Unitod St8t6S
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze FL 32561
EPA-600/3-79-034
March 1979
Retearch and Development
Chemical
Carcinogens in
Bivalve Mollusks
from Oregon
Estuaries
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-79-034
March 1979
CHEMICAL CARCINOGENS IN BIVALVE MOLLUSKS
FROM OREGON ESTUARIES
by
Michael C. Mix
Department of General Science
Oregon State University
Corvallis, Oregon 97330
EPA Grant R804427010
Project Officer
John A. Couch
Gulf Breeze Environmental Research Laboratory
Gulf Breeze, Florida 32561
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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DISCLAIMER
This report has been reviewed by the Gulf Breeze Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. 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.
ii
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FOREWORD
The protection of our estuarine and coastal areas from damage caused by
toxic organic pollutants requires that regulations restricting the introduc-
tion of these compounds into the environment be formulated on a sound
scientific basis. Accurate information describing dose-response relationships
for organisms and ecosystems under varying conditions is required. The EPA
Environmental Research Laboratory, Gulf Breeze, contributes to this informa-
tion through research programs aimed at determining:
• the effects of toxic organic pollutants on individual species and
communities of organisms;
the effects of toxic organics on ecosystem processes and components;
* the significance of chemical carcinogens in the estuarine and marine
environments.
Considerable interest has focused recently on the fate and possible
effects of carcinogens and mutagens in the aquatic environment which usually
is the ultimate receptacle for pollutants. This report describes the fate
and some possible long-term effects of polycyclic aromatic hydrocarbons in
the marine estuarine environment and biota. These data may serve to alert
us to the role of certain carcinogens in the environment generally.
Thomas W. Duke
Director
Environmental Research Laboratory
Gulf Breeze, Florida
111
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ABSTRACT
The research undertaken involved the use of indigenous populations of
bivalve mollusks as monitors for detecting and quantifying environmental
benzo(a)pyrene (BAP) in Oregon estuaries. Short-term and long-term studies
were conducted in order to establish baseline levels of BAP and to identify
seasonal variations in BAP concentrations in shellfish. A presumptive cellu-
lar proliferative disorder, thought possibly to be neoplastic, was also
studied in mussels, Mytilus edulis, from Yaquina Bay.
Certain populations of indigenous bivalve mollusks from two industrializ-
ed Oregon bays contained shellfish with significant BAP body burdens. Highest
levels (f>15 ng/g) were detected in mussels inhabiting the Newport bayfront
area of Yaquina Bay, and in clams, Mya arenaria, collected near the Coos Bay
shipping docks. In general, detectable levels of BAP were found in all pop-
ulations sampled from the two industrialized bays while populations from non-
industrialized bays (Alsea, Netarts) or lightly industrialized bays (Tilla-
mook) contained low or below detectable levels of BAP. BAP body burdens in
M. edulis from Yaquina Bay were lowest during the fall, increased during the
winter to highest levels in the early spring, after which they declined.
Histological studies revealed that mussels inhabiting polluted environ-
ments, and with high BAP body burdens, had an average 6-8% prevalence of the
cellular proliferative disorder while those from clean environments and with
low or undetectable levels, did not have the disorder. The cellular condition
showed a definite seasonal pattern; there was a low prevalence during the
summer and. fall followed by> an increase during the early winter and a peak
prevalence occurred in January-February. The atypical, large cells that
characterize the disorder in M. edulis possess many ultrastructural proper-
ties in common with malignant vertebrate cells.
Further studies are required to evaluate the public health significance
of these results.
This report was submitted in fulfillment of Contract No. R804427010 by
Oregon State University, Corvallis, Oregon, under the sponsorship of the
U.S. Environmental Protection Agency. This report covers the period June 1,
1976, to September 30, 1978.
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CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
Abbreviations and Symbols viii
Acknowledgments ix
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. Materials and Methods 6
Selection of Oregon estuaries 6
Selection of bivalve species for baseline studies 9
Bays and species used for long-term studies 10
Collection and preparation of shellfish samples 11
Chemical analysis for benzo(a)pyrene 11
Histological examination of M. edulis 11
Electron microscopic analysis 12
Statistical treatments 12
5. Results and Discussion 14
Baseline data on BAP body burdens 14
Long-term studies on BAP body burdens 16
Cellular proliferative disorders in M. edulis 26
Electron microscopic analysis of M. edulis cells 27
Public health considerations 27
References 30
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FIGURES
Number Page
1 Oregon bays and estuaries 6
2 Sampling sites in Tillamook Bay, Oregon 7
3 Sampling sites in Netarts Bay, Oregon 7
4 Sampling sites in Yaquina Bay, Oregon 8
5 Sampling sites in Alsea Bay, Oregon 8
6 Sampling sites in Coos Bay, Oregon 9
7 Seasonal differences in BAP body burdens in M. edulis
populations from Yaquina Bay, Oregon 19
8 The amount of rainfall in the Yaquina Bay watershed
and the temperature and salinity during 1976-77 21
9 Rainfall, temperature and salinity during 1977-78
10 Seasonal differences in BAP body burdens, expressed as a
percentage of the mean, in M. edulis from Yaquina bay,
Oregon, 1976-77 ~« • • • 22
11 Seasonal differences in BAP body burdens, 1977-78 23
12 BAP body burdens and the degree of sexual maturity in
M. edulis 25
vi
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TABLES
Number Page
1 Clam, Mussel and Oyster Species Analyzed in the Study 10
2 Baseline Levels of BAP in Bivalve Mollusks from Oregon Estuaries • 15
3 BAP Body Burdens in M. arenaria from Coos Bay, Oregon 17
4 BAP Body Burdens in M. edulis from Yaquina Bay, Oregon 18
5 BAP Body Burdens Calculated as a Percentage of the Mean 20
6 Prevalences of Cellular Proliferative Disorders in M. edulis • • • 26
vii
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ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
ANOVA
BAP
C
CaCl2
DMSO
EM
g
3H-BAP
hr
km
KOH
M
mg
N2
ng
PNAH
RUE
TMP
pm
SYMBOLS
analysis of variance
benzo(a)pyrene
Celsius
calcium chloride
dimethyl sulfoxide
electron microscope
gram
tritiated benzo(a)pyrene
hour
kilometer
potassium hydroxide
molar
milligram
nitrogen
nanogram
polynuclear aromatic hydrocarbon
rate of uptake and elimination
2,2,4-trimethylpentane
micron
Oregon Bays
A
C
N
T
Y
— Alsea Bay
— Coos Bay
— Netarts Bay
— Tillamook Bay
— Yaquina Bay
Bivalve Mollusk Species
B
C
G
M
0
S
— Saxidomus giganteus (butter or Empire clam)
— Clinocardium nuttalli (cockle)
— Tresus capax (gaper clam)
— Mytilus edulis (blue mussel)
— Crossostrea gigas (Pacific oyster)
— Mya arenaria (softshell clam)
viii
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ACKNOWLEDGMENTS
The following individuals were directly associated with the project dur-
ing all or most of the two-year period and their participation and guidance
is gratefully acknowledged: Ms. Diane Bunting, Ms. Debra Early, Mr. Ke.ith
King, Dr. Ronald Riley, Mr. Randy Schaffer, and Mr. Steve Trenholm.
Dr. Bruce Dunn of the University of British Columbia shared his exper-
tise on BAP analytical methods; Drs. Virgil Freed and Donald Buhler gener-
ously permitted the use of their facilities and equipment; Drs. Joyce Hawkes
and Albert Sparks participated in the electron microscope studies; Ms.
Marilyn Henderson provided valuable technical assitance; Ms. Lori Mix helped
transform climatological data; Mr. Paul Heikkala aided in the selection of
sampling sites in Coos Bay; Mr. Dale Snow graciously shared his extensive
knowledge of Oregon shellfish. Their assistance was invaluable and is hereby
acknowledged.
Drs. John Couch, Clyde Dawe, John Harshbarger, Albert Sparks, Hans Stich,
and Mr. Austin Farley made direct and indirect contributions to the initia-
tion and design of this project.
Pergamon Press kindly permitted the use of Figures 1-6, reprinted from,
"Fate and Effects of Petroleum Hydrocarbons in Marine Organisms and Eco-
systems," 1977, pp. 421-431. Mss. Coleen Annis and Gail Siani typed the final
report.
