIONE  CONJUGATION

                                   • SULPHATION
                                                               Reference Modified ham Connull and Miller 1984
     Figure 3. Generalized biolransformation pathways (or exogenous chemicals

(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


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


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):
          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

     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).

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:

     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


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


*.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


(e.g.,  primitive-appearing)  from normal  cells  (i.e.,  they  are  'j.naiffaren-

     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

     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


 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

     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.

     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

     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

     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.


     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


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

     •    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-


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

     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
               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
               Cholangiocellular carcinomas
               Mixed carcinomas.


     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


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

          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.


     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

                                                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

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


     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

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

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.

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


 Scienttfie  Name
 Common  Name









 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

 Coho  salmon
 Sockeye salmon*
 Chinook salmon
 Cutthroat  trout
 Rainbow trout*
 Brown trout
 Lake  trout
                                                            Zebra fish*
Channel catfish

Walking catfish





 si 1verside


TABLE 2.  (Continued)
Scientific Name
Common Name


Channa punctatus
Qghiocephalus punctatus

Oicentrarchus labrax

Lepomus cyanellus
Asian catfish


Green sunfish
Lepomus macrochirus
Lepomus imcrolophus
Leiostomus xanthurus
Mugil auratus
Trichogaster fasclatus
Parophrys vetulus
Platlchtnys flesus
Pseudopleuronectes americanus
Trlnectes maculatus
Redear sunfish
English sole
Winter flounder
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-
H 12-
O 10 —
01 OD
1 1


p- '•



»*••-• •








- 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.

(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


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


Puget Sound, WA

Fox River, IL

Black River, OH

Torch Lake, MI

Hudson River, NY
Boston Harbor, MA

Deep Creek
Lake, MD
Elbe Estuary,
Gull mar Fjord,
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
Falkmer et al . 1976

Engl ish sole
English sole
Starry flounder
English sole
Rock sole
Pacific staghorn
English sole
English sole
English sole
English sole
Brown bullhead
Brown bullhead
Brown bullhead
Brown bullhead

Atlantic tomcod
Winter flounder

White sucker


Atlantic hagfish

Samel e












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.

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

     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


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-

     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


      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.

     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


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.


     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

 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.


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

     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.


     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

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-


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.


     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-

     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

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).

     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.

     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

     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


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

     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


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.

     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.


3.1.1  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

                             I ABLE 4.  OIARACIERISMCS  OF  FISHES  FOUND  10 HAVE  ELEVAJED
                                 PREVALENCES OF HEPAIiC NEOPLASMS  IN FIELD STUDIES9

(bullhead catfishes)
£ Percidae
(right eye flounders)
Common Name
Atlantic hagfish
White sucker
Brown bullhead
Atlantic tomcod
Pacific staghorn
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 llomuuj
Prey Ability.'-
Bl Yes
F Yes
Bl Yes
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.

     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

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


                        H«oatle  Ntoolumi In
                          Atlantic Hagllsh
    40    60    30

                             Hepatic Moduli*
                                 In Ruff*
                         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*
                             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).

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

     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.

     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

     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.


                                              AGE 3
         0 J // .  . T* . '   —.      —r
         u  //I  I  I  I  I  I  I  I ^^ | T
                                              AGE 7
                     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.

     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


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





300 -

280 -
















  Rgure 7.  Sample size required to detect one individual affected with a
            lesion with 95% confidence, given various population sizes

            and prevalences.

 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


 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



FISH wir Hour
Figure 8. Example of a 2 x 2 contingency table.

(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

     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

     r    -    number of rows in the r x s contingency  table  (r-2 in a 2 x 2

     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

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)
     A = the noncentrality parameter
     f • degrees of  freedom.

     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,

     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

        m   0.2-
                                                   PREVALENCES  (%)
                                                      REFERENCE  TEST
                             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%




                           O  0.4 -
0.2 -



      SITE     SITE

-O-     5        10

-•-5        15

•*•     5


                                           40       80       120      160      200       240

                                                SAMPLE  SIZE  (MARGINAL SUM)
                                                                            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

     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


                                70 T   SAMPLE  SIZE
00      2.5      5.0      7.5     100     125     150    17.5

                                                                            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.

