United States

              Environmental Protect.on                 EPA-600/9-83-005
4>EPA       Research and
              Development
             EFFECTS  OF CARCINOGENS, MUTAGENS,
             AND  TERATOGENS ON NONHUMAN
             SPECIES (AQUATIC ANIMALS )
             FOURTH ANNUAL REPORT
             NCI/EPA COLLABORATIVE PROGRAM
             Prepared  for

             National Cancer Institute

             Office of Pesticides
             and Toxic Substances
             Prepared  by

             Environmental Research
             Laboratory
             Gulf Breeze FL 32561

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                                 DISCLAIMER
   This report has been reviewed by  the U.S.  Environmental  Protection Agency,
and approved for publication.  Approval does  not  signify that the contents
necessarily reflect the views  and policies of the U.S.  Environmental
Protection Agency, nor does mention  of trade  names  or  commercial  products
constitute endorsement or recommendation  for  use.

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                Fourth Annual Report
            NCI/EPA Collaborative Program
Fiscal Year 1982 (October 1981 - September 30, 1982)
                      Project 3
          EFFECTS OF CARCINOGENS, MUTAGENS,
             AND TERATOGENS ON NONHUMAN
             SPECIES (AQUATIC ANIMALS).
                         by
  John A. Ccuch, Ph.D., Coordinator, Pathobiologist
          W. Peter Schoor, Ph.D.  Biochemist
            Will Davis, Ph.D., Biologist
                         and
         Lee Courtney,  Research Toxicologist
         U.S.  EPA,  ERL, GULF BREEZE, FLORIDA

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TABLE OF CONTENTS

II.      Abstract	2
III.     Introduction 	  4
IV.      Objectives	5
V.       Methodological Approaches  	  5
VI.      Major Findings and Results 	  7
VII.     Significance to Biomedical Research and
         Program needs of NCI and EPA	13
VIII.    Future Plans	14
IX.      Date Contract Initiated and
         Period of Contract Planned 	 14
X.       Contractors Project Director and
         Project Officers 	 14
XI.      Progress Reports on Specific Extramural
         Cooperative Agreements 	 14
XII.     Publications Resulting from Program  	 39

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                EFFECTS  OF  CARCINOGENS,  MUTAGENS,  AND  TERATOGENS
                     ON  NON-HUMAN  SPECIES-AQUATIC  ANIMALS

           John A.  Couch,  Coordinator;  W.  Peter Schoor,  Biochemist;
          Will  Davis, Biologist; Lee  Courtney, Research  Toxicologist

                  United  States  Environmental  Protection  Agency

                           Gulf  Breeze,  Florida  32561


     Aquatic  systems and organisms are  under  both  laboratory  and  field  study in
order to  develop  indicator,  screening,  and  modeling capabilities  for  detection
and  evaluation  of risks  of carcinogens,  mutagens,  and  teratogens.   Studies
include both  Gulf Breeze laboratory projects  and  complementary, extramural
projects.  In the fourth year  of the  program,  several  advances were made  in  the
development of  laboratory  and  field carcinogen assays, utilizing  fishes such  as
the  sheepshead  minnow (liver lesions  via benzidine  and aflatoxin  exposures),
Menidia peninsulae (liver  lesions  via aflatoxin exposures), and freshwater cat
fish (papillomatous-like lesions via  chlorinated  effluent  exposures).   Emphasis
is still  placed on the  development and  utilization  of  critical life stage
exposures (e.g.,  embryo  and  newly  hatched  fry  exposures) in order  to  expedite
carcinogen tests  and minimize  time required for tumorogenic responses.
Preneoplastic hepatic lesion development in Menidia at 12  weeks suggests
promise for this  species and exposure method.  A  novel approach has shown that
tiger salamanders may be good  biochemical  and  histologic indicators of  the
presence  of certain carcinogens (polycyclic aromatic hydrocarbons  - PAH's).
Skin and  liver  tissues  of  the  salamanders  revealed  induced enzyme  activity (MFO
system) following exposure to  the  PAH,  perylene.   Considerable field  monitoring
work on mollusks  and carcinogenic  PAH's  along  the  coast  of Oregon  has revealed
a positive correlation  between  prevalence  of cellular  proliferation disorders
in shellfish  and  higher  concentrations  of  certain  PAH's  in natural water.

     Emphasis in  biochemistry  for  the last year has been directed  mostly toward
the  elucidation of the  metabolism  of  the mixed-function  oxidases  in marine
organisms.  From  continuing  work on the  mullet (Mugil  cephalus), we have found
that benzo(a)pyrene (B(a)P)  is  converted mainly to  3-hydroxybenzo(a)pyrene,
9-hydroxybenzo(a)pyrene  and  4,5-,  7,8-,  9,10-dihydrodiols  of  BaP,  in  j_n vitro
systems containing liver microsomes from Aroclor  1254-treated mullet.   Other
metabolites such  as diones of B(a)P and  probably  tri-and tetraols  have  also
been produced.  We have  also developed  a procedure  for the epoxide hydro!ase
enzyme system in  order to  determine the  role of this enzyme on the metabolism
of the polyaromatic hydrocarbons.  Our  studies show that UDP-glucuronosyl
transferase and sulfotransferase were significantly increased in  livers of rats
given phenobarbital (PB),  phenolphthalein,  phenanthrene, BaP  or 3-MC  orally  for
10 to 14 days.  The injection of PB or  3-MC into  rats, sea catfish or mullet  on
long-term schedule resulted  in  increase  in  liver  size, and an increase  in
UDP-glucuronosyl  transferase in livers  of  all  three animal species.   We have
observed  that the liver  microsomal  fraction of the  killifish, Fundulus
qrandis, has  a  high in vitro activity when  injected with a single  dose  of 3-MC,
converting B(a)P  to 1-OH,  3-OH, 5-OH, 6-OH, 7-OH,  (t)  9,10-diol,  (t)

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7,8-diol, 1,6-, 3,6-, and 6, 12 diones, and 4,5-oxide.  There may be  an  optimal
concentration of MADPH, and this concentration may have a significant effect  on
the products of the in vitro reaction, quantitatively as well as
qualitatively.  These studies continue to reveal that fish have metabolic
pathways similar to mammals for disposition of certain carcinogens.   A brief
study was conducted to determine possible interactions between the 9-OH  BaP and
salmon sperm DNA.  The interaction between salmon DNA and 9-OH BaP was shown  by
relative fluroescence studies.  The binding was evidenced by an eight-fold
reduction in relative fluorescence yield.  We are developing methods  to
determine the possible binding of proximal carcinogenic metabolites to DNA's  of
selected aquatic animals.

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                               III.   Introduction

     A major problem faced by  the National  Cancer  Institute  and  the
Environmental Protection Agency  is  that  of  aiding  in  determining the  fate  and
effects of carcinogenic pollutants  in the larger environment.  One way  is  to
study wildlife populations that  are  widespread, but which  live  in  environments
where exposure to ambient pollutants  is  certain.   The  use  of  wildlife
populations -as surrogates for  human  populations may be considered  to  be  a  novel
expansion or logical extension of the use of  laboratory  animals  and animal
models of human diseases as  alternatives to the use of human  subjects.   The
sharing of biologic characteristics  by phylogenetically  diverse  species  makes
certain comparative approaches possible.
     Problems arise, however,  when  such  factors as selection  of  sensitive,
indicative species, geographically  adequate populations, and  representative
segments of the air, land, water biosphere  are considered.   In this regard,
Office of Research and Development  laboratories, such  as Gulf Breeze
Environmental Research Laboratory,  have  exemplary, pilot research  programs that
are investigating the use of aquatic  animal species as indicators  of
carcinogens in the environment.  The  Gulf Breeze pilot research  program  has
been under way since August, 1978 and is supported jointly by the Office of
Research and Development (EPA) and  the National Cancer Institute through an
interagency agreement.  The  Gulf Breeze  studies are based  on  the  premise that
the aquatic portion of the biosphere  (water,  biota, and  sediment)  is the
ultimate "sink" for the runoff,  fallout, and  discharge of  most toxic
pollutants.  In addition, animals living in the relatively efficient solvent,
water, are more intimately exposed  (total exposure through body  surfaces,
giI Is, alimentary tracts) than are  species  living  in  terrestrial or air
environments.  Aquatic species are  also  less  likely to escape a  dissolved  or
carried pollutant.
     At Gulf Breeze, researchers have been  studying species  of fish and
shellfish along the Northern Gulf of Mexico (Florida,  Alabama, Mississippi)  in
order to determine which are good indicators  of the role of  carcinogenic agents
in the environment.  Selected  species of fish have been  used  in  both long-term
and short-term laboratory exposures  to determine their specific  tissue,
cellular, and biochemical responses  to known  chemical  carcinogens  which  may
occur in the environment.  In  addition,  a significant  number  of  cooperative
agreements with principle investigators  from  around the  nation support an
extramural complemental and  supplemental effort in the identification of
aquatic species and systems  that may  serve  as early warning  mechanisms.

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                           IV.  Summarized Objectives

Objective 1:
     Determine fate  and effects  of  carcinogens, mutagens,  and  teratogens  in
     aquatic species  (individuals;  populations).
Objective 2:
     Determine role  of aquatic species  and  systems  in  actual  or  potential
     exposure of man  to carcinogens,  mutagens,  or teratogens  (metabolism,
     accumulators).
Objective 3:
     Develop aquatic  species  as  bioindicator,  sentinel,  and model  systems  for
     use in study of  risks  of carcinogens,  mutagens, and teratogens  in  the
     general environment  (sensitive species  and systems).

                          V.  Methodological  Approaches

     We have underway both  in-house and  complementary  extramural  research
projects.  The overall project is divided into  major disciplinary  approaches:
1)  Pathobiology and  2) Biochemistry.   Therefore, methods  outlined  below  and
results in the next  section will  be reported  under  these two  complementary,
disciplinary headings.  Detailed results  of each cooperative  agreement  in
progress during FY 82 will  be included  at the  end of this  report.   The
disciplinary area of  each cooperative agreement will be  identified  by project
officer (Couch - pathobiology; Schoor -  biochemistry).

     1.  Pathobiological  Methods

         Fish carcinogen  assay system,  toxicology,  and histopathology of
         induced lesions.

              At Gulf Breeze, major emphasis  was concentrated  on  short-term
         critical life-stage carcinogen  exposures of several  estuarine  fish
         species.  Cyprinodon variegatus  (Sheepshead minnow),  Menidia
         peninsulae  (Silverside), and Rivulus  marmoratus were  exposed to  the
         overt carcinogen aflatoxin B^~.   This  compound,  a  natural  product  of
         the metabolic activity  of  fungi  (genus Aspergillus),  has  been  shown  to
         be of high  toxicity  and carcinogenicity in mammalian  studies
         (including  humans) and  work  with freshwater fish  species.   Selection
         of aflatoxin Bi  as a test  compound  was partly based  on  this high
         hazard background, to test marine  species  with  a  proven  and potent
         carcinogen.  As  such, special  precautions  and methods were needed to
         insure safety.

              Exposures were  conducted  under static conditions within  a glove
         box.  Test organisms were  exposed  in  3.Si  chambers  containing  1.5z
         seawater, toxicant,  and carrier, then  rinsed  and  transfered to clean
         holding facilities for  development  and observation.   A  detailed
         description  of the test systems  utilized  is  now in  press  (Courtney  and
         Couch, 1982).  Carrier  controls  were  run under  identical  conditions
         except for  toxicant.  Species,  test  concentrations,  exposure  duration,
         carrier, and life-stage tested  varied.  96 hour embryos  and 120  hour
         newly hatched larvae of _C.  variegatus  were exposed  for  1  hour  to

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         200 ug/i aflatoxin 8}  using  dimethylsulfoxide  (DMSO)  as  a carrier.
         £. variegatus, R_. marmoratus,  and  M_.  peninsulae  were  exposed  to 1000
         ug/i aflatoxin 8-j_  (using  DMSO  and  triethylene  glycol  as  carriers)
         for exposure durations  ranging  from  2  to  43.5  hours.   Holding  and
         observation continues  on  surviving test organisms.
              In extramural efforts,  Hendrix  at Oregon  is  continuing
         histopathologic  evaluation of  tissues  from  rainbow  trout  (Salmg
         gairdnerl) exposed for  18 months to  dietary  benzo(a)pyrene  (BAP:   1000
         ppm), Aroclor 1260 (PCB:  500  ppm),  and toxaphene (50  ppm).  Trout
         embryo exposures to methyl azoxymethanol  acetate  (MAMA)  and benzidine
         dihydoro chloride  (BDHC)  were  conducted and  an  experiment to produce a
         neuroblastoma neoplasm  in coho  salmon  and Shasta  strain  rainbow trout
         was initiated.   Martin, at the  University of Southern  Mississippi,  is
         examining  specimens from  over  thirty  adult  and  embryo  C_.  variegatus
         benzidine  exposures.   Additional work  has been  done with  C_. variegatus
         involving  aseptic embryo  techniques.   Cell  culture  techniques  have
         been investigated using a Fundulus species  and C_. variegatus.
              Grizzle, at Auburn University,  is continuing laboratory and field
         experiments to examine  causation and  development  of oral  papillomas
         found on feral black bullheads  (Ictalurus melas)  from  the final
         oxidation  pond of a Tuskegee,  Alabama  sewage treatment plant.
              Rose  and Anderson  are involved  in a  two part research project
         involving  tiger  salamanders  (Ambystoma tigrinum)  which have shown  an
         unusually  high prevalence of dermal  neoplasia.   Rose  is  studying the
         histopathological effects of perylene  on  the species  and  Anderson  is
         studying metabolic activation  and/or  detoxification of perylene and
         B(a)P by tissue  enzymes of _A.  tigrinum.
              Mix,  at Oregon State University,  is  continuing a  long-term field
         study of mulluscs in Oregon  coastal  waters  and  expanding  laboratory
         research.  Major emphasis this  year  include  1) baseline measurements
         of arsenic, cadmium, and  nickel  levels; 2)  methods  development  for
         trace levels of  polynuclear  aromatic  hydrocarbons  (PNAH)  in seawater;
         3)  developing a crustacean  (barnacle) bioassay  system;  4) studying
         possible viral associations  with Mytijus  edulis  neoplastic disorders;
         and 5) evaluating data  previously  collected  on  neoplastic disorders  in
         M_. edul is.

2.  Biochemical  Methods

         Induction  Studies:

          Aquatic species such  as  mullet, killifish,  flounder,  sea catfish  and
         others have been and will continue to  be  exposed  to inducers of
         microsomal mixed-function oxygenase  (MFO) activity  either by
         intraperitoneal  injection or direct  exposure in  seawater. Since
         periods up to one year  might be necessary to induce MFO  activity by
         water exposure, the direct injection  of inducer  is  used  at the  start
         of the investigations  in  order  to  optimize  other  parameters such as
         metabolite and conjugation reactions.  The  long-term,  low exposure  in
         seawater will follow.   [Schoor,  Melius (CR809493),  Strength
         (CR809673)].

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         Metabolite Identification:

          Metabolites from the MFO reactions  will  be  identified  using  high
         pressure liquid chromatography coupled with  fluorescence  detection  and
         confirmed by stopped-flow fluorescence scanning.  Metabolite  standards
         for benzo(a)pyrene [(BaP)] have been obtained  from  the  Illinois
         Institute of Technology through the  courtesy of NCI.  All  the  phenolic
         compounds have been chromatographed  and their  excitation  and  emission
         spectra have been obtained in appropriate  solvents.   All  spectra will
         be stored on discs for later data manipulation.

         Conjugation and Excretion Studies:

          Rats are being used to make a series of  conjugation  products  in vivo
         by injection of 14C-labelled BaP.  They will  include  glucuronides,
         glutathiones, and sulfates.  Their occurrence  in  fish will then be
         ascertained by comparison to the standards produced  in  the rat.  This
         will  be helpful in determining the final  dispositon of  a
         carcinogen like BaP within the animal and  in what forms the parent
         compound is finally passed back into the  seawater.

