EPA 600-4-82-026
PB82-189523
Polynuolear Aromatic Hydrooarbona and
Cellular Proliferative Disorders in
Bivalve Mollusos from Oregon Estuaries
Oregon State Univ
-------
PB0 2—189523
IPA-6,00/4-82-026
April 1962
POLYNUCLBAR AROMATIC. HYDROCARBON8 AMD OLLOLAR-PeOLXiERATIVe DISORDER* IN
BZVALVI MOLLUSCS FROM OREGON ISTOAMB8
fay
Michael c. Mix
Department of General Science
Oregon State University
Corvallii/ Oregon 97331
SPA Grant R&06224020
Projeot Officer
John A* Couch
Environmental Research Laboratory
U.St Environmental Protection Agency
Gulf Breese, Florida 32561
nVXRONMSNTAL RE8EARCH LABORATORY
optics or riskarcb jor> devilopment
U. 8. ENVIRONMENTAL PROTECTION AGENCY
GULP BREEZE, TLORXDA 32561
-------
(Htm Si? wlm ^t$rttemplttim)
1. RIPOAT NO. *•
EPA-600/4-82-026 ORD Rioort
3. RECIPIENT'S ACCESSION NO.
mm 1A9S2 3
4,t,t^WOTaromatic hydrocarbons and cellular pro-
liferative DISORDERS IN BIVALVE MOLLUSCS PROM
OREGON ESTUARINES
1. RIPORT date
March 1982
1. PERFORMING ORGANIZATION COOE
7'Aura«l C. Mix
S. PIRPORMINO ORGANIZATION REPORT NO.
D. PERFORMING ORGANIZATION NAM! AND ADORES*
Department of General Science
Oregon State University
CorvalUs, Oregon 97331
44. MflfiAAM ILIMINT Nfl,
\\. fiOHTHACT/flHAMT NO.
R806224020
12. SPONSORING AGENCY NAME ANO AOORESS
U.S. Environmental Protection Agency
Environmental Research Laboratory
Office of Research and Development
Gulf Breeze, FL 32561
13. TYPE OP REPORT ANO PSRIOO COVERED
14. SPONSORING AGENCY CODE
EPA/600-4
18. SUPPLEMENT* BY NOTES
li. ABSTfcA&t
Indigenous populations of economically Important bivalve molluscs were
used as monitors for detecting and quantifying environmental PNAH, Including
11 compounds classified as carcinogens, 11 EPA Priority Pollutants and 11
Toxic Pollutants. Baseline levels of PNAH were determined during a two-
vear period for mussels (M. edulls), clams (M. arenarla and T. capax) and
oysters (C. qlqas) from different sites, ranging from relatively pristine to moder-
ately polluted, in Yaquina, Coos and Tillamook Bays, Oregon. Total concen-
trations of 15 unsubstltuted PNAH were 30 to 60 ug/kg 1n shellfish from uncontaminr
ated waters to greater than 1000 yg/kg 1n those from sites classified as contami-
nated. A major effort was made to determine and evaluate certain relationships
between PNAH and their concentrations 1n shellfish. Studies were conducted to:
determine the effects of depuration of PNAH concentrations; Identify seasonal
differences 1n PNAH concentrations; and measure BAP uptake and elimination.
Preliminary studies indicated that mussels may possess a limited ability to
metabolize BAP. Multiple regression and multiple correction techniques were used
to Identify and evaluate interrelationships between PNAH. Certain relationships
may be useful for predictive purposes 1n evaluating environmental PNAH. The
data from these studies Indicate that 1t may be possible to Identify and measure
sianflcant variables to assess total PNAH.
17. K«Y won OS ANO OOCUMINT ANALYSIS
1. DESCRIPTORS
b. 1OSNTIP11RS/OPSN INOiO TERMS
c. COSATi Field/Group
1*. DISTRIBUTION STATIMCNT
Release to public
Unclassified
20. SECURITY CLAM fTMtpf)
Unclassified
22. PRICE
EPA Frm 2220-1 (K»r. 4-77) fakvioui coition is omolctk
-------
DISCLAIMER
This report has bean reviewed by the Gulf Breeze Environmental Research
Laboratory, U. S. Environmental Protection Agency, and approved for publi-
cation. Approval does not signify thai the contents necessarily reflect the
views and policies of the U. 8. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or re-
commendation for use.
a
-------
FOREWORD
The protection of our estuarint ahd ooaatal araaa from damage caused by
toxic organia pollutants raquiraa that regulations raatrloting the introduc-
tion of these compounds into tha environment ba formulated on a sound
sciantifio basis* Accurate information dascribing dose-response
relationships for organisms and ecosysjtems undar varying conditions is
required. The EPA Environmental Research Laboratory, Gulf Breeze,
contributes to this information through research programs aimed at
determining!
the effects of toxic organic pollutants on individual spacies and
communities of organijmnsi
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 polyoyolio aromatic hydrocarbons in
the marine estuarine environment and biota• these data may serve to alert ui
to the role of certain carcinogens in the environment generally.
Henry^T. Enos
HanryV/K Enos
Director
Environmental Research Laboratory
Gulf Breeze, Florida
til
-------
ABSTRACT
Tha research project involved utilising indigenous populations of eco-
nomically- important bivalve molluscs as monitors for detecting and quanti-
fying environmental PNAH, including 11 compounds classified as carcinogens,
11 EPA Priority Pollutants and 11 Toxic Pollutants.
Baseline levels of PNAH were statettnined during a two-year period for
mussels (A/. edulis), clams (M. ca*wia and T. oapax) and oysters (C. gigaa)
from different sites, ranging from relatively pristine to moderately polluted,
in Yaquina, Coos and Tillamook Bays, Oregon. Total concentrations of 15 un-
substituted PNAH were .30-60 vgAg in shellfish from uncontaminated waters to
greater than 1000 yg/kg in those from sites classified as contaminated.
A major effort was made to determine and evaluate certain relationships
between PNAH and their concentrations in shellfish. Studies were conducted
to: determine the effects of depuration on PNAH concentrations; identify
seasonal differences in PNAH concentrations; and measure BAP uptake and
elimination. Preliminary studies indicated that mussels may possess a limited
ability to metabolise BAP.
Multiple regression and multiple Correlation techniques were used to
identify and evaluate interrelationships between PNAH. Certain relationships
may be useful for predictive purposes in evaluating environmental PNAH. The
data from these studies indicate that it may be possible to identify site-
specific, significant variables (individual PNAH) after a suitable period of
sampling and to subsequently measure ohly those key variables for an adequate
assessment of total PNAH. Combined with other approaches, this may result
in considerable cost reductions for lopg-term biological monitoring programs.
Cellular proliferative disorders, resembling neoplastic conditions in
vertebrates, were found in mussels with the greatest PNAH concentrations.
Further studies will be necessary to determine the significance of this
correlation.
This report was submitted in fulfillment of Contract No. R806224020 by
Oregon State University, Corvallis, Oregon, under the sponsorship of the
U. S. Environmental Protection Agency. "his report covers the period
;October 1, 1978]to November 30, 1980.
we
-------
CONTtNTB
Foreword ill-
Abstract lv
Figures . . . vi
Tables • • • vii
Abbreviations and Symbols ix
Acknowledgements x
1. Introduction 1
2. Conclusions 3
3. Recommendations ... ..... . ... t* ,. . . • • •. ^
4. Materials and Methods 6
Sample sites# species utilised and sampling protocol 6
Collection and preparation of shellfish samples 8
PNAH analytical procedure ... 9
Benzo(a)pyrene uptake and elimination ............. 11
Tissue storage sites of benzo(a)pyrene in M. edulis 11
Benzo(a)pyrene metabolism by M. edulie 11
Statistical analyses ................ 12
5. Results and Discussion 13
Identification of PNAH in bivalve molluscs 13
Baseline data on PNAH concentrations in clams from Coos Bay . .13
PNAH concentrations in M. abulia from Yaquina Bay 21
Baseline data on PNAH concentrations in other bivalve molluscs. 31
Cellular proliferative disorders in bivalve molluscs from
Oregon bays , 36
Relationships involving PNAH in shellfish monitors 38
References 46
T
-------
FIGURES
Number Page
1 Oregon bays and estuaries . . . 7
2 Sample sites ,in Tillamook, Yaguina and .Coos', Bays, Oregon 7
3 A. Seasonal differences in PNAH Concentrations in Y1H M. edulia . . 25
I •
B. Seasonal differences in PNAH concentrations in Y2M M. edulis . . 25
=4 Pates' of b~<5nz6 (a)pyrerfe 'uptake~arf<3_elimi"natTo}i~ 26"
5 Renzo(a)pyrene concentrations in gonadal and somatic tissues .... 29 •
6 Prevalence'of. cellular proliferative disorders in M. edulia ... 37
7 PNAH-water(solubility regression relationship .... 44
-------
TABlBS
Number Page
1 Information on Sample Site«, Species and Sampling
Frequency . . . 6
2 Apparatus, Operating Condition* and Procedures for PNAH Analysis . . 10
3 PNAH Analyzed in Shellfish from Oregon Estuaries 14
4 Some Physical and Chemical Characteristics of PNAH 15
5 PNAH Concentrations (ugAg) in T. oapax from Coos Bay, Oregon;
Site C11G 16
6 PNAH Concentrations (ugAg) in M. areruxria from Cogg Bay, Oregon . . 18
7 PNAH Concentrations (yg/Jtg) in M. arenaria from Coos Bay, Oregon;
Site C3S, Before (N) and After Depuration (D) for 24 hours 19
8 A. PNAH Concentrations (ygA?) in M, edulia from Yaquina Bay,
Oregon; Site Y1M 22
B. PNAH Concentrations Ivg/kg) in M. edulia from Yaquina Bay,
Oregon; Site Y2N . . 23
9 Benzo(a)pyrene Uptake and Elimination in M. edulia Maintained
under Ambient (Field) Conditions in Yaquina Bay 27
10 Benzo(a)pyrene Concentrations in the Gonad and Somatic Tissues of
M. edulia from Yaquina Bay, Oregon 30
11 PNAH Concentrations (ng/kg) in C. gigaa from Yaquina Bay, Oregon;
Site Y140 . ..... 32
12 PNAH Concentrations (ugAg) in Bivalve Molluscs from Tillamook
Bay, Oregon . i 33
13 Range of PNAH Concentrations in Shellfish Monitors from Various
Sites in Three Oregon Bays . . . f . .35
14 The Prevalence of Cellular Proliferative Disorders in M. edulia
and M. cveruxria
*£1
-------
TABLES (C6NTXNUBD)
N«nber Page
15 Multiple Regression and Correlation Analyse* 39
16 An Evaluation of Quantitative Relationships Between Individual
PNAH in Tissues of M. edulis from Yaquina Bay, Oregon
17 The Mean Percentage Individual PNAH Contributed to the Total
PNAH for all Sampling Dates (± S.D.)
18 Linear Relationships Between Log s (X), Hater Solubility and
Log CQ (Y), Mean Concentration (%), for 10 PNAH in Shellfish .... 45
-------
ASRREVX ATIOtft AND SYMBOLS
ABBREVIATIONS
3H — tTitiated compound
HPLC ~ high performance liquid chromatography
LOG S ~ water solubility (moleB/X)
LOG Ss — solubility in aeawater (ttoles/L)
LOG Co — PNAH concentration
MW — molecular weight
PNAH — polynuclear aromatic hydrocarbon
METRIC MEASUREMENTS
ff
kg -
L .
mg
- grams
- kilograms
- liter
- milligram
moles
pinoles
&
— nanoooles
— picomoles
— micrograms
— microns
PNAH
PH&N — phenanthrene
FLOOR — fluoranthene
PYR — pyrene
BCP — benzo(c)phenanthrene
TRI — triphenylene
BAA — benzo(a)anthracene
CHRY — chrysene
BJP — benzo(j)fluoranthene
SAMPLE SITES (BAY, SPECIES)
BBF
BKF
BEP
£)BACA
BAP
&BABA
BGHIP
IP
QOR
benzo (b) fluoranthene
benzo(k)fluoranthene
benzo(e)pyrene
dibenz(a,c)anthracene
benzo(a)pyrene
dibenz(a,h)anthracene
benzo(g,h,i)perylene
indeno(1,2,3-c,d)pyrene
coronene
yim — (Yaquina, Mytilua adulia) C11G
Y2M — (Yaquina, Mytilua edulie) TIM
YlYO — (Yaquina, Craaeoatraa gigae) TSS
C3S — (Coos, \Mya armaria) TBC
ess — (Coos, JMya aranaria)
(Coos, Tzv8us oapax)
(Tillamook/ Mytilua adulia)
(Tillamook, Mya arenaria)
(Tillamook, Craaeoatrea gigas)
¥*
-------
ACKNOWLEDGEMENTS
Mr. Randy Schaffer was responsible for developing tha analytical methods
used to measure PMAH and managing tha Sampling program) his assistance was
invaluable and is gratefully acknowledged. Tha following individuals were
directly associated with tha project and their contributions were of great
value: Dr. David L&Touche, Ha. Susan Hemingway and Mr. Keith King.
I am grateful for the assistance and support of the following: Drs.
John Byrne, Robert Krauss, David Willis and Virgil Freed provided generous
support which permitted acquisition ofjHPLC analytical capabilities} Dr.
Bruce Dunn of the. University of British. Columbia.shared Ms_ expertise on
analytical methods; Dr. Donald Buhler permitted the use of his facilities and
equipment; Dr. Cary Chiou provided valuable information about solubility re-
lationships; Ms. Marilyn Henderson and Ms. Lori Mix provided valuable tech-
nical assistance; Ms. Leslie Mix helped transform climatolcgical data; Mr.
Paul Heikkala ..and Mr. Dale Snow shared valuable insights about sampling sites
and shellfish population^ and Ms., Karl* Russell typed the final report.
I am deeply indebted to Dr. John Couch for hia support and wise counsel
during the project period.
The organization and analysis of the data base associated with this in-
vestigation was carried out in part using the PROPHET system, a unique nation-
al resource sponsored by the NIH.
-------
SBCTION 1
lOTRODtJCTION
There has bean much recent interest expressed about the presence of
organic chemical carcinogens and mutagens in opastal estuaries and the
possible effects of these compounds on indigenous organisms that inhabit these
productive environments. Foremost among these chemicals, polynuclear aromatic
hydrocarbons (PNAH) are ubiquitous in the marine environment and may present
the greatest carcinogenic hazard (Kraybill, 1976).
Scientists from several diverse disciplines have advocated using bivalve
molluscs to serve as biomonitors for the detection and quantification of en-
vironmental contaminants, ineluding~cai,einog«ns " (e.g. couch et al., 1974»
DiSalvo et al., 1975; Dunn and Stich, i975; Goldberg, 1975; Mix et al., 1977).
Bivalve shellfish have received the widest support for such an approach
because they are permanent inhabitants of a specific environment and tend
to concentrate toxic substance3 in their tissues.
An additional factor in utilizing'bivalve molluscs in studies of environ-
mental contaminants is related to the discovery that many species, from
several different geographic locations! have been reported to have cellular,
perhaps neoplastic, proliferative disorders (Mix et al., 1979a). Environ-
mental pollutants were implicated as potential causative agents in several of
those reports although no cause-effect;relationships have yet been estab-
lished.
Field studies to determine PNAH concentrations in marine organisms have
only recently been initiated despite earlier suggestions that extensive in-
vestigations of the marine environment, Including chemical identification,
monitoring and surveillance, and identification of fish and shellfish tumors
may play an important role in the epidemiology of cancer' (Kraybill, 1976).
Of the PNAH, benzo(a)pyrene (BAP) has teen the most extensively studied car-
cinogen in the marine environment. Thire have been a number of reports of
BAP concentrations in tissues of indigenous shellfish (Dunn and stich, 1976a,
b; Dunn and Young, 1976» Mix et al., 1977 j Pancirov and Brown, 1977; Joe et
al., 1979; Mix, 1979; Mix and Schaffer, 1979; Risebrough et al., 1980).
Excepting BAP, which has often bean used to indicate the presence of
other PNAH, there is relatively little
or quantities of unsubstituted PNAH in
information available on the presence
tissues of aquatic organisms. Such
information, based on the use of advanced analytical methods capable of
measuring ng quantities, is needed. Considerable emphasis should be placed
on determining the composition of PNAH assemblages in the tissues of organisms
from both pristine and ispacted areas in order to gain an understanding of
the sources__and .fluxesof PNAHthrough' the a_quatic ecosystem (Neff, 1979).
I
-------
Assooiatioito between high tisaue oonoantrationa of PNAH, and other car-
oinogtni* and tija appaaranoa of oalluldx prolifarativa diaordera in ahellfish
populations should alao ba identified And carefully Invaitigatad. The exii-
tanoa of auoh aiiooiationi would atWMt tha Motility of additional atudiaa
to evaluate mord fully„potantial causa and iffact ralationahipa.