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SECTION 1
INTRODUCTION
It has been suggested from analyses of epidemiological data, that 60-90
percent of all cancers occurring in human occupants of industrial societies
are caused by environmental carcinogens (e.g. Higgenson, 1971). To date, no
definitive data are available on the proportion of human cancers which develop
as a consequence of exposure to chemical carcinogens. However, environmental
chemicals are suspected to be etiologic agents on the basis of man's proven
susceptibility to some chemical carcinogens and the wide occurrence of chemi-
cals which are known to be carcinogenic for vertebrate animals or have struc-
tures similar to those of known carcinogens. The effects of chronically
exposing indigenous plant and animal species to environmental carcinogens are
not known.
Benzo(a)pyrene (BAP) and several other polynuclear aromatic hydrocarbons
(PNAH), which are carcinogenic in vertebrate systems, are found in low levels
in crude oil, particularly refined oils, and thousands of kg of these com-
pounds enter the sea each year (NAS, 1975). Also, weathered and partially
degraded oils may contain additional oxidation products of potential carcino-
genicity (Feldman, 1973). However, several recent reports indicate that, in
general, PNAH's found at ppb levels in marine animal tissues are derived from
common combustion sources and not directly from petroleum contamination
(Blumer and Youngblood, 1975; Hites, 1976; Brown and Weiss, 1978). In any
event, it seems clear that PNAH levels in economically important marine or-
ganisms will continue to increase in biologically productive estuaries because
of the inexorable increasing utilization of fossil fuels.
The use of bivalve mollusks for monitoring marine environments in order
to detect and quantitate various pollutants, including chemical carcinogens,
has been advocated by many investigators (e.g. Goldberg, 1975). Indigenous
populations of shellfish seem to be ideal subjects for evaluating carcinogenic
PNAH loads in the marine environment (Lee, 1977; Mix et al., 1977a). They
are sedentary and, excepting the larval planktonic stage, spend their entire
lives in the same location; certain species have cosmopolitan distributions;
they inhabit waters that are polluted by all classes of environmental contam-
inants, including PNAH; they tend to concentrate these contaminants in their
tissues; they do not metabolize PNAH; PNAH persist in their tissues after
long depuration periods; and they are large, relatively easy to locate,
sample, dissect, and prepare for chemical analysis. In addition, bivalve
shellfish are widely utilized as a food resource and are exploited by both
commercial and recreational fishermen. Thus, qualitative and quantitative
analysis of shellfish tissues for specific environmental pollutants consti-
tutes a direct assay method for measuring such contaminants in the marine
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environment and offers the potential of using resulting data to assess the
public health hazard, if any, of consuming contaminated seafoods.
During the past decade, there have been numerous cases of presumptive
sarcomatoid disorders reported in marine bivalve mollusks from areas through-
out the world (see review by Mix et al., 1977b) . Studies to determine the
etiological agent(s) of the putative proliferative disorders have thus far
been equivocal. Recently, there have been reports that tentatively linked
the appearance of atypical large cells in bivalve populations with oil spills
(Barry and Yevich, 1975; Brown et al., 1977; Yevich and Barszcz, 1977), sig-
nificant body burdens of benzo(a)pyrene (Mix et al., 1977a; in press) and
perhaps other aromatic hydrocarbons (Lowe and Moore, 1978), and "adverse
water quality" (Alderman et al., 1977; Couch et al., in press). These re-
ports offer some support to the suggestion that general pollution or point-
source contamination may be related to increases in neoplasia in marine
animals (Kraybill, 1977).
Kraybill (1976) suggested that in order to assess the carcinogenic risk
of chemicals as inducers of neoplasms in aquatic organisms, a systematic
survey must be made to identify and quantitate carcinogens in waterways, and
to determine the body burdens or concentrations in aquatic animals and their
relevance to tumor incidence in these species.
The present study was designed to accomplish the following:
1. Evaluate the use of bivalve shellfish as monitors for identifying
and quantifying chemical carcinogens in estuaries;
2. Measure baseline levels of BAP in several species of bivalve mol-
lusks from pristine and industrialized Oregon bays;
3. Identify sources of environmental BAP;
4. Evaluate and identify environmental parameters that may influence
uptake and elimination and account for any seasonal differences in BAP body
burdens;
5. Determine the prevalence of cellular proliferative disorders in
mussels (M. edulis) that inhabit uncontaminated waters and environments con-
taminated with BAP and possibly other PNAH;
6. Determine if there were seasonal differences in the prevalence of
the cellular conditions in mussel populations;
7. Describe the ultrastructure of large cells in affected M. edulis and
determine whether or not they have features in common with neoplastic, malig-
nant vertebrate cells;
8. To evaluate the public health implications of our findings.
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SECTION 2
CONCLUSIONS
Utilization of a single species of bivalve mollusk for monitoring an
estuary has certain limitations. As a result of these studies, the concept
of using multiple species as estuarine monitors for chemical carcinogens has
been advanced. There are two primary advantages in using two or more species
in monitoring studies. First, it enables investigators to monitor an entire
bay, which is not always possible when using a single species. This is par-
ticularly important for estuaries that are freshwater dominated. It was
found that for Oregon estuaries, softshell clams (Mya arenaria) are useful
monitors for upbay sites, where much of the industry is located, since they
are virtually the only economically important bivalve mollusk that thrives in
these areas of reduced salinity. M. edulis can be used to monitor the lower
bays where the salinities are higher. A second advantage is that by using
two species which occupy different habitats, it may be possible to obtain in-
formation about the routes of PNAH movement (water v. sediments) and reser-
voirs in the marine environment.
Certain populations of indigenous bivalve mollusks from the industrial-
ized Oregon bays, Yaquina and Coos Bays, contained shellfish with significant
BAP body burdens. Highest levels (>15 ng/g) were present in mussels collect-
ed from the Newport bayfront in Yaquina Bay and in Coos Bay clams collected
near the shipping docks adjacent to Highway 101. In general, detectable
levels of BAP were found in all populations sampled from the industrialized
bays while populations from non-industrialized bays (Alsea, Netarts) or
lightly industrialized bays (Tillamook) contained low or below-detectable
levels of BAP. The data suggest that levels of BAP and other PNAH contamina-
tion in shellfish are a direct function of the degree of industrialization
and/or human activities in the watershed drainages. The sources of BAP and
other PNAH in Oregon bivalve mollusks are not precisely known. However,
small fuel or oil spills, marinas, industrial wastewater from fish processing
and other onshore factories, large ships, creosoted pilings, sewage treatment
plants, runoff from storm sewers and watersheds, especially where logging and
subsequent slash burning has occurred, and rainout of PNAH produced from com-
bustion during the winter months are all potential sources.
BAP body burdens in M. edulis from Yaquina Bay tended to be lowest dur-
ing the fall, increased during early winter to the highest levels in early
spring, after which they declined. Factors that may be associated with the
early spring peaks and perhaps a preceding increase in environmental BAP
during the early winter include: reduced water temperatures during the
winter; reduced photooxidation of BAP during the winter; resuspension of BAP
in the sediments caused by winter floods; rainout of higher levels of atmos-
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pheric BAP associated with increased combustion of fuel for heating during
the winter; and greatly increased runoff from watersheds during the winter.
Intrinsic biological mechanisms may also be involved; changes in BAP body
burdens paralleled changes in gonadal maturation during 1977-78.
It should be noted that when BAP body burdens were high, there were also
substantial amounts of other fluorescing compounds that, because of the sep-
aration techniques utilized, are known to be other PNAH. Thus, BAP essen-
tially serves as an indicator PNAH; the quantitative relationship between BAP
concentration and the levels of other PNAH is not yet known.
M. edulis from the moderately polluted Newport bayfront, on Yaquina Bay,
contained significant levels of BAP (>15 ng/g), while those from cleaner
areas contained very low or undetectable levels of BAP. Histological studies
showed that mussels from populations with high BAP body burdens had an
average prevalence of 6-8% while those with ]ow or undetectable levels did
not have the cellular proliferative disorder. The cellular condition in
mussels showed a definite seasonal pattern with a low prevalence during the
summer and fall followed by an increase during the early winter and a peak
prevalence in January-February. It is not yet known whether or not high body
burdens of BAP and other PNAH are directly or indirectly associated with the
appearance of the cellular disorder in mussels.
The atypical large cells that characterize the cellular proliferative
disorder in M. edulis possess many ultrastruetural properties in common with
malignant vertebrate cells. The atypical cells have: large, polymorphic
nuclei; large, multiple nucleoli; altered Golgi complexes; mitochondrial
inclusions; an absence of cytoplasmic differentiation; ribosomes not assoc-
iated with a membrane system; pleomorphic inclusions in the cytoplasm; and
possible asynchronization in the maturation of nucleus and cytoplasm. Never-
theless, alternative explanations concerning their nature can be formulated
and much additional information about their life history is required before
any definitive conclusions can be made.
