v>EPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze FL 32561
Research and Development
EPA-600/S4-82-026
May 1982
Project Summary
Polynuclear Aromatic
Hydrocarbons and Cellular
Proliferative Disorders in
Bivalve Molluscs from
Oregon Estuaries
Michael C. Mix
This research project utilized indig-
enous populations of economically-
important bivalve molluscs as moni-
tors for detecting and quantifying 15
environmental polynuclear aromatic
hydrocarbons (PNAH), including 11
compounds classified as carcinogens,
11 EPA Priority Pollutants, and 11
Toxic Pollutants. Cellular proliferative
disorders resembling neoplasia were
also studied in shellfish populations.
Baseline levels of PNAH were deter-
mined during a two-year period for
mussels (M. edulis), clams (M.
arenaria and T. capax) and oysters (C.
g/gas] from different sites in Yaquina,
Coos, and Tillamook Bays, Oregon.
Total concentrations of 15 unsubsti-
tuted PNAH were 30 to 60 A
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as biomonitors for the detection and
quantification of environmental contam-
inants, including carcinogens. The use
of bivalve shellfish has received the
widest support because these molluscs
are permanent inhabitants of a specific
environment and tend to concentrate
toxic substances in their tissues. In
addition, bivalve molluscs have been
recommended for use in studies of en-
vironmental contaminants because
many species from several different
geographic locations have been reported
to have cellular, perhaps neoplastic,
proliferative disorders. Environmental
pollutants were implicated as potential
causative agents in several of those
reports, although no cause-effect rela-
tionships have yet been established.
Quantitative field studies of PNAH
concentrations in marine organisms
have only recently been initiated despite
earlier suggestions that extensive inves-
tigations of the marine environment,
including chemical identification, moni-
toring and surveillance, and identifica-
tion of fish and shellfish tumors may
play an important role in the epidemi-
ology of cancer. Of the PNAH, benzo(a)
pyrene (BAP) has been the most exten-
sively studied carcinogen in the marine
environment. Excepting BAP, which has
often been used as an index to indicate
the presence of other PNAH, relatively
little information is available on the
presence or quantities of other unsub-
stituted PNAH in tissues of aquatic
organisms. Considerable information,
based on the use of advanced analytical
methods capable of measuring nano-
gram quantities, is needed.
Associations between high tissue
concentrations of PNAH, and other car-
cinogens, and the appearance of cellular
proliferative disorders in shellfish popu-
lations should also be identified and
carefully investigated. The existence of
such associations would suggest the
necessity of additional studies to fully
evaluate potential cause and effect rela-
tionships.
Experimental Procedure
Clams, mussels, and oysters were
sampled periodically from Coos,
Yaquina, and/or Tillamook Bays in,
Oregon. Clams from the three bays
were dug during low, approximately
zero, tides, whereas mussels were
collected during the entire ebb tide
period, depending on location. Oysters
were obtained from commercial growers
and simply removed from the shucking
tables. Immediately after collection, sam-
ples from each site were placed in
labeled plastic bags, put on ice, and
transported back to Corvallis. Animals
were then removed from their shell and
the pooled sample from each site was
weighed. Each pooled sample was
stored at -20°C until it was processed
for PNAH analysis. Mussels and clams
to be prepared for histological examina-
tion were placed in Davidson's fixative,
processed in the usual way, sectioned at
6//m, and stained with hematoxylin and
eosin.
For PNAH analysis, tissues were
saponified inethanol/potassium hydrox-
ide for 1.5 hr and the supernate liquid-
liquid extracted into 2,2,4-trimethylpen-
tane. The organic phase was passed
through a column of partially deactivated
florisil (4% water added w/w). The
PNAH were eluted from the florisil with
benzene and the eluate liquid-liquid
extracted with dimethylsulfoxide. The
sample was then cleaned up by column
chromatography on Sephadex LH-20,
concentrated to 100 fA and analyzed by
reverse phase high performance liquid
chromatography (HPLC).
Only a brief description of the HPLC
operating conditions are included below.