A relatively iimpla rational! oat\ ,ba offarad for conducting atudiaa on
PNAH in marina aboayaeama. Tha environmental levels of" tfiese compounds can
be expected to increase in the biologiqally produotiva coaatal regions. Such
contamination is inevitable becauaa of inoraaiad ahip traffic, eacalating re-
creational demands and expanding industrialisation. Yet, there is a scarcity
of dependable qualitative information dn PNAH in the inhabitants of marine
ecosystems. Finally, little is known ibout the affects of chronic# low-level
contamination with environmental PNAH on-shellfish or the potential public
health hazard associated with their cortsunption.
The present studies were designed to accomplish the following:
1. Develop state-of-the-art methods for identifying and quantifying
tSrisubstituted PNAH in tissues of ftajfine oYgaCBtsKS;
2. To measure baseline levels of PNAH in bivalve molluscs from Oregon
bays;
3. To evaluate critically the practicality of utilizing indigenous
populations of bivalve molluscs for PN^H monitoring and surveillance studies;
4. To identify and evaluate the Quantitative relationships between
individual PNAH in tissues of bivalve molluscs;
5. To determine the prevalence of cellular proliferative disorders ir;
bivalve molluscs that inhabit environments with significantly different
degrees of PNAH contamination;
6. To determine rates of BAP uptake and elimination in bay mussel
(MytiluB edulis) populations under ambient (field) conditions;
7. To identify tissue sinks or storage compartments for BAP in M.
edulia from PNAH-contaminated sites;
3. To determine if there is any association between PNAH body burdens
and the prevalence of cellular proliferative disorders in affected M. edulis
populations}
9. To conduct preliminary studies to determine whether or not M. edulia
is capable of metabolising BAP.
I"
-------
SECTIONS 2 AMD 3
CONCLUSIONS MO RECOMMENDATIONS
An analytical method, utilising HPLC, was developed and used to identify
and quantify 15 unsubstitutad PNAH inoludingi phenanthrena, £luoranthene,
pyrene, benzo(c)phenanthrena, triphenylene, banco(a)anthracene, chrysene,
benzo(b)fluoranthene, benzo(k) fluoranthene* dibens(a,c)anthracene, benzo (a)-
pyrena# dibens(a,h)anthracene, benzo(g>h,i)perylena, indeno(1,2,3-c,d)pyrene
and ooronene. The method resolved moat members of the benzpyreno group.
Perylena was not identified because it did not absorb UV light at the wave-
length used and benzo(j)fluoranthena and benzo(e)pyrena could not be sepa-
rated. Additonal efforts will be required to completely resolve and identify
B(e)P, B(j)F and perylena.
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 contamination. PNAH concentrations in
shellfish from relatively pristine areas ranged from 30-60 pg/kg while those
from industrialized areas contained 500-1500 ygAg* The data collected during
the present study indicate that bivalve molluscs make excellent monitors for
detecting and measuring PNAH in estuaries. Future efforts should be directed
towards fully defining the sampling protocols to be used in monitoring
studies and to identify and evaluate endogenous and exogenous factors that
may influence PNAH concentrations under ambient (field) conditions. The
latter should include studies of potential sources and measurements of PNAH
in water.
Identification and evaluation of quantitative and qualitative relation-
ships between individual PNAH and between PNAH and their concentrations in
bivalve molluscs indicate that a significant potential exists for developing
predictive models for PNAH in aqueous environments and their concentrations
in certain seafood products. Some of the relevant findings and conclusions,
based on thorough statistical analyses of the data from these studies are
summarized below.
1. Quantities of a single PNAH ptesent in shellfish cannot be used to
predict total PNAH.
2. For each site, different independent variables (individual PNAH) were
identified and used to predict total PNAH in bivalve molluscs. It may be
possible to identify site-specific independent variables after a suitable
sampling period'and to subsequently measure only those variables for an ade-
quate assessment of total PNAH. Complete analyses could perhaps be made
periodically to1 confirm the continuing!validity of the established relation-
ship) deviations may indicate new sources of contamination. Such an approach.
-------
may result in considerable ooat reduction for long-Una monitoring programs.
3. Ben«o(a)pyrene was not * significant varlabia for predicting total
PNAH at any sits. Thus, tha oonoapt that BAP oan ba uaad aa an index of PNAH
contamination was not supported by tha raaulta of this study. Prom thia and
othar studies, it seems that tha uaa of BAP for making daoiaiona about tha
quantities and praaanoa or abaanoa of Cther PNAH ahould ba abandonad or
modified.
Data from studies of BAP in W. tdulie suggest that tha dapuration rata
for this compound was exponential with a half-11fa of 8-10 daya while uptake
was linear. Routine depuration procedure* in which shellfish are placed in
clean aeawater for 24 hours, would have little affeot in reducing BAP con-
centrations. Gametogenesis and/or incorporation of BAP, and presumably other
lipophilic PNAH, into the gonad, do not appear to be directly responsible
for seasonal increases of BAP in mussels during winter-spring. BAP storage
occurred primarily in the somatic tissues compared to the gonad, even during
the spring spawning period. It is tentatively concluded, based on incomplete,
preliminary studies, that 7,10- *C-BAP was metabolized by microaomal extracts
from the visceral mass Of M. edutie. Phenolic metabolite* were'the only
measurable BAP metabolites present. More complete studies, utilizing advanced
methods, will be required to fully evaluate tha metabolic capabilities of
M. eaulia for altering BAP and othar PltAH.
Different populations of shellfish ware 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 charac-
terize the conditions. The disorder was present in a significant number
(mean prevalence - 10%) of Yaquina Bay mussels with the highest concentra-
tion of pnah measured in this study while it rarely appeared in a second popu-
lation at a "clean" aite across tha bay. Tha correlation between the degree
of PNAH contamination and tha prevalence of tha cellular disorders may be
significant, but no cause-effect relationship has been established, it re-
mains to be determined if carcinogenic metabolites can ba formed by this
species. If bivalvea are not subject to PNAH-induced carcinogenesis, and the
presence of atypical cella is related to a neoplastic process, then other
causative agenta must be responsible. Assuning the condition is analogous to
neoplasia, it seems evident that this disorder in M, edulia has great poten-
tial for serving as a model for studying cancer-like diseases in an inverte-
brate. The cells have many characteristics in common with malignant con-
ditions in mammals and affected mussels can be obtained easily and on a
regular seasonal basis by procedures developed during this study. Consider-
able future efforts should be directed towards further characterizing the
cells, attempting to establish culture techniques suitable for maintaining
and growing tha calls, and identifying the oausal agent(s).
4. Hhile it was eatablished that quantitative predictiona about total
PNAH could not be made on the basis of individual PNAH measurements, tha
results of this research suggest that certain qualitative relationships
existed which may permit first approximations of individual PNAH concentra-
tions. In general, there were no significant differences between individual
PNAH with 4 rings, or between those with 5, 6 or 7 rings. Phenanthrane. a
1
-------
3-xlng oonpoundf differed lignifioantly from other PNAH. Thua, datactlon of
a certain quantity of P¥R, for axaaqplaj suggested that a similar quantity of
BGP| TRI and BAA waa pvaaantt aaaauraaint of an individual PNAH oonoantratlon
for any B-, 6- or 7-ring PNAH lndicatM that approxiaately the aama oonoan-
tratlon would b« found for any othar unaubatitutad PNAH with 5-7 ringa.
It would ba productive to oonduot thaaa kinda of analyaaa for PNAH data
collect** during futura studies artd fWa dthef, aatabXllTHd biolagiaal moni-
toring programs. Confirmation of tha ralationahlpa ldantifiad during thla
raaaaroh nay eventually load to a aimpllflad monitoring approach and raault
in conaiderable coat reductions•
Statistical analyaaa ravealad an anpirioal relationship between individ-
ual PNAH ooncantrationa and their respective aolubilitiaa. Tha concentration*
in shallfiah wara graatar for tha PNAH iaomar which had tha higher solubility
in water. This finding oontraata with tha obaervation that organic/water
(e.g. octanol/water) partition ooefficienta show an inverae relation to water
solubility. Because the concentration in the organic phaaa (shellfish, in
this study), Cq, is equal to the product of tha partition coefficient (K) and
concentration in water (C*), the data iuggaat that the ratio of the PNAH
concentrations in water would have to be generally greater than the ratio of
their reciprocal partition coefficients or their water solubilities. Direct
measurements of PNAH concentrations in seawater will be necessary to confirm
whether the uptake of PNAH*a by shellfish can be represented by a simple
partition process.
5
-------
MOTION 4
MATERIALS AND METHODS
SAMPLE SITES, SPECIES UTILIZED AMD SAMfcLINQ PROTOCOL
The Oregon coast is characterised toy the presence of generally small bays
and astuarias (Fig. 1)* With tha exception of Ooos Bay and, to a lesser de-
gree, Yaquina Bay, none can be characterized as being industrialized. To
measure PNAH concentrations in species of interest, shellfish were collected
from sites in Coos# Yaquina and Tillambok Bays, the three largest bays in
Oregon. Those three bays were selected beoausa they satisfied criteria adop-
ted during previous studies (Mix, 1979). • They each have major commercial and
recreational shellfisheries with abundant populations of those species to bd
utilized in tha present study. It was also considered desirable that each
bay reflect different degrees of industrialisation and human onshore and
watershed habitation.
Mussels and clams from Yaquina and Coos Bays were also examined histo-
logically to determine whether or not cellular proliferative disorders oc-
curred in their populations and, if so, to calculate the prevalence of the
condition for a 6 month - 1 year period.
Figure 1 identifies the specific sites from which samples were obtained
for measuring PNAH concentrations and identifying cellular disorders. Table
1 lists the species sampled from each Site and the periods during which
samples were collected.
TABLE 1. INFORMATION ON SAMPLE SITES. SPECIES UTILIZED AND SAMPLING FREQUENCY
BAY
SITE
SPECIES COLLECTED
PERIOD OF SAMPLING
PURPOSE(NUMBER)
Coos
C3S
Mya armaria
Bimonthly, 1978-80
PNAH (10-1-10)
CSS
M. arenaria
Bimonthly, 1978-79
PNAH (20)
Tmbub oapax
histology (50)
CIXG
Bimonthly, 1978-80
PNAH (5)
Yaquina
Y1M
Mytilua edulis
Bimonthly, 1978-80
PNAH (30-40)
M. gdulie
histology (50)
Y2M
Bimonthly, 1978-80
PNAH (30-40)
1
2
1
histology (50)
Y140
Quarterly, 1976-80
PNAH (2o)
Tillamook
TIM
M. •duli*
Twice, 1979
PNAH (30-S0)
T8S
M. cavrvxria
Quarterly, 1979
PNAH (20)
SBC
C^gigaa
ZfadLfift*~jL92a
*NAH (201.
B
-------
C All r rtN IA
Figure 1. Oregon bays and estuaries.
TIM*
we.
C%
cno
Figure 2. Sample sites in Tillamook, Yaquina and Coos Bays, Oregon.
9-
-------
Tha rational* for using mora than ona spsoies haa baan discussed else-
where (Mix, 1979). Briefly* utiliaation of a iirigla shellfish spsoies, *ubh
aa M* •duUit Mr monitoring estuaries haa oartain limitation!, a single
speciea ia rarely found to inhabit an antire estuary, particularly if, ai in
Oragon baya, thay ara freshwater dominated during major parioda of tha yaar.
Thua, M, atvMcria ara usoful aa monitors of upbay sites, whara much of tha
industry ia loodtad, ainoa thay ara uaually tha only economically-important
bivalve mollusc that thrivaa in thaaa araaa of low salinity. Mussels are
used to monitor tha lower baya whara atlinitiea ,ara higher. While it is re-
cognized that these 2 species oooupy entirely different habitats, and direct
comparisons in PNAH concentrations have limited value, they have both been
found to be suitable aa biomonitora fo± detecting the presence of PNAH in
estuaries.
The specific sites in each bay were selected because they harbored sub-
stantial populations of the species of interest and each had unique features
relative to anthropogenic impact. Information about each site is included
below. Y1M consisted of a number of weathered pilings that formerly supported
an old railroad trestle. From previous studies (Mix, 1979) it was known that
mussels from this site had very low-concentrations of-BAP-and a low prevalence
of the cellular proliferative disorders. The site no longer exists since the
pilings were removed during the summer of 1980. Mussels from Y2M were removed
from pilings and ladders situated along the Newport bayfront. The creosoted
pilings support cold storage facilities and fish processing plants. The
earlier studies had shown mussels from this site had higher levels of BAP and
cellular proliferative disorders. Oysters were collected from the shucking
tables of a commercial oyster grower at 1140. The C. gigae were grown in
trays suspended from pilings at this site. Softshell clams were collected
from Coos Bay at CSS, an open, muddy area adjacent to timber products indus-
tries along Highway 101, and C3St a mixed sandy mud flat on North Slough ad-
jacent to Highway 101, which is removed from hunan onshore activities. Gaper
clams wcsre collected from CIIG, a small mixed sand-mud flat at Charleston ad-
jacent to the Coast Guard station. Mussels from Tillamook Bay were collected
froci rocks located near the entrance, across from Kincheloe Point, clams from
sand and mud flats near Pitcher Point imd oysters from commercial growing
grounds near Bay City.
COLLECTION AND PREPARATION OF SHELLFISH SAMPLES
Clams from the three bays were ducj during low, approximately zero tides
while 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, samples from each
site were placed in labeled plastic bags, put on ice and transported back to
Corvallis. Animals were then removud from their shell and the pooled sample
from each site Was then weighed. Each pooled sample was then stored at -20°C
until it was processed for PNAH analysis.
To determine the effects of a depuration period on PNAH concentration
in clams, 10 M. arenaria from the C3S sample were placed in a plastic bucket
filled with salt water that contained a small quantity of cornmeal. The
B
-------
buokat was placid in a cooler maintained at 10*C and the olams allowad to de-
purata for 24 hours. Tha dapuratad olama wara than prooaaaad in tha usual
way. This prooadura took plaaa aaoh time olama wara sampled aftar J una, 1979,
Muaaals to ba examined histologically Vara placad in Davidson's fixative
(3i3t2tlil - 95% ethanoltsea-waterifortaliniglyceroltacatio acid addad just
befora uaa), procassad in tha usual way,sectioned at 6ym, and atainad with
hematoxylin and doain.
PNAH ANALYTICAL PROCEDURE
Ona of the major objectives of this study was to develop standard pro-
cedures that could be used confidently for measuring PNAH concentrations in
bivalve shellfish. These methoda, as developed and modified by Mr. Randy
Schaffer, will be described in considerable detail.
Reagents and Standards
The following reagents were obtained from J. T. Baker (Phillipsburg, NJ):
High performance liquid chromatography (HPLC) grade acetonitrile; photrex
grade dimethyl sulfoxide (DMSO); reagent grade 2,2,4-tri-methyl pentane (TMP),
potassiun hydroxide and benzene. Ethanol (USP) was obtained from Central
Solvents (Oregon Liquor Control Commission) and redistilled before use. Water
for the HPLC was prepared in a Milli-Q System (Millapore Corp., Bedford, MA).
The following PNAH standard were purchased as dry powders: fluorane,
phenanthrene, fluoranthene, pyrene, triphenylene, benzo(a)anthracene, chry-
sene, benzo(e)pyrene, dibenz(a,c,)anthracene, benzo(a)pyrene, dibenz(a,h)-
anthracene, benzo(g,h,i)perylene and coronene, from RFR Corp. (Hope, RI);
indeno(l,2,3-c,d)pyrene from Analabs (North Haven, CT); and benzo(j)fluoran-
thene, benzo(k)fluoranthene, benzo(b)fluoranthene and benzo(c)phenanthrene
from Dr. J. E. Meeker (U.S. EPA, Research Triangle Park, NC). Stock solu-
tions of all standards were made with DMSO to prevent evaporation.
Apparatus
A Spectra-Physics Model SP8000 High Performance Liquid Chomatograph,
equipped with a Schoeffel Model 770 variable wavelength UV detector and a
Schoeffel Model 970 variable wavelength fluorescence detector connected in
series, was used throughout the study. Chromatography conditions are sum-
marized in Table 2.
Sample Preparation
Shellfish samples were prepared according to the methods of Dunn (1976)
Approximately 30-40 g of pooled tissue was saponified in ethanol/XOH. The
resulting supernatant was extracted with TMP and the organic phase passed
through a column of partially deactivated florisil. The PNAH were eluted
from the colunn with benzene and the eluate was extracted with DMSO. The
resultant TMP fraction was then passed through a Sephadex LH-20 column
(Pharmacia Fine Chemicals, Inc., Piscataway, NJ)/ tritiated BAP was added to
determine BAP recovery. The end produfct was brought up to lOOyl in DMSO.