                       VI.  Major Findings and Progress

     As noted earlier under Methodological Approaches,  results and  findings  of
studies to date are reported under the two disciplinary areas  of Pathobioloqy
and Biochemistry with extramural cooperative  agreement  reports at  the  end of
this report.

1.  Pathobiology

             Table I summarizes the carcinogen assay/induction studies
         undertaken by the Gulf Breeze staff  and investigators working  under
         cooperative agreements.  This work represents  an  investigative effort
         encompassing a wide range of organisms and several  selected
         carcinogens and suspect-carcinogens.  In  addition to  the  data
         generated, and possibly of even greater value, is the methodology
         developed (both in exposure systems  and support technology).
          The following represents scientific results and  accomplishments of
         the Gulf Breeze staff and associated investigators  during FY  82.
             At Gulf Breeze, the static exposure system utilized in the
         aflatoxin studies has proven to be an efficient method  for short-term
         exposures using high hazard compounds.  Each species  tested performed
         well  and the use of various life-stages presented no  problems.  We
         feel  this system provides a safe and inexpensive  method of high hazard
         single short-term or pulse exposures for  a wide range of  life-stages
         and species.
             Data generated to date include some basic  toxicity  data with
         regard to aflatoxin and the three species  tested,and  two  lesions
         related to the exposures.  Gross necropsy  and  histological examination
         of C_. variegatus exposed to 200 yg/i aflatoxin B^ for 1 hr. have
         revealed a benign hepatic polyp on several specimens.   Exact  nature of
         the lesions is still under investigation  and a  large  number

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SPECIES
OYSTER



OYSTER


SHEEPSHEAD
MINNOW


SHEEPSHEAD
MINNOW


SHEEPSHEAD
MINNOW






RIVULUS



SILVERSIDE



SILVER PERCH
CELL LINE

RAINBOW
TROUT

TIGER
SALAMANDER
AGENT
3-Methyl-
Cholanthrene


Benzo(a)pyrene


Triflural in-
(Herbicide)


Aflatoxin BI



Benzidine



,



Aflatoxin 5\



Aflatoxin BI



Asbestos-
(Crocidolite)

Benzo(a)pyrene


Perylene

EXPOSURE
Flowing seawater,
1-5 yg/z for one
year.

Flowing seawater,
1-5 pg/z for one
year.
Flowing seawater,
5 ug/i, 19 months.


Embyro-fry static
200 yg/£ for one
hour held for 18
months.
Recirculated
seawater, 1 ppm,
for 166 days.





Fry xposure
static, 1 ppm,
2 hours, 8 hours.

Fry exposure,
static 1 ppm, 2
hours, 8 hours.
Held 12 weeks.
Asbestos in
culture medium.

1000 ppm, diet,
12 month feeding.

Injection i.p.

RESULTS
Incipient,
perivascular
blood cell
pro! iferation.
No tissue
response.

Focal , benign,
vertebral
growths-non-
neoplastic.
Hepatic polyps
(on surface of
livers). Benign
neopl asm.
*Prol iferatai ve
lesions in
liver; ductile
pro! iferation;
cholangioma;
adenofibrosis;
oval cell
hyperplasia.
No tumor
response yet.

*
Early altered
foci in livers;
enlarged cells;
preneoplastic.
Nodule formation
in culture around
asbestos fibers.
Hepatocellular
Carcinoma.
*
Hepatic tumor.
j_
 umors or sus-
pect neoplasms
actually induced

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of specimens  still  remain  in  holding.   In  addition,  a  suspect
pre-neoplastic  lesion  has  been  noted  in  histologic  sections  of  M_.
peninsulae exposed  as  fry  for 2  hrs.  to  1000  ug/i  aflatoxin  (using
both DMSO and TEG as carriers).   The  lesions,  noted  in  specimens
examined 12 weeks post-exposure,  consist  of  foci  of  enlarged hepatic
cells.  Additional  specimens  from the  exposure  are  being  held  for
continued observation  and  future  histologic  examination.   No lesions
have been noted  in  those Rivulus  examined  to  date.

    During FY 82 a  final report  summarizing  a two-year  field
biomonitoring study (Aug.  1978  to Aug.  1980)  was  completed.  This  is
now available in manuscript form  entitled  "A  Prospective  Study  of
Infectious and  Noninfectious  Diseases  in  Fishes  and  Shellfishes  in
Relationship  to  Pollutant  Activity in  Three Gulf  Coast  Estuaries".
The main issue  of the  study was  that  certain  diseases of  fishes  and
shellfishes (including  neoplasia)  from coastal  populations have  been
suggested to  be  related to, caused by,  or  enhanced  by pollutant
activity.  Considerable data  have been published  from which  inferences
have been made  that  fishes and  shellfishes inhabiting contaminated
waters are at higher disease  risk  than those  in  cleaner environments.
Most of these studies  from which  data  derived were  retrospective,
epizootiological efforts,  initiated after  the  fact  because certain
fish diseases were  increasingly  observed  or because  fish  kills were
noted or had  recurred  frequently  in specific  limited bodies  of water.
Prospective studies  of  estuaries  without  prior  knowledge  of  disease
prevalence to determine possibly  previously undetected  frequencies and
relationships of diseases  and pollutant  residues  in  fishes and
shellfishes have been  rare.   Therefore,  a  need  existed  to determine
prospectively if populations  of  fishes and shellfishes  at higher
disease risks related  to pollutant activity could  be identified with
standard sampling,  pathological,  and  analytical methods.

    The study of three  northern  Gulf  Coast estuaries, Pensacola  and
Escambia Bays in Northwest Florida, Mobile Bay, Alabama,  and
Pascagoula Harbor in Mississippi  Sound,  Mississippi, was  undertaken  in
August, 1978.   The  specific goals  of  this  prospective study  were to:
1) determine  and compare relative contamination  by  select pollutants
on specific sites in and among  the three  estuarine  areas, 2) determine
frequencies of  known or new diseases,  including  neoplasms, in
shellfish (oysters)  and fishes  at  these  sites  among  the estuaries, and
3) to examine critically any  relationships between  disease frequency
and pollutant contamination among the  selected  estuarine  sites  in
order to assess  the  role of pollutant  activity  in  influencing  disease
prevalences in  fish  and shellfish  populations  in  coastal  regions
characterized by varying degrees  of human  pollutant  activity.   For
each of the three estuaries,  three different  kinds  of samples
(oysters, fishes and sediments)  were  collected  quarterly  for chemical
analysis of pollutant  residues  from August, 1979  through  August, 1980.
One offshore  control station  was  sampled  for  prevalence comparison to
the estuarine stations.  Monthly  samples  of  fishes,  and oysters  were
collected for disease  analyses  and diagnoses  from  one or  more  specific
sites in the  three  estuarine  systems  from August,  1978  through  August,
1980 (25 Month  Period).

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    Results  of  this  study  suggest  that  though far from being pristine,
the estuaries studied  were  not  severely unhealthy environments at the
time that the biota  were  sampled.   However,  because predictions have
been made that  the Gulf Coastal  Plain  of the southeastern U.S. will
probably be  the fastest growing  region  in the nation in the next
decade, this study and others  provide  base lines  and points of
departure and comparison  for  studies  that should  be made,
periodically, on  the health of  coastal  biota in  relation  to increasing
impact of population and  industrial  growth.
    In extramural projects, rainbow  trout exposed to dietary
benzo(a)pyrene  (100  ppm)  have  developed hepatocellular carcinoma at
24% incidence level  (see  Hendricks  attached  cooperative agreement
report no. CR809344-010).   Aroclor  1260 and  toxaphene exposures have
yet to reveal neoplasms;  however, tissue examination is not complete.
Methylazoxymethanol  acetate (MAMA)  and  benzidine  dihydrochloride
exposures have  been  completed,  however,  all  necropsy samples have not
yet been processed.  Preliminary indications are  that MAMA is
carcinogenic to trout.  Mechanical  problems  have  hindered progress on
attempted neuroblastoma induction.
    Martin (see attached  report  no.  CR809347)  has produced liver
lesions, probably adenofibrotic  or  neoplastic,  in C_. variegatus
exposed to 1, 5,  and 10 ppm benzidine  dihydrochloride (BDHC).   Several
experiments  are still  in  progress and  characterization  of the  BDHC
produced lesion is still  underway.   In  BDHC  exposed C_.  variegatus
embryos (50  to  500 ppm exposure  concentrations),  85% exhibited
developmental anomalies compared to  a  0.02%  rate  of anomalies  in
controls.  Aseptic C_.  variegatus techniques  have  developed to  the
point of successful  maintenance  of  embryos for 60 days  or more.   Four
C_. variegatus cell lines  have  been  developed including  three
fibroblastic cell lines,  and  a  striated muscle cell  line, to our
knowledge, the  first such  line  developed from fish  tissue.   In
immunological studies, banding  pattern  differences  have been noted
between BDHC-exposed and  non-exposed C_.  variegatus  and  the
characterization  of  these  differences  is being pursued.   Additional
studies using immunoelectrophoresis, competitive  enzyme immunoassay
and bacteriophage neutralization show much promise  in exploring the
processes occurring  in BDHC exposed C_.  variegatus.
    Grizzle  (see  attached  report no. CR809336-010)  has  produced
lesions in caged  black bullheads (Ictalurus  melas)  confined to a
chlorinated  sewage effluent pond.   The  lesions consisted  of oral
mucosa hyperplasia, developing  after 2  months,  and  papilloma-1ike
lesions in the  mouth fornix, developing during the  second year.   These
lesions were similar to those  found  on  black bullheads  naturally
occurring in the  pond.  Transmission  experiments  and ultrastructural
studies failed  to indicate a viral  etiology  for the papillomas.
Induction of glucuronosyl  transferase  in caged channel  catfish within
the pond and Ames tests with the pond water  indicate the  presence of a
chemical  possibly related  to the Ictalurus melas  papillomas.
    The work of Rose and Anderson  (see  attached report  no.  CR807740)
includes the development  of a  radioisotopic  method  to quantify B(a)P
and perylene (PI metabolism in Ambystoma tigrinum.   Hepatic metabolism
of ^C-BaP and  3H-P was studied  in  normal  and induced A.
tigrinum.   Uninduced animals produced  about  0.2 total   C-BaP
metaboi ites/mg  protein/30 min.   Methylcholanthrene  was  indicated

                             10

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as a AAH inducer.  Metabolism  of  3H-P was  found  greater than that of
14C-BaP, inducers  had  little effect  on  metabolism compared to
noninduced Ambystoma.  Carbon  14-BaP metabolite  profiles  of induced
and non-induced  animals  were compared and  it  was found  that some
inducers, including  P, produced a  metabolite  shift  away from the more
potentially carcinogenic compounds.  This  indicates a  possible
protective action  by inducers,  such  as  P,  for subsequent  BaP exposure,
similar action is  frequently reported in  higher  animals.   The results
of mutagenicity  testing  indicate  that Ambystoma  is  quite  resistant  to
PAH carcinogenesis and suggests that aromatic amines may  be more
appropriate model  carcinogens.  Histopathological  lesions  were found
in several salamanders injected with perylene, one, a  liver cell
neoplasm.  Final evaluation of  this  phase  of  the study  is  not yet
complete.

    The research project of Mix,  at Oregon State University, (See
attached report  no.  CR808000-01-0) has  produced  much data  related to
shellfish populations  along the Oregon  coast  and valuable
investigative methodology.  Baseline measurements  of arsenic, cadmium,
and nickel were  determined for  two species of mussels,  and it was
determined that  the  mussels would  be excellent monitors of these
metals  in freshwater (Margaritifera marqaritifera)  and  marine (Mytilus
edulis) environments.  The evaluation of a long  term study (1976-1981)
of pro!iferative disorders in M_.  edulis has continued  and  produced
much data relating to  occurrence,  geographical distribution,
prevalence, seasonality,  and nistopathology.   Extensive investigation,
employing a variety  of techniques  and methods, failed to  reveal  the
presence of any  RNA  tumor virus in M.. edulis  exhibiting neoplastic
disorders.  Additionally, methods  for PNAH determination  in seawater
and a crustacean bioassay system  utilizing Pollicipis  polymerus were
investigated.  The bioassay system exhibits great  potential  for
testing environmental  carcinogens  and mutagens.

    Studies of teratogenic responses have  arisen from a broader
research program formerly supported  by  EPA Energy Effects  funding
(R805469).  The  objective of the  investigation was  to  develop an
experimental test  with marine  animals suitable for  assessing the
by-products of chlorination arising  from biofouling control  and
disinfection of  coastal  waters.   An  additional task was requested by
EPA to test specific compounds.   Phthalate tests revealed  significant
increase in endematous cardiac  malformations  in  embryos,  as well  as
abnormal development of  vertebral  and other skeletal elements among
surviving fishes.

    A colony of  the  fish  Rivulus  marmoratus was  transferred from Bears
Bluff Field Station, S.C. and the  College  of  Charleston to Gulf Breeze
EPA laboratories in  1981.  With the  previous  studies as a  foundation,
we have been concentrating on  refinement of screening tests for
teratogenic effects.  Since Rivulus marmoratus is  a self-fertilizing
hermaphrodite, it  provides natural clones  which  allow data tracking on
individuals (temporal  variations), as well  as lineages  and clones and
representatives  of wild  populations.  Our  research  designs incorporate
all stages of gametogenesis as  well;  as  post-fertilization
                              11

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         developmental  stages.

             This  strategy  is  noteworthy  inasmuch as  during initial  screening
         one does  not know  the  breadth  of  potential  "windows  of vulnerability"
         to teratogenic  effects.   Thaiidomide,  for example, is teratogenically
         dangerous to the embryo  for  only  very  brief  periods.   Rodent  tests
         were  run  for several  years before they "modelled"  established human
         effects.

             The number  of  potential  responses  of Rivulus  are  broad  and
         apparently clearly distinguishable.  Classical  teratogenic  studies   ,
         used  fishes, and focused mainly  on expressed gametes  just  prior to, or
         soon  after fertilization.  The ability to "scan"  broader periods of
         gametogenesis,  and have  the  relative economy of fish  are among the
         obvious advantages of  this species.  Studies presently underway will
         refine testing  procedures, and compare results  from  n-dibutyl
         phthalate, thalidomide,  and  hexazinone (a herbicide).   Other  reports
         presented at the NCI  (Koenig,  et  al.)  Bethesda  meeting report  general
         biology and tumorgenic responses.
2.  Biochemistry
              Induction  of  transferases  (UDPGA  and  sulfate)  was  observed  in
         mullet and  in  channel  catfish.   The  rate  of  conjugation  was  confirmed
         by enzyme assay with  conjugation using  p-nitrophenol  as  substrate.
         This was determined spectrophotometrically and  by  TLC  separation of
         the  respective conjugates  of p-nitrophenol.  Metabolites formed  by  MFO
         in microsomes  derived  from both  control and  induced  animals  were
         separated by HPLC  and  identified.  Microsomes  incubated  with both BaP
         and UDGPA enabled  us  to  ascertain  the extent of conjugation  in the
         presence of the oxidation  system.
              In addition to finding BaP metabolites already reported, we
         believe we  have found  some "cis" diols  as well  as  "trans"  diols.  The
         evidence is based  on  agreement of retention  times  of standards  and
         unknown metabolites.   Nanogram amounts  of the  "cis"  diols  have  not  yet
         allowed more positive  identification.   In order to establish a  kinetic
         sequence for the metabolites, we have slowed the MFO reaction at 0°C;
         however considerable  activity  remains at  that  temperature.
             MFO activity has  been  studied  in  the  sea catfish,  Aries  felis.
         collected from coastal waters  in N-W  Florida.   The oxidase activity
         was  induced with 3-MC  injections and  tissue/body mass  ratios, hepatic
         microsomal  protein components  and  in  vitro BaP  metablism were all
         studied.  The  effect of  3-MC was measured over  a 5-40  mg 3-MC/kg body
         mass range  for 7 days  and  at  a 20  mg  3-MC/kg body  mass dose  level  for
         1, 3, 5, and 7 day periods.  Liver/body and  kidney/body  mass ratios
         increased with increasing  3-MC dose  levels and  treatment period.
         Cytochrome P-450,  epoxide  hydratase  and cytochrome b5  reductase  all
         increased significantly  with  increasing doses  of 3-MC  up to  40 mg
         3-MC/kg body mass  and  with increasing time periods.   A linear response
         was obtained in the Ames test with S. typhimurium  TA 98  up to 3-MC
         dose level  for 20  mg.  In  the Ames test at a 20 mg 3-MC  dose level, we
         obtained a  linear  response over  a  period  of  7 days.  The hepatic
                                       12

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         microsomal fraction  from the  3-MC  induced  sea  catfish  metabolized BaP
         to the usual metabolites including  the  3-and 9-  phenols,  4,5-,  7,8-
         and 9,10-dihydrodiols  and  quinones.  Metabolite  production  was
         enhanced by butylated  hydroxyanisole and  inhibited  by  6-aminochrysene,
         adenosine-21-phosphate and l,2-epoxy-3,3,3-trichloropropane.