Chemical carcinogenesis has recently become a major public health con-
cern. It is known that some chemicals, including BAP and other PNAH, enter-
ing the marine environment are either direct carcinogens or carcinogenic if
they are metabolically activated by appropriate enzymes. We found detectable
levels of BAP in shellfish from 38 of 44 sampling sites, indicating that BAP
is rather widespread in Oregon estuaries. Many of the sites were on or near
commercial or recreational clam beds. However, a great amount of additional
data is required before it is possible to fully assess the public health
implications of our findings.
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SECTION 3
RECOMMENDATIONS
It has been suggested that the presence of neoplastic cellular disorders
in feral populations of marine organisms may be indicative of the presence of
carcinogens in the environment. It is now established that such disorders
occur in Yaquina Bay mussels and our data suggest that such disorders may be
linked with environmental contaminants. The implications relative to water
quality standards and public health are potentially important. The tentative
association between environmental pollution, as indicated by the presence of
BAP in biological monitors, and the appearance of proliferative disorders in
molluscan populations requires further study. Substantial amounts of addi-
tional data are necessary to fully evaluate this potential cause and effect
relationship.
Many PNAH, other than BAP, are potent carcinogens. In future studies,
efforts should be directed toward: establishing baseline levels of PNAH in
monitoring species from pristine and industrialized bays; determining the
quantitative relationship between BAP and total PNAH body burdens; identify-
ing specific PHAH present in the tissues of economically important marine
organisms; identifying PNAH storage sites in molluscan tissues; and identify-
ing seasonal differences in PNAH body burdens. In order to assess potential
public health problems, it is particularly important to expedite the accumul-
ation of this information for marine organisms which are exploited as food
sources by recreational and commercial shellfishermen.
An issue that is not yet resolved concerns the exact nature of the large
cells that characterize the cellular conditions in bivalve mollusks that have
been diagnosed as being neoplastic. The failure to clarify this problem is
primarily related to the paucity of information about the complete life his-
tory of the cells. In M. edulis, such cells have several cytomorphologic
features that are characteristic of malignant vertebrate cells. However,
additional studies are required to determine their precise nature.
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SECTION 4
MATERIALS AND METHODS
SELECTION OF OREGON ESTUARIES
Oregon has a large number of bays and estuaries (Fig. 1). Three criteria
were employed for selecting the 5 estuaries to be studied: they must have
major commercial and/or recreational shellfisheries; a variety of bivalve
species had to be present in, or on, different substrates; and each bay had
to reflect varying degrees of industrialization and human onshore and water-
shed habitation.
Young's Bay
Necanicum
Nehalera '
Tillamook
Netarts
Sand Lake
Nestucca
WASH'NGTON
CALIFORNIA
Figure 1. Oregon bays and estuaries.
The following bays were selected for the first studies to determine base-
line levels of BAP: Tillamook (Fig. 2); Netarts (Fig. 3); Yaquina (Fig. 4);
Alsea (Fig. 5); and Coos (Fig. 6), (accompanying information from Percy,
et al., 1974).
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Figure 2. Tillamook Bay, with a total sur-
face area of 8,660 acres of which 50-60%
are tidelands, drains a basin of 540 square
miles with a high freshwater yield. Major
industries: timber, agricultural products,
fish and seafoods, tourism. Not considered
to be highly industrialized.
Figure 3. Netarts Bays, with a total surface area
of 2,200 acres of which 65-90% are tidelands, drains
a basin of 14 square miles with a very low fresh-
water yield. Manufacturing companies are lacking
completely; the bay is considered to be relatively
pristine.
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Figure 4. Yaquina Bay, with a total surface area of 4,000 acres of which 35-
61% are tidelands, drains a basin of 253 square miles with a medium fresh-
water yield. A major industrial estuary, the bay is a center for lumbering
and commercial fishing activities. Toledo is the focal point of the forest
industry processing facilities for the entire Mid-Coast Basin. Newport is
the center for commercial fishing activities and there are numerous fish
processing plants along the bayfront. Numerous marinas are scattered through-
out the bay.
Figure 5. Alsea Bay, with a total surface area of 2,140 acres of which 45-
50% are tidelands, drains a basin of 474 square miles with a high freshwater
yield. Lumber-related activities, tourism and agriculture are of major
economic importance. Little industrial use of the bay.
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Figure 6. Coos Bay, with a total surface of 10,000 acres of which 50% are
tidelands, drains a basin of 605 square miles with a very high freshwater
yield. Coos Bay is the most heavily industrialized of all Oregon bays.
Timber, fish resources and agricultural activities are of major economic
importance. Many major lumber manufacturers are located in the bay and
there is heavy shipping traffic concentrated around Coos Bay.
SELECTION OF BIVALVE MOLLUSCAN SPECIES FOR BASELINE STUDIES
The following criteria were applied for selecting the shellfish species
to be analyzed for baseline BAP body burdens. They had to be: economically
important (utilized as a food resource by commercial and/or recreational
shellfishermen) ; present in sufficient numbers to withstand sampling for sev-
eral years; present in at least two of the bays; cosmopolitan, so comparisons
could be made with other researchers; representative of certain types of sub-
strates and/or specific habitats (e.g. soft mud, low salinity); available for
sampling during the entire year. Table 1 summarizes the species analyzed
from the various bays.
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TABLE 1. (CLAM, MUSSEL, AND OYSTER SPECIES USED IN THE STUDY)
Species
Common Name
Habitat, substrate;
salinity
Bays
Tresus capax
Gaper clam (G)
Saxidomus giganteus Butter or
Empire clams (B)
My a arenaria
Softshell clam (S)
Clinocardium nuttalli Cockle (C)
Mytilus edulis Mussel (M)
Crassostrea gigas Pacific oyster (0)
Soft, sandy and
rocky mud; high
Rocky mud; high
Soft or sandy
mud; low
Sandy or rock mud
Pilings, rocks;
low to high
Held in trays, on
Sticks, moderate
T,N,Y,A,C
N,C,Y
T,Y,A,C
T,N,Y,C
T,N,Y,A,C
T,Y,C
The abbreviations used in this table: T - Tillamook Bay; N - Netarts Bay;
Y - Yaquina Bay; A - Alsea Bay; C - Coos Bay.
The abbreviations used to designate the sampling sites (Figs. 2-6) are
read as follows: first letter refers to the bay; the number indicates the
specific site and the last letter the species. For example, C2S refers to
Coos Bay, site 2, softshell clam.
BAYS AND BIVALVE SPECIES UTILIZED FOR LONG-TERM STUDIES
In order to determine temporal or seasonal differences in BAP body
burdens, M. arenaria from Coos Bay and M. edulis from Yaquina Bay were
studied for two years, from June, 1976 through June, 1978. Mussels and clams
from the following sites were assayed for BAP during this period: C2S C3S
C4S, and C19S; YlM, Y2M, Y3M, Y4M, Y5M, Y6M, Y7M, Y8M, Y10M, Y11M Y12M
Y13M, and YUM.
A brief description of each Yaquina Bay collecting site is included be-
low. YlM: mussels were collected from a number of weathered, heavily creo-
soted pilings that formerly supported an old railroad trestle. Y2M Y3M
Y4M: creosoted pilings that support cold storage facilities and fish process-
ing plants; several nearby marinas. Y5M: creosoted pilings. Y6M: creosoted
pilings supporting a small boat dock. Y7M: creosoted floating dock support-
ing a gas pump; near a large boat basin; Y8M: creosoted floating dock
Y10M: creosoted piling supporting a large ship dock. Y11M: rocks near'the
liquid natural gas storage tanks. Y12M: creosoted pilings supporting a
10
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marker buoy in the main channel. Y13M: iron pilings near a marina. YUM:
creosoted piling.
COLLECTION AND PREPARATION OF SHELLFISH SAMPLES FOR CHEMICAL ANALYSIS
Clams and cockles from the various bays were dug during low, usually
minus, tides while mussels were collected during the entire tidal cycle, de-
pending on their location. Oysters were obtained from commercial growers
and simply removed from the shucking tables. Excepting _T. capax, at least
10 shellfish were collected from each sampling site; only" 5 of the large
gaper clams were sampled. Immediately following collection, the shellfish
from a single site were placed in labeled plastic bags, put on ice contained
in coolers and transported back to our laboratory in Corvallis. Individual
animals were sized, shucked (removed from the shell) and the pooled sample
from each site was then weighed. Each pooled sample was then stored in a
plastic bag at -20QC until it was analyzed for BAP.