0
-------
PNAH Analysis
The HPLC analyses war* conduotad fcccording to procedures described in
Table 2.
TABLE 2. APPARATUS, OPERATING CONDITIONS AND PROCEDURES FOR PNAH ANALYSES.
LIQUID CHROMATOGRAPHY« 8pectra-Physics 8000 with data system^ Valoo injector,
model cv-6-Ul+Pa-N60, with lOyl loop.
COLUMNS I Parkin Elmer HC-ODS-PAH number 258-0082, 0.26 x 25cm connected in
series with a 0.32 x 10cm guard column slurry packed with Vydac 201 TP.
MOBILE PHASE» Acetonitrile(MeCN)/water gradient, constant flow mode 0.8
ml/min at 20 ®C.
Time(mln) % MeCN % H2Q
0 60 40
4 60 40
22 100 0
45 100 0
55 20 80
65 20 80
70 60 40
95 60 40
DETECTOR 1: Schoeffel Model 770 variable wavelength UV detector, 296 nm
range 0.02 and 254 range 0.02.
DETECTOR 2: Schoeffel Model 970 variable wavelength fluorescence detector,
326 ran excitation > 412 nm emission cutoff-type filter; range 0.1, sensitivity
580.
Identification and Quantification
Four methods were used to identify individual PNAH. (1) A preliminary
identity was assigned to each sample peak by comparing its retention time to
that of a known standard. Each sample; was chromatographed at two UV wave-
lengths, 296 nm and 254 nm, and one fluorescent wavelength combination, 326
excitation greater than 412 nm emission. Precise identification of PNAH was
then possible by calculating ratios of response at different wavelengths;
(2) UV peak area at 254 nm/UV peak area at 296 nm, (3) UV peak area at 254
nm/fluorescent peak area, and (4) UV peak area at 296 ran/fluorescent peak
area.
.Individual PNAH were quantified by use of an internal data system in the
HPLC; essentially, a comparison is made between a calibration standard run
and an analytical (sample) run. A concentration factor, calculated in the
calibration mode, was used to determine a concentration for the sample peak
nearest the retention time of the calibration peak. Concentrations were
10
-------
determined vaing a program designed fo? an HP-85 oomputar.
A portion of th« sample was analykad for 'h by liquid aointillation
counting in ordir to determine BAP raoovary. That raoovary figura was uiad
to convart all ?NAH quantitiaa to 100%'.
BENZO(A)PYRENE OPTAXE AND ELIMINATION
An appropriate number of M. edulie from Y1M and Y2M wara collected and
placed in fiberglass mesh bags designed to hold one sample of 20-30 mussels.
Individual bags were then placed inside 46 x 92 cm nylon mesh drawstring bags
and suspended at the +2.0 foot tide level.
For the uptake study, 10 samples were collected from Y1M and transferred
to the more contaminated site, Y2M. Ah equal number of Y2M mussels were col-
lected and hung next to the transferred TIM mussels to serve as a control to
measure background fluctuations in BAPiconcentrations. For the depuration
study, 10 samples of BAP-contaminated mussels were collected from Y2M and
transferred to Y1M. An equal number of Y1M-mussels-were-collected and hung-*-
near the transferred Y2M mussels to senre as a control. Animals were sampled
biweekly or weekly and analyzed for BAP using Dunn's techniques (Dunn, 1976? :
Mix and Schaffer, 1979).
TISSUE STORAGE SITES OF BENZO(A)PYRENE: IN M. EDULIS
An initial effort was directed towards resolving the question of whether
or not the gonad is a major tissue repository for BAP.
Site Y2M was selected for this study since it was known to harbor mussels
with significant BAP concentrations (Mix and Schaffer, 1979). High BAP con-
centrations were necessary for detection when small gonad samples (<5g pooled
net weight)' were analyzed. Prior to initiating the study, 12 samples, con-
sisting of at least 30 mussels, were collected from the pilings at Y2M. Each
sample was placed in fiberglass mesh bags and then collected in a large,
nylon mesh bag and suspended at the same location and tide level from which
they were collected. Mussels were acclimated for two weeks before sampling
began; samples were collected at approximately 10 day intervals from 9 Jan 79
to 25 May 79 when spawning had been completed.
Mussels in each sample were separated into gonadal tissue and somatic
tissue fractions. Each tissue fraction was blotted dry on paper towels,
placed in plastic bags, labeled, and held at -10°C until analyzed. Concen-
trations of BAP were determined by Dunn's TLC methods (Dunn, 1976).
BENZO(A)PYRENE METABOLISM BY M. EDULIS
Only studies of a preliminary nature were conducted during the present
period. The basic techniques and protocol followed those developed by
Anderson (1978) for American oysters (Cvas80Btrea virginica). The method
11
-------
involvea um of ia ••naltiva radioiaotopie ayatem that permit* quantification
of and watar-aolubla BAP matabolitna produced by digaative
gUnd microaomai. High parformanca liquid chromatography waa uaad to iden-
tify tha matabolitaa.
STATISTICAL ANALYSES
The data gathered during thia rasaaron vara aucjaotaa to extonsive sta-
tistical analyses using the PROPHET aystem. PROPHET is a National Computer
Resource of the Division of Research Resources, National Institute of Health.
It consists of two FDP-10 computers, located at ADP Network Services, Inc.,
Waltham, Mass. and appropriate software developed by Bolt; Beranak and Newman,
Inc., Cambridge, Mass. HP-25C and HP-41C programmable calculators were also
used for certain procedures. The information below is summarized from the
PROPHET manuals.
After the basic measures were computed, it was general practice to pur-
sue a course for drawing conclusions or making decisions about the data under
study. The general method was to assume a testable hypothesis about a popu-r
lation and then use the data in an appropriate test of the hypothesis.
The appropriateness of any statistical test rests on certain underlying
theoretical assumptions about the sample and the population. Parametric
procedures were used when it could be determined that samples wore obtained
from populations whose distributions were normal. Parametric tests were
always used where appropriate since they tend to be more efficient in detec-
ting departures from the null hypothesis. Por certain samples, where the
assumption of normality was clearly violated, nonparametric procedures were
used.
12
-------
SECTION 5
RESULTS AND DISCUSSION
IDENTIFICATION OF PNAH IN BIVALVE MOLLUSCS
Table 3 includes a list of unsubstituted PNAH identified and quantified
in indigenous populations of bivalve mblluscs during this study and provides
a svmmary of their activities according to several classification systems.
There is not yet universal agreement about the carcinogenic potential of
several PNAH, yet, except for coronene, all of the PNAH identified during the
present study have obvious environmental importance.
The identification of this large group of individual PNAH in indigenous
organisms represents a significant advance in studying levels of PNAH in ma-
rine organisms. The presence of PNAH in water, aquatic sediments and organ-
isms has been recognized for more than 20 years (Neff, 1979). Yet, depend-
able high resolution techniques, such as those utilized in this study, have
only recently been developed and a more precise information base can now be
expected to develop.
Table 4 summarizes available information on certain physical and chemical
characteristics of PNAH. Complete data on PNAH solubilities will be required
for a full understanding of the behavior of these compounds in marine eco-
systems.
BASELINE DATA ON PNAH CONCENTRATIONS IN CLAMS FROM COOS BAY
While there have been numerous reports of PNAH concentrations, particu-
larly BAP, in mussels there is little similar information available for clam
species. Tables 5-7 contain data on PNAH concentrations in 3 clam populations
from Coos Bay, Oregon, sampled during a 2 year period.
Gaper clams, T. capax, are large ((10-15 cm), suspension-feeding, bay
clams that are found in sand or sand-mud bottoms, 25-70 cm below the surface.
They are heavily dug by recreational fishermen and fried or minced for in?or-
poration into chowder. PNAH concentrations in T. oapax from Charleston were
the lowest of any clams from Coos Bay, ranging from 31.4-109.7 yg/kg. It is
apparent from examining Table 5, that PNAH concentrations fell into two
narrow ranges, 37-51 vgAg and 104-110 vgfkg. Regression analyses (day
ntmber vs. total PNAH) indicated that there were no seasonal differences, nor
was there any apparent relationship between concentration and reproductive
cycle. The differences in concentration may be due to variation in the
samples-analyzed, or higher concentrations may have been„caused_.by local
13
-------
TABLE 3. POLYNUCLEAR AROMATIC HYDROCARBONS ANALYZED IN SHELLFISH FROM OREGON ESTUARIES.
CLASSIFICATION
PNAH
Phenanthrene (PHEN)
Fluoranthene (FLUOR)
Pyrene (PYR)
Benzo(c)phenanthrene (BCP)
Triphenylene (TRI)
Benzo(a)anthracene (BAA)
Chrysene (CHRY)
Benzo(b)fluoranthene (BBF)
Benzo(k)fluroanthene (BKF)
Dibenz(a,c)anthracene (DBACA)
Benzo (a) pyrene (BAP)
Dibenz (a,h) anthracene (DBAHA]
Benzo(g,h,i)perylene (BGHIP)
Indeno(l,2,3-c,d)pyrene (IP)
Coronene (COR)
EPA
PP
PP,TP
PP
PP,TP
PP,TP
PP,TP
PP,TP
TP
PP,TP
PP,TP
PP
PP,TP
NAS
+
±
NIOSH
NEO
CAR
CAR
CAR
CAR
CAR,NEO
CAR
CAR, NEO
CAR,NEO
CAR
IARC
IARC
C
C
C
C
1EPA classification: PP-Priority pollutant; TP-Toxic pollutant (Keith and Telliard, 1979>
2
NAS classification: - not carcinogenic, ± uncertain or weakly carcinogen!c, + carcinogenic,
++, +++, ++++ strongly ceircinogenic (NAS, 1972)
aNIOSH classification: CAR-carcinogenie effects in animals, NEO-neoplastic effects in
animals (Christensen et al., 1975)
"iARC classification: C - evidence of carcinogenicity in experimental animals (IARC, 1972)
5IARC classification: C - sufficient evidence of carcinogenicity in experimental aniials
(IARC, 1979)
-------
TABLE 4. SOME PHYSICAL AND CHEMICAL CHARACTERISTICS OP PNAH.
NUMBER
MOLECULAR
WATER
SOLUBILITY, S,
(mg/L)
-LogSs
,5%.
-LogSs/
15%.
-LogSsr
30%»
PNAH
OF RINGS
WEIGHT
MfiS1
(-LoqS) 1
MAY
(-LogS)3
MSS
MAY
M&S
MAY
HtS
MAT
PHEN
3
178.2
1.29
(5.14)
1.002
(5.25)
5.14
5.25
5.21
5.32
5.29
5.39
FLOOR
4
202.3
0.26
(5.89)
0.206
(5.99)
5.92
6.02
5.97
6.08
6.06
6-17
PYR
4
202.3
0.135
(6.18)
0.132
(6.19)
6.20
6.21
6.25
6.26
6.32
6.33
BCP
4
228.3
TRI
4
228.3
0.043
(6.73)
0.0066
(7.54)
6.74
7.56
6.78
7.59
6.83
7.65
BAA
4
228.3
0.014
(7.21)
0.0094
(7.39)
7.25
7.42
7.30
7.48
7.40
7.57
CHRY
4
228.3
0.002
(8.06)
0.0018
(8.10)
8.09
8.13
8.15
8.19
8.23
8.28
BJF
5
252.3
BBF
5
252.3
BKF
5
252.3
BEP
5
252.3
DBACA
5
278.3
0.0055
(8.75)
BAP
5
252.3
0.0038
(7.32)
DBAHA
5
278.3
BGHIP
6
276.3
0.0026
(9.03)
IP
6
276.3
COR
7
300.3
0.00014
(9.33)
1 MacKay and Shiu, 1977.
2 May, 1980 S x 10~3g/L
3 Log S, in moles/L, determined from the formula, LogS MW s
* Log Sg, solubility in saltwater, determined for various salinities by the formula. Log — = *s^-s» "here
S and Ss are the concentrations of the solute in freshwater and saltwater, respectively? Kg is the
Setschenow constant for the PNAH and Cs is the molar salt concentration for a specific salinity (after
May, 1980).
-------
TABLE 5.
PNAH CONCENTRATIONS (yoTVeri1 IN T, GAPAX FROM COOS BAY, OREGON,
SITE CI1Q.
DATE
SAMPLED
PHEN
FLUOR
PYR
BCP
TRI
BAA
CHRY
BB T
9/29/78
25.9
20.3
11.5
9.9
8.9
10.3
9.8
4.3
12/2/78
20.3
21.2
12.5
10.9
9.0
9.3
10.2
4.9
2/9/79
16.1
8.3
2.0
4.3
4.6
8.3
2.4
0.9
4/18/79
14.4
7.0
2.1
4.0
4.0
6.4
2.1
0.8
6/23/79
11.0
6.3
2.1
3.5
2.1
5.3
2.0
1.0
8/13/79
23.1
20.0
11.5
9.8
9.0
9.6
9.8
4.7
10/6/79
8.3
5.3
2.0
2.5
1.8
4.1
1.9
1.0
2/22/80
24.4
20.8
12.5
10.4
9.1
10.0
10.0
4.7
4/25/80
9.5
6.3
3.0
1.8
2.0
6.5
3.1
0.9
6/14/80
10.5
7.3
4.1
2.7
3.0
7.1
3.5
1.1
X
16.4
10.0
6.3
6.0
5.4
7.7
5.5
2.4
(s.d.)1
(6.6)
(12.3)
(4.9)
(3.8)
(3.3)
(2.1)
(3.9)
(1.9)
DATE
TOTAL
SAMPLED
BKF
DBACA
BAP
DBAHA
BGHIP
IP
COR
PNAH
9/29/78
3.9
1.4
1.0
0.9
0.3
0.2
0.3
108.2
12/2/78
3.0
1.2
1.1
1.0
0.3
0.2
0.3
105.6
2/9/79
0.8
0.6
1.0
0.8
0.3
0.3
0.3
51.0
4/18/79
0.6
0.9
1.0
0.8
0.4
0.1
0.2
44.8
6/23/79
0.5
1.0
1.3
0.6
0.6
0.3
0.1
37.7
8/13/79
3.1
1.4
1.1
1.0
0.3
0.2
0.4
104.9
10/6/79
0.4
0.9
1.2
,0.8
0.6
0.4
0.2
31.4
2/22/80
3.2
1.4
1.2
jl.O
0.3
0.3
0.4
109.7
4/25/80
0.4
0.9
1.0
1.0
0.6
0.3
0.2
37.5
6/14/80
0.5
0.9
1.0
1.5
0.3
0.4
0.3
44.2
X
1.6
1.1
1.1
0.9
0.4
0.3
0.3
67.5
(s.d.)
(1.5)
(0.3)
(0.1)
(0.2)
(0.1)
(0.1)
(0.1)
(34.5)
1one standard deviation
16
-------
•venta related to boat or flah procaailng plant aotlvitiaa.
Two populations of aoft-ahall dams (W. artnarla) vara atudlad during a
two yaar period. Total PNAH conoantrationa in olama from CSS wara muah
greater than thoaa from C3S (Table 6).j The' two aitea repreaant considerably
different environments. Site C38 ia in a relatively priatine aaotion in
North Slough with little evident impact from hunan onahore activitiea. Claini
from CSS inhabited a aoft mud flat located adjaoent to the induatrial water-
front complex at Cooa Bay. Potential aoureea of PNAH at the latter site in-
clude, but are not limited to, timber produota industries, fuel atoraga
facilities, boats and pilings. Zt ia noted that clams from C3S are dug by
recreational fishermen while those from CSS are not because of access problems
and difficultiea in digging in aoft mud. A paired t statistic was used to
test the null hypotheses, Hot Vxi ¦ wyi where x « concentrations from C3S
clams, y « concentrations from CSS clams and i represents individual, and
total, PNAH. For 10 PNAH and total PNAH, the H was rejected; calculated
values were greater than t .01.5 ¦ 4.03. However, there were no significant
differences in the mean concentrations of DBACA, DBAHA, BGHIP, IP or COR
during the one-year period. There were no large differences in PNAH concen-
trations during the 6 bimonthly sampling periods. However, since all samples
consisted of 20 pooled clams, no statistical analyses could be done in in-
dividual animals. Only by obtaining additional samples during each collec-
tion date would it be possible to use statistical tests to identify signifi-
cant seasonal differences. The present results suggest that such an effort
would not be warranted since the differences, if they existed, were small
and the additional costs would be substantial. It is concluded that the
means and standard deviations for individual and total PNAH represent an
accurate baseline for concentrations of those compounds in M. arenaria from
two geographical locations in Coos Bay, Oregon.