                 VII.  Significance to Biomedical Research  and
                         Program Needs of NCI and EPA

     The following contributions to the  needs of NCI and  EPA were  made during
FY 82:

     1.  The responsiveness of  a number  of  species  to environmental
       carcinogens has been demonstrated.   The sheepshead minnows  have
       developed proliferative  liver  lesions  (oval  cell hyperplasia,  possibly
       adenofibrosis) resulting from  benzidine exposures  and benign  hepatic
       polyps related to aflatoxin  exposure.  A  pre-neoplastic  condition,  also
       related to aflatoxin exposure,  has been produced in Menidia livers.
       Finally, rainbow trout developed  overt hepatocellular carcinoma
       following treatment with benzo(a)pyrene.  All three  species have  high
       potential to be used as  routine carcinogen  assay organisms.
       Additionally, the black  bullhead  has  been successfully used to
       demonstrate a cause and  effect  relationship  to an  existing  pollution
       situation.  Laboratory and field  responsiveness  have  shown  it to  be a
       good indicator organism. Finally, the tiger salamander  (Ambystoma  sp.)
       is particularly interesting  as  an assay or  indicator  organism because  of
       its cutaneous and liver  enzyme  responses  to  the  PAH,  perylene.  The fact
       that this species has  shown  cutaneous tumor  development  in  contaminated
       environments heightens its significance as  a possible indicator
       species.

     2.  Results from the Field biomonitoring study (Report  entitled:  "A
       Prospective Study of Infectious and  Non-infectious Diseases in Fishes
       and Shellfishes in Relationship to Pollutant Activity in Three Gulf
       Coasts Estuaries") indicate  the following:   First, a  low tumor epizootic
       in fishes suggests low risks to environmental carcinogens at  locations
       and times sampled.  Therefore,  future  studies should  be  retrospective  in
       regard to neoplasia as an indicator  of carcinogen  contaminants; i.e.
       studies should be based  on an  a priori indication  of  tumors in certain
       species at certain sites.  Secondly,  findings  indicate that oysters may
       be more sensitive indicators of environmental contamination and
       especially may provide a more  representative response to focal
       contamination in coastal  regions. Future studies  should include  careful
       monitoring for tissue  and physiologic  changes  indicative of chemical
       induced stress in oysters.   Furthermore,  several diseases of  oysters  and
       fish were detected during the  study.  At  least two of these,  a
       rickettsia found in oyster digestive gland  cells and  ichthyosporidosis
       from several fish species, are  important  in  terms  of possible impact
       related to effects on  biota  and human  health.  These  diseases  and their
       role in the Gulf coast environment should be investigated further.
       Finally, the overall study suggests  that, while  being far from pristine,
       the study area was not a severely unhealthy  environment  at  the time of
       investigation.  This study and  others now provide  a  base line  for


                                       13

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       future  comparison.   Studies  should be periodically made to monitor the
       impact  of  population and  industrial  growth  along  this  valuable coastal
       region,  a  region  predicted  to  be  one of the fastest growing areas of the
       country during  the  next decade.

     3.  Biochemical  and correlated structural  responses of fish  liver systems
       seem to be  relatively similar  to  responses  of  mammals  to  certain
       carcinogens.   Recent results show that the  mixed  function  oxidases are
       induced in  fish as  well as  the conjugating  enzymes that aid in the
       detoxification  and  excretion of compounds  such as B(a)P.   This permits
       future  comparative  studies  to  determine if  biochemical  methods may be
       incorporated  in early warning  or  sentinel monitoring projects  with fish.
       The biochemical studies suggest that fish may  serve as  animal  models in
       carcinogen  (preneoplasia) studies to complement mammalian  studies.

                      VIII.   Proposed  Course - Future  Plans

     1.  The NCI/EPA  collaborative  project  officially concludes October  1982
       and no  new  projects  will  be  undertaken.  During FY83,  efforts  will  be
       made toward concluding research  projects already  underway  at Gulf Breeze
       and by  investigators under  cooperative agreements.   Major  emphasis will
       be directed to  preparing  final  reports (due October 1983)  and  collating
       reprints produced as a result  of  the research  period.

     2.  An in-house  study, at Gulf Breeze, will be conducted  on  the  effects of
       nltrosamines on fish species using test systems developed  during  the
       NCI/EPA research  program.   The study will focus on endpoints of tumor
       induction  and  possible other effects (histopathological, physiological,
       etc.).

          IX.  Date Contract Initiated and  Period  of  Contract  Planned

Initiated                                              Expiration Date

October 1978                                           September  30,  1982

                       X.   Contractors Project Director

                    Dr. Henry F. Enos, Laboratory  Director
                              Gulf  Breeze, ERL, EPA

                        Project Officers for NCI or EPA

NCI:                            Dr. Herman  Kraybill
EPA/ORD:                        Dr. Wayne Galbraith
Prinicipal Investigator:        Dr. John A.  Couch, Gulf  Breeze, ERL,  EPA

          XI.  Cooperative  Agreements  Funded - Progress  Reports FY 82

Title:  Rainbow Trout:  A Model for Environmental  Carcinogenesis
Prinicipal Investigator:  Jerry D.  Hendricks
Cooperative Agreement  Number:  CR-809344-010
Project Officer:   John Couch
                                       14

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Introduction
     Work  is  continuing  on  the histopathological  evaluation of tissues from
fish exposed  for  18  months  to  dietary benzo(a)pyrene (BAP), Aroclor 1260 and
toxaphene.  The data from the  BAP  dietary study and the BAP intraperitoneal
injection  study is complete and a  manuscript  is in the final  stages of
preparation.
     Trout  embryo exposures to methyl azoxymethanol acetate (MAMA) and
benzidine  dihydrochloride (BDHC) were conducted in December,  1981 and the
finger!ings are now  8 months of age.   An attempt  to produce a rare
neuroblastoma neoplasm in coho salmon fry was  unsuccessful  but will be
conducted  again during the  next year.
Current Progress
     Dietary  exposure of trout finger!ings to  1000 ppm BAP  for 18 months
resulted in a 24% incidence of liver  neoplasms at 18 months (table 2).   Average
body weight for these fish  at  18 months  was 364 g (table 1).   Average
conversion  of dry diet to fish weight for rainbow trout is  1:1 indicating that
the average fish  would have consumed  364 g of  dry diet and  consumed 364 mg of
BAP.   In another  experiment, we injected, IP,  a group of 50 trout with 1 mg BAP
in .4  ml of propylene glycol monthly  for 12 months and held them an additional
6 months before killing  and examination.  The total  dose in  this  case was only
12 mg/fish  but the incidence of hepatic  tumors was 50% (table 3).  This may
indicate that BAP is a more potent carcinogen  to  trout than dietary exposure
demonstrates.  It is possible  that much  of the highly water insoluble BAP is
not absorbed  from the trout gut, making  the effective dose  much  lower than what
was put in  the diet.,
     Gross  examination did  not reveal any neoplasms in fish fed  either Aroclor
1260 (500  ppm) or toxaphene (50 ppm)  for 18 months, however,  a histological
examination of all tissues  is  befng conducted.

     The first objective of the new project was to determine  the effects of two
potential  carcinogens on rainbow trout  embryos as a continuing effort to assess
the suitability of rainbow  trout as indicators of environmental  carcinogenesis.
We are interested in determining the  carcinogenicity of two rodent carcinogens,
methylazoxymethanol  acetate (MAMA) and  benzidine  dihydrochloride (BDHC), in
rainbow trout embryos as well  as the  effects  of prior exposure of trout embryos
to the environmental  contaminant,  Aroclor 1254 (PCB), on the  carcinogenicity of
MAMA and BDHC.

     The 3 year-old  female  brood fish were removed from the usual OMP brood
ration and  placed on our semi purified test diet containing  200 ppm Aroclor 1254
two months  prior  to  spawning.   Two months later these fish  were  each given two
injections of commercially  prepared salmon pituitaries and  induced to spawn.
Eggs from  seven fish were available on  the same spawning date and were pooled
to provide the source of PCB-exposed  embryos for  subsequent embryo exposure.
Eggs from  the same number of OMP-control  brood females hormonally induced to
spawn  on the  same day were  used for comparative control embryo exposures.  Egg
samples from  both sources taken on the  day of  spawning, day of carcinogen
exposure (day 21  of  incubation) and periodically  thereafter were prepared for
PCB analysis  to follow PCB  depuration.   On day 21 of incubation, 12 groups of
200 viable embryos from  both the PCB  and  control  groups were  exposed for 24
hours  to MAMA and BDHC at the  0.1, 1, 10, 100  and 1,000 ppm levels.  Both MAMA
and BDHC were lethal   to  trout  embryos at  the 1,000 ppm level  for 24 hours.
                                       15

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                                               Table  1

                Body weight, liver weight/body  weight ratios and mortalities, within
         groups of trout sampled at 6,  12,  and  18-months of diet exposure to benzo(a Jpyrene.

                                                                     Liver weight   10Q
Sample time       Diet                 Body weight  (g)a     Mort      Body weight

                  CD                    50  ± 10  (23)        17        1.13 ± 0.13  (23)

6 months          CD + 1000 ppm BP      41  ± 10  (29)        11        1.20 ± 0.12  (29)
                  CD                   195  ±  49   (32)         8        0.73 ± 0.11  (32)

12 months         CD + 1000 ppm BP     158  ±  43   (33)         7        0.83 ± 0.15  (29)b
                  CD                   425  ±  153  (109)       11        0.66 ± 0.16 (109)

18 months         CD + 1000 ppm BP     364  ±  125  (Til)        9        0.69 ± 0.17  (89)



aData presented as mean ± SD (no.  of fish used  1n determining mean represents pooling of
 duplicate tanks).

 n used 1n determining mean does not correspond to n used for computing mean body weight, I.e.
 data is not biased by Including weights of livers with large tumors.

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                                               Table  2
                 Histologlcal Incidences of liver tumors  1n  trout sampled following
                        12 and 18 months of diet exposure to  benzo(a)pyrene.
Sample time
12 months
Diet
CD
CD + 1000 ppm BP
Foci of basophll
0/32
4/33
- 1
1c cells0
0%
2%
Carcinomas
0/32 - 0%
1/33 - 3%
                 CD                            0/109  -  0%                   0/109 - 0%
18 months
                 CD + 1000 ppm BP              5/111  -  4.5%                22/111 - 20*
alncludes fish having only basophillc foci.

 Data presented as no. of fish with at least one  tumor/total  no. of fish sampled.

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                                               Table 3
                     Hlstological Incidences of tumors 1n trout sampled 6 months
     after 12 monthly Intraperitoneal inoculations of benzo(a )pyrene (BP) In propylene  glycol  (PG)
Exp. group
PG controls
PG + BP
Tumor type
Carcinoma
Carcinoma
Basophilic foci
Flbrosarcoma
Papllloma
Organ
Liver
Liver
Liver
Liver
Swlmbladder
Incidence
 1/27 - 4%
13/28 - 46%
 1/28 - 4%
 1/28 - 4%
 1/28 - 4%
                                                                                                           co
 Data presented as no. of fish with at least one tumor/total  no.  of fish.
 Includes fish having only basophillc foci.

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The 100 ppm MAMA dose was  also  highly  toxic,  resulting in  high  mortalities.
After hatching and swimup,  100  fry  from  the  control  and  three  highest  tolerated
dose levels,  (0.1, 1, 10 ppm  for MAMA  and  1,  10,  100 ppm for BDHC)  were kept
and are being fed our semipurified  control diet  for  the  12-18  month tumor
development period.  A 20  fish  sample  will be taken  9 months after  treatment
(September, 1982) to assess tumor development.   If tumors  are  present  the
experiment will be terminated at 12 months.   If  not, another 20 fish will  be
killed at 12 months and a  decision  made  at this  time on  whether to  terminate
the experiment or continue for  another 6 months.  A  few  fish survived  the 100
ppm MAMA treatment and were kept to determine tumor  development.  These fish
grew very poorly, average  weight was only  3.3 g  after 8  months, and mortalities
were frequent so the remaining  15 fish (PCB  and  control  groups  combined)  were
killed.  All  fish, but one from the control  group, had grossly  observable liver
tumors.  This information  gives a preliminary indication that MAMA  is
carcinogenic  to trout.  Dose  response  and  the effects of PCB will be determined
at later samples.

     The second objective  in  our proposal  was to  reenact the conditions which
apparently led to an inadvertent epizootic of neuroblastoma  in  juvenile coho
salmon reared in chlorinated-dechlorinated river  water,  the  intent  being  to
reproduce this tumor with  subsequent investigation of the  causative factors.
The experiment was begun as proposed and involved exposure of  coho  salmon  and
Shasta strain rainbow trout embryos and  sac  fry to chlorinated-dechlorinated
(sodium thiosulfate) McKenzie River water  brought into our laboratory.
However, an unfortunate mechanical  failure of our water  recirculating  pump
resulted in complete loss  of  the coho  salmon  fry.  The trout,  having reached
the swimup stage, had been moved elsewhere and were  not  in the  closed  system
when the failure occurred.  Consequently,  the trout  are  being monitored for
tumor development as proposed.  The experiment using both  coho  salmon  and
rainbow trout will be repeated  during  the  second  year of this  cooperative
project.

Title:  Teleost Carcinogen Assay Systems
Principal Investigator:  B. J.  Martin, University of Southern Mississippi
Cooperative Agreement Number:   CR809347
Project Officer:  John A.  Couch

     During the year we have  conducted or  have in progress over thirty (30)
whole organism experiments  exposing £. variegatus to benzidine  dihydrochloride
(BEN).  These experiments  represent our  efforts  to reproduce and more
accurately characterize the proliferative  liver  lesions  previously  induced by
long-term exposure of C. variegatus to BEN.

1)  Benzidine Exposures. C_. variegatus.  To  date, we have  histologically
    examined  the livers from  44 fish that  have been  exposed  to  1 ppm BEN  for at
    least 200 days and the lesion was  evident in  5 individuals.  In an
    experiment in which C.  variegatus  are  being  exposed  to 5 ppm BEN,  4 fish
    have been examined hTstologically  and  the liver  lesion was  evident in  2
    individuals.  One of these  was  sacrificed after  62 days  of  exposure,  and
    the other after 132 days.   Seven fish  have been  examined from an experiment
    involving exposure to  BEN at 10 ppm  and  one  individual had  the  lesion.
    Also, some £. variegatus  that survived 96-hour exposure  at  high
    concentrations to BEN  during LD-50 experiments were  maintained  in  the


                                       19

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    laboratory  and  the  liver  lesions  were evident  in one of the 2 individuals
    examined histologically.   Since a  number of the experiments from which
    these data  are  derived  are still  in  progress,  we feel  that we will  be able
    to accomplish our goal  of  characterizing the lesion and more accurately
    determining  its  incidence  and  latency period by the end of the project
    period.