CHEMICAL ANALYSIS FOR BENZO(a)PYRENE
Prior to analysis, each sample, excepting the mussels, was homogenized
in an electric, plastic meat grinder. Mussel samples were prepared similarly;
however, because of the small size of the individual animals, no homogeniza-
tion was necessary. An aliquot of each sample was analyzed according to the
method of Dunn (Dunn, 1976). A 30-40 g sample was digested by refluxing in
an ethanol-KOH solution. Following digestion, the ethanol-KOH supernatant
was extracted with 2,2,4-trimethylpentane (TMP) and the organic phase passed
through a column of partially deactivated florisil. The PNAH were eluted
with benzene and, after removal of the benzene, the eluate was cleaned up by
DMSO extraction in TMP.
BAP was isolated by preparative thin layer chromatography on 20% acety-
lated cellulose, made to volume in hexadecane and the concentration of BAP
determined by spectrophotofluorimetry. Recovery of BAP by the extraction
procedure was determined by spiking the original digestion with an aliquot
of G-%BAP and counting an aliquot of the final hexadecane solution.
Analysis of wood (piling) samples was accomplished similarly. Approxi-
mately 100 mg of the ovter 0.2 cm of the piling was dried at 60°C for 24 hr
and then placed in a pre-cooled porcelain mortar, liquid N2 added, and the
wood pulverized to a fine powder with a porcelain pestle. Ten mg of the
powdered wood was then analyzed according to the procedures outlined pre-
viously.
COLLECTION AND PREPARATION OF M. EDULIS FOR HISTOLOGICAL EXAMINATION
After preliminary analyses to measure BAP concentrations in mussels from
14 Yaquina Bay sites, four sites were selected for prevalence studies.
Mussels from two of the sites, Y1M and Y12M, initially contained very low or
11
-------
undetectable levels of BAP (<1 ng/g) while mussels from Y2M and Y4M contained
high levels (>15 ng/g). Mussels from all sites were removed from creosoted
pilings that had undergone varying degrees of weathering. BAP levels in wood
from these pilings were also determined during preliminary studies. The four
sites were:
Y1M. This site consists of a number of weathered, heavily creosoted pil-
ings that formerly supported an old railroad trestle. The pilings are locat-
ed slightly south of the main channel and are subjected to heavy tidal cur-
rents.
Y2M and Y4M. Mussels from these sites were removed from creosoted pil-
ings that support two fish processing plants along the bayfront; there are
also several marinas nearby. Tidal flows in the bayfront area are minimal.
Y12M. Mussels were removed from creosoted pilings that support a marker
buoy in the main channel. This site is also subjected to heavy tidal flows.
Mussels were collected bimonthly from each of the four sites. Immediate-
ly after collection, they were separated according to site, placed in labeled
plastic bags, put on ice in coolers, and transported to our laboratory in
Corvallis. Mussels to be examined histologically were placed in Davidson's
fixative (3:3:2:1:1 - 95% ethanol:seawater:formalin:glycerol:acetic acid
added just prior to use), processed in the usual way, and sectioned at 6 urn.
Tissue slides, prepared for each specimen, were examined microscopically by
two or three investigators to determine if they possessed the large cells
that characterize cellular proliferative disorders.
ELECTRON MICROSCOPIC ANALYSIS OF THE LARGE M. EDULIS CELLS
Portions of the visceral mass of living mussels from the Y2M site and
several downbay sites were excised and fixed in 0.75% glutaraldehyde, 3%
formalin, 0.5% acrolein in 0.1 M sodium cacodylate buffer with 0.02%
CaCl2*2H20, and 5.5% sucrose. Initial fixation was followed by a buffer
wash and post-fixation in 1% osmium tetroxide. Tissues were dehydrated,
embedded in Spurr medium (Spurr, 1969), and sectioned with either a glass or
diamond knife. Thin sections for electron microscopy were triple stained
with lead citrate, uranyl acetate, and lead citrate and examined with a
Philips EM-301.
STATISTICAL TREATMENTS
A summary of the statistical tests is included below. A Monroe 1980
Programmable Calculator was used for most of the analyses. A CDC computer
was used to determine the quadratic equation from the seasonal prevalence
data.
1. Determination of the seasonal-year correlation for BAP body burdens:
two way analysis of variance (two way ANOVA).
2. Determination of the seasonal effects on BAP body burdens for a one
year period: one way ANOVA.
3. Evaluation of season-BAP body burden correlation: Student Newman-
Keuls multiple comparison test.
12
-------
4. Determination of site-BAP body burden correlation: one way ANOVA.
5. Evaluation of site-BAP body burden correlation: Student Newman-
Keuls multiple comparison test.
6. Determination of site-prevalence (cellular proliferative disorder)
correlation: one way ANOVA.
7- Evaluation of site-prevalence correlation: Student Newman-Keuls
multiple comparison test.
8. Determination of seasonal-prevalence correlation: one way ANOVA.
9. Test seasonal-prevalence correlation to determine if the variation
was linear or curvilinear: linear regression analysis.
10. Evaluate seasonal-prevalence correlation: multiple regression
analysis.
13
-------
SECTION 5
RESULTS AND DISCUSSION
BASELINE DATA ON BAP BODY BURDENS
The concentrations of BAP found in bivalves from collecting sites in the
5 bays are included in Table 2. Highest levels (>15 ng/g) of BAP were found
in mussels from one site (T3M) in Tillamook Bay (pilings near a gas pump at a
marina), and 2 sites (Y2M, Y4M) in Yaquina Bay (pilings beneath fish process-
ing factories and near marinas). Lower levels (>5 ng/g) were found in mussels
on pilings near a large ship dock in Yaquina Bay (Y10M) and in softshell
clams, also near large ship docks, from Coos Bay (C4S). Shellfish from
Netarts and Alsea Bays did not contain detectable levels of BAP.
Although high levels of BAP were found in mussels from Tillamook and
Yaquina Bays that were taken from pilings near marinas and fish processing
factories, the sources of BAP at these sites have not been clearly estab-
lished. Because of the suggestion (Dunn and Stich, 1976) that creosote may
be the major source of environmental BAP in mussels, the pilings from which
they were attached were analyzed from 4 sites. The results are summarized
below {site; BAP level in mussels (ng/g); BAP levels in the piling (ng/g)}.
Y1M; 0.22; 265,512
Y2M; 30.30; 137,204
Y3M; 3.25; 6,695
Y4M; 15.00; 107,097
The results of these analyses do not permit the formulation of any firm
conclusions. It is interesting to note that the pilings with the highest
level of BAP (265,512 ng/g) harbored mussels with one of the lowest levels
(0.22 ng/g). However, other factors, such as the age of the piling and
proximity to strong tidal currents, must also be considered.
Clam species have not previously been utilized as biomonitors for study-
ing environmental carcinogens. They are buried in the substrate and it may
be possible that, because of various physico-chemical processes operating in
sediments, food chain decay,"utilization of different food sources, or differ-
ent metabolic capabilities, they will possess lower levels of BAP than mus-
sels even though they inhabit a highly contaminated environment.
The present results suggest that BAP is a ubiquitous contaminant in
shellfish inhabiting industrialized Oregon bays. Similar results have also
been reported for mussels from Vancouver Harbor (Dunn and Stich, 1975) and
14
-------
Southern California (Dunn and Young, 1976). With the exception of Coos Bay
and portions of Yaquina Bay, no Oregon bay can yet be characterized as being
heavily industrialized. However, further industrialization and the concom-
itant increase in fuel combustion, ship traffic, dredging activities and the
number of smaller supporting industries may lead to greater environmental
contamination and subsequent increases in PNAH concentrations in economically-
important shellfish. Thus, the continuation of monitoring studies in indus-
trialized bays supporting major shellfisheries seems warranted.
TABLE 2. BASELINE LEVELS OF BENZO(a)PYRENE IN BIVALVE MOLLUSKS FROM OREGON
BAYS
Bay site
TILLAMOOK
TIM
T2M
T3M
T4M
T5M
T6G
T7G
T10S
T110
NETARTS
NIG
YAQUINA
Y1M #
Y2M //
Y3M //
Y4M //
Y5M //
Y6M //
Y7M #
Y8M
Y9M
Y10M #
YUM #
Y12M //
Y13M #
Y14M #
Y15G
Y16G
Y17G
Species
SIS IS
M.
M.
T.
T.
M.
C.
T.
M.
M.
M.
M.
M.
M.
M.
M.
M.
M.
M.
M.
M.
M.
T.
T.