To determine the effects of depuration on PNAH concentrations in M.
ajpenajpicit samples from C3S were divided, beginning with the 6/23/79 collec-
tion. One group ("D") was placed in seawater containing corn meal and al-
lowed to depurate for 24 hours; the control group (N) was simply placed in
a refrigerator; both were maintained at the same temperature. After 24 hours,
all animals were shucked and analyzed. Table 7 provides a summary of the
data from the depuration study. A paired t statistic was used to determine
if there were significant differences in PNAH concentrations between clams
that had been depurated and those that had not. For all 3 and 4 ring PNAH,
there were significant differences between the clams that had been depurated
and those that had not; depurated clams had significantly lower concentra-
tions of individual 3 and 4 ring PNAH and total PNAH. In contrast, concen-
trations of all 5, 6 and 7 ring compounds, which included most of the car-
cinogenic PNAH, were not statistically different in the two groups.
While it is not yet possible to fully explain these results, differences
in water solubility of the PNAH may at least partially account for the ob-
served differences between depurated and non-depurated clams. In general,
the lower molecular weight (MW) PNAH are more soluble in water than those
with greater MW,(May, 1980). Previous depuration studies in shellfish (e.g.
Stegeman and Teal, 1973) have produced results which suggest that PNAH are
stored in two compartments. One of the compartments, perhaps fluid and/or
17
-------
TABLE 6.
OATS
SAMPLED
9/30/78
12/1/78
2/9/79
4/18/79
6/23/79
8/13/79
X
(a.d.)
DATE
SAMPLED
9/30/78
12/1/78
2/9/79
4/18/79
6/23/79
8/13/79
X
(s.d.)
DATE
SAMPT.Fn
9/30/78
12/1/78
2/9/79
4/18/79
6/23/79
8/13/79
X
(s.d.)
DATE
SAMPLED
9/30/78
12/1/78
2/9/79
4/18/79
6/23/79
8/13/79
X
(s.d.)
PNAH CONCENTRATIONS (wg/kg) IN N. ARENARIA FROM C008 BAY, OREGON
BITS C38
PHEN
FLUOR
PVR
BCF
TRI
BAA
CHRY
BBF
13.9
10.8
5.4
2.9
6.3
2.8
8.9
1.4
17.4
9.6
6.3
3.0
5.6
2.7
8.7
1.9
9.4
7.7
5.3
3.0
4.8
2.0
6.3
0.9
10.5
12.0
6.8
3.1
5.9
2.9
7.9
1.0
9.9
8.7
6.0
3.0
5.5
2.7
7.9
1.3
14.2
7.9
3.6
19.5
4.5
5.9
2.3
12.2
10.5
6.3
3.1
7.9
2.9
7.6
1.5
(3.4)
(2.4)
(1.0)
(0.2)
(5.7)
(0.8)
(1.2)
(0.5)
BKF
DBACA
BAP
DBAHA
BGH1P
IP
COR
TOTAL
3.2
2.8
3.3
5.4
6.4
5.8
1.5
80.8
3.0
2.1
3.1
5.0
6.0
5.5
1.4
81.3
2.0
1.0
2.3
1.5
3.2
4.3
0.9
54.6
3.1
2.1
3.4
4.7
6.0
6.4
1.5
77.3
3.4
2.1
3.3
4.9
4.6
7.5
1.6
72.4
1.1
12.8
5.5
6.7
6.4
1.0
91.4
2.6
3.8
3.5
4.7
5.4
5.9
1.3
76.3
(0.9)
(4.4)
(1.0)
(1.7)
(1.3)
(1.2)
(0.3)
(12.3)
SITE CSS
PHEN
FLUOR
PYR
BCP
TRI
BAA
CHRY
BBF
"
¦
158.4
121.2
58.6
65.4
41.8
29.5
28.6
13.2
144.4
111.2
47.3
51.5
38.6
25. 3
21.5
10.5
152.4
119.6
49.8
58.4
40.3
27.5
24.1
11.6
149.7
89.9
63.9
49.0
44.3
65.4
25.6
11.0
161.3
103.7
98.8
54.5
49.4
71.5
38.9
11.4
162.3
118.5
51.2
52.8
45.4
31.0
24.7
14.8
154.8
110.7
61.6
55.3
43.3
41.7
27.2
12.1
(7.1)
(12.1)
(19.1)
(5.9)
(3.9)
(20. 9)
(6.2)
(1.6)
BKF
DBACA
BAP
DBAHA
BGHIP
IP
COR
TOTAL
10.9
9.9
8.5
6.4
3.4
2.0
1.4
559.2
8.8
8.3
7.4
5.2
2.1
1.4
1.0
484.5
9.9
9.5
8.0
5.3
2.0
1.3
1.0
520.7
8.5
6.0
9.4
8.3
7.0
7.9
6.0
551.9
8.8
9.1
11.4
9.5
8.0
8.7
5.4
650.4
10.4
8.3
7.6
6.6
3.0
1.9
1.2
539.7
9.6
8.S
8.7
6.9
4.2
3.9
2.7
551.1
(1.0)
(1.4)
(1.5)
(1.7)
(2.6)
(3.4)
(2.4)
(55.5).
18
-------
TABLE 7. PNAH CONCENTRATIONS (ygA?) IN M. ARSNARIA FROM COOS BAY, OREGON,
SITE C3S, BEFORE (N) AMD AFTER DEPURATION (D) FOR 24 HOURS.
DATS
PHENANTHRENE
FLtJORANTHENl
PYRENE
SAMPLED
N
D
N
D
%
N
D
%
6/23/79
9.9
7.5
-24.2
8.7
5.3
-39.1
6.0
1.0
-83.3
8/13/79
...
...
14.2
0.8
-94.4
7.9
0.8
-89.9
10/6/79
12.5
9.5
-24.0
9.8
7.8
-20.4
6.3
3.0
-52.4
2/22/80
13.5
5.4
-60.0
9.8
1.4
-85.7
5.4
1.4
-74.1
4/25/80
11.5
6.4
-44.4
9.6
7.7
-19.8
5.5
1.3
-76.4
6/14/80
10.1
5.3
-47.5
5.4
0.9
-83.3
4.7
1.1
-76.6
9/12/80
8.7
5.1
-41.4
4.8
1.3
-72.9
3.7
2.0
-46.0
X
11.0
6.5
-40.2
8.9
3.6
-59.4
5.6
1.5
-72.2
(s.d.)
(1.8)
(1.7)
(14.0)
(3.1)
(3.2)
(32.1)
(1.3)
(0.8)
(16.1)
t value2
t -
5.39
t «
2.74
t -
6.59
sig. level
.01
.05
.01
DATE BEN2Q(C)PHENANTHRENE
SAMPLED N D %
TRIPHENYLENE
6/23/79
8/13/79
10/6/79
2/22/80
4/25/80
6/14/80
9/12/80
3.0
3.6
3.0
3.0
3.5
2.7
3.1
0.9
0.4
0.9
0.6
0.8
0.6
0.8
-90.0
-88.9
-70.0
-80.0
-77.1
-77.8
-74.2
N
5.5
19.5
4.9
5.4
5.6
9.5
6.4
0.4
3.1
2.4
0.9
1.4
3.5
3.8
-92.7
-84.1
-51.0
-83.3
-75.0
-63.1
-40.6
BENZO(A)ANTHRACENE
N D %
2.7
4.5
2.0
2.4
2.3
2.0
2.2
1.4
1.2
1.0
1.4
1.0
0.9
0.5
-48.2
-73. 3
-50.0
-41.7
-56.5
-55.0
-77.3
X
3.1
0.7
-76.9
8.1
2.2
-70.0
2.6
1.1
-57.4
(s.d.)
(0.3)
(0.2)
(6.5)
(5.2)
(1.3)
(19.1)
(0.9)
(0.3)
(13.2)
t value
t -
' 15.6
t -
3.25
t -
4.94
sig. level
•
01
.1
05
.
01
DATE
CHRYSENE
BEN20(B)FLUORANTHENE
BENZO(K)FLUORANTHENE
SAMPLED
N
D
*
N
D
%
N
D
%
6/23/79
7.9
1.3
-83.5
1.3
1.5
15.4
3.4
3.0
-11.8
8/13/79
5.9
2.3
2.1
-8.7
1.1
1.1
0.0
10/6/79
5.4
3.3
-38.9
1.4
1.1
-21.4
3.0
3.1
3.3
2/22/80
6.9
5.8
-15.9
1.1
1.1
0.0
3.0
2.8
-6.7
4/25/80
5.8
2.9
-50.0
1.2
1.0
-16.7
4.0
3.9
-2.5
6/14/80
2.7
1.1
-59.3
1.4
1.3
-7.1
3.0
3.0
0.0
9/12/80
2.8
1.0
-64.3
1.9
1.6
-15.8
3.1
2.9
-6.4
X
5.3
2.6
-52.0
1.5
1.4
-7.8
2.9
2.8
-3.4
(s.d.)
(2.0)
(1.9)
(23.0)
(0.4)
(0.4)
(12.4)
(0.9)
(0.8)
(4.8)
t value
t -
3.27
t -
1.89
t -
1.80
sig. level .05
nsd
nsd
x 100
1Determined by:
2Paired t-statistic, to test Ho:
N-D
N
lip » for all dates sampled.
19
-------
TABLE 7. (continued)
DATE
DIBBNZ(A,C)ANTHRACENE
BBNfeO(A)PYRKNE DIBBNZ(A,H)ANTHRACENE
SAMPLED
N
D
%
N
0
I
N
D
%
6/23/79
2.1
2.1
0.0
3.3
3.3
0.0
4.9
5.0
2.0
8/13/79
12.8
2.7
-78.9
S.S
2.4
-56.4
6.7
10/6/79
2.3
2.4
4.4
2.0
2.1
5.0
3.9
3.9
0.0
2/22/80
2.5
2.4
-4.0
3.2
3.1
-3.1
5.0
5.3
6.0
4/25/80
3.6
3.0
-16.7
2.9
2.7
-6.9
5.1
5.0
-2.0
6/14/80
2.0
1.9
-5.0
2.5
2.6
4.0
4.7
4.9
4.3
9/12/80
2.8
2.1
-25.0
2.5
2.3
-8.0
4.1
3.0
-26.8
X
4.0
2.4
-17.9
3.1
2.6
-9.3
4.9
4.5
-2.8
(s.d.)
(3.9)
(0.4)
(28.7)
(1.1)
(0.4)
(21.3)
(0.9)
(0.9)
(12.1)
t value
t -
1.16
t -
1.11
t -
-0.48
sig. level nsd
nsd
nsd
DATE
BENZO(G»H,I)PERYLENE
INDENO (1,2,3-
•C,D)PYRENE
CORONENE
SAMPLED
N
D
1
N
D
%
N
D
*
6/23/79
4.6
5.0
8.7
7.5
6.5
-13.3
1.6
1.4
-12.5
8/13/79
6.4
9.6
33.3
—-
1.0
2.1
110.0
10/6/79
5.0
5.8
16.0
4.0
4.0
0.0
2.0
1.7
-15.0
2/22/80
5.1
6.0
17.6
6.0
3.3
-45.0
2.4
1.9
-20.8
4/25/80
5.0
4.6
-8.0
5.8
5.7
-1.7
1.9
1.9
0.0
6/14/80
3.0
2.4
-20.0
3.1
2.9
-6.4
1.1
1.1
0.0
9/12/80
3.6
2.8
-22.2
4.6
3.1
-35.4
1.6
1.0
-37.0
x
(s.d.)
t value
slq.
4.7
(1.1)
t ¦
level nsd
5.2 3.6
(2.4) (20.9)
-0.97
5.2 4.2
(1.6) (1.5)
t - 2.16
nsd
-17.0
(18.8)
1.7 1.6 -3.5
(0.5) (0.4) (48.6)
t - 0.33
nsd
TOTAL PNAH
N
D
%
6/23/79
72.4
45.6
-37.0
8/13/79
78.8
26.3
-70.0
10/6/79
67.5
52.0
-23.0
2/22/80
74.7
42.8
-42.7
4/25/80
73.3
49.3
-32.7
6/14/80
57. *5
33.5
-42.1
9/12/80
56.1
33.3
-40.6
X
68.7
40.4
-41.2
(s.d.)
(8.7)
(9.5)
(14.5)
t value
t -
6.36
Big, level .01
20
-------
hsmoly&ph* is oharaotarisad by tha rapid turnover of PNAH whila tha othar,
nor* «table, ooapartmant is oharaetarlaad by alow turnover. Thus, mora
soluble PMAH» tha 3 and 4 ring maobara, may ba aisociatad primarily with tha
fluid ccmpartaant in H, armaria and ware eliminated by a measurable degree
during a 24 hour dapuration period. Tha 5# 6 and 7 ring PNAH which hava low
aqueous aolubilitiea nay hava been praaant primarily in tiaauaa and vara not
released in significant quantities. Claaranoa of tha gut oontanta may hava
alao baan associated with tha decrease in PNAH oonoantrationa• However, if
tha organio contents of tha gut contained substantial quantities of pnah,
seasonal differences in ratea of decrease, or percent change* would perhaps
be anticipated since M. arenaria do not pump actively during the winter
months. No such seasonal differences wen evident for this study and re-
gression analyses (day nrnber vs. PNAH concentrations) confirmed that the
amounts of PNAH decreases in depurated clams were independent of season
(P - 0.0 < F.os(7.is^ ¦ 3.52). Additional studies will be necessary to iden-
tify the underlying mechanisms responsible for the observed differences dis-
cussed above.
There are essentially no reports of PNAH concentrations in clams col-
lected from their native environment; thus, comparisons of results with those
other studies are not possible. Joe et al. (1979) reported on BAA, BAP,
FLOOR, PYR, BBF and TRX concentrations in a single M. apenapia collected from
an oil spill site. The total value was approximately 5X the mean concentra-
tion of the same PNAH for soft shell clams sampled from C3S (136 vgAg vs.
29.8) but only half that for CSS clams (265.9 Pg/kg). No conclusions are war-
ranted from this comparison because of the unacceptable sample size in that
study.
PNAH CONCENTRATIONS IN M. EDUUS FROM YAQUINA BAY
Baseline Concentrations of PNAH
Mussels were sampled monthly or bimonthly frcra two separate populations
in Yaquina Bay. M. edulis from Y1M were collected and analyzed from 10/2/78
until 6/17/80, when the site was destroyed during construction of a marina.
Since the population at Y2M was smaller, sampling did not begin until January,
1979, to insure that PNAH measurements could be made for an entire year
(1979). Mussels were also collected and analyzed for the first 4 months of
1980 before their ntctber was reduced to a level where it was no longer con-
sidered possible to obtain random samples from a normal population.
Tables 8A and 8B include data on PNAH concentrations in those two popu-
lations. It is evident that there were substantial differences in PNAH levels
between the two populations. The average total concentration in Y1M mussels
was 283.6 ligAg compared to 986.2 UgAg in mussels from Y2M. Individual
PNAH measurements also reflected these; differences. Paired t-tests were used
to determine if there were significant differences in individual PNAH and
total PNAH concentrations between the two sites for each sample collected on
the same day (10/2/78, 4/20/79, 5/21/80, 6/2/80 and 6/17/80 data for Y1M
were not included in any of the comparative tests). For all tests, cal-
culated t > t.o5,n ¦ 2.2? therefore, Ho* WiYlM - ViY2M was rejected; con-
centrations of each individual PNAH (i) and total PNAH were-obviously
21
-------
TABLE 8A.
PNAH CONCENTRATIONS (Vg/kg)' IN M. EDUtdS FROM YAQUINA BAY, OREGON;
SITE Y1M.
DATE
8AMPLED
PHEN
FLUOR
PVR
BCF
TRI
BAA
CHKY
BBF
10/2/78
207.8
90.9
20.9
52.0
46.4
15.2
l
1.6
1/24/79
135.3
62.1
24.6
28.9
30.8
49.6
4.0
2/13/79
146.8
64.8
45.6
38.2
37.0
48.9
5.1
3/21/79
84.8
40.3
23.6
18.7
20.2
32.3
2.5
4/20/79
178.4
66.8
17.2
48.2
40.9
14.0
1.5
5/31/79
100.8
46.4
28.8
23.9
24.5
39.2
3.2
7/2/79
165.4
59.8
17.0
46.4
42.1
7.0
0.2
7/30/79
45.9
10.7
25.7
24.9
22.9
7.7
-—
0.8
10/8/79
140.6
67.5
15.4
33.1
30.6
10.4
1.2
11/14/79
76.4
42.2
20.5
13.1
18.0
35.7
--—
2.9
1/14/80
103.2
46.9
28.2
22.6
23.0
37.5
—-
3.0
2/11/80
86.4
39.4
24.6
19.6
21.4
33.1
2.7
3/10/80
92.4
42.4
24.9
20.0
25.5
37.8
-—
3.0
4/23/80
100.4
42.5
27.0
21.3
27.3
40.2
2.6
5/21/80
89.5
40.4
25.5
21.0
24.3
34.4
2.6
6/2/80
87.5
39.7
25.1
20.0
20.5
33.8
2.6
6/17/80
88.4
39.1
25.4
20.5
21.1
32.6
2.3
X
113.4
49.5
24.7
27.8
28.0
30.0
2.5
(s.d.)