2)  Benzidine Exposures. £. variegatus Embryos.  Embryos were exposed to BEN at
    concentrations  ranging  from 5  to  500 ppm,  and  effects  were observed  at
    concentrations  of 50 ppm  or higher.   To  date,  ca.  600  embryos have  been
    exposed to  BEN  at 50 ppm  or higher and only 15% developed normally  beyond
    the hatching stage.  In our 300 control  embryos, 77% developed normally
    beyond the  hatching stage  and  only 0.02% exhibited abnormalities;
    therefore,  the  detrimental  effects of BEN  at these concentrations are
    rather obvious.

    Approximately 85% of the  embryos  exposed to BEN at 50  ppm or higher
    exhibited anomalies.  These anomalies, in  order of frequency of occurrence,
    are:  1) Tubed  heart syndrome  with distended pericardia,  2) Poor
    circulation, 3)  Sparse  distribution  of melanophores around yolk, 4)
    Inability to hatch, 5)  Abnormal head morphology, 6) Scoliosis, 7) Faint RBC
    'pigmentation.

    Efforts are  in  progress to examine these anomalies histologically,  and
    experiments  are  planned to study  the effects of BEN on the early
    post-hatching stage of  development.

3)  Aseptic Embryo  Technique.   Our main  efforts have been  to  find a suitable
    food  regime and  environmental  conditions to maintain healthy £.  variegatus
    fry for an  amount of time  sufficient to  study  the  effects of exposure to
    carcinogens  in  a sterile  environment.  The foods we have  tried were
    ethylene oxide-treated  Mono Lake  Flakes,  autoclaved Tetramin,  autoclaved
    Artemia eggs, and chlorox-treated  Artemia  eggs.  We also  conducted
    experiments  with reduced  quantities  of antibiotics and no antibiotics in an
    effort to determine the effects of antibiotics on  mortality.

    Chlorox-treated  Artemia eggs seem  to have  provided the best food regime
    since more  animals  remained healthy  when fed this  diet.   Also, fry
    consistently lived  longer  when maintined in'an antibiotic-free sterile
    environment; however, as  one might expect, the preparations without
    antibiotics  are  much more  likely  to  become contaminated.   Thus,  it was
    possible to  consistently maintain  healthy  embryos  on chlorox-treated
    Artemia eggs in  an  antibiotic-free sterile environment for 60+ days.

    Obvious problems associated with  the use of Artemia are that they are
    another living organism in the system and, since as yet we have no method
    of excluding the non-viable Artemia  from the system, the  unhatched  eggs and
    uneaten Artemia  accumulate in  the  preparation.  This problem can be
    somewhat ameliorated by underfeeding and more  frequent water changes.

4)  C. variegatus Cell Line Development.  We have  developed four new cell lines
    Trom C. variegatus:  A  large cell  fibroblastic line (through passage 8), a
                                       20

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    small cell fibroblastic  line  that  forms  cellular  aggregates  in  culture
    (through passage 10),  a  small  cell  fibroblastic  line  that  does  not
    aggregate in culture  (through  passage  11),  and a  striated  muscle  cell  line
    (through passage 12).  The  striated  muscle  cell  line  is  particularly
    interesting since, to  our knowledge,  it  is  the first  striated muscle cell
    line to be developed  from fish  tissue.

    We plan to use all four  of  these cell  lines  in toxicity  and  cardnogenicity
    studies during the coming project  year.

5)  Primary Hepatocyte Cell  Culture Technique.   Because of our initial  success
    in developing primary  hepatocyte cell  cultures from Fundulus. we  felt  the
    development of such cultures  from  £.  varlegatus would  not  be difficult.
    However, after a year  of effort in this  regard with very  little  success, we
    have decided to work  again  with Fundulus.   Our recent  emphasis  has  been  the
    determination of media and  environment  conditions  that will  provide healthy
    and morphologically differentiated cells.   Although the  cells do  not  appear
    completely differentiated with  respect  to morphology,  we  are conducting
    tests to determine their state  of  biochemical differentiation.   If  these
    cells prove to be sufficiently  differentiated to  be considered  "functional
    hepatocytes," we will  begin to  use them  in  carcinogen  assays.

6)  Immunological Studies.   Agarose and  polyacrylamide serum  electrophoresis
    has disclosed differences in  the banding patterns  of BEN-exposed  and
    non-exposed Cyprinodon varlegatus.   Densitometer  scans of  the agarose
    electrophoresis preparations  indicate  that  both the number and  size of the
    peaks from BEN-exposed fish differ from  non-exposed individuals.  PAGE
    (polyacrylamide gel electrophoresis)  provides a much better  resolution of
    the  serum differences, and  they are  clearly  visable with  the naked  eye.
    When the Bio-Rad silver  stain  is used, we can detect  significant
    differences by using  a 1:50 dilution  of  only lug  of serum;  thus,  allowing
    the conservation of serum from  each  individual fish for  additional  tests.

    By immunoelectrophoresis (Graber-Wil1iams),  we are able  to demonstrate
    differences in precipitation  arcs  generated  from  exposed  and non-exposed
    fish.  This technique  is not  quantitative,  but it  demonstrates  that our
    antiserum is sufficiently potent for  use in  crossed and  tandem  crossed
    rocket procedures.

    A competitive enzyme  immunoassay has  been developed and  employed
    successfully- with non-fish  antibody.   We are presently miniaturizing  the
    procedure so that it  can be used with  the yl quantities  of blood  available
    from C_. variegatus.

    A bacteriophage neutralization  procedure has been  adapted  for use  in the
    study of the ability  of  £.  variegatus  to generate  neutralizing  factors.
    Results show a consistently higher level of neutralization in BEN-exposed
    fish (fish are immunized with MS-2 phage).
                                       21

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Title:  Causes of Paplllomas  on  Fish  Exposed to Chlorinated Sewage Effluent
Principal  Investigator:John M.  Grizzle
Cooperative Agreement  Number:  CR809336010
Project Officer:  William Davis

     Laboratory  and  field experiments are  in progress  to determine the cause of
oral papillomas  on black  bullheads  in the  final  oxidation pond of the Tuskegee,
Alabama, sewage  treatment plant.  Wild,  adult black  bullheads in the final
oxidation  pond had a 73%  prevalence of oral  papillomas  between December 1979
and July 1980.   A gill-net sample of  black  bullheads from this pond on 28 May
1982 had an oral papillona prevalence of  57%.  The occurrence of tumors in  this
recent  sample  indicated  a continuation of  the original,  tumor inducing
conditions.

Exposure of fish to  water in  the final  oxidation pond.   Black bullheads
(Ictalurus melas), brown  bullheads  (Ictalurus nebulosus),  yellow bullheads
(Ictalurus natal is), and  channel  catfish  (Ictalurus  punctatus) were placed  into
cages in the final oxidation  pond of  the Tuskegee, Alabama,  sewage treatment
plant.  Control  fish were kept  in cages  in  an earthen  pond at the Auburn
University Agricultural Experiment  Station.   The approximately 1-nr cages
were allowed to  rest on the pond bottom  (sinking cages)  or suspended off of the
bottom  by  floats (floating cages).  Cages  were placed  at three locations in the
final oxidation  pond:  near the  inlet (inlet A), approximately 60 meters from
the  inlet  (inlet B), or  near  the pond outlet (outlet).   Most  cages were stocked
with 50 fish.  Table 1 summarized all  cage  exposures including the percentage
of fish with grossly visible, oral  mucosa  hyperplasis  or papilloma-like
lesions.

     The oral  lesions  on  fish confined to  cages  in the  final  oxidation pond
were in the same location occupied  by oral  papillomas  in wild black bullheads
from this  pond.  The hyperplasic lesions were most prevalent  during the spring
and  regressed  during the  summer.  Black  bullheads  had  a  higher prevalence of
oral musoca hyperplasia  than  the other species.   Lesion  prevalence was higher
in black bullheads stocked during FY  81  than in  those  stocked the following
year after similar lengths of exposure.   Incidence of  hyperplastic lesions  was
also higher in black bullheads  near the  outlet than  in  those  near the  inlet,
but  low fish survival  near the  inlet  could  have  influenced this  result.   No
difference was found between  fish in  floating and  sinking cages.

     During their second  year of exposure  to the final  oxidation pond, some of
the black  bullheads  developed oral  lesions  that  grossly  resembled papillomas.
These lesions  were in  the same  locations  in  the  mouth  fornix  as  the
hyperplastic lesions developing  during the  first year  of exposure.   The
appearance of  the lesions that  developed during  the  second year  is described in
a later section  of this  report.

Lesions on caged fish.  Lesions  in  both  fornices of  the  mouth were examined
from a 175-mm  total  length black  bullhead  confined for 537 days  to a sinking
cage near  the  outlet of  the final oxidation  pond.   Grossly,  these lesions
appeared similar to  papillomas  on wild black bullheads  in this pond and to  the
lesions on other caged black  bullheads with  a similar  length  of  exposure.  The
surface dimensions of  the lesions were approximately 8 mm by  5 mm,  and each
                                       22

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Table 1.  Sumrary of exposures of caged fish to  the Tuskegee, Alabama, final oxidation pond.
Species
Black bullheads



Yellow bullheads
Brown bullheads
Channel catfish

Stocking Dates
21 Oct 80
23 Feb 81
29 Oct 81
9 Apr 82
12 May 81
29 Oct 81
16 Jan 81
29 Oct 81
Days of exposure
(as of 14 Jun 82)
603
476
226
66
398
228
514
231
Maximum percentage
with oral lesions
95
50
18
0
0
0
25
0
Maximum percentage with
paplllcraa-like lesions
8U
50
0
0
0
0
U
0
Maxinun percent
of control fish
with oral lesions
4
4
0
0
14
0
8.3
0
                                                                                                                                      CM

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lesion was  raised  approximately  1.5 mm above the surrounding oral  mucosa.  In
paraffin sections  of  the  lesion,  the maximum thickness of the connective tissue
between the  epidermis  and  mucosa  was 1.5 mm compared to a normal  thickness of
approximately 0.2  mm.   The maximum  thickness of the mucosal  portion of the
sectioned lesion was  1.2 mm compared to a normal  thickness of approximately 0.2
mm.  The connective tissue component of the lesion extended  from  the hyperemic
submucosa into the epithelial  portion of the lesion, but the connective and
epithelial  tissues was  less intermingled than in larger oral  papillomas of wild
bullheads.   These  lesions  were different than the hyperplasic lesions appearing
soon after  exposure because these papilloma-like lesions were larger, there was
more folding of the lesion surface, and the connective tissue was  more
extensive and intermingled with  the epithelial  tissue than in the  hyperplastic
lesions.

Ultrastructure of  papillomas in  black bullheads.   The oral papilloma of wild
black bullheads from  the final oxidation pond has epithelial  cells resembling
normal oral  mucosa cells and a central  connective tissue column that was
continuous  with the submucosa.   Near the tumor surface there were  mucous cells,
and plasma  membranes  of the epithelial  cells adhered closely with  those of
adjacent cells.  Desmosomes occurred less frequently between the  cells than in
normal mucosa.  Mitochondria,  endoplasmic reticulum, and ribosomes were
concentrated in the peripheral cytoplasm of the epithlial  cells.   These surface
cells often  contained  glycogen particles among the tonofilaments.   The mid to
near basal  portion of  the  papilloma consisted of stellate epithelial  cells
having more  tonofilaments, fewer  organelles in the cytoplasm, and  more
intercellular space than epithelial  cells near the surface.   Desmosomes
occurred more frequently between  the cells in the basal  area than  near the
surface.  Alarm substances cells'occurred in the mid portion of the epithelium
and were surrounded by stellate  cells.   Numerous lymphocytes were  present in
the basal region of the tumor.   The basal  epithelial  cells contained  numerous
mitochondria, endoplasmic  reticulum, and ribosomes in the cytoplasm.   Plasma
membrances  adhered to  the  adjacent  cells by desmosomes.   Intercellular space
occurred less frequently in the  basal  region than in the middle portion of the
tumor.

Mutagenicity of the final  oxidation pond water.   Extracts of the  pond water
were tested  for mutagenicity with the Ames test (Ames et al., 1975,  Mutat.  Res.
31:347).  An acid  fraction was prepared by acidifying the water to pH 1.0,
extracting three times  with 200 ml  of 25% ether and 75% hexane per liter of
water, and  then distilling to  remove the bulk of the solvents.  The last 20 ml
were evaporated under  a stream of nitrogen.   A basic fraction was  prepared  in
the same manner except  that the  water was adjusted to pH 12-13 before
extraction.  Both  fractions were  tested with and  without S-9  (Aroclor-induced
rat liver enzymes).  Benzo(a)pyrene was used as a positive control  mutagen and
was tested with and without S-9.

     The acid fraction  was mutagenic to Salmonella typhimurium tester strains
TA98 and TA100 if  S-9  was  present,  and  there was  a positive  dose  response (Fig.
1 and 2).  The mutagenicity of the  acid fraction was higher  on some dates than
on others (Fig.3)  and  may  be seasonal.   The mutagenicity of  the water_at the
inlet and outlet was  compared  on  two dates:   there was no difference in
February 1982, but mutagenicity was higher at the inlet in March  1982 (Fig.

                                       24

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                         JOO -
                         4CO i
                        §200
                         100 ••
                                            AC:OIC -.3-9
                                            AC:OIC
                                     LO    LS   to
                                             to
                                             Tc
                                                        10
Figure 1.  Response  of  Salmonella typhimurium  strain TA 98 in Che Ames test  to
           an acid organic solvent extract of  the  final oxidation pond water.
           The water was collected at the pond outlet on 15 July 1981.
                          JOO •-
                                 OJ
Figure 2.  Response  of  Salmonella
                                                 strain TA 100 in the Ames t«st  to
            an acid  organic solvent extract  of  the final oxidation pond water.
            The water  was  collected at the pond outlet on 29 June 1981.  The
            results  for cwo trials are given on this graph.
                                         25

-------
                    JAN a  MX; st   SOT a  ocr at  NOV &  rta at  MAA az
    fcesponse of Salmooella typhimurium strain TA  100  «„  PK  A
    « add organic solvent extract of the finlt  ilY  ,     ^S CeSt t0
    The number of revertanc coloniL f            oxidation pond water.
    colonies on aedLawith the Stract STSl,-  ^  """*•* °f
    medium vith extract onl7                S 9 aiQUS Che
                   S.8+
                          "CSNTSO.
                   ua-f.«
                     i occ a   i ra «  i
                                OAft
k.   Glucurososyl craasferaae accivlcy la liver  caicrosomes of chapel
    cacfish confined  to  cages ta the final  oxidation pond dr a control
    pond.
                              26

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3).  The number  of  revertant  colonies  on  plates  with  acid fraction but without
S-9 was similar  to  the  number of  colonies  on  negative control  plates (DMSO or
S-9 only); therefore, the  mutagenicity of  the samples in Fig.  3 was expressed
as the difference in the number of  revertant  colonies on plates with and
without S-9.  The basic fraction  was  not  mutagenic  when  tested with Salmonella
typhimurium strains TA98 and  TA100.

Hepatic enzyme induction.   UDP-glucuronosyl  transferase  and sulfotransferase
activities in livers of black bullheads,  brown bullheads, and  channel  catfish
from cages in the final oxidation pond were  measured  and compared to control
fish.   Dr. D. R. Strength  and colleagues  determined the  enzyme activity of
these fish as part  of a separate  EPA cooperative agreement.  Glucuronosyl
transferase activity in exposed fish was  generally  higher than in control  fish
with channel catfish having the greatest  induction  response (Figs.  4-6).  The
difference between  sulfotransferase activity  in  exposed  and control  fish was
not consistent.  The induction of glucuronosyl  transferase in  fish confined to
the final oxidation pond indicated  exposure  to a toxicant.   Similar induction
occurs in rats and  fish given oral  or  intraperitoneal doses of carcinogens.