Liquid chromatography: Spectra-
Physics Model 8000 with data system,
Valco injector with 10 pi loop;
Column: Perkin Elmer HC-ODS for
PNAH, 0.26x25 cm, 10pm Ci8 packing
with an Alltech Cis guard column,
0.46x1 Ocm;
Mobile phase: acetonitrile/water gra-
dient constant flow mode, 0.8 ml/min.
temp. 20°C (Time-%MeCN-%H2O were
as follows: 0 min-60-40, 4 min-60-40,
20 min-60-40, 45 min-100-0, 55 min-
20-80, 65 min-20-80, 70 min-60-40,
and 80 min-60-40);
Detector 7/Schoeffel Model 770 vari-
able wavelength UV detector, 296 nm,
range 0.02 and 254 nm, range 0.02;
Detector 2: Schoeffel Model 970
variable wavelength fluorescent detec-
tor, 326 nm excitation, greater than 412
emission (cutoff filter), range 0.1.
Known standard concentrations were
made and the data stored by the HPLC.
Subsequently, those data were used to
calculate the concentrations of PNAH in
the samples. Sample losses were ac-
counted for by using an internal spike of
3H-BAP. Total PNAH reported in the
results represent the sum of the concen-
trations of phenanthrene, f luoranthene,
pyrene, benzo(c)phenanthrene, tri-
phenylene, benz(a)anthracene, chry-
sene, benzo(b)fluoranthene, benzo(k)
f luoranthene, dibenz(a,c)anthracene,
BAP, dibenz(a,h)anthracene, benzo(g,h,i)
perylene, indeno(1,2,3-c,d)pyrene, and
coronene.
Conclusions
Baseline levels of PNAH in indigenous
bivalve molluscs used as biomonitors
reflected the degree of human onshore
activity at the various sample sites and,
presumably, the level of water contami-
nation. PNAH concentrations in shellfish
from relatively pristine areas ranged
from 30 to 60 yug/kg, whereas those
from industrialized areas contained 500
to 1500/ug/kg. The data collected during
the presehf study tend to confirm that
bivalve molluscs make excellent biomon-
itors for detecting and measuring PNAH
in estuaries.
Multiple regression and multiple cor-
relation techniques were used to identify
and evaluate relationships between
PNAH. Identification and evaluation of
quantitative and qualitative relation-
ships between individual PNAH and
between PNAH and their concentrations
in bivalve molluscs indicated that a
significant potential exists for develop-
ing predictive models for PNAH in
aqueous environments and their con-
centrations in bivalve molluscs. Statis-
tical analyses also indicated that concen-
trations of individual PNAH were greater
for the isomer which had the greatest
solubility in water.
Different populations of shellfish were
examined histologically for the presence
of cellular proliferative disorders. Clams
from Coos Bay and mussels from
Tillamook Bay were not found with the
large, abnormal cells that characterize
the conditions. The disorder was present
in a significant number (mean preva-
lence = 10%) of Yaquina Bay mussels
with the highest concentrations of PNAH
measured in this study, whereas it rarely
appeared in a second population at a
"clean" site across the bay. The corre-
lation between the degree of PNAH
contamination and the prevalence of
the cellular disorders may be significant,
but no cause-effect relationship has
been established. It remains to be deter-
mined if carcinogenic metabolites can
be formed by this species.
Discussion
An analytical method, utilizing HPLC,
was developed and used for qualitative
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and quantitative determination of 15
unsubstituted PNAH isomers. The
method resolved most members of the
benzpyrene group. Perylene was not
identified because it does not absorb UV
light at the wavelength used and benzo(j)
fluoranthene and benzo(e)pyrene could
not be separated.
The identification of multiple PNAH in
indigenous organisms represents a sig-
nificant advance in studying environ-
mental levels of PNAH in marine orga-
nisms. The presence of PNAH, particu-
larly BAP, in water, sediments and
organisms has been recognized for over
20 years. Yet, dependable, high resolu-
tion techniques, such as those utilized
in this study, have only recently been
developed and a more precise and
complete information base can now be
developed.
A major effort was made to identify
and interpret statistically significant
relationships between PNAH and their
concentrations in shellfish and between
individual PNAH isomers. Certain of the
relevant findings from these studies are
summarized below:
1. Quantities of a single PNAH pres-
ent in shellfish cannot be used to
predict total PNAH.
2. For each site, different independ-
ent variables (individual PNAH)
could be identified and used to
predict total PNAH in bivalve mol-
luscs. It maybe possible to identify
multiple site-specific independent
variables after a suitable sampling
period and subsequently to meas-
ure only those key variables for an
adequate assessment of total
PNAH. Complete analyses could
perhaps be made periodically to
confirm the continuing validity of
the established relationship; devia-
tions may indicate new sources'of
contamination. Such an approach
may result in considerable cost
reduction for long-term monitoring
programs.