(42.0)
(17.6)
(6.7)
(11.6)
(8.6)
(13.7)
(1.2)
DATE
SAMPLED
BKF
DBACA
BAP
DBAHA
BGHIP
IP
COR
TOTAL
10/2/78
nd2
nd
1.9
1.1
0.3
0.0
0.8
438. 9
1/24/79
3.4
2.6
1.2
2.8
0.6
0.3
0.9
347.1
2/13/79
4.2
3.1
1.4
3.6
0.9
0.4
1.2
401.2
3/21/79
2.0
1.5
0.8
1.8
0.4
0.2
0.6
229.7
4/20/79
nd
nd
1.7
1.0
0.2
0.4
0.6
370.9
5/31/79
2.7
2.2
0.9
2.3
0.6
0.2
0.7
276.4
7/2/79
nd
nd
1.6
1.0
C.2
0.4
0.6
341.7
7/30/79
nd
nd
1.0
0.5
0.2
0. 3
0.4
141.0
10/8/79
nd
nd
1.2
0.8
0.3
0.4
0.6
302.1
11/14/79
nd
nd
2.7
3.2
0.4
0.5
0.9
216.5
1/14/80
2.9
2.1
0.9
2.1
0.5
0.2
0.7
273.8
2/11/80
2.1
1.5
1.1
2.5
0.5
0.2
0.5
235.6
3/10/80
2.0
1.6
1.3
2.7
0.4
0.3
0.7
255.0
4/23/80
2.2
1.6
1.6
2.9
0.4
0.4
0.6
271.0
5/21/80
2.2
1.4
1.2
2.4
0.4
0.3
0.5
246.0
6/2/80
2.0
1.4
1.2
2.4
0.4
0.3
0.5
237.4
6/17/80
2.1
1.3
1.1
2.2
0.4
0.2
0.5
237. 2
X
2.5
1.8
1.3
2.1
0.4
0.3
0.7
283.6
(s.d.)
(0.7)
(0.6)
(0.5)
(0.9)
(0.2)
(0.1)
(0.2)
(75.1)
1not identified
2below lines of detection
22
-------
TABLE 8B.
PNAH CONCENTRATIONS (Uq/kq) IN EDULIS PROM YAQUINA BAY, OREGON,
8ITB Y2M.
DATE
SAMPLED
1/24/79
2/13/79
3/21/79
5/31/79
7/2/79
7/30/79
10/8/79
11/14/79
1/14/80
2/11/80
3/10/80
4/23/80
X
(s.d.)
DATE
SAMPT.fti
1/24/79
2/13/79
3/21/79
5/13/79
7/2/79
7/30/79
10/8/79
11/14/79
1/14/80
2/11/80
3/10/80
4/23/80
X
(s.d.)
PHBN
FLUOR
PVR
BCP
284.3
242.2
142.3
107.3
256.6
213.7
131.9
105.2
237.5
198.7
122.4
104.3
205.4
175.3
111.2
94.6
202.2
162.1
99.4
91.2
186.4
131.3
81.2
76.9
173.3
127.5
75.7
68.5
191.3
146.4
92.3
81.3
206.4
183.2
105.4
96.4
231.3
193.2
117.6
100.4
222.5
199.4
120.6
105.3
198.4
185.3
111.1
97.5
216.3
179.9
109.3
94.1
(31.8)
(33.8)
(19.8)
(12.5)
TR1
BAA
CHRY
BBF
123.9
154.3
137.6
22.4
119.8
141.2
102.5
23.6
120.6
103.5
98.7
24.4
109.4
91.3
87.6
20.1
95.5
87.6
65.5
17.6
71.3
62.4
51.2
14.4
70.3
58.3
47.6
10.4
92.5
60.4
61.2
12.3
112.6
92.6
98.4
17.3
122.4
98.4
96.5
18.0
125.5
99.4
100.8
19.2
102.4
si: 5
87.4
16.3
105.5
94.2
86.2
18.0
(19.6)
(29.6)
(25.8)
(4.3)
BKF DBACA
22.5 14.3
21.6 14.2
20.4 13.9
18.0 10.3
14.3 9.8
9.6 7.4
8.5 6.3
9.5 8.3
20.2 12.3
21.7 13.5
22.5 13.7
19.8 10.3
17.4 11.2
(5.4) (2.8)
bap dbaha
31.5 12.4
29.5 10.5
33.1 11.5
24.4 9.4
20.4 7.6
18.7 5.3
17.0 4.9
24.5 6.0
28.5 11.5
3Q.8 11.9
31.6 12.6
24.6 11.3
26.2 9.6
(5.4) (2.9)
BGHIP IP
10.4 9.4
11.0 9.0
10.9 8.5
8.4 5.4
5.4 3.2
3.2 1.0
2.4 1.0
4.3 1.1
9.9 8.8
10.3 7.5
11.4 7.6
9.9 5.5
8.1 5.7
(3.3) (3.3)
COR TOTAL
8.9 1323.7
6.6 1196.9
7.6 1116.0
4.3 975.1
2.0 883.8
1.0 721.3
1.1 672.8
2.5 793.8
6.5 1010.0
5.8 1079.3
5.4 1097.5
3.2 964.5
4.6 986.2
(2.6) 193.8
23
-------
graatar in Y2M rfuasala. Thaaa findings agraa with raaulta of an aarlier
study (Mix and flohaffar, 1979) in whioh it vaa shown that concentrations of
BAP vara aignificantly greater in muaaala from Y2M during a two yaar pariod.
It ia alao ralavant that tha maan BAP concentrations did not diffar signifi-
cantly at aithat aita batwaan 1976-78 |Y1M - 1.9 ygAgi Y2M - 25.6 pg/kg) and
1978-80 (Y1M » 1.3 yg/fcg> Y2M ¦ 26.2 v£/kg) at indicated by a two-aample
t-test (calculated t F.os,i8t22i ¦ 1.69 and for Y2M,
F ** 38.0 > F. 05#n,i56 ™ 1.85. Therefore, the null hypotheses were rejected
at P < .01» there were differences in the percent PNAH concentrations that
were related to sampling date or season.
Figure 3A and 3B describe the seasonal fluctuations in PNAH percent con-
centrations. The means and standard deviations were calculated by summinq
the % contribution for each PNAH for a specific day of year ("season") and
dividing by the nunber of total PNAH (n ¦ 14 for YIM and 15 for Y2M; see
Tables 8a and 8B). There were no general seasonal trends observed in mussels
from Y1M. Only two values deviated measurably from the mean during the 20
month sampling period» mean PNAH concentrations were significantly higher
during February, 1979 and lower during'July-August, 1979. Mussels from Y2M
showed a clear pattern that is interpreted as being seasonal. Highest con-
centrations were present during the laie winter-early spring after which they
declined to low concentrations during the sinner and fall.
-------
Y1M
1978
1979
1980
400
200
o
o
2
O
o
16
Y2M
— 1979
—1980
X
<
2
a.
1200
2
<
Ui
2
800
o MEAN CONC. <%) t S.O.
¦ TOTAL CONC. (^fl/Kg)
400
I J I F I M I A | Ml J I J I A I SI 0 I N I 0 I
DAY OF YEAR
Figure 3. A. Seasonal differences in PNAH concentrations in M. edulia from
Y1M, 1978-80. B. Seasonal differences in PNAH concentrations in M. edulis
from Y2M, 1979-80. The left Y-axis indicates the mean which was derived by
converting each concentration value (right Y-axis) to a percentage of the
total PNAH for the entire period. Total PNAH concentrations are shown on
the right Y-axis and correspond precisely to the mean percent value. The
bars represent ± one standard deviation for total PNAH concentrations.
Excepting BAP, there are limited data available on PNAH concentrations
in M. edulia. Dunn and Young (1976) found BAP levels at or near 0 in M,
edulis and M. californianua collected from relatively pristine areas of
Southern California. Elevated levels, up to 108 yg/kg were typical of
mussels sampled on or near creosoted pilings. Dunn and Stich (1976a) re-
ported BAP concentrations in mussels from various Vancouver sites that
ranged from 2-8 and 10-35 ug/Xg depending on location. Some values were as
high as 215 vg/kg in mussels sampled from creosoted pilings. They observed
seasonal variations with a tendency towards lower levels during the summer.
Finally, Risebrough et al. (1980) measured concentrations of 3 PNAH in M.
edulia from San Francisco Bay. They reported levels of 2-6, 8-26 and 4-7
yg/g for phenanthrene, fluoranthene and BAP, respectively, in mussels from
metal and cement pilings. For mussels from wooden pilings, they found
concentrations of 1,000, 4,200 and 2,000 ug/kg for those 3 PNAH. The
latter concentrations are 1-3 orders o'f magnitude greater than those
23
-------
rtporttd for H. tdulia collected from wooden piling* in Yaquina Bay and
Vanoouver Harbor (Mix* 1979) Mix and Bchaffar, 1979) Dunn and Stich, 1976a),
Tha raaion for thaaa oonaidarabla difference! ie not yat olaar.
BAP Uptake and Elimination
Tha raaulta of the BAP uptake and alinination atudiaa ara aummariaed in
Table 9 and Figure 4. Tha control muaaali maintained atYlM showed no sig-
nificant changee in BAP concentration during tha experimental period (n - 50
days). Tha V2M mussels that initially contained 23 UgAg BAP reached back-
ground, Y1M, levels by approximately 15-20 daya. The rate of BAP release was
exponential and beat described by the following formula, using 4 parameters:
Y ¦ 23.7~°*l2X + 4.52 ~°*1>X (R2 - . 96j significance level - .005). By in-
spection of Figure 4, it can be determined that the BAP half-life, or time
required for depuration of 50% of the BAP initially present, was 8-10 days.
These results suggest that levels of available BAP in the immediate environ-
ment at Y1M remained low and stable during the experimental period.
B 23.7-0.12X + 4.52-0.13X
**• o.t«
X OBSERVED VALUES
— FITTED VALUES
X OBSERVED VALUES
— FITTED VALUES
KV 7V »w ,» '» JV
DAY NUMBER DAY NUMBER
Figure 4. Rates of benzo(a)pyrene uptake and elimination. A. Decrease in
BAP concentrations in contaminated musiels moved from Y2M and allowed to
depurate at Y1MJ B. Rate of BAP elimination determined by linear regression
analysis. C. Uptake of BAP by uncontfcninated Y1M muasela moved to Y2M.
D. Rate of BAP uptake determined by linear regression analysis.
26
-------
TABLE 9. BEN20(A)FYKENE UPTAKE AND ELIMINATION ZN W. EDVLIS MAINTAINED UNDER
AMBIENT (FZ3LD) CONDITIONS X$ YAQUINA BAY.
BAP CONCENTRATION (pgAg)
SAMPLE DATE
DAY NUMBER
Y1 at Yl1
Y2 at Yl*
Y2 at Y21
Yl at Y2S
10/16/78
1
1.0
23.0
23.0
1.0
10/19/78
4
*
20.4
14.7
0.2
10/24/78
8
0.9
14.8
19.7
2.9
10/27/78
11
0.8
4.0
11.1
1.4
10/31/78
15
1.5
2.0
8.5
2.9
11/7/78
22
0.9
1.5
17.6
8.0
11/14/78
29
1.3
* .
3.2
7.5
11/23/78
38
1.6
0.5
11.1
5.7
11/28/78
43
0.6
0.3
19.4
6.8
12/5/78
50
*
*
25.3
*
1control
2designed to measure BAP elimination
3dasigned to measure BAP uptake
*no measurement made
BAP uptake by Y1M mussels transferred to Y2M was linear where Y - 0.93
+ .164X (R ¦ .68} significance level ¦ .006).- As indicated by the data, BAP
levels in Y2M mussels maintained at that site seemed to fluctuate widely.
Those variations were not correlated with changes in salinity and/or tempera-
ture. There are at least two potential explanations for this pattern. First,
sample variance within the population may have been substantial enough to
account for the observed fluctuations, although this did not appear to be the
case in other samples. Second, since it is thought that BAP, and other lipo-
philic PNAH, are isolated in two comportments within the mussel (see subse-
quent discussion), the fluctuations may reflect real changes in the short-
lived compartment. That may occur if available BAP levels in the environment
changed rapidly during the experimental period. Such differences may have
been reflected by rapid changes in the short-lived compartment but not the
long-lived compartment which would be assuned to be saturated. The BAP uptake
pattern of Y1M niussels would also be consistent with this hypothesis? in those
mussels, the long-lived compartment was not yet saturated. Thus, since
tissues in those animals had not yet equilibrated with the levels of environ-
mental BAP, they would not be expected to reflect rapid changes in BAP levels
in the environment. It must be emphasized that this is all speculative, but
is compatible with existing theories of FNAH uptake and storage in bivalve
molluscs (Stegeman and Teal, 1973).
27
-------
Despite the\ variable pattern of BAP concentration in Y2M mussels, thara
imm<1 to be a general decrease during the first phase followed by a period
when BAP levels increased* Such a pattern is consistent with our earlier
finding that BAP concentrations increased during tha early winter in M, edulia
from Yaquina Bay (Nix end 6chaffer, 1979)*
It is interesting to note that BAP levels in both mussel populations
maintained at Y2M inoreaaed and/or fluctuated at a time of year when they
Were not engaged in gametogenesis. Thl|s nay indicate that increased BAP
and PNAH concentrations observed during the winter are related primarily to
increased levels in the environment and not necessarily to metabolic
processes associated with gametogenesiSj*
Our results on BAP uptake and elimination under ambient conditions are
isimilar to those obtained by others (Stegeman and Teal, 1973; Fossato, 1975;
Dunn and Stich, 1976b; Fossato and Cansonier, 1976). In common with all
those studies, tie observed a biphasic mode of release which was initially
rapid, then decreased quickly and was constant. The VAP half-life in the
present study, in which the ambient temperature ranged from 15*C on day 1 to
9*C at tetmiftatibri, was 8-10 days. This compares with 16 days in M. edulis
maintained at 7-9#C (Dunn and Stich, 1976b) and 18 days for C. Vrrginica.
maintained at 12*C. The differences may be attributed to lower water
temperatures in the latter studies, although it has been reported that
depuration in M, edulis are independent of temperatures between 7-26*C
'(Fossato, 1975).
It has been hypothesized previoulsjy that biphasic depuration is
indicative of PNAH storage in two compartments within shellfish (Stegeman and
Teal, 1973). One of the compartments, (perhaps fluid and/or hemolymph, is
characterized by! the rapid turnover of PNAH while the other, more stable,
compartment ia characterised by low turnover; the latter compartment
evidently contains pentane-extractable lipids. The exact location of either
compartment has not yet been established by definitive experiments. However,
results of other, studies seem to suggest that, the digestive gland
(hepatopancreas). is a primary candidate; (Lee et al,, 1972; Dunn and Stich,
1976b; Couch et al., 1979). The underlying CT and gonad may also serve as
storage sites since it has been demonstrated that C -BAP (Couch et al.,
1979) is possibly transported from digelstive tubules to those tissues in C.
virginica Perhaps spawning would bo expected to account for sudden decreases
in PNAH concentrations during the spring if the gonad is a major sink in
shellfish. The next study attempted to determine if this occurred in M. edulie.
Tissue Storage Sites for BAP
The purpose of this study were to measure the concentration of BAP in
somatic and gonadal cissues of M. edulik and determine if changes in levels
in these tissues' could account for sessional differences in BAP concentra-
tions; the high January-February concentrations were of special interest.
Mussels from V2M were used because higher BAP levels insured that this PNAH
bould be quantified in small amounts of tissue. Table 10 contains the
quantitative data from the 6 month study. Figure 5 illustrates the seasonal
Variation, in percent, in weights of sortie an& gonadal tisAUfi find the.
26
-------
mount of BAP associated with eaoh of thai
»
100
75
% WT. SOMATIC
% BAP IN SOMATIC
Z
LlI
u 50
QC
UJ
0.
% BAP IN GONAO
•/. WT. OF GONAD
50
75
100
125
150
I JAN I FEB I MAR I APR I MAY I
DAY OF YEAR
Figure 5. Benzo(a)pyrene concentrations in gonadal and somatic tissues. BAP
concentrations in the 2 tissue groups are expressed as a percentage of total
PNAH in whole animals. The weights of gonad and somatic fractions are also
expressed as a percentage of total weight.