Analysis of water from  the sewage pond.   Between September 1981 and May 1982,
water leaving the final oxidation pond had 0.1 to 1.0 mg/1  ammonia-nitrogen
(average 0.55 mg/1) and a  pH  between 5.1  and  7.0.   During this same period,
total  residual chlorine of water  leaving  the  chlorine contact  chamber  before
entering the final  oxidation  pond was  0.2  to  1.0 mg/1 (average 0.53 mg/1).  The
residual chlorine concentration  in  the effluent  has been lower during  the
experimental studies in this  pond than during 1979  when  the papillomas were
first found  in the  wild black bullheads.

     Water samples  from the inlet to the  final  oxidation pond  were collected  on
22 January 1982  and 3 March 1982.   The Environmental  Health Administration
Laboratory, Montgomery, Alabama,  examined  the samples with gas chromatography.
Total  volatiles, herbicides,  pesticides,  and  PCB's  were  not detectable.

Transmission of  papillpmas by cell-free tumor homogenate.  Two attempts  were
made to transmit papillomas by injecting  homogenized, filtered papillomas  into
the mouth fornix of healthy black bullheads.   The first  group  of fish  was
adults and was observed for 14 months  after  injection.   The second  attempt  was
with one-year-old fish  that were  observed  for 11 months  after  injection.   No
signs of papillomas or  hyperplastic lesions  were found  in any  of the injected
fish.

Embryo Exposures.   Brown bullhead embryos  were exposed  to aflatoxin  B^ (0.5
or 1.0 mg/i) or  a concentrate of  the final oxidation  pond water.   Controls  were
exposed to uncontaminated  pond water or to the solvents  used in the  chemical
exposures (dimethylsulfoxide  and  ethanol).   Groups  of 200 embryos were exposed
to each treatment for one  hour on the  fourth  day after fertilization.   Embryos
were kept in a closed,  recirculating system  and  after hatching, were
transferred to 3-meter  diameter plastic pools.   Fish  were sampled after 3  and 6
months, and all  remaining  fish were fixed  after  8 months.  These fish  are
currently being  examined.   No evidence of  tumor  induction has  been found.
                                       27

-------
3).  The number of  revertant  colonies  on  plates  with  acid fraction but without
S-9 was similar to  the  number of  colonies on negative control  plates (DMSO or
S-9 only); therefore, the  mutagenicity of the samples in Fig.  3 was expressed
as the difference in  the  number of  revertant colonies on plates with and
without S-9.  The basic fraction  was  not  mutagenic when tested with Salmonella
typhimurium strains TA98  and  TA100.

Hepatic enzyme induction.  UDP-glucuronsyl  transferase and sulfotransferase
activities in livers  of black bullheads,  brown bullheads, and  channel  catfish
from cages in the final oxidation pond were measured  and compared to control
fish.  Or. D. R. Strength  and colleagues  determined the enzyme activity of
these fish as part  of a separate  EPA  cooperative agreement.   Glucuronosyl
transferase activity  in exposed fish  was  generally higher than in control  fish
with channel catfish  having the greatest  induction response  (Fig. 4-6).  The
difference between  sulfotransferase activity in  exposed and  control  fish was
not consistent.  The  induction of glucuronosyl  transferase in  fish confined to
the final oxidation pond  indicated  exposure to a toxicant.   Similar induction
occurs in rats and  fish given oral  or intraperitoneal doses  of carcinogens.

Analysis of water from  the sewage pond.   Between September 1981 and May 1982,
water leaving the final oxidation pond had 0.1 to 1.0 mg/1  ammonia-nitrogen
(average 0.55 mg/1) and a  pH  between  5.1  and 7.0.   During this same period,
total residual chlorine of water  leaving  the chlorine contact  chamber before
entering the final  oxidation  pond was  0.2 to 1.0 mg/1 (average 0.53 mg/1).  The
residual chlorine concentration in  the effluent  has been lower during the
experimental studies  in this  pond than during 1979 when the  papillomas were
first found in the  wild black bullheads.

     Water samples  from the inlet to  the  final  oxidation pond  were collected  on
22 January 1982 and 3 March 1982.   The Environmental  Health  Administration
Laboratory, Montgomery, Alabama,  examined the samples with  gas chromatography.
Total volatiles, herbicides,  pesticides,  and PCB's were not  detectable.

Transmission of papilloms  by  cell-free tumor homogenate.   Two  attempts were
made to transmit papillomas by injecting  homogenized, filtered papillomas  into
the mouth fornix of healthy black bullheads.  The first group  of fish  was
adults and was observed for 14 months  after injection.   The  second attempt was
with one-year-old fish  that were  observed for 11 months after  injection.  No
signs of papillomas or  hyperplastic lesions were found  in any  of the injected
fish.

Embryo Exposures.   Brown  bullhead embryos were exposed  to aflatoxin B.  (0.5
or 1.0 mg/1) or a concentrate of  the  final  oxidation  pond water.  Controls were
exposed to uncontaminated  pond water  or to the solvents used in the chemical
exposures (dimethylsulfoxide  and  ethanol).   Groups of 200 embryos were exposed
to each treatment for one  hour on the  fourth day after fertilization.   Embryos
were kept in a closed,  recirculating  system and  after hatching, were
transferred to 3-meter  diameter,  plastic  pools.   Fish were  sampled after 3 and
6 months and all remaining fish were  fixed after 8 months.   These fish are
currently being examined.  No evidence of tumor induction has  been found.
                                       28

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Laboratory  exposures  of black  bullheads  to sediment extracts.   One-year-old
black bullheads  were  acclimated  to  static-water,  40-liter aquaria,  one fish per
aquarium.   Sediment  from the  final  oxidation pond was collected near the inlet
on 14 June  1982.   Samples  were extracted for organic compounds  with methylene
chloride  and  acetone  according to  the EPA protocol  for extraction of priority
pollutants  from  sediments.
     Two  types of  skin  exposure  to  the sediment  extract will  be used:
subcutaneous  injection  and  topical  application.   The sediment  extract  was
injected  on 30 July  1982,  and  the  fish will  be observed for at  least one year.
The topical applications will  begin during August 1982.

Title:  Perylene as  a Possible Environmental  Carcinogen:   Hydrocarbon
Metabolism  and Mutagenesis  in  Ambystoma  tigrinum
Principal Investigator:   Robert  S.  Anderson  and Francis L.  Rose
Coopertive  Agreement  Number:   CR807740
Project Officer:   John  A.  Couch

     The  unusually high prevalence  of various forms of dermal  neoplasia  in  a
population  of tiger  salamanders  (A.  tigrinum) inhabiting  a  sewage lagoon whose
water and sediment are  unusually rich in perylene has prompted  this study of
the possible  role  that  this polycyclic aromatic hydrocarbon (PAH) might  have in
the etiology  of  these lesions.   The work is  divided into  two  portions:   1)  the
histopathological  effects  of  perylene (P)  as  a carcinogen or cocarcinogen on A.
tigrinum. under  the  direction  of Dr.  F.  Rose  and  2) the metabolic activation
and/or detoxification of P, and  a  related  environmental  carcinogen
benzo(a)pyrene (BaP), by tissue  enzymes  of £. tigrinum.  under the direction of
Dr. R. Anderson.   The progress  of this effort through the first  two years 1s
summarized  in this report.

     Methodological  developments.   A radioisotopic  method used  to measure BaP
metabolism  in mammals was  adapted for use  with A. tigrinum  to quantify BaP  and
P metabolism.  Basically,  the  parent  compounds are  converted to  metabolites  and
conjugates  which can  be extracted based  on their  solubility in  water and/or
NaOH.  This technique is useful  to  obtain  an  indication  of  the  total rate of
metabolism  of these  substrates.  Once the  in  vitro  conditions of. reaction had
been optimized this  reaction  procedure was used to  obtain metabolite extracts
in ethyl  acetate which  were used for HPLC  separation of metabolites  on a
Perkin-Elmer HC  ODS/SilX reverse-phase column.  Metabolites of   H-P  or
14C-BaP were eluted  in  a 60-80%  MeOH-H20 gradient,  the eluted samples
were collected for radioactivity determinations and UV-absorbance was monitored
continuously.  A complete  set  of BaP  authentic standards  were used  to  identify
by coelution all  of the BaP metabolites; unfortunately a  set of  P metabolite
references  is not  currently available.

     Hepatic metabolism of 14C-BaP  and 3H-P  in normal  and induced A.
tigrinum.   Liver homogenates from tiger  salamanders mediate the  in  vitro
metabolism  of both BaP  and P.  We studied  the effect of two known aryT
hydrocarbon hydroxylase (AHH)  inducers:  aroclor 1254 (PCB)  and
methylcholanthrene (MC),  and P on the metabolism  of BaP  and P.   Both the
metabolites in the aqueous phase (conjugates  and  macromolecule-bound species)
and the metabolites  in  the NaOH  phase (certain dihydrodiols and  the phenolic
derivatives) were  quantified;  their  sum  allowed us  to estimate  the  total
nmoles/ml liver  protein/30 min produced  under the various conditions.

                                       29

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Induction procedures used were those  found  effective  for  standard  laboratory
rodents:  25 mg/kg MC ip, 48 hr; 500  mg/kg  PCB  ip, 96  hr; 60 mg/kg  P  ip, 48 hr
(this dosage was based on that commonly  used  for  its  isomer BaP);  olive  oil was
the vehicle in all cases.

     Table 1 summarizes the data on ^C-BaP metabolism.   Uninduced  animals
produced about o.2 total 14C-8aP metabolites/mg protein/30 min; the
alkali-soluble metabolites were produced  in quantities more than twice that of
aqueous phase metabolites.  MC was the only chemical  tried that produced
induction, this induction would have  been highly  statistically significant if
the variation in the induced data had been  somewhat reduced by having a  larger
sample size.  Interestingly in the MC-induced animals  aqueous phase > NaOH
phase metabolites, an effect also seen in the PCB-treated salamanders, although
PCB injection at this level did not produce an  over all AHH induction.
Perylene injection had no significant effect  on metabolite partitioning  and was
not an effective AHH inducer.  14C-BaP metabolism by  Mutazyme™ (a
commercial preparation of aroclor induced rat hepatic  microsomes designed for
the Ames bacterial mutagenesis assay) was measured for reasons of  comparison.
Its activity per mg protein was  100-fold greater than the uninduced  salamander
with aqueous phase = NaOH phase metabolites.
Table 1.
                     HEPATIC 14C-BaP METABOLISM*
Ambystoma
 Inducer
                   NaOH
                         Metabolites
                        Aqueous phase
                        Total
Rat
None

Aroclor

MC


Perylene



Aroclor
 0.131 +_ 0.148

 0.055 + 0.052

 0.228 +_ 0.230


 0.118 + 0.117



10.104 + 1.098
0.059 + 0.045

0.089 + 0.038

0.318 + 0.339


0.056 + 0.016



8.044 + 1.925
                                                             0.190 + 0.130  (6)

                                                             0.144 i 0.072  (6)

                                                             0.546 + 0.562  (4)
                                                                 (P<0.2)

                                                             0.174 + 0.116  (3)
                                                             18.148 i 3.021  (3)
*Nmoles/mg protein/30 min: X _+ SO  (n).
                                      30

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Table 2.
Rat
   Aroclor
     HEPATIC 3H-PERYLENE METABOLISM*
Ambystoma
Inducer
None
Aroclor
MC
Perylene
Metabolites
NaOH
0.
0.
0.
0.
005
192
144
000
± °-
± °-
± °-
+ 0.
010
289
184
000
Aqueous phase
1.
1.
1.
2.
302
305
365
477
+ 0.528
i 0.873
+ 0.527
+ 0.288
1
1
1
2
Total
.307
.498
.509
.477
1 °-
± °-
t °-
+ 0.
529 (6)
733 (5)
641 (6)
288 (3)
16.425 + 1.280
                                                                 (p<0.01)
56.169 i 19.014   72.593 + 18.483 (3)
 Nmoles/mg  protein/30  min;  X jf SO  (n).


     When assays  using the  same protocol  as  those  for ^C-BaP  metabolism
were run using  3H-P  as substrate an estimate of perylene  metabolism was
obtained (Table 2).   In both induced and  uninduced salamanders the  majority  of
P metabolites were  extracted in the aqueous  phase.  Perylene was  more
extensively metabolized than BaP,  -9-fold more  for uninduced A. tigrinum and
* 4-fold more for  PCB-induced rat.   Although  neither PCB nor  MC treatment caused
significant changes  in overall  3H-P metabolism, both treatments produced a
significant increase in the production  of NaOH-soluble metabolites.  Perylene,
while not a BAE-OHase  inducer (Table 1),  did significantly increase  the  rate of
metabolims  of   H-P  in  hepatic  homogenates.   This inductive event  was
accomplished by a marked  increase  in the  production of aqueous-phase
metabolites.  As  was the  case  for  BaP-OHase, the specific  activity  of _A.
tigrinum P-metabolizing enzymes was much  less (-70-fold)  than  those  of the
rat.

     Effects of inducer on  14C-BaP metabolite profiles by HPLC.   In Table 3
the specific BaP  metabolites produced by  uninduced and induced salamanders are
listed.  Metabolites marked with an "X" were produced during the  30-min  j_n
vitro incubation, the  number of "Xs" for  a given metabolite  indicates the
numbers of  animals  in  a given  group that  produced  that metabolite.  Metabolites
that were not only  produced by  the majority  of  animals within  a group, but were
also quantitatively  the major metabolite(s)  of their class (diols,  quinones,
phenols) for that group of  animls,  are marked with an arrow.   Basal  animals
produced many readily  identified BaP dihydrodiols, quinones, and  phenolic
derivatives.  The major diol  is the 7,8-diol, which can serve  as  a  precursor of
the ultimate carcinogenic BaP  metabolite, the 9,10-epoxide of  the
7,8-dihydrodiol.  In uninduced  A.  tigrinum the  major monohydroxylated
metabolite  appears  to  be 7-OH-BaP,  in contrast  to  mammals  which produce  mainly
3-OH and 9-OH BaP.   In PCB- and MC-induced salamanders 7,8-diol is  also  a major
                                       31

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                   14C-BaP METABOLITES PRODUCED BY AMBYSTOMA LIVER:   RESPONSE  TO AHM  INDUCERS
BASAL (4)
AROCLOR 1254 (3)
MC (3)

PERYLENE
60 mg/kg (2)

PERYLENE
20 mg/kg (1)

X
X
X

X
X
X
X
x


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                     9,10  4,5  7,8     4,5   1,6   3,6   6,12     (11  or  12)  (4  or  5)   6   9  (8 or 10)   2   7   1   3

                      Dihydrodiols            Quinones                          Phenolic  derivatives
                                           BaP  METABOLITES  IN ORDER OF ELUTION

-------
  diol,  as  is 9,10-diol  in the case of MC induction.  The  pattern  of  3aP
  metabolism is shifted toward 3-OH-BaP production  by PCS  and MC,  and  1-OH-BaP  in
  MC-induced animals.  Attempts to induce with 20 mg P/kg  produced  no  significant
  alteration from the normal metabolite profile, but treatment with 60 mg  P/kg
  caused an apparent shift away from dihydrodiol production.
  Table 4.   EFFECTS OF AHH INDUCERS ON AMBYSTOMA 14C-BaP METABOLITE  PROFILES  (HPLC)
Basal
(4)
Dihydrodiols

Qui nones
HO-BaPs
54

14
30
.8

.8
.4
i 13.5

+ 14.2
+ 10.9
Aroclor 1254
(3)
52

11
36
.3

.3
.4
± 6-

± 6-
+ 4.
0

4
7
40

11
48
MC
(3)
.3 •»•
(p<0.
.7 +
.0 +
Perylene
60 mg/kg (2) 20 mg/k<
4.0 0
2)
5.0 20.5 +_ 10.6
3.6 79.5 + 10.6
31.0

12.0
57.0
                                                                (p<0.025)
X% total    C-BaP metabolites in each category _+ SD  (n),  peak  identity  confirmed by
coelution with authentic metabolites standards.
       Another way to look at the effect of putative AHH inducers on   C
  metabolite profiles is presented- in Table 4.   In this table the actual amount
  of metabolites produced from each major class  is presented as  the % of total
  metabolites in each category.  For example, in basal animals approximately  55%
  of the total BaP metabolites produced are dihydrodiol s, 15% are qui nones, and
  30% are phenolic derivatives.  Of the three inducers tested, only MC produced
  significant overall BP-Phase induction (Table  1); however, both MC and P cause
  alterations in BaP metabolite profiles (Table  4).  Aroclor 1254 had no effect
  on relative composition of the metabolite categories.  MC-treatment tended  to
  cause a shift from diol production to phenol production.  This effect was much
  more pronounced in the case of 60 mg P/kg, where virtually no  dihydrodiols  were
  generated and -80% of the metabolites were phenols.  The distribution of
  metabolites started to revert to normalcy when the dose was reduced to 20 mg
  P/kg.  It would appear that P treatment might  have a protective effect in the
  case of subsequent BaP exposure because it shifts £. tigrinum  metabolism away
  from the production of potentially carcinogenic epoxides and diol -epoxides
  toward generation of comparatively inactive phenol derivatives.  AHH induction
  in higher animals also is frequently reported  to be  protective.