T.
edulis
edulis
edulis
edulis
edulis
capax
capax
arenaria
gigas
capax
edulis
edulis
edulis
edulis
edulis
edulis
edulis
edulis
edulis
edulis
edulis
edulis
edulis
edulis
capax
capax
capax
Type of
substrate
Rock
Rock
Dock
Log
Concrete
Gravel
Sandy mud
Soft mud
Mud
Rocky Gravel
Piling
Piling
Piling
Piling
Piling
Dock
Dock
Dock
Piling
Piling
Rocks
Piling
Iron piling
Dock
Sand
Sandy mud
Soft mud
Date of
collection
6-29-76
6-30-76
6-28-76
6-28-76
6-30-76
6-30-76
6-10-76
6-10-76
6-29-76
6-29-76
6-15-76
6-15-76
6-15-76
6-15-76
6-15-76
6-15-76
6-15-76
6-15-76
6-15-76
6-15-76
6-15-76
6-16-76
6-16-76
6-17-76
6-15-76
6-15-76
6-15-76
B(a)P levels
ng/g (ppb)
*
0.40
16.86
0.28
0.10
0.16
3.21
0.30
*
*
0.12
30.20
3.15
14.90
0.87
3.00
4.13
0.39
0.77
5.23
0.51
0.44
0.37
4.30
0.56
0.25
0.25
15
-------
TABLE 2. Continued
Bay site
Species
Type of
substrate
Date of
collection
B(a)P levels
ng/g (ppb)
ALSEA
A1G
A2M
A3S
_T. cap ax
M. edulis
M. arenaria
Rocky sand
Dock
Soft mud
7-09-76
7-09-76
7-09-76
*
*
COOS
CIS
C2S #
CSS #
C4S #
CSS
C6S
C7S
CSS
C9G
C10G
C11G
C12G
C13G
C16B
C17B
C19S #
M.
M.
M.
M.
M.
M.
M.
M.
T.
T.
T.
T.
T.
S.
S.
M.
arenaria
arenaria
arenaria
arenaria
arenaria
arenaria
arenaria
arenaria
cap ax
cap ax
cap ax
cap ax
cap ax
giganteus
giganteus
arenaria
Sandy mud
Sandy mud
Soft mud
Rocky mud
Soft mud
Sandy mud
Soft mud
Soft mud
Sandy mud
Soft mud
Sandy mud
Sandy mud
Sandy mud
Sandy mud
Sandy mud
Soft mud
6-28-76
6-28-76
6-28-76
7-15-76
7-20-76
7-15-76
7-28-76
7-15-76
7-29-76
6-29-76
7-29-76
6-29-76
6-29-76
7-29-76
7-25-76
0.57
0.33
6.66
0.33
0.56
0.58
1.91
0.51
0.44
0.33
0.14
0.35
1.05
0.29
* Not detectable, less than 0.1 ppb
// Analyzed bimonthly for two years
— Not analyzed
LONG-TERM STUDIES ON BAP BODY BURDENS
To evaluate the seasonal influence and temporal fluctuations in BAP body
burdens, M. arenaria from 4 Coos bay sites and M. edulis from 13 Yaquina Bay
sites were assayed for two years (Tables 3 and 4).
M. arenaria from Coos Bay did not show any consistent patterns of season-
al maximums or minimums. For 1976-77, BAP body burdens were lower in the fall
and higher in the spring; in 1977-78, that trend was reversed. None of the
bimonthly deviations were statistically significant. Clams sampled from the
industrial bayfronts of Coos Bay (C19S) and North Bend (C4S) had greater BAP
concentration than those sampled from non-industrialized areas (C2S, C3S)
during the two year period.
BAP concentrations varied considerably in mussels from different sites
and during different times of the year. Mussels collected along the Oldtown
bayfront (Y2M, Y3M, Y4M) generally had the highest BAP concentrations through-
16
-------
out the two year period. The reasons for this are not known nor are the
sources of BAP. It is assumed that these mussels inhabit a more contaminated
environment because of their proximity to creosoted pilings, marina, boats
and fish-processing plants. Local sources of BAP may include creosote, fuel
from boats and marinas, wastewater from fish-processing plants, and/or run-
off after periods of heavy rainfall.
There were occasional periods when mussels from certain sites, typically
with low BAP concentrations, appeared with unusually high body burdens (e.g.
Y6M on 6/29/77). Although analytical error or inadvertent contamination dur-
ing processing cannot be ruled out, this seems unlikely since other samples,
handled in precisely the same manner, did not show such deviations. Small
gas or oil spills may have been associated with the sporadic high concen-
trations .
The data on BAP body burdens in _M. edulis from Yaquina Bay were subject-
ed to extensive statistical analyses. To determine if there were significant
differences in BAP concentrations in mussels from Y1M-Y14M over the two-year
study period, the data were analyzed using a one-way ANOVA. It was found
that there were significant differences between sites (F=8.15>F.01 [l3,137j=
1.80). A Student Newman-Keuls Multiple Range Test showed that only site Y2M
differed significantly from the others (F=10.55>F.01 [13,137]=2.26). Mussels
from Y2M had significantly higher BAP body burdens than mussels from other
sites during the two-year sampling period.
TABLE 3. BAP BODY BURDENS (ng/g) IN M. ARENARIA FROM COOS BAY, OREGON
Date
++ Not sampled
C2S
SITE
C3S
CAS
C19S
6/28/76
10/20/76
12/17/76
2/14/77
4/08/77
6/17/77
8/12/77
10/14/77
12/09/77
2/06/78
3/30/78
6/24/78
0.33
0.85
1.27
1.06
3.58
1.43
0.86
1.89
2.96
++
0.54
2.75
5.56
0.38
0.42
0.50
0.69
0.53
0.00
1.43
0.28
0.63
0.07
0.82
6.66
3.63
5.31
9.57
8.51
4.17
4.86
12.08
6.24
8.11
2.88
3.31
-H-
12.71
18.32
22.52
31.40
32.54
25.68
7.63
24.98
17.17
13.08
13.92
-------
TABLE 4. BAP BODY BURDENS (ng/g) IN M. EDULIS FROM YAQUINA BAY, OREGON
00
Date
6/15/76
7/22/76
9/24/76
11/16/76
12/16/76
2/03/77
4/08/77
6/29/77
8/29/77
10/13/77
12/08/77
2/03/78
4/28/78
6/24/78
AVERAGE
BODY
BURDEN
SITE
Y1M
0.12
4.72
0.68
0.62
8.41*
3.77
1.72
6.32
1.19
0.82
1.24
3.12
0.70
0.83
1.99
Y2M
30.10
67.88
33.84
40.20
12.23
32.97
21.89
15.39
5.14
4.97
15.68
27.73
20.70
29.27
25.57
Y3M
8.15
4.50
15.74
8.46
7.27
71.93*
7.91
3.54
2.82
5.35
NS
NS
NS
NS
6.53
Y4M
15.00
6.73
6.88
8.95
7.47
170.13*
12.67
5.43
2.24
1.86
6.38
13.72
5.52
17.50
8.49
Y5M
0.87
4.37
1.15
2.71
0.61
0.93
1.48
2.14
5.64
1.22
4.73
7.72
1.16
NS
2.67
Y6M
3.01
2.26
1.90
17.39
6.07
8.08
4.44
50.52*
4.42
3.16
3.07
32.59
4.04
NS
7.54
Y7M
4.13
2.37
14.27
19.07
3.81
1.71
NS
3.51
5.78
4.24
36.99
NS
27.42
NS
11.21
Y8M
0.39
0.82
0.86
NS
NS
1.97
NS
1.46
NS
0.52
8.14*
2.34
2.68
NS
1.38
Y10M
5.23
10.70
6.30
3.60
2.80
3.04
3.77
NS
NS
4.19
9.42
10.59
NS
NS
5.96
YUM
0.51
0.65
NS+
0.45
NS
0.74
0.69
1.96
0.41
0.13
NS
2.98
NS
NS
0.95
Y12M
0.44
0.70
0.82
0.45
0.33
0.46
0.50
0.36
0.00
0.24
0.00
1.18
0.10
NS
0.40
Y13M
0.37
0.33
0.80
0.90
0.06
0.20
NS
0.00
2.57
0.37
NS
NS
NS
NS
0.7
Y14M
4.29*
0.53
0.26
0.57
0.36
0.18
0.38
0.35
0.33
0.34
NS
NS
NS
NS
0.37
* Data not included in statistical analyses.
+NS Not sampled or not yet analyzed.
-------
Each piece of data from Table 3 was then transformed into a "percentage
of the mean" in order to obtain a universal unit that could be used in analy-
zing the data. For example, the average body burden of BAP in mussels from
Y1M and Y2M during the two years was 1.99 and 25.57 ng/g, respectively. The
fact that on 4/8/77, mussels from Y1M contained 1.72 ng/g BAP whirls those
from Y2M had 21.89 ng/g means only that those from Y2M were more contaminated;
such data alone could not be used to measure seasonal differences. However,
1.72 ng/g is 86.43% of the mean (1.99 ng/g) body burden in mussels from Y1M
or -13.57% of the mean; similarly, mussels from Y2M had a -14.39% of the mean
body burden. These percentages of the mean could then be used to evaluate
seasonal differences. Table 5 contains the transformed data.