The results of this study suggest that gamstogenesis and/or incorpora-
tion of BAP and presumably other lipophilic PNAH, were not directly respon-
sible for the seasonal increases in BAP during January-February, 1979. There
were no measurable increases in either gonad weight or the % BAP contributed
by the gonad (Fig. 5). It is evident that BAP storage occurred primarily in
the somatic tissues compared to the gonad, even during the spring spawning
season. This is in agreement with Lee et al. (1972) and DiSalvo et al.
(1975) who reported that somatic tissues, especially the hepatopancreas,
contained higher concentrations of aromatic hydrocarbons than the gonad.
Examination of Figure 5 reveals that during later April-early May, there was
a slight increase in the weight of the gonad and a single period, May 3, when
a more substantial amount of BAP was contained within the gonad than at any
other time during the study. This peak was followed by a sharp decrease in
% BAP in the gonad. Corresponding inverse changes occurred in the somatic
29
-------
¦CABLE 10.
BENZO (A) PYRENE CONCENTRATIONS IN THE GONAD AND SOMATIC TISSUES OF M. EDULIS
FROM YAQOINA BAY, OREGON.
GONAD SOMATIC WHOLE MUSSEL (GONAD + SOMATIC)
(A)
(B)
(C)
(D)
(E)
DATE
WEIGHT
BAP CONC.
WEIGHT
BAP CONC.
BAP CONC.
tWEIGHT
% BAP
% WEIGHT
» BAP
SAMPLED1
(GRAMS)
(UQ/kq)
(GRAMS)
(uq/kq)
(U
-------
tissues during tha aim* time. Zt haa been auggaatad that spawning may
oonatituta a potential ralaaaa machanito for avomatio hydrocarbona oontainad
within the ova of M, edulia (DiSalvo at al.f 1975). Perhaps tha April-May
ohangaa may have bean aaaooiatad with ipawning of tha Y2M muaael population
although thia prooaaa normally oooura taring February-April in Yaquina M.
edulie, If ao» spawning, and tha oonoomitant loaa of PNAH in gamataa« may
account for the lowered lata apring early aonmar levela. Additional field
studiM will be naeeaaary to mora fulljrivfluata tha ralationahip batwaan
seasonal concentrations of PNAH and fluxaa involving somatic and gonadal
tissues.
BAP Metabolism in M. edulie
The finding that BAP is concentrated, at least intially, in the diges-
tive tissues may be significant since microsomal fractions from those tissues
in C. virgirtioa and perhaps M, edulia are capable of metabolizing BAP
(Anderson, 1978). The enzymes responsible for this conversion and the meta-
bolites produced have not yet been identified completely. A related point is
that bap depuration in bivalve molluscs may not be an entirely passive pro-
cess. Metabolic alteration, conjugation and-subsequent excretion may play a
minor or major role in the release of these compounds from shellfish.
Only preliminary studies on BAP metabolism in M. edulia have been con-
ducted during this investigation) the first results must be considered tenta-
tive. Briefly, it was found that 7,l6-1*C-BAP was metabolized by microsomal
extracts from the visceral mass of M, edulie, from both Y1M and Y2M. Phenolic
metabolites, identified by HPLC methods as 3-hydroxybenzo(a)pyrene (3-OH) and
9-hydroxybenzo(a)pyrene (9-OH), were the only measurable BAP metabolites
present. The rates of formation were low, ranging from 27.3-76.7 x 10~12
pmoles BAP/min/mg microsomes.
These preliminary results are important since this species has been re-
ported to be incapable of metabolizing PNAH (Lee et al., 1972; Vandermeulen
and Penrose, 1978; Payne and May, 1979). Mora complete studies utilizing
advanced methods will be required to fully evaluate the metabolic capa-
bilities of M. edulia for altering BAP and other PNAH.
BASELINE DATA ON PNAH CONCENTRATIONS IN OTHER BIVALVE MOLLUSCS
PNAH concentrations were measured;in oysters (C. gigoa) from Yaquina Bay
(Table 11) to provide some comparison With PNAH levels in M. edulia and be-
cause they are utilized as a food source. PNAH concentrations were also
measured in M. edulia j M. avenaria and C. gigas from Tillamook Bay, Oregon,
to compare levels in indigenous shellfish from a relatively pristine bay
with those inhabiting more developed baya (Coos, Yaquina)(Table 12). It is
clear that Pacific oysters from Yaquina Bay contained much lower concentra-
tions of PNAH than muaaela from tha same bay. Tha oyatara were sampled at
a site approximately 7-8 lam upbay from M. edulie at a site far removed from
the downbay mussel sites where there ia a considerable amount of industrial-
ization and nunerous potential point sources (a.g. marinas, fish processing,
plants, condominiums). The simplest explanation for the differences is that
-------
TABLE 11»
PNAH CONCENTRATIONS (Vfl/kgl XH C, OIGAS FROM YAQUINA BAY, OREGON;
8ZTB T140.
DATE
SAMPLED
12/16/76
2/17/77
8/29/77
12/9/77
4/28/78
1/24/79
3/21/79
4/20/79
5/31/79
6/28/79
7/30/79
11/14/79
l/14/r.o
3/10. 3J
X
(s.d.)
DATE
SAMPLED
12/16/76
2/17/77
8/29/77
12/9/77
4/28/78
1/24/79
3/21/79
4/20/79
5/31/79
6/28/79
7/30/79
11/14/79
1/14/80
3/10/80
X
(s.d.)
PHEN
6.4
6.7
5.4
4.9
7.3
5.4
5.9
6.1
7.1
7.3
7.1
5.3
6.2
6.4
6.2
(0.8)
BKF
1.6
1.7
1.4
1.1
1.2
0.9
1.0
1.2
1.2
1.3
1.1
0.8
•1.1
1.2
1.2
(0.2)
FLUOR
7.6
8.0
6.8
5.6
6.5
4.3
4.8
5.3
6.3
6.6
6.2
4.4
4.5
4.7
5.8
(1.2)
DBACA
1.2
1.4
1.3
1.1
1.4
0.9
1.0
1.2
1.3
1.4
1.2
0.6
1.0
1.3
1.2
(0.2)
PYR
4.4
4.4
3.2
3.0
3.6
3.0
3.1
4.0
4.5
4.8
4.6
3.0
3.2
3.5
3.7
(0.7)
BAP
1.6
1.7
1.5
1.0
1.3
1.1
1.2
1.3
1.5
1.6
1.4
1.0
1.3
1.4
1.4
(0.2)
Mcp
4.3
5.8
4.7
4.4
4.9
3.5
3.6
4.1
4.3
4.2
4.3
3.4
3.6
3.7
4.3
(0.7)
DBAHA
0.5
0.6
0.4
0.3
0.4
0.3
0.3
0.4
0.5
0.4
0.5
0.3
0.4
0.5
0.4
(0.1)
TRI
2.0
2.4
2.0
2.0
3.1
2.0
2.2
2.8
2.9
3.0
3.0.
2.1
2.4
2.8
2.5
(0.4)
BGHIP
0.3
0.4
0.3
0.3
0.3
0.3
0.3
0.4
0.3
0.3
0.3
0.3
0.4
0.4
0.3
(0.0)
BAA
4.3
4.6
4.3
4.2
5.1
5.1
5.6
5.9
5.9
5.8
5.6
5.0
5.4
5.7
5.2
(0.6)
IP_
0.3
0.3
0.3
0.2
0.3
0.2
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
(0.0)
CHRY
3.2
4.0
3.9
3.0
4.1
3.8
4.0
4.2
4. 3
4.4
3.2
3.6
3.8
4.0
3.8
(0.4)
COR
0.4
0,4
0.4
0.3
0.2
0.2
0.3
0.4
0.4
0.4
0.4
0.3
0.5
0.5
0.4
(0.1)
BBF
1.5
1.3
1.1
1.0
0.9
0.8
1.0
1.2
1.3
1.4
1.2
1.0
1.2
1.2
1.2
(0.2)
TOTAL
40.6
43.7
37.0
32.4
40.8
31.8
34.6
38.8
42.1
43.2
40.4
31.4
35.3
37.6
37.8
(4.2)
32
-------
TABLE 12. PNAH CONCENTRATIONS (Ug/Hg) IN BIVALVE MOLLUSKS FROM TILLAMOOK BAY,
OREGON.
DATE
SAMPLED
SPECIES
PHEN
FLUOR
PYR
BCP
TRI
BAA
CHRY
BBF
1/24/79
M.
edulia
15.5
10.4
0.8
5.3
3.5
2.6
4.4
1.4
4/9/79
M.
edulia
12.4
8.5
6.0
4.3
3.0
2.4
3.1
0.9
1/24/79
M.
arenaria
17.4
15.1
12.1
3.2
5.4
2.0
1.4
1.0
4/19/79
M.
armaria
9.3
5.0
3.0
2.0
4.2
1.9
1.0
0.8
6/14/79
M.
arenaria
9.0
5.3
4.1
2.3
4.6
1.9
1.1
1.0
8/23/79
M.
arenaria
10.0
6.3
3.0
2.9
4.7
1.8
1.5
1.1
1/24/79
C.
gigaa
3.1
6.5
3.3
4.8
4.6
2.4
3.8
1.3
4/19/79
C.
gigaa
10.9
7.1
3.6
5.1
4.2
2.6
3.8
1.0
6/14/79
c.
gigaa
10.1
5.4
4.1
3.6
4.9
2.2
3.7
1.1
DATE
SAMPLED
SPECIES
BKF
DBACA
BAP
DBAHA
BGHIP
IP
COR
TOTAL
1/24/79
M.
edulia
1.0
0.6
0.4
0.3
0.2
0.2
0.2
54.8
4/9/79
M.
edulia
0.6
0.4
0.2
0.2
0.1
0.1
0.1
42.3
1/24/79
M.
arenaria
0. 3
1.4
0.5
0.7
0.9
0.0
0.1
61.5
4/19/79
M.
arenaria
0.0
1.7
0.4
0.6
0.8
0.0
0.0
30.7
6/14/79
M.
arenaria
0.0
1.1
0.6
0.8
0.9
0.1
0.0
32.8
8/23/79
M.
arenaria
0.1
0.9
0.5
0.7
1.0
0.1
0.0
34.6
1/24/79
C.
gigaa
1.6
1.0
1.0
1.0
1.1
0.9
0.6
37.0
4/19/79
C.
gigaa
1.6
0.8
1.1
0.9
1.1
0.9
0.5
45.2
6/14/79
C.
gigaa
1.3
1.6
1.1
0.6
1.1
0.3
0.6
41.7
33
-------
yvi'iM
vatar at tha doifnbay musaal aitaa vta ihora contaminated than tha water at the
mora pristina araa where oyatara ara giown in Yaquina Bay. It ahould ba
notad that oyatara ara grown in traya iuapandad from oraoaotad boarda
attaohad to oraoaotad piling*. Diffartncea in feeding maohaniama would not
appaar to ba a faotor ainoa musaala* oyatara and aoftahell olama ara all
filtar feedera.
Thara vara no apparent aaaaonal difference* in PNAH concentration* in
syaters front Yaquina Bay auoh aa occurred in M, tdulie. That may be due to
Jiffarancaa in matabolio capabilitiea,;aa&ional availability of PNAH at tha
diffarant aitaa or reproductive patterns. C, gigaa mature 8axually but do
not apawn in Yaquina Bay and, thus, thara would be no release of PNAH asso-
ciated with gametea during spawning that would result in summer and fall de-
creases. That nay or may not occur in M. adulia. If oysters function meta-
bolically in a way similar to M. adulia during the winter, these data would
suggest that runoff from the upper watershed may not contribute in a major
way to PNAH burdens in oysters and mussels or account for seasonal differences
in the latter.
The levels of PNAH in C. gigaa rrom Yaquina Bay wera generally compar-
able to those reported for C. virffinioa from Galveaton Bay for a smaller
nunber individual PNAH (Fazio, 1971). ;No BAP was detected in oysters in that
study. Cahnmann and Kuratsuna (1957) reported considerably higher levels,
700-1300 ugAg total PNAH, in C. virginioa from a polluted harbor area in the
vicinity of Norfolk, Virginia. Murray1et al. (1980) detected only 0.07-0.14
ppb PNAH in C. virffinioa from Galveston Bay while Bravo et al. (1978) re-
ported that C. virginiaa from 10 stations along the Mexican coast had total
mean PNAH concentrations of 2080-9160 Wg/kg. The latter values included both
unsubstituted and substituted PNAH but the levels have been, questioned as
being nearly 2 or 3 orders of magnitude too high for shellfish from a rela-
tively pristine area (Neff, 1979). Since at least some of the samples were
collected near an oil producing area, some of the oysters may have been con-
taminated by wastes which reached the lagoons (Bravo et al., 1978).
The data in Tables 11 and 12, while not as complete as that from similar
studies on M. edulie from Yaquina Bay (Tables 8A and 8B) and M. arenaria from
Coos Bay (Tables 6 and 7), provide soma statistical basis for evaluating
differences between species and sitea and formulating tentative conclusions
about PNAH concentrations in bivalve molluscs. One-way analyses of variance
or t-tests were used to compare differences between populations. The limited
nunber of samples precludes precis* interpretation; the results are con-
sidered indicative but not definitive. Statistical analyses revealed the
following relationships:
1. Tha mean concentration of total PNAH in C. gigaa from Yaquina Bay
did not differ during tha 5 separate yeara, 1976-1980 (F **0.2 < F. "
14,5), i.e. tha mean did not differ significantly for any single year.
2. The mean concentration of total PNAH in C. gigaa from Yaquina Bay
differed significantly for M. edulia from Y1M (t ¦ 12.2 > t,ox,29 " 2.7) and
Y2M (t ¦ 18.4 >,t.oi,2* " 2.8) for all sampling periods.
-------
3* TM aaan concentration for total PNAH did not differ signifioantly
in C. gigm from Yaquina Bay and TlllaAook lay (t ¦ 1.3 < t,oi,n ¦ 2.95) for
•11 amplas.
4. The aaan oonoantrationa of total PNAH did not dlffae aignifioantly
In *duli8t tf. avncarta or C, gigas tram Tillamook Bay (*"0.4 t.oi,« ¦ 3.36;
for CSS, t ¦ 17.66 > t(oi#• ¦ 3.36).
The lowest PNAH concentrations measured in this research were recorded
in mussels, clams and oysters from Tillamook Bay, the most undeveloped of the
three Oregon bays. These data support tha view that PNAH concentrations in
shellfish monitors will reflect the degree of industrialisation, human on-
shore habitation and/or relative pollution levels (Goldberg, 1975j Dunn and
Stich, 1975; Mix et al., 1977; Risebrough et al., 1980). PNAH concentrations
in the three bivalve species from Tillamook Bay did not differ even though
they came from sites separated by several kilometers, and ranged from upbay'
to downbay locations. One interpretation of this finding is that all filter-
feeding bivalve molluscs will contain baseline concentrations of PNAH in well-
mixed estuaries that can be considered as background. Deviations above these
levels may then be indicative of the relative degree of pollution. Table 13
categorizes the PMAH concentrations measured in shellfish monitors during
this study with observations about tha degree of contamination at the various
sample sites.
TABLE 13. RANGE OP PNAH CONCENTRATIONS IH SHELLFISH MONITORS FROM VARIOUS
SITES IN THREE OREGON BAYS.
PNAH
BAY
SITE
SPECIES
1
!•
1
DEGREE OF INDUSTRIALIZATION
Tillamook
TIM
M.
edulie
40-60
relatively pristine
Tillamook
TSS
M.
arenaria
30-60
relatively pristine
Tillamook
TBC
C.
gigaB
35-45
relatively pristine
Yaquina
Y140
C.
gigas
30-45
relatively pristine
Coos
C3S
tf.
arencaria
70-90
relatively pristine; near
highway
Coos
C11G
T.
oapax
30-110
light; nearby marinas, fish
proceasing plants
Coos
CSS
AT.
arenaria
460-650
heavy; shipping docks, wood
products industry, marinas
Yaquina
Y1M
M.
adults
140-440
light; shipping docks
Yaquina
Y2M
M.
edulia
675-1325
heavy; marinas, fish pro-
cessing plants, recrea-
tional developments
The values in Table 13 indicate tnat ahellfish in relatively pristine
areas of tha three bays have a baseline PNAH load of approximately 50 ygAg.