       Mutagenicity of BaP, P, and aromatic amines in  the Ames bacterial system.
  We have been able to identify and quantify numerous  BaP metabolites produced by
  A. tigrinum enzymes, and quantify P metabolism in the same species (although
  The exact identity of the P metabolites is unknown).  However, the biological
  activity of the metabolites produced by salamanders  has not been determined
  using conventional testing systems.  Therefore, we tested the  ability of A_.
  tigrinum hepatic enzymes to activate BaP, P, and some carcinogenic aromatic
                                        33

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amines using the Ames Salmonella  typhimurium  tester  strains.   The  results  are
given in Table 5.

     Microsomal enzyme  preparations  from  the  livers  of  Aroclor-induced
salamanders mediated the production  of  mutagenic  aromatic  amine  metabolites.
These preparations were generally  less  active,  on the  basis  of protein
concentration, than comparable  rat S9 preparations,  with the  exception  of
2-aminofluorene and 4-aminostilbene.  Therefore,  higher substrate
concentrations were usually  required to get significant mutagenesis  with
Ambystoma S9; in no case were the  concentrations  used  highly  toxic  or  directly
mutagenic to the bacterial tester  strains.  All data presented in Table 5  have
been corrected for this background,  as  well as  for spontaneous revertants.
After the appropriate corrections, it was  seen  that  salamander mixed-function
oxygenase (MFO) activated 2-aminoanthracene,  2-aminofluorene,
2-acetylaminofluorene,  4-ami no-trans-sti1bene,  and mono- and  diacetylbenzidine;
however, benzidine and  4-aminobiphenyl  were poorly mutagenic  in  this system.
As was expected, the frameshift mutagen detector  strains TA 1538 and TA 98  (TA
1538/pK M101) were most sensitive  to the  activated aromatic amines.  Strain TA
100 (TA 1535/pK M101) was also  useful because it  detects frameshift, as well  as
base-pair substitution  mutagens.

     The generation of  mutagenic  aromatic  amine metabolites by Ambystoma
microsomes was totally  inhibited  by  prior  heating of the S9 to 56° C for 30
min.  The reaction did  not proceed in the  absence of the NADPH-generating
system usually included in the  S9 mix.

     Rat microsomal enzyme induction with  phenobarbital  produced a marked
increase in the mutagenesis  by  benzidine,  N-acetylbenzidine,  and
N,N-d1acetylbenzidine.  However,  phenobarbital  was not  a more  effective inducer
than Aroclor in the activation  of these substrates,  or  other  aromatic amines,
by Ambystoma enzymes.

     In this study there was  no evidence  that either BaP or perylene were
mutagenic after reaction with Ambystoma liver microsomes.  Strains TA 98 and  TA
100 were mutated by BaP with Mutazyme^,  as  had  already been  reported.
Another frameshift detector, TA 1537, was  most  useful  in perylene activation
studies using rat S9.   Although data from  only  1  concentration of BaP or
perylene + Ambystoma S9 are  given  in Table 1, concentrations  ranging from  5-250
ug/plate were tried, without  any  sign of  mutagenesis.   BaP was neither  toxic
nor directly mutagenic  for TA 98  or  TA  1537 over  this  range;  it  was  slightly
toxic over 100 ug/plate for TA  100.  Ambystoma  enzymes  produce no BaP
activation as measured  by TA 98, TA  100 or TA 1537.  Likewise  perylene
concentrations of 5-250 ug/plate  were tested  against TA 1537,  the only  tester
strain to give a good response  to  rat S9-activated perylene.   No perylene
concentration tested was directly  toxic or mutagenic;  reaction with  Ambystoma
microsomes had no effect.

     In summary, a number of carcinogenic  aromatic amines  when activated by
liver microsomes from a salamander,  Ambystoma tigrinum,  are mutagenic for
Salmonella tester strains sensitive  to  frameshift mutagens.   However, 2
polyeyelie aromatic hydrocarbons  (PAH)  (BaP and perylene)  that are  rendered
mutagenic by mammalian  microsomes  are not  activated  by  Ambystoma mixed-function
                                       34

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                         TABLE  5
COMPARATIVE ACTIVATION OF CARCINOGENS
Compounds*
Aromatic Amines
Mg
         S9b
his' revertants/platec
                   TA 1538
             TA 98
Polycyclic aromatic
   hydrocarbons
Mg
          S9b
TA 1537
TA98
            TA100
2-Aminoanthracene (PC)

2-Aminofluorene (PC)

2-Acetylaminofluorene (PC)

4-Aminobiphenyl (PC)

4-Amino-rron5-stilbene (PC)

Benzidine (PC)



A/-Acetylbenzidine (NT)
,

NJV'-Diac«tylb«nzidine (NT)



25
5
10
10
100
5
500
100
50
50
500
500
500
500
200
10
10
200
20 1000 revertants/plate).
                                       35

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 xidases.   These results tend  to  support the observation that  amphibians  are
 uite resistant to PAH carcinogenesis  and suggest that aromatic  amines  may be
 :ore appropriate model carcinogens;   possibly because amphibian  mixed-function
 xidases carry out N-oxidation more  efficiently than C-oxidation.

    From the point of view of environmental carcinogenesis  in relation  to
 jiibystoma, it is of interest that  the  recognized carcinogen  benzidine  can be
 ojjndln water supplies.  In addition  to benzidine and its salts,
 .-aminobiphenyl and 2-acetylaminofluorene are included in a  list  of
 :arcinogenic substances for which  regulatory standards were  promulgated  by the
 iccupational Safety and Health Administration.

 listopathological  Response of  Ambystoma to Perylene:

    Determination of maximum  tolerated dose.  Three groups  of 10  neotenes each
 /ere injected ip with 10 mg, 50 mg,  or 100 mg P/0.5 olive oil.   No lethality or
pther harmful effects noted after  60 days.  Autopsy showed much  residual  P in
 leritoneal cavity, especially  in  higher dose groups; P frequently  triggered
 jranulomatous reactions.

    Injection of perylene.  A control  group of 19 neotenes  each  received 2  ml
 )live oil  ip; 19 experimental  were injected ip with 100 mg P/2 ml  olive oil.
hnimals in both groups were maintained for about 3 months.  Only 2 of the
 :ontrols died during this period;  one  died almost immediately  (probably of
 :auses unrelated to the study), the  other died at about 2 months.  None of the
 :ontrol animals had tumors of  any  kind.  Thirteen of the experimental group
 iied during the course of the  study.  Of 11 perylene-treated animals sent to
 )r. John Harshbarger for study, two  had epidermal papillomas and one had  an
 jnusual hepatocellular carcinoma  (differential diagnosis:  cholangio
 :arc1noma).  This  is the first unequivocal liver cell  neoplasm reported in
 salamanders.  A commonly observed  reactive lesion characteristic of the
 experimental group was diagnosed  as  a  lipid granuloma arising  in response to
 the olive oil vehicle, although they were uncommon in control  animals which
Deceived olive oil alone.  These  lesions were composed of mixed  populations  of
 :ells in which foamy histiocytes  and collagen-producing fibrocytes
 predominated.  These granulomas were often found on the surface  of the lungs,
 liver, and kidneys.

    Another major study of the effects of P, and P and BaP  in combination,  is
 :urrently underway; results are not  yet available because of the length of time
 required in the carcinogen-testing protocols.  Attempts to transplant cutaneous
 lelanomas are also in progress.
                                      36

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Title:   Carcinogens  and  Neoplasia  in  Indigenous  Populations  of  Aquatic
Organisms
Principal  Investigator:  Michael C. Mix
Cooperative Agreement Number:  CR808000-01-0
Project Officer:  John A. Couch

     Major research  activities during  the  past year  were  directed  toward:  (1)
measuring baseline levels of arsenic  (As),  cadmium  (Cd) and  nickel  (Ni)  in
indigenous populations of bivalve  molluscs; (2)  developing methods  for
measuring trace levels of polynuclear  aromatic hydrocarbons  (PNAH)  in seawater;
(3) developing a crustacean (barnacle) bioassay  system that  could  be utilized
for testing environmental levels of carcinogens  and/or mutagens;  (4)
determining whether  or not viruses are associated with the neoplastic disorders
of Mytilus edulis from Yaquina Bay; and  (5) evaluating data, gathered during
the past five years, on  the proliferative  disorders  in M. edulis.   Results from
studies in each of these areas is  summarized below.

Baseline Levels _of Inorganic Carcinogens (As, Cd, NI) ijn  Shellfish
     Freshwater mussels  (Margaritifera margaritifera) and bay mussels (Mytilus
edulis) were examined monthly or bimonthly  during a  one-year period and  tissue
concentrations of As, Cd, and Ni were determined by  either flame atomic
absorption or neutron activation analysis.  For M_. margaritifera. the
concentration ranges of  metal (yg/g, dry weight) were:  Cd, 0.9-6.2; and Ni,
1.6-19.4.  These levels  are similar to those reported in  organisms  from
non-industrial streams and less than, or in the  lower range of, those from
industrialized rivers.   For M_. edulis, the  concentrations of As ranged from
9-15 ug/g (dry weight),  Ni, 1.0-6.8 yg/g and Cd, 7.0-12.5 ug/g.  Both species
were judged to be excellent monitors for evaluating  levels of these important
metals in freshwater (M_. marqaritifera) and marine  (M_. edulis) environments.

Measurement _of PNAH  in Seawater
     The U.S. EPA's  Method 610 (U.S. EPA 1979) Polynuclear Aromatic
Hydrocarbons - Method 610.  Federal Register, 44(223), (69514-69517) was
modified for detecting and quantifying phenantFrene, fluoranthene,  pyrene,
benzo(c)phenanthrene, tnphenylene, benzo(a)anthracene, chrysene,
benzo(b)fluoranthene, benzo(k)fluoranthene, dibenz(a,c)anthracene,
benzo(a)pyrene, dibenz(a,h)anthracene, benzo(g,h,i)perylene,
indeno(l,2,3-c,d)pyrene  and coronene in seawater.  This method  is  applicable to
the determination of PNAH in water and was  designed  to be used to meet the
monitoring requirements  of the National Pollutant Discharge Elimination
System.

     Procedures described in EPA Method 610 and  an  alternative extraction
process using C\Q mini-columns (Sep-Paks; Waters Assoc. Inc.) were
investigated for application.  Further evaluations of various analytical
components are currently being completed.   These studies  were undertaken to
evaluate the use of Sep-Paks for extracting PNAH from water  since they seemed
to offer certain procedural advantages.  Experiments were designed  to determine
percent recoveries of individual PNAH, reproducibility and an optimum flow
rate.   A system to pump  water through the Sep-Paks was also  developed, tests
and evaluated for adsorptive loss of PNAH.
                                      37

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Development of _a Crustacean  Bioassay  System
     In this  study,  an  in  vitro  system  for culturing  the eggs  and larvae of the
gooseneck barnacle,  PolTTcipes polymerus.  was  developed and tested in two
bioassay experiments  using the phthalate  ester plasticizer dibutyl  phthalate.
The culture system  employed  aereated  culture chambers composed of chemically
inert substances and  designed to be  inexpensive,  convenient to use,  and to
minimize external contamination  of  the  cultures.

     Pollicipes eggs  were  grown  in  synthetic seawater at 14° +_ 1° C  under
lighting conditions  of  alternating  darkness and very  low light.   Water was
changed every 24 hours.  Antibiotics  were  not  necessary if prescribed methods
were employed.  An  overall mean  hatching  success  index of 75.5 was achieved
with egg masses not  exposed  to dibutyl  phthalate  (DBF).  Experiments  indicated
that allowing 86-96  hours  after  the  first  hatch before ending  the experiment
resulted in sufficient  stage II  larvae  to  be able to  draw conclusions regarding
molting success.

     One of the DBP  experiments  showed  there was  a significant decrease in
molting success in  larvae  from eggs  exposed to 1000 ppb DBP from a week prior
to first hatching to the end of  the  experiment.  Several  phthalate esters were
found to be persistent  contaminants  in  synthetic  sea  salts.

     The results of  these  studies indicate that the £_.  polymerus assay system
has great potential  for testing  environmental  carcinogens and  mutagens.

Viral Studies

     Because  of a recent report  (Oprandy,  et jil_.  1981.   J_.  Invertebr. Pathol..
38:45-51) which indicated  that henric  proliferative disorders in  Mya  arenaria
may be caused by a B-type  retrovirus, studies  were undertaken  to determine
whether or not such  a virus  was  associated with the neoplastic disorders in M_.
edulis.  Extensive  investigation, employing a  variety of techniques  and methods
did not reveal the  presence  of any  RNA  tumor virus in either affected or
control M_. edulis.