        20 -
   «-.   10-

                                     HEPATIC  NEOPLASMS

                                     P>0.05,  G-tast

..•• '

              JANUARY   APRIL     JULY   OCTOBER

                (182)      (95)      (112)     (162)


                         (Sample   Size)
                                     Reference: Modified from McCain tt al 1982.
Figure 12.  Seasonal variation of hepatic lesions in English sole from

           the Duwamish River, WA.

     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

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


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-

3.2.1  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.

     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

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

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.


     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).


     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


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

     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-


     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


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.

     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

after  laboratory  exoosure  to  kepone  and  heavy metals  (Sinaermann  5t  oi.

     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

      •    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.


 3.3.1  Tissue  Processing


      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.

     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-

     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


 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


     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-

     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.


     Following sectioning,  tissues are mounted  onto glass microscope  slides
 (Luna  1968).   This  procedure involves floating  tissue  sections in a warm-


 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.


     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.

     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-



     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).

 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


•;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,


: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


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).

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.

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


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.


     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


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

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.

     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:


                                    ,\    /_* __  _" __ A
                                    V    Kl-N21   N12'N22  V
                                                                        (  }
     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

     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

          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


       o.io n
    O  0.08

        0.04 H
   0.02 -
                                                          UNCORRECTED VALUES

                                                          NOMINAL TYPE I ERROR




                                       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

     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.


     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.

     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).

                               d.O  SUMMARY

     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.


     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

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

     •    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

     •    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
               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


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

      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-


•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:

     •    .-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.

 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-

     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.

     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-

     •    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


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

     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

•    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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 Pliss,  G.B.,  and  V.V. Khudoley.   1975.   Tumor induction by  carcinogenic
 agents in aquarium fish.  J. Natl. Cancer  Inst.  55:129-136.

 Racicot, O.G., M. Gaudet,  and  C. Leray.   1975.   Blood  and  liver  enzymes  in
 rainbow trout (Salmo gairdneri  Richardson) with  emphasis on their diagnostic
 use:   study  of  CCL.  toxicity  and  a  case of  Aeromonas  infection.
 J. Fish. Biol.   7:825-835.                            	

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 Neptune, NJ.

 Reichenback-KIinke, H.-H.   1975.  Lesions  due to drugs,   pp.  647-656.   In:
 Pathology  of  Fishes.   W.E. Ribelin and  G.  Mlgaki   (eds).   University  of
Wisconsin Press,  Madison, WI.

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Can. J. fish. Aauat. Sci.  -2:1870-1380.

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                                S.O  GLOSSARY
        1 ic



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
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
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.


reticulum (ER)

the  substance  of  a cell  exclusive  of  the nucleus.   :t
contains  various  organeMes  and  inclusions within a
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
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.


Hemosiderosis  -


Heoatocarcin-  -

Hepatocellular -




Hyalinization  -







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

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

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.












fragmentation of  the nucleus  whereoy its  cnromatin  •=
distributee  irregularly  tnrougnout  the  cytoplasm.   A
stage of necrosis usually followed by 
MitotK figure -















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

 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.

Pleomorphi sm
    i lothermic

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
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
containing bundles of fibers traversing the  substance of
a structure.
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.

        APPENDIX A




Lake trout
Coho salmon
Lake trout
Rainbow trout
Rainbow trout
Rainbow trout
Brown trout
Asian fish sp.

Focal heoatocyte vacuoiation
Focal heoatocyte degeneration
Fatty change
Pen portal necrosis
Disorganized architecture
Fatty change
Hepatocelluar cell carcinoma
Hepatic neoplasm
Cholangiocel lular neoplasm
Hepatic neoplasm
Nuclear hypertrophy
Hepatocyte vacuolation
Severe necrosis

Hepatocyte hypertrophy
Hepatocyte degeneration

Eller, unouol
.n Coucn 1975
Walsh and Ribel \
Wai sh and Ribi
Halver ct al .
Hendricks 193Z
Halver 1967
King 1962
King 1962
Weis 1974
Mathur 1962
?       Zebra fish