Increased concentrations occurred in direct relation to the degree of indus<
35
-------
trialiiation and huaan onahora activity,
It would be beneficial to hava data ralating PNAH maaauramanta in ahell-
fiah monitors with ambient water concentrations. However, aoata and statisti-
cal eonaidarationa praoluda obtaining thia aort of information on a routina
baaia. Perhaps futura afforta can ba diraetad toward! ralating PNAH concen-
trations in watar and ahellfish monitors# solubilities, octanol-watar parti-
tion coaffieiants and bioacoumulation faotori, Ultimataly, auoh information
could parhapa ba inoorporatad into pradlotiva modals that oould ba uaad for
pradiotiona about tha anvironmantal behavior of PNAH (e.g. raviaw by Kenaga
and Goring> 1980).
CELLULAR PROLIFERATIVE DISORDERS IN BIVALVE MOLLUSCS FROM OREGON BAYS
Mussels from Y1M and Y2M were examined histologically for the presence
of abnormal cells during the fall and Winter of 1979-80 and M. arenama
during a four quarter period during 1978-79 from Coos Bay (Table 14). In
addition, 50 mussels were examined during February and May, 1980 from
Tillamook Bay.
TABLE 14. THE PREVALENCE OF CELLULAR PROLIFERATIVE DISORDERS IN M. EDULTS
FROM YAQUINA BAY AND M. ARENAFIA FROM COOS BAY, OREGON.
YAQUINA BAY COOS BAY
DATE
Y1M(%)
Y2M(%)
DATE
CSS(%)
C3S(%)
9/10/79
0/47
(0.0)
2/47 (4.3)
9/30/78
0/48 (0.0)
0/46 (0.0)
10/8/79
0/43
(0.0)
5/46(10.9)
12/1/78
0/46 (0.0)
0/42 (0.0)
11/14/79
1/49
(2.0)
4/44 (9.1)
2/19/79
0/47/(0.0)
0/47 (0.0)
12/17/79
1/54
(1.8)
4/18/79-
0/46 (0.0)
0/41 (0.0)
1A 4/80
0/49
(0.0)
5/45(11.1)
2/11/80
—
—
6/49(12.2)
TOTAL
0/187(0.0)
0/176(0.0)
3/10/80
0/50
(0.0)
——
TOTAL
2/292
(0.7)
22/231(9.5)
No clams from Coos Bay or mussels from Tillamook Bay were found with the
large, abnormal calls that characterize one type of cellular proliferative
disordara in ahallfiah from Oregon and alaawhara (Mix et al., 1979a). The
condition has appeared in a significant nunber of muasals from Y2M during a
4-year period from 1976-1980 while it occurred rarely in mussels from Y1M
(Mix, 1979» Mix at al., 1979b). The correlation between tha degree of PNAH
contamination and tha pravalance of tha condition at Y2M la obvious but no
cause-effect relationship has bean establiahad.
I
Although there have been nmerous reporta of apparent eorrelationa be-
tween the appearance of abnormal cells in shellfish inhabiting oil-contami-
nated environments, there have been no published reports of cancer induction
in.bivalva molluscs by axpoaura to, or injection with, PNAH (Couch at al. , 1979).
Significant questions about the affects of PNAH on bivalve molluscs and fehe
36
-------
fcttabolio capabilitiaa thaaa apaoiaa h*va for altering PNAH romain unanswered.
Thara have boon nunaroua raporta that feivalvai cannot mataboliza PNAH, yat
tha avidanoa prasantad ia by no maani dafinltiva (Stagaman and Teal, 197 3)
and racant raaulta indicata that at laiat ioma apaciaa can metabolize dap
(Andaraon, 1978). It ramaina to ba daiarminad if carcinoganta metabolites
can ba formed by thaaa spaoias. "If bivalvai ara not aubjoct to PNNi-inducad
carcinogenaaia, and tha cellular abnotwalitiaa ara raiatad to a neoplastic
procaaa, than othfir cauaativa <*glnta mtiaetfci reiponaibla.
Figure 6 shows the prevalence-Beaion relationship of these conditions in
M. edutie from Y2M over a 5-year period/ 1976-1981) the prevalenco data waa
from Mix (1979) and the present report! In addition, one sample waa taken in
1981f the prevalence waa 20.0% on 1/16/81 (50 of 250 mussels wero positive).
For the 5 year period, 192/1911 mussels were diagnosed histologically and
found positive for the condition} the average prevalences was 10.05%. Exami-
nation of Figure 6 reveals the seasonal occurrence of the disorders. There
was a consistent pattern characterized;by highest prevalences during the
later winter followed by a period of decline to lowest prevalences during the
suruner and fall after which there waa i subsequent increase. The data were
;subjected to curve-fitting analyses and it was determined that the following
'quadratic equation is quite good (R2 -!.71) for describing the seasonal-
}prevalence relationship (£ = prevalence; X =» day of year) :
1
Y = 18.70 - 0.14X + 0.0001X2
25
Y « 18.70-O.I4XfO.OOOIX"
20
o
5 15
30
60
130
90
120
180 210
DAY OF YEAR
240
270
J00
5 JO
Figure 6. Prevalence of cellular proliferative disorders in M. cdulio. The
individual prevalence points represent jvalues determined by histological
examinations of mussels collected at various timeB from 1976-1901. The
fitted line wa3 determined by regression analyr "s.
The significance of the prevalence pattern Is not known. It has been
observed that the seasonal occurrence of the di 'era correlator, with the
concentrations of PNAH (Mix et al., 1979bj this report) and the suggestion
has been made that the appearance of atypical cells may constitute some sort
of cellular response to toxic substances in the environment (Mix et al.,
1979a,b). The importance of determining the metabolic capabilities, if any.
37.
-------
of W. •dull* in modifying PNAH will ba nacawary to further clarify ¦•agonal
relationships. The raoant disoovery that a virus may ba a cauaal agant re-
sponsible for similar condition! in ta^natia from tha aaat coast (Dr. P.
Chang. Univ. Rhode Island, personal oowmunioation) dictates that future
raaaaroh afforta ba diraotad towards detraining whether or not viruses ara
associated with thaaa conditions in U% tdulitt
RELATIONS HI PS INVOLVING PNAH IN SHELLFISH MONITORS
Tha data on PNAH concentrations in bivalva molluaca reported here are
more detailed them those from any other similar study. There have been
several reports of PNAH in tissues of aquatic organisms, but most available
data are restricted to BAP concentrations (see review by Neff, 1979). Only
a limited amount of information is available on the concentrations of other
unsubstituted PNAH. Also, the data included in some of those reports are
compromised by limitations related to the analytical methods used and/or
small sample sizes.
It has been commonly reported, without much justification, that quanti-
tative and qualitative measurements of BAP can be used as an index of con-
tamination for other PNAH. Information relative to the predictive use of
such a BAP index has been generally absent or predictive models have lacked
the precision necessary for use in environmental studies. Dunn (1980) found
that BAP concentrations in his samples were correlated with the levels of
PNAH containing 3 or more rings in Fuous. Ha concluded that BAP levels could
be used as em index of the contamination of marine samples by carcinogenic
PNAH. No details about the index nature or quantitative specifications
were presented. Others have cautioned that BAP may serve as only a very
rough index of PNAH contamination (Baum, 1978) and that, while it is im-
portant and one of the most ubiquitous PNAH carcinogens, it generally con-
stitutes only between 1-20% of the total carcinogenic PNAH (Suess, 1976).
Nevertheless, BAP has been used by the EPA and other3 as an indicator or
marker compound for total PNAH content (refer to 2 citations in Brown et al.,
1980; "Atmospheric Polycyclic Organic Matter (POM) s Sources and Population
Exposure." Draft EPA Report, by Energy and Environmental Analyses, Inc.,
Arlington, VA> and, "Preferred Standards Path Report by Polycyclic Organic
Matter," U. S. EPA, Durham, NC, Oct. 1974).
Data on PNAH concentrations in M. edulia from Y1M, Y2M and Y140 were
used in two statistical approaches to identify significant relationships be-
tween individual PNAH and total PNAH concentrations. Multiple regression and
multiple correlation techniques were used in considerations of interrelation-
ships between variables (individual PNAH). In these calculations, Y, the
dependent variable, was considered to be the total PNAH concentration for
one sample period and the were independent variables. The general formula
for multiple regression is
Y • a + biXii + baXii***** bnXni,
implying that one variable, Y (total PNAH), is linearly dependent upon mul-
tiple variables, Xi, X2, etc. (individual PNAH). bi, bz, etc., are partial
38
-------
regreaaion coefficients! bi expresses how muoh V would change for a unit
change in Xi if Xt, Xi, etc.» wera held oonatant. Tha Y intercept, a, is
tha valua of Y whan all X values ara itro. Analyses of varianca and t-tests
wara oaloulatad during aaoh analyiia ih ordar to datarmina tha Xi'a that con-
tributed significantly to RJ, tha coefficient of determination uaad to indi-
eata tha proportion of total variabiliiy in Y attributable to tha dapendanca
of Y on all Xi (Zar, 1974). Tabla 15 iixunarisea tha results of the statisti-
cal analyses for PNAH data from aaoh site.
TABLE 15. MULTIPLE REGRESSION AND CORRELATION ANALYSES.
SITE
R2
VARIABLE
(PNAH)1'2
REGRESSION
COEFFICIENT(b)
t-test
of b
Y1M
.99
BAA
1.19
33.1
PHEN
1.05
26.3
PYR
1.07
20.5
FLUOR
0.93
18.4
TRI
1.09
14.0
BCP
0.87
8.2
BGHIP
9.00
3.6
Y2M
.99
FLUOR
3.67
8.8
DBACA
15.42
4.2
Y140
.99
PYR
3.24
7.2
DBACA
8.32
6.6
IP
23.16
3.4
1 only those variables that were significant in determining the multiple
correlation coefficient (R2) are included.
2 the listed order of variables indicates the relative contribution to R2
for each site; the first listed PNAH contributed most, the last listed,
least.
Examination of Table IS indicates that for two sites, Y2M and Y140, only
two or three PNAH, or independent variables, were necessary to complete the
regression formula. For Y2M, the formula is
TOTAL PNAH (Y) - 986.23 (a) + 3.67 (FLUOR) + 15.42 (DBACA)
where Xi and Xj are the quantities of fluoranthene and dibenz(a,h)anthracene,
respectively. Quantities of seven PNAH yield partial regression coefficients
that contributed significantly to the multiple correlation coefficient for
Y1M«
The primary purpose of these analyses was not to generate multiple re-
gression formulas to be used for predictive purposes, although that was done.
Such an approach may or may not be particularly useful for making general
predictive statements about quantitative relationships for environmental PNAH.
Further research will be necessary to determine the accuracy of such formulas^
39
-------
For the preaant atudiM, tha fomulai Wart llmitad to describing tha rela-
tionships of individual PNAH and total PNAH for a particular Bite during n
definite period of intermittent aampling. Cartain conclusions, tuimarizsd
below, can b« formad aftar examining tha raaults of thasa analyses.
1. For aaoh site, different indapandant variables (individual pnah)
were used to predict Y (total PNAH).
2. No aingle PNAH was a significant variable for predicting total PNAH
at all three sites.
3. BAP 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 contamina-
tion is not supported by the results of this study.
4. In monitoring programs* it nay be possible to identify the signifi-
cant variables (PNAH) after a suitable period of sampling and to subsequently
measure only those variables for an adequate assessment of total PNAH. Com-
plete analyses could be made periodically to confirm the continuing validity
of the established regression. Such an approach may result in considerable
cost reduction for long-term monitoring programs.
To further evaluate the interrelationships between PNAH, additional
statistical analyses were conducted. Multiple comparison tests were used to
compare pairs of samples. Student-Newman-Keuls tests were used for Y1M re-
sults and Friedman's test for Y2M results. The latter test was used because
Levene's statistic showed that the variances were not homogenous at P < 0.01
for Y2M. The results of these analyseb are summarized in Table 16.
There are two tentative conclusions that are supported by the informa-
tion in Tible 16. In general, the concentrations of individual 4-ring PNAH
did not differ from each other; fluoranthene was a major exception since it
was concentrated to higher levels in Yaquina Bay mussels. Except for BAP and
coronene in Y2M mussels, there were no significant differences in PNAH con-
centrations between all 5-, 6- and 7-ring PNAH. Thus, while it was estab-
lished that quantitative predictions about total PNAH cannot be made on the
basis of individual PNAH measurements, the results from Table 16 indicate
that general qualitative relationships existed in the present study. For
example, detection of a certain quantity of PYR suggested that a similar
quantity of BCP, TRI and BAA were present. Meaourement of an individual
PNAH concentration for any 5-, 6- or 7-ring PNAH indicated that approximately
the same concentration would be found for any other unsubstituted PNAH with
5-7 rings. It may be productive to conduct these kinds of analyses for PNAH
data collected from other established biological monitoring programs. Con-
firmation of the relationships identified during the present study may even-
tually lead to a simplified monitoring approach. Eventually, analysis of
only a small mxnber of PNAH may be necessary to produce acceptable results
suitable for predictive purposes.
In these studies where PNAH measurements .have been made in bivalve
molluscs, it was evident that those with lower molecular weights (MW) were
generally present in greater concentration than those with higher MW (Table
17). Such a pattern has also been evident in most other studies (Cahnmann
and Kuratsune, 1957; Fario, 1971; Neff et al., 1976; Pancirov and Brown,
1977; Joe et al., 1979). However, onegroup (Risebrough et al., 1980) re-
40
-------
TABLE 16. AN EVALUATION OF QUANTITATIVE RELATIONSHIPS BETWEEN INDIVIDUAL PNAH IN TISSUES
OF M. EDUHS FROM YAQUINA BAY, OREGON.
—
; 3
PHEN
H
FLUOR
H
PYR
—*
BCP
H
TRI
•»
BAA
5
BBF
" 5-
BKF
S "
DBACA
BAP
DBAHA
(
BGHIP
»
IP
7
COR
?HEN
X
*
*
*
*
*+
*+
*+
*+
*+
*+
*+
*+
TLOOR
*
X
•
?YR
*
*
X
*
*
*+
*
*+
*+
*+
*+
3CP
*
' *
X
*
*
*
*
*+
*+
*+
*+
PRI
*
*
X
*
*
*+
*
*+
*+
*+
*+
3AA
*
*
X
*
*
*
*
*
*+
*+
*+
3BF
*+
*+
*
*
*
*
X
3KF
*+
*+
*
*
*
*
X
DBACA
*+
*+
*+
*
*+
*
X
SAP
*+
*
*
# *
*
*
X
+
)BAHA
*+
*+
*+
*+
*+
*
X
3GHIP
*+
*+
*+
*+
*+
X
EP
*+
* +
*+
*+
*+
X
!X>R |
*+
*+
*+
*+ 1 *+
*+
+
X
•indicates that means are not equal at site
^indicates that means are not equal at site
3~'indicate the number of rings in the PNAH.
Y1M (e.g. reject V AT P<.01).
Y2M.
-------
TABLE 17.
THE MEAN PERCENTAGE INDIVIDUAL PNAH CONTRIBUTED TO THE TOTAL PNAH
FOR ALL SAMPLING DATES (i 8.D.).
BAY
SITE
SPECIES
n
PKEN
FLUOR
PYR
BCP
TRI
YAQUINA
Y1M
1
17
39.115.„
17.013.0
9.313.3
9.712.8
10.011.9
YAQUINA
Y2M
2
12
22.2±2.0
18.310.4
11.110.3
9.710.7
10.710.7
YAQUINA
Y1Y0
3
14
16.511.0
15.312.1
9.810.9
11.311.4
6.510.8
COOS
CSS
A
6
28.212.0
20.313.3
11.012.2
10.111.3
7.910.3
COOS
C3S
5
5
16.6±3.3
13.411.5
8.311.1
4.210.8
7.710.7
COOS
C11G
6
10
25.614.3
17,611.6
8.313.0
8.411.7
7.511.5
TILLAMOOK
TIM
7
2
28.810.7
19.610.8
15.211.3
10.010.4
6.810.5
TILLAMOOK
TSS
8
4
28.711.2
18.814.0
12.715.0
6.811.3
12.612.4
TILLAMOOK
TBC
9
3
18.919.1
15.412.3
8.910.9
11.012.2
11.211.6
BAY
SITE
SPECIES
n
BAA
CHRY
BBF
BKF
DBACA
YAQUINA
Y1M
1
17
11.115.1
0.910.4
0.910.1
0.710.1
YAQUINA
Y2M
2
12
9.411.2
8.611.1
1.810.2
1.710.3
1.110.1
YAQUINA
Y1Y0
3
14
13.811.9
10.211.2
3.010.4
3.210.4
3.010.4
COOS
CSS
4
6
7.413.3
4.910.6
2.210.3
1.810.2
1.610.3
COOS
C3S
5
5
3.610.2
10.910.5
1.710.4
4.010.4
2.710.6
COOS
CllG
6
10
12.813.4
7.412.0
3.211.1
2.011.0
1.810.7
TILLAhOOK
TIM
7
2
5.210.7
7.610.5
2.410.4
1.610.3
1.010.1
TILLAMOOK
TSS
8
4
5.111.3
3.310.8
2.610.7
0.210.2
3.411.4
TILLAMOOK
TBC
9
3
5.910.6
9.211.0
2.810.7
3.610.6
1.110.4
42
-------
TABLE 17.