Statistical Evaluations of Proliferative  Disorders _UT, M.  edulis
     The occurrence,  prevalence, seasonality and  histopathological progression
of a cellular disorder, thought  to  be a hemic  neoplasm, were studied  in
subpopulations of Mytilus  edulis inhabiting different sites in Yaquina Bay
Oregon, from 1976-1981.There were  significant differences in the occurrence
of the disorder that  were  related to  geographical  location. In  the
subpopulation with  the  highest levels of  the disease, the prevalences ranged
from 0.0% to 20% with a five-year mean  of  9.8%.  There was a statistically
significant relationship between prevalence and season.  During the  five-year
study period, there  was a  consistent  pattern characterized by  highest
prevalences during January through  March  followed by  a period  of decline to
lower levels during  the summer and  early  fall, after  which there was  an
increase.  Data analyses revealed that  there was  no seasonal histopathological
progression of the disorder.  Numbers of  stage 1  (early), 2, 3 and 4 (advanced)
cases were not related  to  season but, rather,  occurred in a random manner
throughout the entire year.
                                       38

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                   XII.  Publications, Reports and Abstracts
                         from Program - Cumulative list
                         (1978 - 1981)
        *
Anderson, Robert S. 1978.  Benzo(a)pyrene metabolism in the American Oyster,
     Crassostrea virginica.  Ecol. Res. Series, EPA-600/3-78-009.  pp. 18.
Anderson, R.S., Doos, J.E., Rose, S.L. 1982.  Differential ability of Ambystoma
     tiqrinum hepatic microsomes to produce mutagenic metabolites from
     Polycyclic Aromatic Hydrocarbons and Aromatic Amines.
Bunting, O.B. 1979.  An evaluation of benzo(a)pyrene metabolism in an Oyster
     (Ostrea edu1is)-Bacteria System. M.S. (Thesis) Oregon State.
Coker, S. 1980. Benzo(a)pyrene metabolism in the Sea Catfish (Arius fell us).
     Ala. Acad. Sci. Meeting, March 21, 1980.
Couch, J.A. 1979. Vertebral dysplasia in young fish exposed to the herbicide
     Trifluralin.  Journal of Fish Diseases. 2:  35-42.
Couch, J.A. 1979. Pollution ecology of Penaeid Shrimps.  In:  Pollution Ecology
     of Estuarine Invertebrates, pp. 236-258.  Hart/Fuller (eds.) Academic
     Press, New York.
Couch, J.A. 1979.  Carcinogens in the Aquatic Environment.  Interagency
     Collaborative Group on Environment Carcinogenesis, NIH, Bethesda,
     Maryland January 10,  1979.  (Minutes of Meeting).
Couch,, J.A., Lee Courtney, James T. Winstead and Steven Foss.   1979.  The
     American Oyster (Crassostrea virginica) as an indicator of carcinogens in
     the Aquatic Environment.  In:  Animal Models and Wildlife as Monitors, pp.
     65-84.  National Academy of Sciences, Washington, D.C.
Couch, J.S. and W.P. Schoor.  1979.  First EPA/NCI Annual Report.  Effects of
     Carcinogens. Mutagens. and Teratogens on Non-Human Species (Aquatic
     Animals).  February,  1980, NCI Publication.
Couch, JA. and W.P. Schoor.  1980.  Second Annual Report to NCI/EPA.  Effects
     of Carcinogens. Mutaqens and Teratogens on Non-Human Species (Aquatic
     Animals).  April, 1981. NCI Publication.
Couch, J.A. and James T. Winstead.  1979.  Concurrent Neoplastic and Protistan
     Disorders in the American Oyster (Crassostrea virginica).  Haliotis, 8,
     1977-  pp. 249-253.
Couch, J.A., Frank G. Lowman and Ford A. Cross. 1980. Biomonitoring of Coastal
     Waters, - An Overview.  In:  Biological Monitoring for Environmental
     Effects, Douglas L. Wolf (ed.), Lexington Books, Mass.  pp. 93-95.
Couch, J.A., Lee A. Courtney and Steven S. Foss. 1981.  Laboratory of Symposium
     of Marine Fishes as Carcinogen Assay Subjects.  Proceedings of Symposium
     of Princess Takamatsu Cancer Research Fund, Phyletic Approaches to Cancer,
     C.J. Dawe et al. (eds), Japan Sci. Soc. Press, Tokyo, (In Press).
Couch, J.A. 1982.  Aquatic Animals as Indicators of Environmental Exposures,
     Journal Envion. Sci.  Health, A17(4) 473-476.
Couch, J.A., Clyde Dawe. 1982. Mouse vs Minnow:  The Future of Fish in
     Carcinogenicity Testing.  A Symposium on the use of Small Fish Species in
     Carcinogenicity Testing.  National Cancer Institute Monograph.  In press.
Courtney, L.A., J.A. Couch. 1982. Usefulness of Cyprinodon variegatus and
     Fundulus grandis in Carcinogenicity Testing"!  Advantages  and Special
     Problems.  A Symposium on the use of Small Fish Species in Carcinogenicity
     Testing. National Cancer Institute Monograph. In Press.
                                      39

-------
Dunn, J.E. 1982.  Development of a Marine Bioassay System for Priority
     Pollutants Using Larvae of the Gooseneck Barnacle, Pollicipes polymerus:
     A Feasibility Study. M.S. Thesis.
El am, D., M.V. Kilgore, B. Tan and W.P. Schoor.  1979. Mixed Function Oxidase
     Inducibility in the Mullet and Killifish.  4th Intl. Symp. on PAH,
     Columbus, Ohio, October 4, 1979.
Elam, D., M.V. Kilgore and W.P. Schoor.  1979.  Induction of Mixed Function
     Oxidase and Metabolism of Polyaromatic Hydrocarbons in Marine Organisms.
     llth Intl. Congress of Biochemistry, Toronto, Canada, July 11, 1979.
Elnenaey, Elsayed, and W. Peter Schoor. A Simple High Performance Liquid
     Chromatography Method for the separation of benzo(a)pyrene metabolites.
     Presented at American Association for Clinical Chemistry 32nd Annual
     Meeting, Kansas City, Missouri, July 18-23, 1981.
Gregory, P.E., P.M. Howard-Peebles, R.D. El lender and B.J. Martin. 1980.
     C-Banding of chromosomes from three established marine fish cell lines.
     Copeia, 3:  545-547.
Gregory, P.E.% P.N. Howard-Peebles, R.D. Ellender and B.J. Martin.  1980.
     Analysis of a marine fish cell line from sheepshead:  chromosomal
     alterations in Archosargus probatocephalus. J. Heredity 71: 209-211.
Grizzle, J.M., T.E. Schwedler, A.L. Scott. 1981. Papillomas of Black Bullhead
     (Ictalurus me!as Rafinesque) Living in a Chlorinated Sewage Pond.  J. Fish
     Diseases.
Harshbarger, John D., Elliot R. Jacobson, Charles E. Smith and John A. Couch.
     1980. Hematopoietic Neoplasms in  Invertebrates and Cold Blooded
     Vertebrates.  In:  Advances in Comparative Leukemia Research. 1979. David
     S. Yohn, Boris A. Lapin and James R. Blakeslee (eds.) Elsevier/North
     Holland.  New York/Amsterdam/Oxford,  pp. 223-225.
Hemingway, S.J. 1979. (1) Storage Sites of Benzo(a)pyrene in contaminated Bay
     Mussels (Mytilus edulis) inhabiting different sites in Yaquina Bay,
     Oregon. (2) Uptake and depuration of Benzo(a)pyrene in contaminated Bay
     Musses! (Mytilus edulis) inhabiting different sites on Yaquina Bay. M.S.
     Thesis, Oregon State University.
Hendricks, J.D. 1980. Chemical carcinogenesis in fish.  In:  Aquatic
     Toxicology.  L.Weber (ed.). Raven Press, New York. (In press).
Hendricks, J.D., J.H. Wales, R.O. Sinnhuber, J.E. Nixon, P.M. Loveland and R.A.
     Scanlan. 1980.  Rainbow Trout (Salmo gairdneri) embryos:  A sensitive
     animal model for experimental carcinogenesis.  Fed. Proc. (In press).
Hendricks, J.D., R.O. Sinnhuber, P.M.  Loveland, N.E. Pawlowski and J.E. Nixon.
     1980.  Hepatocarcinogenicity of gland!ess cottonseeds and refined
     cottonseed oil to rainbow trout (Salmo gairdneri).  Science (In press).
Hendricks, J.D., and T.R. Meyers.  Ganglioneuroblastomas in cono salmon reared
     in chlorinated-dechlorinated river water.  J. Natl. Cancer Inst. (In
     review).
Hendricks, J.D., Meyers, T.R., Shelton, D.W., and Sinnhuber, R.O. (1982).
     Liver neoplasia and induction of  hepatic mixed function oxidase enzymes in
     the rainbow trout following dietary exposure to benzo(a)pyrene. Proc.
     Amer. Assoc. Cancer Res. 23:58.
Hendricks, J.D., Meyers, T.R., and Shelton, D.W. Histologic progression of
     hepatic neoplasms in rainbow trout (Salmo qairdneri).  Nat!. Cancer  Inst.
     Monogr. (In press).
                                      40

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Hendricks,  J.D., Meyers,  T.R.,  Casteel,  J.L.,  Nixon,  J.E.,  Loveland,  P.M.,  and
     Bailey, G.S.   Rainbow  trout  embryos:   Advantages and  limitations for
     carcinogenesis  research  Natl.  Cancer  Inst.  Monogr.  (In  press).
Hendricks,  J.D., Meyers,  T.R.,  Shelton,  D.W.,  and  Casteel,  J.L.   The
     hepatocarcinogenicity  of benzo(a)pyrene to  rainbow  trout  by  dietary
     exposure  and  intraperitoneal  injection.   (Final  stages  of preparation).
Hillebert,  S.A., B.J. Martin  and  R.D.  Ellender.  1980. Chronic exposure of  a
     teleost cell  line  to suspect carcinogens.   J.  Miss. Acad.  Sci.  (In press).
     Kilgore,  M.V.,  and 0.  Elam.  1979.   Mixed-Function Oxidase Inducibility in
     Marine Organisms.  Ala. Acad.  of Sciences  Meeting, April 14,  Florence,
     Ala.
Kilgore, M.V.,  and D. Elam.   1979.  A  comparatiave study of  induction of mixed
     function  oxidase activity  in the  rat,  mullet  and killifish.   Ala. Acad.
     Sci. Meeting, March  30,  Florence, Ala.
Koenig, Christopher  C., M.  Chasar 1982.  Usefulness of Hermaphroditic Marine
     Fish Rivulus marmoratus  as a Test Animal  for  carcinogenicity  testing.  A
     symposium on the use of  small  fish  species  in  carcinogenicity testing.
     National  Cancer Institute Monograph.   In  Press.
Koening, Christopher C.,  C. Abel,  C.W. Klingensmithe,  M.B. Maddock, 1982.
     Usefulness of the  self-fertilizing  Cyprinodontid fish, Rivulus marmoratus
     as an  experimental animal  in  studies  involving carcinogenesis,
     teratogenesis and  mutagenesis. EPA  Research Assistance Grant  (R805469) 129
     pp.
LaTouche, Y.D.  1981. Metal  levels  in a population  of  Mytilus edulis from
     Yaquina Bay, Oregon. PH.D. Thesis,  Oregon State  universiTyT
LaTouche, Y.D., and  M.C.  Mix. 1981.  Seasonal  variations in trace  metal levels
     in a Mytilus edulis  population in Yaquina Bay  (Newport, OR).  The 21st
     Hanford Life Sciences  Symposium.
LaTouche, Y.D., C.W. Bennett  and M.C. Mix.  1981.   Determination of Vanadium in
     a marine  mollusk using a chelating  ion exchange  resin and neutron
     activation.  Bull. Environ.  Contam. Toxicol., 26: 224-227.
LaTouche, Y. D. and  M.  C. Mix.  1982.  The  effects  of depuration,  size and sex
     on trace  metal  levels  in  bay mussels.  Marine Pollution Bulletin, 13:27-29
LaTouche, Y. D. and  M.  C. Mix.  In  Press.   Seasonal variations of  arsenic and
     other  trace elements in  bay  mussels (Mytilus  edulis).  Bulletin  of
     Environmental Contamination  and Toxicology.   1982.
Long, R.L., and B.J. Martin.1980.Morphology  of  peripheral  blood cells of
     Cyprinodon variegatus.   ABSTR. J. Miss. Acad. Sci., Vol.  XXV, Suppl:
     122.
Martin, B.J. 1980.   Effects of petroleum compounds  on estuarine fishes.  Ecol.
     Res. Series.  EPA-600/3/80-019.  January, 1980.
Martin, B.J.,  and W.W.  Greenwich.  1980.  Exposure  of  two teleost species to
     polycyclic aromatic  hydrocarbons.   ABSTR. J. Miss. Acad.  Sci., Vol. XXV,
     Suppl:  120.
Martin, B.J.,  and W.W.  Greenwich.  1981.  Benzidine Toxicity. J. Miss.  Acad. Sci.
     26 (Suppl.):  127.
Martin, B.J.,  and W.W.  Greenwich.   The Induction of Proliferative Lesions in
     the Sheepshead  Minnow, Cyprinodon variegatus, with Benzidine
     Dihydrochloride.   (In  preparation).
                                      41

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Martin, B.J., R.D., Ellender, S.A. Hillebert, and M.M. Guess.  Primary Cell
     Cultures from  the Teleost, Cyprinodon variegatus:  Culture Establishment
     and Application  in Carcinogen Exposure Studies.  National Cancer Institute
     Symposium  on the Use  of Small Fish Species  in Carcinogenicity Testing,
     Bethesda, MD.  December 8-10, 1981.
Meador, C.B., B.L.  Middlebrooks,  and B.J. Martin.  Serologic Alterations in
     Carcinogen-Exposed Teleosts:  Procedures for Preparation and Analysis of
     Samples From Small Fish.  National Cancer Institute Symposium on the Use
     of Small Fish  Species  in Carcinogenicity Testing, Bethesda, MD.  December
     8-10, 1981.
Melius, P., B. Tan, M. Kilgore, D. Elam, W.P. Schoor. 1979.  Product pattern
     and kinetic analysis  of benzo(a)pyrene metabolism in Marine Organism by
     HPLC.  ACS 31st Southeast Regional Meeting, Florence, Ala. April 14,
     1979.
Melius, P., D. Elam, M.V.  Kilgore, B. Tan and W.P. Schoor. 1979.
     Mixed-Function Oxidase Inducibility and Polyaromatic Hydrocarbon
     Metabolism in  Mullet,  Sea Catfish  and Gulf Killifish.   In:  Polynuclear
     Aromatic Hydrocarbons:  Chemistry  and Biological Effects, Bjorseth and
     Dennis (eds.)i PP« 1059-1075.  Batelle Press, Columbus, Ohio.
Melius, P., D. Elam, M.V.  Kilgore and W.P. Schoor. 1979.  Induction of
     Mixed-Function Oxidase and Metabolism of Polyaromatic Hydrocarbons in
     Marine Organisms.  Eleventh  International Congress of Biochemistry,
     Toronto, Canada.  July 7-11.
Melius, P., B. Tan, M.V. Kilgore, and D. Elam. 1979.  Metabolites of B(a)P in
     Aroclor-induced fish.  Fourth ASTM Symposium on Aquatic Toxicology,
     Chicago, Illinois.  October  16-17.
Meyers, T.R. and J.D. Hendricks.  Case  reports of spontaneous neoplasms in
     feral and  laboratory-reared-salmonids.  J. Fish Dis. (In review).
Meyers, T.R. and Hendricks, J.D.  Histopathological methods.  In Principles of
     Aquatic Toxicology-   (Rand, G.M. and Petrocelli, S.R.,  ed!7).  Hemisphere
     Publishing Co., New York (In press).
Meyers, T.R. and Hendricks, J.D.  A summary of tissue lesions in aquatic
     animals induced by controlled exposures to environmental contaminants,
     chemotherapeutic agents and  potential carcinogens.  Marine Fish. Review
     (In press).
Meyers, T.R. and Hendricks, J.D.  Histopathology of four spontaneous neoplasms
     in three species of salmonid fishes.  J. Fish Dis. (In  press).
Meyers, T.R. and Hendricks, J.D.  A limited epizootic of multi-differentiating
     neuroblastoma  in coho  salmon reared in chlorinated-dechlorinated water.
     Submitted to J. Natl.  Cancer Inst.
Mix, M.C., R.T. Riley, K.I. King, S.R.  Trenholm and R. L. Schaffer.  1977.
     Chemical carcinogens  in the  marine environment.  Benzo(a)pyrene in
     economically important bivalve mollusks from Oregon estuaries.  In:  Fate
     and Effects of Petroleum Hydrocarbons in Marine Organisms and Ecosystems,
     D. A. Wolfe (ed.), pp. 421-431.  Pergamon Press, New York.
Mix, M.C., J.W. Hawkes and  A.K. Sparks.  1978.  The ultrastructure of cells
     associated with proliferative disorders in mussels (Mytilus edulis) from
     Yaquina Bay, Oregon.   Proceedings  of a Colloquium on Invertebrate
     Pathology:  Neoplasms  in Invertebrates.
                                      42