Figure 7 portrays graphically the data included in Table 5. There were
no obvious deviations during 1976-77 while there was a noticeable increase in
BAP body burdens during the late winter and early spring of 1977-78.
200
DC
DO
S
0
DO
0.
200
JUNl JUl lAUGISEP 10CT IftlOVl DEC I JAN 1 FEB I MAR I APR I MAY I JUN I JUl I AUG I SEP I OCT I NOV I DEC I JAN I FEB iMARl APR I MAY'
1976 1977 1978
FIGURE 7. Seasonal differences in BAP body burdens in M. edulis populations
from Yaquina Bay, Oregon. Each dot represents the BAP body burden, expressed
as a percentage of the mean during the two-year period, from a single site.
Each open circle is the mean of the data points for each sampling period
(e.g., June 1976, October 1977).
19
-------
TABLE 5. BAP BODY BURDENS CALCULATED AS A PERCENTAGE OF THE MEAN
Date Day of SITE
the year Y1M Y2M Y3M Y4M Y5M Y6M Y7M Y8M Y1QM YUM Y12M Y13M Y14M
6/15/76 167 -94.0 17.7 -51.8 76.7 -67.4 -60.1 -63.2 -71.7 -12.2 -46.3 10.0 -47.1
7/22/76 204 137.2 167.5 -31.1 -20.7 63.7 70.0 -78.9 -40.6 79.5 -31.6 75.0 -52.9 43.2
9/24/76 268 -65.8 32.3 138.0 -19.0 -56.9 -74.8 -27.3 -37.7 5.7 105.0 14.3 -29.7
11/16/76 321 -68.8 57.2 29.6 5.4 1.5 130.6 70.1 -39.6 -52.6 12.5 28.6 54.0
12/16/76 352 * -52.2 11.3 -12.0 -77.2 -19.5 -66.0 -53.0 -17.5 -91.4 -2.7
2/03/77 44 69.4 28.9 -65.2 7.2 -84.8 42.8 -49.0 -22.1 15.0 -71.4 -51.4
4/08077 98 -13.6 -14.4 21.1 49.2 -45.6 -41.1 -36.7 -27.4 25.0 2.7
NJ 6/29/77 180 217.6 -39.8 -45.8 -38.4 -19.8 -68.7 5.8 106.3 -10.0 -100.0 -5.4
o
8/29/77 241 -40.2 -79.9 -56.8 -73.6 111.2 -41.4 -48.4 -56.8-100.0 267.1 -10.8
10/13/77 286 -58.8 -80.6 -18.1 -79.1 -54.3 -58.1 -62.2 -62.3 -29.7 -86.3 -40.0 -47.1 -8.11
12/08/77 342 -37.7 -38.7 -24.8 77.2 -59.3 230.0 58.0 -100.0
2/03/78 34 56.8 8.4 61.6 189.1 332.2 69.6 77.7 213.6 195.0
4/28/78 87 -64.8 -19.0 -35.0 -56.6 -46.4 144.6 94.2 -75.0
6/24/78 175 -58.3 14.5 106.1 -100.0
* Blanks indicate that either no data were available because the site was not sampled or the samples were
not analyzed, or the data were not transformed.
-------
There were dramatic differences in weather during the two-year study; the
5-month period from October, 1976 - February, 1977 was the driest ever record-
ed, while the same period for 1977-1978 was generally normal. Figures 8 and
9 contain information for 1976-77, 1977-78, on the amount of rainfall on the
Yaquina Bay watershed and the temperature and salinity of the estuary where
the mussels were sampled.
MAY
1976 -1977
Figure 8. The amount of rainfall (broken line) in the Yaquina Bay watershed
(each point represents 1 week) and the temperature (dotted line) and salinity
(solid line) of Yaquina Bay adjacent to the O.S.U. Marine Science Center dur-
ing 1976-77. Each point for the salinity and temperature profiles represents
the weekly maximum and minimum.
Z
JUN ' JUL ' AUG ' SEP ' OCT
NOV ' DEC ' JAN ' FEB
MAR ' APR ' MAY
1977 -1978
Figure 9. The same information for 1977-78
21
-------
The most significant difference between the 2 years was the virtual ab-
sence of rain during the winter (Nov.-Feb.) of 1976-77. The amount of rain-
fall during the winter of 1977-78 was rather typical for the Oregon coast.
The influx of freshwater resulted in depressed temperatures and salinity in
the estuary during this period.
As a result of the fluctuations in seasonal and environmental parameters
during the two years, a two way ANOVA was used to determine if there were
differences in seasonal-year correlation for BAP body burdens between 1976-77
and 1977-78. It was determined that there was a significant difference be-
tween the two years (F.01-7.06>F.01 [5,80]=2.33). As a result of that sta-
tistical test, further analyses were conducted separately for 1976-77 and
1977-78. Figures 10 and 11 show the fluctuations in BAP body burdens during
those 2 years. The percent mean body burdens were recalculated using only
the data for the respective year.
z 150-
UJ
^ 100-
o
* 50-
i
^ <&
S Y
a
3
™ 50
§ 100-
Q-
00 150
. e
^
.x>->- • ---^v- :
r « : : !
i t
* • _
* .
.IIIN 1 JIM 1 Ann sfp nnr wnu nrr JAN FFR MAR i APR i MAY
1976-1977
Figure 10. Seasonal differences in BAP body burdens, expressed as a percen-
tage of the mean, in M. edulis from Yaquina Bay, Oregon.
A one way ANOVA was used to determine if there were seasonal effects in
BAP body burdens for each of the two years. There were no seasonal effects
during 1976-77 (F=0.26F.01 [5,40]=2.45). A Student Newman-Keuls Multiple
Comparison test was used to identify which "seasons" were significantly
22
-------
different. Each of the 6 sampling dates (i.e., June, 1977; December, 1977,
etc.) was considered to be a season. Only the February, 1978 season differed
significantly from the other seasons at the 0.01 level. It should be noted
(Fig. 11) that the decline in BAP concentrations during the fall, while not
statistically significant, seemed to be a characteristic of all the different
mussel populations as evidenced by the tight clustering of data points.
JUN I JUL I AUG I SEP I OCT I NOV I DEC I JAN I FEB I MAR I APR I MAY I
1977 -1978
Figure 11. Seasonal differences in BAP body burdens, expressed as a percen-
tage of the mean, in M. edulis from Yaquina Bay, Oregon.
There are several factors that may account for the general seasonal
trends of 1977-78. Various mechanisms may be expected to influence the en-
vironmental availability of BAP and thus, the amount found in mussels at any
particular time. Photo-oxidation of BAP occurs in the presence of sunlight;
the greater the amount of sunlight the greater the quantities of BAP oxidized
by this process. Thus, the decrease in BAP body burdens during the summer
and fall may be related to the fact that August and September are generally
months with the most days of sunshine along the Oregon coast. A similar ex-
planation may partially account for the increases in BAP in mussels during
the winter months when there are prolonged periods without direct sunlight.
The amount of BAP in the atmosphere would be expected to be higher dur-
ing the winter since more organic material (oil, coal, gas, wood) is burned
to provide heat. An increase of combustion products, including BAP, coupled
with an increase in rainfall may result in rainout of atmospheric BAP, thus
increasing the environmental levels of BAP in estuaries.
23
-------
Urban sewage and sewage sledges may contain considerable quantities of
PNAH (Harrison et al., 1975). Dunn and Stich (1976) reported that discharges
from sewage treatment plants represented a major source of BAP contamination
in Vancouver Harbor. Three small towns are situated on Yaquina Bay or its
major tributaries, the Yaquina and Elk Rivers. BAP contamination of Yaquina
Bay from Elk City (septic tanks) and Newport sewer systems, which empty into
the ocean, would be expected to be minimal or nonexistent. Unknown, but
probably small, quantities of BAP may be introduced during the winter from
Toledo storm sewers which empty into the bay; inundation of the secondary
treatment plant may also result in untreated water being introduced into the
bay during periods of heavy rainfall.
Freshwater runoff from the watershed may also increase environmental
levels of BAP in three ways. First, it has been suggested that forest fires
contribute significant amounts of BAP to nearby ecosystems via atmospheric
deposition or runoff of residual BAP (Blumer and Youngblood, 1975; Kites,
1976; Clark and McLeod, 1977). While there were no major forest fires in the
Yaquina Bay watershed during the study, there was a considerable amount of
slash burning in logged areas, particularly during the fall. Thus, the heavy
rains of November-December, 1977, may have transported residual BAP from the
burned areas of the watershed to the estuary. Second, heavy runoff of fresh-
water results in the resuspension of sediments in receiving bodies of water.