W       Various fishes

W       Lake trout
Hepatocyte necrosis

Decreased hepatocyte  size
Glycogen loss

Hepatocyte pleomorphism
Cytoplasmic vacuoles

Fatty change
Congestion of  sinusoids and
  hepatic veins
                                                                        Weis  1974
Mathur 1965
                                                                         Walsh  and Ribe

TABLE A-l.   (Continued)



Coho salmon
Lake trout
Rainbow trout
Asian catfish
Rainbow trout
Fatty cnange
Congestion of sinusoids ana
hepatic /ems
Hepatocyte hypertrophy
Vacuotar degeneration of
Vacuolar degeneration of
Localized necrosis
Fatty change
Hepatocyte degeneration
Suggestive preneopiascic
Focal necrosis of nepatocytes
Hepatocyte inflammation
Glycogen loss
Lipid loss
Fatty change
Reduced cytoplasmic vacuol-
Hepatocyte hypertrophy
Hypertrophy of hepatocyte
Centrolobular necrosis
Perilobular vacuolation
Heavy bile pigment deposits
Hepatocyte degeneration
Deposition of bile pigments
«aisn and Ribei m
Mathur 1975
Mathur 1975
Walsh and Ribelm
Wood, unpubl . ,
in Couch 1975
E1ler 1971
Lowe 1965
Mount 1962
Grant and MehrU
Sastry and
Sharma 1978
wood, unpubl..
in Couch 1975
Andrews et
al. 1965

TABLE A-l.   (Continuea)
Hexachlorocyclo-   W
hexane (beta isomer)

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
                Vacuolar degeneration of
                Hepatocyte necrosis
                Hepatocyte atrophy
                Loss of normal cord pattern

                Cytoplasmlc alterations
                Margination of nuclear
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
of cord archi-
                                                                           ai.  L966
                                                Wester et  al.
                                                Goodman et  a).
                       Wood,  unouoI..
                       in  Couch  1975;
                       Walsh  and  Ribe

                       Matnur 1975
                       Mathur  1975
                       Walsh and  Ribe

                       Cope 1966

                       Kennedy et
                       al.  1970
                                                                           lakota  et  al.  I97f
                       wood, unpubl.,
                       in Couch 1975;
                       Walsh and Ribel

TABLE A-l.   (Continued)
           h                -. 
TABLE A-l.   (Continued)
. EiDOSurg
Contaminant Route Species
PCB-Aroclor 1248 V Lake trout
Focal neoatocyte degeneration
Cytoplasmic vacuolation
Eller, unouol . ,
in Couch 1975
PCB-Aroclor 1254   W
PCB-Miscellaneous  0
                           Rainbow trout

                           Rainbow trout

                           English sole

                           Rainbow trout
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
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.

                                               Hacking et al ,
Sivarajah et
al. 1978

Llpsky et al.
                                                                           Hawkes  1580

"ABLE  A-l.  (Continuea;
b r
Contaminant 3oute
tetracnlonde IP
Rainbow trout
Rainbow trout
Prol i feration of ER
Bizarre whorls of RER
ana SER
Hepatocyte vacuolation
Compression of sinusoids
Hepatocyte necrosis
Eosinopmlic degeneration
Hydropic degeneration of
Hmton et al.
Oaunig et al .
Racicot et
al. 1975
Gingencn et
al. L978
                           Rainbow trout
                Pyknosis  and  coagulative
                  necrosis  in subcapsular  areas
                Liquefactive  necrosis  and
                  karyolysis  in  centrilooular

                Focal  and laminar necrosis

Rainbow trout   Hepatic neoplasm
Monochlorobenzene  IP.U    Rainbow trout


Abate (temeohos)    w       Bluegill
Ofazinon (Spec-    W

Olmethoate (Cygon) w
Asian catfish

Asian catfish
                Pericentral  necrosis
                Fatty infiltration

                Pencentral  necrosis
                Hydropic degeneration
Distortion of muralia
Variability of stain of
Large foci of edema
Hepatocyte necrosis

Granular dystrophy
Cytoplasmic vacuolation

Granular dystrophy
Cytoplasmic vacuolation
                                Statnam et al.