(continued)
BAY
SITE
SPECIES
n
BAP
DBAHA
BGHIP
IP
COR
YAQUINA
Y1M
1
17
0.510.2
0.810.4,
0.210.0
0.210.0
0.210.1
YAQUINA
Y2M
2
12
2.710.2
1.010.2
0.810.2
0.510.3
0.410.2
YAQUINA
Y140
3
14
3.610.3
1.110.2
0.910.1
0.810.1
1.010.2
COOS
CSS
4
6
1.610.2
1.210.2
0.810.4
0.710.5
0.410.4
COOS
C3S
5
5
4.210.3 ,
5.711.7
6.910.8
8.111.4
1.910.2
COOS
C11G
6
10
2.011.0
1.710.9
0.810.6
0.610.4
0.410.1
TILLAMOOK
TIM
7
2
0.610.1
0.610.1
0.410.1
0.310.1
0.310.1
TILLAMOOK
TSS
8
4
1.310.4
1.910.6
2.410.6
0.210.2
0.010.1
TILLAMOOK
TBC
9
3
2.610.2
2.010.6
2.710.3
1.710.9
1. 4±0. 2
SPECIES
1 M. edulisj 2 M. edulis, 3 C. gigas, 4 M. arencriaj 5 M. axenaria,
6 7 8 9
T. aapaxj M. edulis, M. arenariaj C. gigaa.
ported highest levels of fluoranthene with approximately equal concentrations
of phenanthrene and BAP? only those three PNAH were measured.
It seems apparent that some mechanisms may exist to account for the
commonly observed pattern of uptake. These may include differential rates
of uptake and/or elimination or greater biological availability of lower MW
PNAH because of their higher water solubilities (Fazio, 1971). To identify
significant relationships between PNAH concentrations and their water solu-
bility, the data in Table 17 were converted to log values and regressed
against their respective solubilities (Log S values; Table 4). Figure 7
illustrates the results of those analyses. Linear regression analyses were
conducted for each of the 9 sites used in this study and the results are pre-
sented in Table 18.
Separate linear regression analyses were performed using solubility
values from MacKay and Shiu (1977) and from May (1980) for phenanthrene,
fluoranthene, pyrane, triphenylene, benzanthracene and chrysene with the
common values for dibenz(a,c)anthracene, benzo(a)pyrene, benzo(g,h,i)perylene,
and coronene from MacKay and Shiu (Table 4). For MacKay and Shiu's values
for all 10 PNAH, calculated P - 235.4 > F^oSflc.87 - 3.95; R2 « 0.73. The
regression formula used to calculate Y from X where Y » log mean PNAH con-
centration and X ¦ -log S is
43
-------
CO
CD
Y = 3.46-0.38 X
R2 » 0.73
CO
-LOG S
Figure 7. PNAH-water solubility regression relationship. See text for a
complete explanation.
Y - 3.46 - 0.38X.
For May's values, calculated F « 205.0 > F^oSfl/87 ¦ 3.95; R2 - 0.70 and
$ - 3.48 - 0.38X.
Thus, an empirical relation between log CQ (log % concentration in
shellfish) and log S is observed for PNAH, as shown in Figure 7. It is
interesting to note that the concentrations in shellfish were greater for
the PNAH isomer which had the higher solubility in water. This is in con-
trast to the observation that the organic/water (e.g. octanol/water) parti-
tion coefficient shows an inverse relation to water solubility (Chiou et al.,
1977). Because the concentration in the organic phase (in this case, shell-
fish) , CQ is equal to the product of the partition coefficient (k) and con-
centration in water (C^,), the data suggest that the ratio of the concentra-
tions of these PNAH in water would have to be generally greater than the
ratio of their reciprocal partition coefficients or their water solubilities.
Direct measurements of the PNAH concentrations in seawater will be necessary
to confirm whether the uptake of PNAH's by shellfish can be represented by a
simple partition process.
44
-------
TABLE 18.
LINEAR RELATIONSHIPS BETWEEN LOO S (X) , WATER SOLUBILITY, AND
LOO C0 (Y) MEAN CONCENTRATION (%), FOR 10 PNAH1 IN SHELLFISH
FROM YAQUINA (Y) , C008 (C), AND TILLAMOOK (T) BAYS.
CALCULATED LINEAR
SITE
SPECIES
REGRESSION
FORMULA
F2
R
Y1M
W.
edulie
A
Y
¦
4.58
- 0.55X
93.6
.93
Y2M
M.
edulia
*
-
3.83
- 0.43X
36.5
.82
Y1Y0
C.
gi>ga8
m
2.83
- 0.28X
21.5
.73
CSS
M.
arencria
y
-
3.68
- 0.41X
82.5
. 91
C3S
M.
arenaria
A
Y
-
2.00
- 0.16X
10.1
. 56
C11G
T.
oapax
*
-
3.90
- 0.45X
23.8
. 75
TIM
M.
edulia
A
Y
-
4.05
- 0.48X
41. 2
.84
TSS
M.
arenaria
Y
m
3.77
- 0.43X
20.6
.72
TBC
C.
gig as
A
Y
-
2.74
- 0.27X
28.7
. 78
1 PNAH include phenanthrene, fluroanthene, pyrene, triphenylene, benzo(a)an-
thracene, chrysene, dibenz(a,c)anthracene, benzo(a)pyrene, benzo(g,h,i)pery-
lene and coronene.
2 F_o5,i.9 ¦ 5.12; F#oi,i,9 ¦ 10.56. All regressions, except C3S, were
significant at P = .01} C3S was significant at P * .05.
45
-------
REFERENCES
Anderson, R. S* Benso(a)pyrene Metabolism in the American Oyster, Craiioitrta
virginlea. KPA-600/3-78-009. U.S. Environmental Protection Agency, Gulf
Breeze, Florida, 1978* 26 pp.
Baum, K» Jt Occurrence and Surveillance of Polyoyclic Aromatic Hydrocarbona.
Zni Polyoyolic Aromatic Hydrocarbons and Cancer, H. V. Oelboin and P. 0.
Ta'o, ada. Academic Press, N.Y., 1978. pp. 45-70.
Bravo, H., 8. Salaear, A. Botello and 8. Mandelli. Polyaromatic Hydrocarbons
in Oysters from Coastal Lagoons Along the' Eastern Coast of the Gulf of
Mexico, Mexico. Bull. Environ. Contain. Toxicol., 19:171—176, 1978.
Brown, J. M., S. A. Wise and W. E. May. Determination of Benzo(a)pyrene in
Recycled Oils by a Sequential HFI£ Method. J. Environ. Sci. Health,
Al5:613-623, 1980.
Cahnmann, H. J. and M. Kuratsune. Determination of Polycyclic Aromatic
Hydrocarbons in Oysters Collected in Polluted Water. Anal. Chem.,
29i1312-1317, 1957.
Chiou, C. T., V. H. Freed, D. W. Schmedding and R. L. Kohnert. Partition
Coefficient and Bioaccumulation of Selected Organic Chemicals. Environ.
Sci. Technol., 11t475-478, 1977.
Christensen, H. E., T. T. Luginbyhl and B. S. Carroll. Suspected Carcinogens
—A Subfile of the NIOSH Toxic Substances List. NTIS, Springfield, Va.
1975. 342 pp.
Couch, J. A., G. Gardner, J. C. Harshbarger, M. R. Tripp and P. p. Yevich.
Histological and physiological evaluations in some marine fauna. Int
Marine Bioaasays, Proceedings of Workshop, Marine Technology Society,
Washington, D.C., 1974. pp. 156-173.
Couch, J. A., L. A. Courtney, J. T. Winstead and S. F. Foss. lhe American
Oyster (Crassostrea vlrglnlca) as an Indicator of Carcinogens in the
Aquatic Environment. Int Animals as Monitors of Environmental Pollu-
tants, National Academy of Sciences, Washington, D.C., 1979, pp. 65-84.
DiSalvo, L. K., H. E. Guard and L* Hunter* Tissue Hydrocarbon Burden of
Mussels as Potential Monitors of Snvironmental Hydrocarbon Insult.
Environ. Sci. Technol., 9i247-251, 1975.
Dunn, B. P. Techniques for Determination of Benzo(a)pyrene in Marine
Organisms and Sediments. Environ. 8ci. Technol., 10)1018-1021, 1976.
Dunn, B. P. Determination of PAHs in Marine Samples by Liquid Chromatography*
Chromatog* Wewsl., 8i10-12# 1980*
46
-------
Dunn, B. P. and H. P. Stioh. The U«« of Muisala In Batimating Benzo(a)pyrene
Contamination of the Marina Environments Proo. Soc. Expt. Biol. Mad.,
150«49-51, 1975.
Dunn, B. P. and H. F. Stich. Monitoring Prooaduraa for Chamical Carcinogens
in Coaatal Waters. J. Fiah. Rea. Bd. Can., 33t2040-2046, 1976a.
Dunn, B. P. and H. F. Stioh. Ralaaaa of Carainogen Ben«o(a)pyrene from
Environmentally Contaminated Mussels. Bull. Environ. Contam. Toxicol.,
15:398-401, 1976b.
Dunn, B. P. and D. R. Young. Baseline Levels of Benzo(a)pyrene in Southern
California Mussels. Mar. Poll. Bull., 7:231-234, 1976.
Fazio, T. Analysis of Oyster Samples for Polycyclic Hydrocarbons. Proc.
7th Natl. Shellfish Sanit. Workshop. Washington, D.C.: FDA, Div.
Shellfish Sanit. 1971. pp. 238-243.
Fossato, V. U. Elimination of Hydrocarbon by Mussels. Mar, Poll. Bull.,
6:7-10, 1975.
Fossato, V. U. and W. J. Canzonier. Hydrocarbon Uptake and Loss by the
Mussel Mytilus edulis. Marine Biol., 36:243-250, 1976.
Goldberg, E. D. The Mussel Watch. A First Step in Global Marine Monitoring.
Mar. Poll. Bull. 6:111, 1975.
International Agency for Research on Cancer. Volune 3. Evaluation of the
Carcinogenic Risk of Chemicals to Man: Certain Polycyclic Aromatic
Hydrocarbons and Heterocyclic Compounds. 1972. 271 pp.
International Agency for Research on Cancer. Chemicals and Industrial Processes
Associated with Cancer in Hunans. IARC Monographs Supplement 1. Lyon,
France, 1979. 71 pp.
Joe, F. L., E. L. Roseboro and T. Fazio. Survey of Some Market Basket
Commodities for Polynuclear Aromatic Hydrocarbon Content. J. Assoc.
Off. Anal. Chem., 62:615-620, 1979.
Keith, L. H. and W. A. Telliard. Priority Pollutants. I—A Perspective View.
Envirorm. Sci. Technol., 131416-423, 1979.
Kenaga, E. E. and C. Goring. Relationship between Water Solubility, Soil
Sorption, Octanol-water Partitioning, and Concentration of Chemicals in
Biota. In: Aquatic Toxicology, ASTM STP 707, J. G. Eaton, P. R.
Parrish and A. C. Hendricks, eds,, 1980. pp. 78-115.
Kraybill, H. F. Distribution of Chemical Carcinogens in Aquatic Environments.
Prog. Exptl. Timor Res., 20:3-24, 1976.
47
-------
Lee, R. P.» R. flauerheber and A. A. Benson. Petroleun Hydrocarbons Uptake
and Diaoharge by th« Marina Muaial Mytllua adulla. Balance, 177i344-346,
1972.
MacKay, D. and W. Y. Shiu. Aquaoua Solubility of Polynuolaar Aromatic Hydro-
carbona. J. Cham. Bngn. Data, 22t399-402, 1977.
May, W. B. Tha Solubility Bahavior of Polyoyolio Aromatic Hydrocarbons in
Aquaoua Syatama. Ini Advancaa in Chamiatry Sariea, No. 185» Petroleum
in tha Marina Environment, L. Petrakia and F. T. Weiss, eds., 1980.
pp. 143-192.
Mix, M. C. Chemical Carcinogens in Bivalve Molluaks from Oregon Estuaries.
EPA-600/3-79-034. U.S. Environmental Protection Agency, Gulf Breeze,
Florida, 1979. 33 pp.
Mix, M. C. and R. L. Schaffer. Benzo(a)pyrene Concentrations in Mussels
(Mytilus edulls) from Yaquina Bay, Oregon During June 1976-May 1978.
Bull. Environm. Oontam. Toxicol., 23t677-684, 1979.
Mix, M. C., R. T. Riley, K. I, King, S. R. Trenholm, and R. L. Schaffer.
Chemical Carcinogens in the Marine Environment. Benzo(a)pyrene in
Economically Important Bivalve Molluska from Oregon Estuaries. In:
Fate and Effects of Petroleum Hydrocarbons in Marine Organisms and
Ecosystems, D. A. Wolfe, ed., Pergamon Press, Oxford, 1977. pp. 421-431.
Mix, M. C., J. W. Hawkes and A. K. Sparks. Observations on the Ultrastructure
of Large Cells Associated with Putative Neoplastic Disorders of Mussels,
Mytllua edulis, from Yaquina Bay, Oregon. J. Invertebr. Pathol., 34:
41-56, 1979a.
Mix, M. C., S. R. Trenholm and K. I, King. Benzo(a)pyrene Body Burdens and
the Prevalence of Proliferative Disorders in Mussels (Mytilus edulis) in
Oregon. In: Animals as Monitors of Environmental Pollutants, National
Academy of Sciences, Washington, D.C., 1979b. pp. 52-64.
Murray, H. E., G. S. Neff, Y. Hrung and C. S. Giam. Determination of Benzo-
(a)pyrene, Hexachlorobenzehe and PentacHlorophenol in Oysters from Gal-
veston Bay, Texas. Bull. Environm. Contain. Toxicol., 25:663-667, 1980.
National Academy of Sciences. Biologic Effects of Atmospheric Pollutants.
Particulate Polycyclic Organic Matter. Washington, D.C., 1972. 361 pp.
Neff, J. M. Polycyclic Aromatic Hydrocarbons in the Aquatic Environment.
Sources, Fates and Biological Effects. Applied Science Publishers, Ltd.,
London, 1979. 262 pp.
Neff, J. M., J. W. Anderson, B. A. Cox, R. B. Laughlin, Jr., S. S. Rossi and
K. E. Tatixo. Effects of Petroleum on Survival, Respiration and Growth
of Marine Animals. Int Sources, Effects and Sinks of Hydrocarbons in
the Aquatic Environment. American Institute of Biological Science, 1976
pp. 516-533.
48
-------
Panoirov# R. 3, and R. A. Brown. Polynuolaar Aronatio Hydrocarbons in Marini
Tissual. Environ. 8ci. Taohnol., lit989-992* 1977.
Payna, J. F. and H. Hay. Further Studies on tha Effaot of Petroleum Hydro-
oarbona on Mixed-Fvmction Oxidases in Hasina Organisms. Xm Psstioide
and Xanobiotio Mataboliam in Aquatia Organisms# M, Xahn, J. Lach and
J. Mann, ads., American Chemical Society# Washington* D.C., 197S».
pp. 339-347.
Risebrough, R. W., B. K. da Lappa# I. f. LeHerman, J. L. Lane, M. Fireatona-
Gillis, A. M. Springer and W, Walker II. California Muaaal Watohj
1977-78. Vol. Ill - Organic Pollutants in Mussels, Mytilua californianus
and M. edulis, along tha California Coast. Water Quality Monitoring
Report No. 79-22, 1980. 108 pp. + Appendices.
Stegeman, J. J. and J, M. Teal, Accunulation, Ralaaaa and Retention of
Petrolean Hydrocarbons by the Oyster Crassoatrea virginlca. Marine
Biol., 32J37-44, 1973.
Suess, M. J. rhe Environmental Load and Cycle of Polycyolic Aromatic Hydro-
carbona. Sci. Total Environs.# 6t239-250, 1976.
Vandermeulen, J. H. end W. R. Penrose. Absence of Aryl Hydrocarbon Hydroxy-
lase (AHH) in Three Marine Bivalves. J. Pish. Res. Bd. Canada, 35s 643-
647, 1978.
Zar, J. H. Biostatistical Analysis. Prentice-Hall, Inc., Englewood Cliffs,
N.J., 1974. 620 pp.
&
------- |