Since sediments serve as a sink for BAP in estuaries, resuspension may also
increase environmental BAP. However, Roesijadi et al., (1978) found that
PNAH bound to particulate matter was less available for uptake than that frac-
tion released to the surrounding water and Neff (in press a) concluded, after
reviewing relevant studies, that sediment-absorbed PNAH are not readily
assimilated, at least by benthic animals. Third, unknown, but perhaps sub-
stantial, quantities of petroleum hydrocarbons are deposited in urban areas
from a variety of sources such as oil heating systems, fallout and the opera-
tion of motor vehicles. Rainfall and runoff may flush petroleum materials
either into storm drains or directly into rivers or bays (NAS, 1975).
Thus, there are four physical or chemical processes—photo-oxidation,
rainout, runoff, resuspension—that may be associated with seasonal fluctua-
tions in the environmental availability of BAP and BAP body burdens in M.
edulis. In addition, there may be certain intrinsic biological processes
that account for seasonal differences. Figure 12 shows the relationship be-
tween BAP body burden and a "gonad index." The gonad index was determined by
examining tissue sections of mussels collected from YlM, Y2M, Y4M, and Y12M
(100 mussels/month/site) and measuring their degree of sexual maturity; a
value of 0 indicates no gametes in the follicle while 5 is a sexually mature
mussel. The apparent relationship may be artifactual since there was no simi-
lar relationship in 1976-77. Nevertheless, 1977-78 was a typical year with
respect to environmental parameters while 1976-77 was not. If temperature
and salinity are involved in synchronizing the spring spawning cycle, then
1977-78 would be expected to be normal with respect to the sexual maturation
of mussels. DiSalvo et al. (1975) found that mussels maintained at, or
transferred to, a polluted site showed ratios of aromatics in gonadal to
somatic tissue of near unity. Contaminated mussels, in a reciprocal transfer,
showed a great reduction in this ratio which was attributed to a discharge
of gonadal material during spawning, although this was not confirmed. Sexual
24
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maturation involves the activation of lipid synthesizing pathways and perhaps
results in an increase in lipid pools containing BAP that is then routed into
tissue sinks (gametes). Thus, the spring peaks of BAP may reflect its in-
creasing concentration in gametes; a decrease would then occur after spawning.
IX
Q_
<
00
APR I MAY | JUN I JUL I AUG I SEP I OCT I NOV I DEC I JAN I FEB I MAR I APR I MA
-too-1-,
O
J>
a
o
m
X
1977-1978
Figure 12. BAP body burdens and the degree of sexual maturity, as measured
by the gonad index, in ^4. edulis from Yaquina Bay, Oregon. Squares represent
the mean degree of sexual maturity in mussels from 4 sites; circles indicate
the average percentage of the mean BAP body burden in mussels from 4 sites.
Finally, it should be noted that temperature and salinity of the immed-
iate environment affect many physiological functions in marine organisms.
These factors also affect solubility, adsorption-desorption kinetics,
octanol/water partition coefficients, etc. of PNAH in water (Neff, in press
a). In general, PNAH uptake is greater at reduced temperatures while
changes in salinity have little or no effect (e.g. Fucik and Neff, 1977).
Thus, increased BAP concentrations in mussels during the winter may be re-
lated to low water temperatures.
In summary, it is possible that the 1977-78 spring peaks were associated
with increases in environmental BAP, intrinsic physiological factors, and/or
the effects of endogenous factors on PNAH uptake and incorporation. Dunn and
Stich (1976) suggested that seasonal BAP variations in mussels from Van-
couber Harbor were attributable to seasonal changes in pollution rather than
variations in BAP uptake, retention with water temperature or breeding cycle.
Data from the present study neither support nor refute that interpretation.
25
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CELLULAR PROLIFERATIVE DISORDERS IN M. EDULIS
Table 6 summarizes the data on the prevalence of cellular proliferative
disorders in M. edulis from four sites in Yaquina Bay. Y1M and Y12M represent
populations of mussels that always had low or undetectable BAP concentrations
while Y2M and Y4M were sites where mussels always had high BAP body burdens
(Mix et al., in press a).
TABLE 6. THE PREVALENCE OF CELLULAR PROLIFERATIVE DISORDERS IN
M. EDULIS FROM YAQUINA BAY
Date
sampled
6/15/76
7/22/76
8/22/76
10/21/76
12/16/76
2/03/77
4/18/77
6/29/77
8/29/77
10/13/77
12/08/77
2/03/78
4/28/78
Y1M
No. %
0/10
0/10
0/45
0/194
0/101
0/100
0/43
1/48
0/48
1/39
0/49
0/48
0/50
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2.1
0.0
2.6
0.0
0.0
0.0
SITE
Y2M Y4M
No. % No. %
0/10
0/10
0/42
NS*
12/97
2/46
11/98
4/48
1/48
0/48
3/51
0/50
2/30
0.0
0.0
0.0
12.4
4.4
11.2
8.3
2.1
0.0
5.9
0.0
6.7
1/10
0/10
6/161
16/199
13/90
13/51
7/50
0/38
1/49
2/44
7/51
3/49
6/50
10.0
0.0
3.7
8.0
14.4
25.5
14.0
0.0
2.0
4.6
13.7
6.1
12.0
Y12M
No. %
0/10
0/10
NS
NS
0/100
0/97
0/95
0/49
0/46
0/45
0/50
0.48
0/50
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
TOTAL
2/785 0.25 35/578 6.1
75/852
8.8
0/600 0.0
*NS, Not sampled.
Statistical analyses revealed that: (1) there was a site-prevalence cor-
relation; (2) mussels from sites Y2M and Y4M (contaminated sites) had signif-
icantly higher prevalences than those from Y1M and Y12M (uncontaminated sites;
(3) there was seasonal-prevalence correlation; and (4) the seasonal-prevalence
correlation was curvilinear. The seasonal-prevalence correlation was sub-
jected to multiple regression analysis for 2 variables. The data fit a
quadratic equation, Y=AX2+BX+C where Y=0.0003X2-0.1231X+15.7914 (Y=prevalence;
X=day of the year), and provides an acceptable curvilinear fit to these data
(R2=0.388).
Although it is not yet possible to formulate any definitive conclusions,
it seems significant that, with two exceptions (n=l,385), no mussels with low
body burdens of BAP from the Y1M and Y12M sites had the cellular disorders,
whereas substantial numbers (110/1,430) were found in mussels collected from
the Y2M and Y4M sites with high body burdens of BAP. However, numerous
26
-------
inorganic and organic chemicals can induce neoplasia in sensitive species and
polluted aquatic environments frequently contain a wide variety of potential
carcinogens (Bergel, 1974). A salient question which remains to be answered
is whether BAP or any other PNAH can cause neoplasia or the malignant equiva-
lent in bivalve mollusks. The evidence accumulated to date indicates that
few, if any, bivalve mollusks possess the enzymes necessary to metabolize BAP
to its reactive intermediates (Lee et al., 1972; Neff and Anderson, 1975;
Payne, 1977; Vandermeulen and Penrose, 1978; Neff, in press b) although
Anderson (1978) detected very low levels of BAP oxidizing enzymes in oysters
(C_. virginica). If metabolic conversion does not occur, it is necessary to
conceive of an alternative metabolic or physiological mechanism by which BAP
or other carcinogenic PNAH's could cause neoplastic cell disorders in these
organisms, if these cells have, in fact, been transformed.
ELECTRON MICROSCOPIC ANALYSIS OF THE ATYPICAL, LARGE M. EDULIS CELLS
Studies on the ultrastructure of large cells associated with putative
neoplastic disorders of mussels from Yaquina Bay have recently been completed
(Mix et al., in press b) . Atypical large cells had an average diameter of
15 urn and a nuclear to cytoplasmic ratio of about 1-1.5. Briefly, the large
M. edulis cells possess many ultrastructural properties that are characteris-
tic of certain malignant vertebrate cells: large, polymorphic nuclei; large,
multiple nucleoli; altered Golgi complexes; mitochondrial inclusions; an
absence of cytoplasmic differentiation; ribosomes not associated with a mem-
brane system; pleomorphic inclusions in the cytoplasm; and possible asyn-
chronization in the maturation of nucleus and cytoplasm. Nevertheless, much
additional information about their life history is required before any defini-
tive conclusions can be formulated.