                                Halver 1967

                                Sastry and Agraw«

                                Gingench and
                                Oahch 1978;
                                Oalich et al.
                                               Eller. unpubl., i
                                               Couch  197S
Anees 1976
Anees 1976

"ABLE  A-l.  (Continued)
Dursoan fcnlor-    W
jyn fosi

Oylox |Fri-        w
Ma lathi on
Methyl  paratnicn   W
                          fatty cnanqe
Rainbow trout   Hepatocyte cytoplasmic

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
;n Coucn 1975

Batten ana
LaHam 1969

«'ood, unouol.,
Walsh ana Ribe
                                                                           Walsh  ara
                                                                           Wal sh  ana  Ribe! in

                                                                           wood,  unoub). ,   .<
                                                                           Wal sh  ana  Ribel in

                                                                           Anees  1976
Aldicarb (Teimk)

Lake trout
Cono salmon
Intense vasodilation
EosinophiHc cytoplasm
Cytoplasmic vacuolation
Fatty change
Fatty change
Propoxur (Baygon)  W
                                           Hepatocyte degeneration
                                                                           Kumar and
                                                                           Pant 1984

                                                                           Coucn 1975
                                                                           Walsh and Ribelin

                                                                           Walsh and Ribeli

                                                                           Lakota et at.  19

Acrolein           W       Cono salmon
                          Separation of hepatocytes
                            within muralia
                                                                           Hendricks 1979

TABLE A-l.   (Continuea)


Hydrothol  191
Cono salmon     Hydropic degeneration of
                Diffuse coaculatwe  necrosis
                  of hepatocytes

Coho salmon     Diffuse necrosis  of  paren-
                  chyma)  eel Is

Cono salmon     Foci of degenerate parenchyma)
                Foci of necrotic  parenchyma1

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

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

TABLE  A-l.   (Continued)
b r
'.ontaminant 3oute
7-t2 Oimetnyl- w
Oiled sediments w
Copper sul fate w
Oiethylstil- 0
besterol (OES)
Sul famethazine 0
Thiabendazole W or 0
Aflatoxin BI 0
English sole

Rainbow trout
Chinook salmon

Rainbow trout
Rainbow trout
Rainbow trout
Rainbow trout
Rainbow trout
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
Vascular congestion

Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Hepatic neoplasm
Hepa*. c neoplasm
Hepatic neoplasm
Schultz ana
Schultz 1381
Schultz and
Schultz 1982b
Schultz and
Shultz 1984
McCain et al . 1'

Klinke 1975
Halver 1967
Wood et al. 195
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-i.   (Continued)
toute      Soectes
                  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
                          Hepatic  neoplasm
  0       Brook trout     Hepatic  neoplasm

  0       Sockeye salmon  Hepatic  neoplasm

 0,E      Rainbow trout   Hepatic  neoplasm

 O.E      Rainbow trout   Hepatic  neoplasm
                          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

al. 1980f

Hatanaka et a).

Wol f and Jackson

wales and Sinnhui

Ayres et al. 197

Hendncks et al

Hatanaka et al .

Hendncks et al

Slnnhuber et a

Hendricks et i

Hendrlcks et

Hendricks et

et al. 1981

rABLE A-i.   (Contmueo)
Contaminant 3outeC
Ocnratoxm A-8

•/ersi colorin A

Cycad nut meal

Fatty acids
Sainoow trout
Rainbow trout
Rainbow trout
Rainbow trout
Rainbow trout
Zebra fish
Rainbow trout
Rainbow trout
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
Ooster et al .
Hendncfcs et a
HendMcks et a
et al. 1975
Hatanaka et al .
Hendricks et al
Hendncks et al
Stanton 1966
Hawkins et al .
Hendncks et al
Stanton 1966
Sinnhuber et al
1976; Hendncks
et al. 1980c;
                                                                   Scnoennaru et
                                                                   al.  1981

 TABLE A-l.   (Continued)
ntetnanoi acetate
Tannic acid


Rainbow trout
Foci of  fatty cnanqe
Bizarre  nuclei
Hepatocellular regeneration
Heoatocyte necrosis arouna
  bile ducts
Inflammation of penductal
Generalized deposition of
  ceroid  pigment

