United States
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze FL 32561
Research and Development
EPA-600/S3-82-087 Feb. 1983
&EPA Project Summary
Causes of Papillomas on Fish
Living in Chlorinated
Sewage Effluent
John M. Grizzle and Paul Melius
This research was initiated to deter-
mine the cause of oral papillomas on
black bullheads (Ictalurus melas) from
the final oxidation pond of theTuskegee,
Alabama, sewage treatment plant. The
water in this pond was chlorinated
effluent from the sewage treatment
plant. Ames-test mutagenicity of a
pond-water concentrate indicated the
presence of a chemical carcinogen in
the pond water. However, water and
sediment analysis did not identify sub-
stances suspected of causing the
tumors. Cytoplasmic inclusion bodies
were present in some papilloma cells.
but attempts to find virions in the tumor
via electron microscopy and to transmit
the papillomas by means of injection of
cell-free tumor homogenate into black
bullheads were not successful. Juvenile
black bullheads, yellow bullheads
(Ictalurus natalis), and channel catfish
(Ictalurus pu net at us) were confined to
cages in the oxidation pond and in a
control pond. Most caged black bull-
heads in the oxidation pond developed
focal, oral hyperplasia and stomatitis in
the same mouth locations occupied by
papillomas in wild black bullheads from
this pond. These hyperplastic lesions
healed in most fish during additional
exposure. Mucosal hyperplasia also
occurred, but at a much lower incidence,
in other test species and in all control
species. A similar incidence of hyper-
plastic lesions in black bullheads in
floating and sunken cages indicated
that contact with the sediment or inges-
tion of benthic food organisms did not
affect pathogenesis of the lesion.
This Project Summary was developed
by EPA's Environmental Research Lab-
oratory, Gulf Breeze, FL, to announce
key findings of the research project that
is fully documented in a separate report
of the same title (see Project Report
ordering information at back).
Introduction
A population of black bullheads in the
1.0-hectare, final oxidation pond of the
Tuskegee, Alabama, sewage treatment
plant had a 70% prevalence of oral
papillomas.'These fish provided an oppor-
tunity to study the etiology of fish papil-
lomas, especially as related to chlorinated
sewage effluent.
Sewage entering this treatment plant
did not include discharge from industries
or institutions, but run-off water from
residential and agricultural areas entered
the plant after rains. The chlorination
treatment of sewage after it passed
through an aeration basin and a settling
basin was considered a possible source of
carcinogens.
The only previous report of a high
prevalence of neoplasms in aquatic ani-
mals living in chlorinated sewage effluent
involved a pond at Reese Air Force Base,
Texas. In that case, tiger salamanders
(Ambystoma tigrinum) had neoplastic and
non-neoplastic lesions with a peak fre-
quency of 53%. Chemical carcinogens,
especially perylene, were suspected of
causing the lesions.
Papillomas occur on many species of
fish and are associated with pollution and
tumorigenic viruses. Other fish tumors
have a genetic origin. All of these factors
were considered possible causes of the
papillomas in fish from the Tuskegee
sewage treatment plant.
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Juvenile black bullheads, yellow bull-
heads, and channel catfish were placed
in floating and sunken cages in the final
oxidation pond of the Tuskegee Sewage
Treatment Plant and in a control pond to
determine the effects of chronic exposure
to the chlorinated effluent. Feral and
caged fish from the oxidation and control
ponds were examined for lesions. Per-
sistence, histology, and ultrastructure of
papillomas on wild black bullheads were
studied. Transmission of the papillomas
to healthy black bullheads was attempted
by means of injecting cell-free tumor
homogenate. Water from the oxidation
pond was analysed for metals using
atomic absorption spectrophotometry and
for organic compounds by gas chromatog-
raphy, Concentrates of the oxidation pond
water were tested for mutagenicity using
the Ames test.2
Results and Conclusions
The results and conclusions from this
study are:
1. Water analyses did not reveal pre-
sence of any chemical in sufficient
concentration to be cited as the cause
of papillomas on black bullheads in
the oxidation pond. The possibility of
temporal changes in composition of
the waters, however, was not ade-
quately considered during this study.