It should be noted that many of these properties are also characteristic
of normal cells that are engaged in rapid proliferation (Maile, 1972). This
last point is particularly important since it suggests another way that high
body burdens of BAP, and presumably other PNAH, may be associated with the
cellular disorders. It became evident during the analyses that BAP is only
one of many PNAH's in the mussels analyzed during this study. Six to ten
fluorescent bands were consistently observed in the thin-layer chromatography
(TLC) plates which may represent individual PNAH or groups of PNAH's. There
was also a direct correlation between the concentration of BAP and the number
and size of the fluorescent bands. Therefore, BAP body burdens may represent
only a small fraction of the total chemical load associated with PNAH and
possibly other anthropogenic environmental insults. Perhaps then, the large
cells represent a response to chronic environmental stress with possible
functional capabilities associated with detoxification. Such a cellular
response may be entirely non-neoplastic, initially non-neoplastic with sub-
sequent neoplastic transformation, or entirely neoplastic.
PUBLIC HEALTH CONSIDERATIONS
The finding that certain indigenous populations of bivalve mollusks con-
tain substantial body burdens of BAP indicates that a potential public health
27
-------
problem may exist. However, a review of relevant data indicates that there
are numerous problems in estimating the hazard to man from consuming seafood
with elevated PNAH content. Gerarde (1960) reported that PNAH's are poorly
absorbed by the mammalian GI tract and that 40-97% of BAP was excreted. It
is apparently still a matter for debate whether there is any dose-response
relationship in man, or a threshold dose below which carcinogens do not induce
cancer (WHO, 1972). If there is an effective threshold for oral intake of
PNAH in man, the critical question becomes whether this threshold could be
exceeded by the PNAH content in contaminated seafood (GESAMP, 1977). Except
for the possible association between heavily smoked fish and a high incidence
of stomach cancer in Iceland and Slovenia (Wynder et al., 1963), no epidemio-
logical studies have linked GI cancers in man with the ingestion of marine
fish or shellfish. The presence of ppb concentrations of BAP and other PNAH
in a number of common foods such as fresh vegetables, meat, fish and poultry
has led to the suggestion that trace amounts of carcinogenic PNAH do not
constitute a health hazard (Friedman, 1974); obviously, this opinion is not
universally accepted by all scientists. Clearly, a great deal of additional
information is required to accurately assess the risk of consuming seafood
contaminated with varying levels of PNAH's.
There are several other questions to consider before it can be determin-
ed whether or not a human health hazard exists.
Those questions, with partial answers, are listed below.
1. Is the shellfish species economically important (i.e., is it exploit-
ed as a food source)? M. edulis, the species with the greatest body burdens,
is not heavily exploited by Oregon shellfishermen. M^. arenaria is subjected
to moderate pressure while _T. cap ax, S_. giganteus, and C^. nuttalli are heav-
ily exploited. M. arenaria from Coos Bay was the only clam species with sig-
nificant concentrations of BAP. Oysters (C_. gigas) did not have significant
body burdens of BAP.
2. Are the heavily contaminated species found in areas accessible to
and harvested by shellfishermen? Heavily contaminated M. edulis from Yaquina
Bay and M. arenaria from Coos Bay are accessible to the public; however, the
former species is not generally utilized while the latter species is not sub-
jected to harvesting pressure at the highly contaminated sites (C4S and C19S).
Lightly contaminated clams from T6G, T7G, T10S, Y15G, Y16G, Y17G, CSS, C7S,
C9G, C10G, C11G, C12G, C13G, C16B, and C17B are all accessible and subjected
to moderate to heavy digging pressure throughout the year as low tides permit.
3. What is the tissue storage time for BAP and other PNAH? Relatively
little information is available concerning the extent to which BAP and other
PNAH are accumulated and eliminated by shellfish. Research on rates of up-
take and elimination (RUE) have generally been conducted in the laboratory
(Lee, et al., 1972; Stegeman and Teal, 1973; DiSalvo, et al., 1975; Neff, et
al., 1976; Roesijadi, et al., 1978); few field studies have been conducted
Dunn and Stich, 1976). Results from the various studies seem to suggest that
BAP may be stored in two compartments by clams, oysters, and mussels. Most
BAP may initially be contained within a fluid compartment where environmental
equilibration would be expected to occur rapidly. The rate of loss from this
28
-------
compartment would be quite rapid (hours to days) and many laboratory studies
have probably analyzed BAP depuration from this depot. Lesser amounts of BAP
appear to be stored in a tissue compartment. Intrinsic lipid/water partition
coefficients favor the rapid transfer from the aqueous phase into lipophilic
compartments (e.g. membranes, macromolecules) (Neely et al., 1974). The
precise storage sites of accumulated PNAH are not known but are generally
thought to include the lipid stores of the tissues (Stegeman and Teal, 1973;
Vandermeulen and Penrose, 1978). However, both environmental equilibration
and the rate of loss from tissues would take considerable time (weeks to
months), so storage in this compartment would be expected to be associated
with any chronic effects of environmental contamination. Few studies have
addressed all these aspects of PNAH contamination of shellfish. Generally,
the available results indicate that BAP will be relatively persistent in
tissues of exposed bivalves (Roesijadi, et al., 1978).
4. Is BAP, and other PNAH, metabolically altered by the bivalve species?
As indicated previously, the evidence accumulated to date indicates that bi-
valve mollusks do not possess the complicated microsomal enzyme systems nec-
essary for metabolic activation of PNAH. Thus, the absence of such enzyme
activity may result in the retention of unaltered PNAH in shellfish. Since
humans possess the enzymes necessary to convert unaltered PNAH to highly muta-
genic or carcinogenic derivatives, retention of unaltered PNAH, if present in
significant quantities, could pose a public health problem.
5. What is the total body burden of all PNAH and possibly other chemi-
cal carcinogens, in shellfish contaminated with BAP? As indicated previously,
BAP seems to serve as an indicator PNAH. Thus, shellfish that are heavily
contaminated with BAP will likely have high concentrations of other PNAH. A
full understanding of the quantitative aspects of this relationship will be
necessary to evaluate the potential public health problem associated with the
consumption of contaminated shellfish.
6. What are the sources of environmental BAP and PNAH in contaminated
marine organisms? With respect to the current study, potential sources in-
clude, but are not limited to: small fuel or oil spills, creosote, fish
processing factories, marinas, large ships, pleasure craft, wood products
industries, sewage treatment plants, runoff through storm sewage, runoff from
logged areas where slash burning has occurred (BAP produced from combustion)
and rainout of atmospheric BAP produced from combustion of organic material.
There is no clear risk to users of the shellfish resources of Oregon at
the present time. Future studies should be directed toward obtaining more
completed answers to these questions in order to assess the potential public
health hazard.
29
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33
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
:PA-6QQ/3-79-034 March 1979
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
CHEMICAL CARCINOGENS IN BIVALVE MOLLUSKS FROM
OREGON ESTUARIES
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Michael C. Mix
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of General Science
Oregon State University
Corvallis, Oregon 97330
10. PROGRAM ELEMENT NO.
IEA615
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
U. S. Environmental Protection Agency
Office of Research and Development
Gulf Breeze, Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
Final. June 1. 1976-Sept. 30.19
78
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The research undertaken involved the use of indigenous populations of bivalve mollusks
as monitors for detecting and quantifying environmental benzo(a)pyrene (BAP) in Oregon
estuaries. Short-term and long-term studies were conducted in order to establish base-
line levels of BAP and to identify seasonal variations in BAP concentrations in shell-
fish. A presumptive cellular proliferative disorder, thought possibly to be neoplastic
was also studied in mussels, Mytilus edulis, from Yaquina Bay.
Histological studies revealed that mussels inhabiting polluted environments, and with
high BAP body burdens, had an average 6-8% prevalence of the cellular proliferative
disorder while those from clean environments and with low or undetectable levels, did
not have the disorder. The cellular condition showed a definite seasonal pattern,
there was a low prevalence during the summer and fall followed by an increase during
the early winter and a peak prevalence occurred in January-February. The atypical,
large cells that characterize the disorder in M. edulis possess many ultrastructural
properties in common with malignant vertebrate cells.
Further studies are required to evaluate the public health significance of these
results. This report was submitted in fulfillment of Contract No. R804427010 by Oregon
State University, Corvallis, Oregon.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
monitoring
carcinogens
polycyclic aromatic
hydrocarbons
benzo(a)pyrene
mullusks
neoplasia
Disease survey
Oregon Coast
06/F
8. DISTRIBUTION STATEMENT
UNLIMITED
19. SECURITY CLASS (This Report)
unclassified
21. NO. OF PAGES
33
20. SECURITY CLASS (This page)
unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
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