Hepatic  neoolasm
                                           Hepatic neoplasm
                                           Cholangiocellular neoplasm
Hepatic neoplasm
Intense eosinophilia
Nuclear aberrations
Mlcrodroplet fatty change
Hepatocyte necrosis
Focal hepatocyte regeneration
Fibrosis in hepatic parencnyma
Veno-occlusive disease in the
  centrolobular and hepatic
 Herman  1970
 Aoki  ana  Matsuoai
 1977;  Aoki  ana
 Matsudaira  1980:
 Hatanafca  et  a I.
 1982;  Hawkins
 et al.  1933
Aoki and Matsuaair

Hawkins et al.

Hendncks et  al.
Rainbow trout   Hepatic neoplasm
                                Halver 1967


ethylamine (OCN)
Hepatic neoplasm
Hepatic neoplasm
Simon and Lapis
Khudoley 1971. 197:
Pliss and Khudoley

TABLE A-L.   (Cantinueal
Contaminant       Souts       Species
w      Guooy

W      Topminnow

W      Medaka
                           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

                           Rainbow trout   Hepatic neoplasm
                         Hepatic neoplasm
                         Cholangiocellular neoplasm

TABLE  A-L.   (Continuea)

guantdine (MNNG)
moronoline (NM)



Zebra fisn
Zebra fisn
Rainbow trout
Zebra fish
Zebra fish
Rainbow trout
Zebra fish
Rainbow trout
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
31 i ss ana 
TABLE  A-l.   (Continued)


Zebra fish
Heoatic neoplasm
3l ' ss ana 

Halver 1967

Ermer 1970

Pllss and Khudoie)

0       Rainbow trout   Hepatic neoplasm

D       Rainbow trout   Hepatic neoplasm
                                                Halver 1967

                                                Halver 1967

TABLE A-l.   (Continued)
(OMSO)             IP
Methylmercunc     IP
Rainbow trout
Rainbow trout
Chinook salmon
Coho salmon
Sockeye salmon

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
Disorganization of muralia

Focal necrosis
Christie and
Battle 1963
Mukherjie and

Cadmium chloride   0       Carp

                   IP      Goldfish
                Enlarged lysosomes
                Glycogen loss

                Formaclan of macrophage
                                Koyama et al.

                                Tafane 111 and
                                Summerfelt 1975

rABLE  A-l.  (Continued)
Contaminant      3outeC
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
                Increased glycogen  levels

Cupnc chloride  w.IP

Cupnc sulfate     W

01 sodium arsenate  w
Asian catfish   Glycogen loss
Wa Ik ing

English sole

Green sunfish
Lipid gain
Cholesterol  gain

Hepatocyte necrosis
Hepatocyte regeneration
Hepatocyte karyomegaly

Focal necrosis
                                Larsson  ana
Oubale and Shan

TABLE  A-l.   (Continued)
Lead nitrate
Mercuric chloride  w
Sodium arsenlte
                                                         Sastry and
                                                         Guota I978a
Asian catfish   Disorganization of muralia      Sastry and
                focal  Heoacocyte necrosis.      Quota 19786
                  esoecially  in centnlooular
                Portal and  cenlobular  infil-
                  tration of  inflammatory cells
                Perivascular  fibrosis
                Dilation of intranepatocyte
                Deposition  of lipofuscin
                  granules  in hepatocyce

Asian catfish   Perilobular necrosis
                Centnlobular necrosis
                Hepacocyte  glycogen  loss
                Disarray of muralia
                Lipid deposition with  infil-
                  tration of phagocytic in-
                  flammatory cells  in  vascu-
                  lature and  intercellular

Lisa            Proliferation of dilated        Estaolier et
                  vascular  elements             al.  L978a
                Vacuolar degeneration  of
                Disorganization of  muralia

Robalo          Hepatocyte  vacuolation         EstaDHer et
                Hepatocyte  degeneration        al.  1978b
                Congestion  of capillaries

Bluegill        Fatty infiltration              Gilderhaus
                Focal necrosis                 1966

*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.