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Mix, M.C. and R.L. Schaffer.  19797.  Benzo(a)pyrene concentrations  in mussels
     (Mytilus edulis) from Yaquina Bay, Oregon during June, 1976 - June, 1978.
     Bull. Environ. Contain. Toxicol., 23: 677 - 684.
Mix, M.C., J.w. Hawkes, and A.K. Sparks. 1979.  Observations on the
     ultrastructure of large cells associated with  putative neoplastic disorder
     of mussels, Mytilus edulis, from Yaquina Bay,  Oregon.  J. Invert. Path.,
     34:  41-56.
Mix, M.C. 19797.  Chemical Carcinogens  in Bivalve Mollusks from Oregon
     Estuaries EPA Ecological Research  Series, EPA600/3-79-034. pp.  33.
Mix, M.C. 1979.  Seasonal variation  in  benzo(a)pyrene in bivalve mollusks from
     Oregon estuaries.  Symposium on Carcinogenic Polynuclear Aromatic
     Hydrocarbons in the Marine Environment.  U.S.  EPA., Gulf Breeze, Florida.
Mix, M.C. 1979.  Leukemia-like disorders in bay mussels (Mytilus edulis) from
     Yaquina Bay, Oregon, U.S.A.  Proceedings of a  Symposium on Comparative
     Leukemia and Related Diseases.
Mix, M.C., D.L. Bunting and D.T. Abbott.  1979.  Preliminary studies to
     evaluate the potential of using embryo and larval  stages of the goose
     barnacle, Pollicipes polymerus, for marine bioassays.  Proceedings of the
     Second Biennial Crustacean Health  Workshop, Texas A M Univ.,
     TAMU-SG-79-114:361-381.  College Station, Texas.
Mix, M.C., S.R. Trenholm and K.I. King. 1979.  Benzo(a)pyrene body burdens and
     the prevalence of cellular pro!iferative disorders in mussels, Mytilus
     edulis, from Yaquina Bay, Oregon.  In:  Animals as Monitors of
     Environmental Pollutants.  52-62.  National Academy of sciences,
     Washington, D.C.
Mix, M.C., R.L. Schaffer and Y.D. LaTouche. 1980.   Environmental  contaminants
     and the occurrence of certain pathological conditions to bivalve mollusks.
     Proceedings of the Society for  Invertebrate Pathology.
Mix, M.C., and W.P- Breese.  1980.   A cellular proliferative disorder in Ostrea
     chilensis from Chilor, Chile, South America.   J. Invert. Pathol.
     36:123-124.
Mix, M.C. 1980.  Utilization of bivalve mollusks. for monitoring carcinogenic
     polynulclear aromatic hydrocarbons in estuarie environments.  The Eleventh
     International Symposium of the  Princess Takamatsu Cancer Research Fund.
     (In press).
Mix, M.C.  Polynuclear Aromatic Hydrocarbons in Bivalve Molluscs from Oregon
     Estuaries.  EPA Ecological Research Series.  (In press).
Mix, M.C., R.L. Schaffer and S.J. Hemingway.  Polynuclear aromatic hydrocarbons
     in bay mussels (Mytilus edulis) from Oregon.   In:  Proceedings  of the
     Eleventh International Symposium of the Princess Takamatsu Cancer Research
     Fund.  (In press).
Mix, M.C.  Cellular prol iferatiave disorders in bivalve mollusks from
     contaminated marine environments.  To be submitted to Journal  of
     Environmental Pathology and Toxicology.
Mix, M. C., S. J. Hemingway and R. L. Schaffer.  1982.  Benzo(a)pyrene
     concentrations in somatic and gonad tissues of bay mussels, Mytilus
     edulis.  Bulletin of Environmental Contamination and Toxicology,
     28:46-51.
                                      43

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Mix, M. C. and R. L. Schaffer.   In Press.   Concentrations  of  unsubstituted
     polynuclear  aromatic  hydrocarbons  in  bay mussels, Mytilus  edulis,  from
     Oregon.  Marine Environmental Research.
Mix, M. C. and R. L. Schaffer.In Press.Concentrations  of  unsubstituted
     polynuclear  aromatic  hydrocarbons  in  softshell  clams  (Mya  arenaris)  from
     Coos Bay, Oregon.  Marine  Pollution Bulletin.
Mix, M. C.   In Press.   The neoplastic disease of  bay mussels, Mytilus edulis,
     from Oregon:   occurrence,  prevalence,  seasonality and histopathological
     progression.   Journal  of Fish Diseases.
Moreno, M.,  and B.J. Martin. 1979.   Embryologic  development of  sheepshead
     minnow,  (Cyprinodon  variegatus).   ABSTR. J.  Miss. Acad.  Sci. Vol.  XXIV,
     Suppl :  116.
Porter, R.D., and B.J.  Martin.  1980.  The  histology  of the postpharyngeal
     digestive tract of the sheepshead  minnow, Cyprinodon  variegatus. ABSTR. J.
     Miss. Acad.  Sci.,  Vol. XXV.   Suppl: 123.
Riley, R.T.  1978.   The  effects  of  chemical  perturbation  by naphthalene  in
     glucose metabolism in the  European flat  oyster, Ostrea edulis:  An  in vivo
     kinetic analysis.  Ph.D. Thesis Oregon State University.
Riley, R.T., and  M.C. Mix. 1980.   Thin  layer  separation  of citric acid  cycle
     intermediates, lactic acid,  and the amino acid  taurine.  Journal of
     Chromatography, 189:  286-288.
Riley, R.T., and  M.C. Mix. 1981.   An  ion -exchange technique  for the
     concentrating  of ammonia in small  volumes of seawater.   Marine Chemistry.
     10:  159-160.
Riley, R.T., M.C. Mix,  R.L. Schaffer and D.L. Bunting.   Uptake  and  accumulation
     of naphthalene by  the oyster, Ostrea  edulis, in a flow-through system.
     Marine  Biology.   (In press).
Riley, R.T., and  M.C. Mix.  The effects of naphthalene on  glucose metabolism
     in the  European flat oyster,  Ostrea edulis.  Marine Biology, (In press).
Riley, R.T.  Stimulatory effect  of naphthalene on glucose transport  in the
     oyster. Submitted to Nature.
Riley, R.T., and  M.C. Mix.  The pathways  of glucose  metabolism  in the oyster,
     Ostrea  edulis. To be submitted to Comparative  Biochemistry and
     Physiology.
Schaffer, R.L.,  and M.C.  Mix.   A method for determining  environmental levels of
     polycyclic  aromatic  hydrocarbons  in  bivalve mollusks  by  reverse phase
     liquid  Chromatography.  To be submitted  to  Analytical Chemistry.
Schneider,  S.R.,  J.D. Hendricks, G.H.  Constantine and R.E. Larson.  1980.
     Tobramycin  nephrotoxicity  in coho  salmon at subtherapeutic doses.
     Toxicol. Appl. Pharmacol.  (In press).
Schoor, W.P. 1979.   Fluorimetric confirmation of metabolites  of benzo(a)pyrene
     from a  marine  fish.   Fourth International  Symposium on Polynuclear
     Aromatic Hydrocarbons, Columbus,  Ohio, October  2-4, Battelle Institute.
Schoor, W.P., and J.A.  Couch.  1979.   Correlation of  mixed-function  oxidase
     activity with  ultrastructural  changes in the liver  of a  marine fish.
     Cancer  Biochemistry  and Biophysics,  4: 95-103.                          _
Schoor, W.P., Elsayed Elnenaey  and Barrie  Tan.  1981.  Benzo(a)pyrene Metabolism
     in 3-Methylcholanthrene-treated sea catfish.  Submitted  to Carcinogenesis.
Schoor, W.P. Exposure  of Fishes to  Benzo(a)pyrene   and Source Aspects  of
     Analysis of Metabolites,  J. Natl.  Cane.  Inst.   in press.
                                       44

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Sigismondi, L.A. 1982.  The Accumulation  of Cadmium, Copper  and Nickel  in
     Freshwater Mussels (Marqaritifera marqaritifera),  Sediment and Algae
     (Cladophora sp.) from Big Elk River, Oregon.  fT.S. Thesis.
Smith,~A.C., and M.C. Mix. 1978.  The effects  of  sodium chloride  concentration
     on electrophoretic patterns of adductor muscle proteins from bivalve
     mollusks.  Comparative Biochemistry  and Physiology (Part B), 61: 169-171.
Strength, R., H.H. Daron, J.L. Aull, J.F. Wilson  and W.P. Schoor. 1980.  The
     induction of glucuronide and sulfate transferases  by phenobarbital and
     polycyclic aromatic hydrocarbons.  Fed. Proc. 39:  1694.
Strength, D.R., K.V. Sarambadal, S.L. Wang, H.H.  Darron, and W.P. Schoor,
     Oxidation and Conjugation of BaP in Mullet.  Fed.  Proceed.,
     41,, 1147(1982).
Strength, D.R., H.H. Darron, and W.P. Schoor.  Comparison of Polycyclic
     Aromatic Hydrocarbon Metabolism in Rodent and Fish.  J. Natl. Cane. Inst.,
     in press.
Tan, B. 1979.  Product Pattern and Kinetic analysis of  benzo(a)pyrene
     metabolism in marine organisms by HPLC.   S.E. Regional A.C.S. meeting,
     Roanoke, Va.  October 25, 1979.
Tan, B. 1980.  Indirect atomic absorption assay of epoxide hydrase.  Ala. Acad.
     Sci., March 21.
Tan, B. 1980.  Indirect atomic absorption assay of epoxide hydrase activity and
     determination of 1,2-diols via digested lead periodate.  English Society
     of Analytical Chemists, 5th Intl. Symp.,  Lancaster, England, July 20-26.
Tan, B. 1980.  Benzo(a)pyrene metabolism  in PAH and Biphenyl treated Tilapia.
     Presented October 28-30, 5th Intl. Symp.  on  PAH's, Columbus, Ohio.
Tan, B., and J.M. Grizzle. 1980.  The use of benzo(a)pyrene metabolism as a
     biochemical indicator in control and tumored bullhead catfish.  October
     28-30, 5th Intl. Symp. on Polynuclear Atomic Hydrocarbons, Columbus,
     Ohio.
Tan, B., P. Melius, M. Kilgore, 0. Elam and W.P.  Schoor. 1979.  Metabolites of
     benzo(a)pyrene  in Aroclor-induced fish.   XI  International Congress of
     Biochemistry, Toronto. Canada, July  8-13.
Tan, B., and P. Melius. 1980.  Responses  of Hepatic Enzymes of Tilapia to PCS
     and PAH modifiers.  American Assoc.  Biol. Chemists Meeting, New Orleans.
     June 2-5. Abstract published in Fed. Proc.
Tan, B., P. Melius and M.V. Kilgore. 1980.  Determination of 1,2-diols by
     direct atomic absorption with digested lead  periodate.  Anal. Chem.
     52:602-604.
Tan, B., D. Elam, E.V. Kilgore and W.P. Schoor. 1979.   Metabolites of
     Benzo(a)pyrene  in Aroclor 1254 treated mullet.  4th ASTM Symp. on Aquatic
     Toxicology.  October 16-17, 1979.  Chicago,  Illinois.  (In press).
Tan, B.  Responses of hepatic enzymes of  Tilapia  to biphenyls and polyaromatic
     hydrocarbons.  Submitted to Biochem.  Pharmacology.
Tan, B.  Comparative metabolism of polyaromatic hydrocarbons in marine
     organisms.  Invited review article being  prepared  for Journal of
     Comparative Biochemistry and Physiology.
Tan, B., and P. Melius.  Kinetic product  patterns and mechanistic pathways of
     benzo(a)pyrene metabolism in Aroclor-treated mullet.  Submitted to
     Biochem. Biophys. Acta.
                                       45

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Tan, B., D. Elam, M.V. Kilgore, and W.P. Schoor.  Mixed function oxidase
     inducibility and polyaromatic hydrocarbon metabolism in mullet, sea
     catfish and gulf killifish.  4th Intl. Symp. on Polynuclear Aromatic
     Hydrocarbons Chemistry and Biological Effects.  Battelle Press. (In
     press).
Tan, B., M.V. Kilgore, D. Elam  and W.P. Schoor.  Metabolites of benzo(a)pyrene
     in Aroclor 1254 treated Mullet.  4th Symp. on Aquatic Toxicology,  ASTM (In
     press).
Tan, B. 1981.  Indirect Atomic Absorption Spectrometric Assay for Epoxide
     Hydrolase.  Analytical Letters, 14(85), 311-322.
Tan, B., P. Melius, and J. Grizzle. 1981.  Hepatic Enzymes and Tumor
     Histopathology of Black Bullheads.  Polynuclear Aromatic Hydrocarbons
     Chemical Analysis and Biological Fate 5th International Symposium.
     Battelle Press.
Tan, B. and P. Melius. 1981.  Responses of the Hepatic Enzymes of a Teleost
     Fish to trans-Stilbene Oxide Treatment.  Bull. Environm. Contam. Toxicol.
     26, 801-806.
Tan, B., P. Melius, and P. Zieglor. 1982.  A Simple Gas Chromatographic Method
     for the Study of Organic Solvents:  Moisture Analysis,  Hygroscopicity, and
     Evaporation.  Journal of Chromatographic Science, Vol.  20. 213-217.
Trenholm, S. and M.C. Mix.  The lethal  and sublethal effects of ionizing
     radiation of juvenile Pacific oysters (Crassostrea gigas).  Radiation
     Research.   (In press).
Trenholm, S.R. and M.C. Mix. 1978.  Regeneration of radiation damaged digestive
     tissue in juvenile Pacific oyster  (Crassostrea gigas).   J. Invert. Pathol.
     32:  249-257.
Trenholm, S.R. 1978.  Effects of X- and Gamma  irradiation on the juvenile
     Pacific oyster, Crassostrea -gigas.  M.S. Thesis Oregon State University.
                                       46

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i  ERL.GB 0267
       TECHNICAL REPORT
:fi, -U' /•( Ju ,'•. 'r.,<:;::-i>ts on i!'.: .-(.". t-/Tc
 1 rIEPO"0" \C
 EPA-600/9-83-005
   Problem Oriented Report
EFFECTS  OF CARCINOGENS,  MLTAGENS, AND TERATOGENS ON
 «)NHUMAN SPECIES--AQUATIC ANIMALS
                                6. PERFORMING ORGANIZATION CODE
  AUTHOR(S)

  .A.  Couch,  W.P. Schoor,  W.  Davis, and Lee Courtney
                                8. PERFORMING ORGANIZATION REPORT \O
 9. PERFORMING ORGANIZATION NAME AND ADDRESS
                                                             10 PROGRAM ELEMENT NO
                                                            •11 CONTRACT'GRANT NO.
 12. SPONSORING AGENCY NAME AND ADDRESS
 U.S.  Environmental Protection Agency
 iivironmental Research Laboratory
 Dffice of Research and Development
 Gulf  Breeze, FL 32561	'	
                                                             13. TYPE OF REPORT AND PERIOD COVERED
                                14. SPONSORING AGENCY CODE
 15. SUPPLEMENTARY NOTES
 16, ABSTRACT
    This report describes the fourth year of a joint U.S. Environmental Protection
    Agency (EPA) and  National Cancer Institute (NCI) effort  to  develop laboratory and
    field assays, using fishes as indicators of the presence of certain carcinogens
    (polycyclic aromatic hydrocarbons).   The study has revealed that fish have
    metabolic pathways  similar to mammals for disposition of certain carcinogens.
 17.
                                 KEY WORDS AND DOCUMENT ANALYSIS
                   DESCRIPTORS
                                                b. IDENTIFIERS-OPEN ENDED TERMS
                                                 COSATi F-ield/Croup
 ,'18. DISTRIBUTION STATEMENT

 !   Release to public
                  U9. SECURITY C._ ASS . 7~'iu
                  !  Unclassified 	
                                               ; 20 SECL/RIT  _ _  __
                                               i  Unclassified
 EPA rorm 2220-1 (Rev. J-T
                        PREVIOUS E - i
                                       n 9 s c - E - s

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