3. The presence or absence of benzo-
(a)pyrene was not a significant
variable for predicting total PNAH
at any site. Thus, the concept that
BAP can be used as an index of
PNAH contamination was not sup-
ported by the results of this study.
From this and other studies, it
seems that the use of BAP for
making decisions about the quan-
tities and presence or absence of
other PNAH should be abandoned
or modified.
4. While it was established that
quantitative predictions about total
PNAH could not be made on the
basis of individual PNAH measure-
ments, the results suggest that
certain qualitative relationships
existed which may permit first
approximations of individual PNAH
concentrations. In general, there
were no significant differences be-
tween individual PNAH with 4
rings, or between those with 5, 6,
or 7 rings. Phenanthrene, a 3-ring
compound, differed significantly
from other PNAH.
Statistical analyses revealed an em-
pirical relationship between individual
PNAH concentrations and their respec-
tive solubilities. The concentrations in
shellfish were greater for the PNAH
isomer which had the higher solubility
in water. This finding contrasts with the
observation that organic/water (e.g.,
octanol/water) partition coefficients
show an inverse relation to water solu-
bility. Because the concentration in the
organic phase (shellfish, in this study),
C0> is equal to the product of the partition
coefficient (K) and concentration in
water (C*), the data suggested that the
ratio of the PNAH concentrations in
water would have to be generally greater
than the ratio of their reciprocal parti-
tion coefficients or their water solubil-
ities. Direct measurements of PNAH
concentrations in seawater will be
necessary to confirm whether the up-
take of PNAH by shellfish can be repre-
sented by a simple partition process.
Although there have been numerous
reports of apparent correlations between
the appearance of abnormal cells in
shellfish and their habitation in polluted,
primarily oil-contaminated, environ-
ments, there have been no published
reports of cancer induction in bivalve
molluscs by exposure to, or injection of,
PNAH. Significant questions about the
effects of PNAH on bivalve molluscs and
the metabolic capabilities these species
have for altering PNAH, remain unan-
swered. There have been numerous
reports that bivalve molluscs cannot
metabolize PNAH, yet the evidence
presented is by no means definitive, and
recent studies indicate that at least
some species can metabolize BAP. It
remains to be determined if carcinogenic
metabolites can be formed by these
species. If bivalves are not subject to
PNAH-induced carcinogenesis, and the
cellular abnormalities are related to a
neoplastic process, then other causative
agents must be responsible. Assuming
the condition is analogous to neoplasia,
it seems evident that this disorder in M.
edulis has greater potential for serving
as a model for studying cancer-like
diseases in an invertebrate. The cells
have many characteristics in common
with malignant conditions in mammals,
and affected mussels can be obtained
easily and on a regular seasonal basis
by procedures developed during this
study.
Recommendations
Future efforts should be directed
towards fully defining the sampling
protocols to be used in monitoring
studies and toward identifying and eval-
uating endogenous and exogenous
factors that may influence PNAH con-
centrations under ambient (field) condi-
tions. The latter should include studies
of potential sources and measurements
of PNAH in water.
It may be productive to conduct similar
statistical analyses for PNAH data col-
lected during future studies and from
other established biological monitoring
programs. Confirmation or extension of
the types of relationships identified
during this research may eventually
lead to simplified monitoring approaches
and result in substantial cost reductions.
Further efforts should be made to
characterize the large, abnormal, pre-
sumably neoplastic, cells; establish cul-
ture techniques suitable for maintaining
and growing the cells; and identify the
causal agent(s) of these disorders in
bivalve molluscs.
While required data are not yet avail-
able or are incomplete, initial attempts
should be made to establish acceptable
or unacceptable levels of PNAH in sea-
food products consumed by humans.
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The EPA author Michael C. Mix is with the Environmental Research Laboratory,
Gulf Breeze, FL 32561.
John A. Couch is the EPA Project Officer (see below).
The complete report, entitled Polynuclear Aromatic Hydrocarbons and Cellular
Proliferative Disorders in Bivalve Molluscs from Oregon Estuaries," (Order No.
PB 82-189 523; Cost: $9.00, 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
Gulf Breeze, FL 32561
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