2. The acidic organic solvent extract of
the oxidation pond water was muta-
genic to Salmonella typhimurium
strainsTA98 andTAI 00 when Aroclor-
induced rat liver enzymes (S-9) were
included in the agar but was not
mutagenic without S-9. The basic
organic solvent extract was not
mutagenic with or without S-9.
3. No tumors developed on adult or
juvenile fish injected with cell-free
tumor homogenate. The adult fish
were observed for 14 months before
they were necropsied. The juveniles
have bee n observed for 140 days a nd
observation will continue for several
more months.
4. The papillomas on adult, wild black
bullheads from the oxidation pond
contained cytoplasmic inclusion
bodies, usually in cells near the tumor
surface, that were eosinophilic, PAS
positive, and weakly Feulgen positive.
No evidence for the presence of a
virus was found during ultrastruc-
tural examination of the papillomas.
Histologically and ultrastructurally,
these black bullhead papillomas were
similar to papillomas in brown bull-
heads and eels.
5. Twenty-eight adult black bullheads
were taken from the oxidation pond,
placed in clean water in the control
pond, and observed for papilloma
persistence. All 28 fish retained the
lesions, even the six that survived for
more than a month. Persistence of
the lesions after removal of the fish
from the oxidation pond indicates
that the lesions are neoplastic rather
than hyperplastic.
6. During 168 days in the oxidation
pond, 91% of the caged black bull-
heads developed focal, oral hyper-
plasia and stomatitis in the same
mouth location as the papillomas
observed in wild black bullheads from
this pond. Because no pathogens
were consistently associated with
these lesions, the lesions in caged
fish may be related to chemicals in
the water. The hyperplastic lesions
healed in most fish during additional
exposure. Mucosal hyperplasia also
occurred in other test species and in
all control species, but at a maximum
frequency of 14%. A similar incidence
of hyperplastic lesions in fish con-
fined to floating or sunken cages
indicated that contact with the sedi-
ment or ingestion of benthic food
organisms did not affect pathogen-
esis of the lesions. Papillomas did not
develop in caged fish kept in the
oxidation pond. Whether additional
exposure time will lead to develop-
ment of papillomas can be deter-
mined only by continuing this experi-
ment.
Recommendations
1. Analyse water from the final oxida-
tion pond for carcinogens. Future
investigations should include collec-
tion of water samples taken during
different times of the year to deter-
mine temporal changes in composi-
tion of the water.
2. Test oxidation pond water for
mutagenicity using the Ames test.
Seasonal variation in mutagenicity .
should be determined. Additional 1
fractionation of the water before
testing could indicate the chemical
nature of mutagens present in the
water.
3. Enzyme induction in caged fish
should be determined, compared to
results of other parts of this study,
and used as an indication of the
presence of a chemical carcinogen in
the water.
4. Black bullheads injected with cell-
free tumor homogenate should con-
tinue to be observed for development
of tumors.
5. Fish placed in cages during this study
should be maintained in cages to
determine if tumors develop after
longer exposure. Additional caged
fish should be placed in the oxidation
pond at different times of the year to
determine if the etiological agent and
development of hyperplastic lesions
is seasonal.
6. Histological and ultrastructural
studies of caged fish should be
continued to document tissue
changes in fish confined to oxidation
pond water.
References
1. Grizzle, J. M., T. E. Schwedler, and A.
L. Scott. 1981. Papillomas of black
bullheads, Ictalurus me/as (Rafines-
que), living in a chlorinated sewage
pond. Journal of Fish Diseases,
4:345-351.
2. Ames, B. N., J. McCann, and E.
Yamasaki. 1975. Methods for detec-
ting carcinogens and mutagens with
the Sa//7w»e//a/mammalian-micro-
some mutagenicity test. Mutation
Research, 31:347-364.
J. M. Grizzle and P. Met/us are with Auburn University, Auburn, AL 36849.
J. A. Couch is the EPA Project Officer (see below}.
The complete report, entitled "Causes of Papillomas on Fish Living in Chlorinated
Sewage Effluent," (Order No. PB 83-111 039; Cost: $8.50. subject to change)
will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
Sabine Island
Gulf Breeze, FL 32561
•ft- U S GOVERNMENT PRINTING OFFICE; 1983 659-OI7/O896
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Environmental Protection
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Center for Environmental Research
Information
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Penalty for Private Use $300
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