EPA-600/3-77-070
june 1977 Ecological Research Series
EFFECTS AND INTERACTIONS
OF POLYCHLORINATED BIPHENYL (PCB)
WITH ESTUARINE MICROORGANISMS
AND SHELLFISH
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. ‘Special” Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical lnforma-
tion Service, Springfield, Virginia 22161.
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EFFECTS AND INTERACTIONS OF POLYCHLORINATED BIPHENYL (PCB)
WITH ESTUARINE MICROORGANISMS AND SHELLFISH
by
Rita R. Colwell and Gary S. Sayler
University of Maryland
College Park, Maryland 20742
Grant No. R-803300-01-0
Project Officer
A. W. Bourquin
Environmental Research Laboratory
Gulf Breeze, Florida 32561
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DISCLAIMER
This report has been reviewed by the Office of Research and Development,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Envirotunental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
ii
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FOREWARD
The protection of our estuarine and coastal areas from damage caused by toxic
organic pollutants requires that regulations restricting the introduction of
these compounds into the environment be formulated on a sound scientific ba-
sis. Accurate information describing dose—response relationships for organ-
isms and ecosystems under varying conditions is required. The Environmental
Research Laboratory, Gulf Breeze, contributes to this information through
research programs aimed at determining:
•the effects of toxic organic pollutants on individual species and com-
munities of organisms;
•the effects of toxic organics on ecosystem processes and components;
•the significance of chemical carcinogens in the estuarine and marine
environments.
The role of microorganisms in the mobilization, transport, and possible re-
moval of organic pollutants is an important aspect for consideration in the
proper regulation of these compounds in the ecosystem.. Additionally, micro-
organisms from sewage outf ails, etc., can and do act as pollutants to shell-
fish in the estuarine environment. The secondary effects of toxic organic
pollution on the accumulation and depuration of enteric bacteria by shellfish
is an important area of research given little attention. This report con-
tributes to our knowledge on the interactions of biotic and abiotic pollutants.
Al W. Bourquin, Ph.D.
Research Microbiologist
lii
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BSTRA CT
The role of estuarine bacteria in the mobilization, transport, and removal of
polychiorinated biphenyls (PCB) was investigated in estuarine environx ents.
A main objective of this investigation was to determine a secondary impact of
PCB contamination of estuarine systems. The specific secondary effect was
the PCB-stress-induced accumulation and depuration of enteric bacteria by
shellfish, i.e., the Chesapeake Bay oyster, Crassostrea virginica .
For this report, bacteria uninhibited by PCB, but capable of growth in the
presence of PCB, are defined as P -resistant. In this regard, PCB-resistant
bacteria were found to be distributed ubiquitously throughout estuarine and
marine environmsnts sampled in this study. The residence tine of PCB in es—
tuarine and marine environn nts is concluded to be sufficiently long to in-
duce stress upon estuarine animals.
This study was completed October 31, 1975. The project was supported by EPA
Grant R-803300--O1-O, Maryland Departi nt of Natural Resources, Westinghouse
Agency Contract No. 34-A-03427, and National Oceanographic Atmospheric Admin-
istration Sea Grant No. 04-5—15811.
iv
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CONTENTS
FOREWARD. . . . iii
ABSTRACT iv
FIGURES
TABLES . . . V ii
ACKNOWLEDGMENTS . . . . . I x
SECTION I INTRODUCTION . . . . I
SECTION II CONCLUSIONS . . . 3
SECTION III RECOMMENDATIONS . . . 4
SECTION IV MATERIALS AND METHODS 5
SECTION V RESULTS . 13
SECTION VI SUMMARY . . 42
REFERENCES 43
V
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F IGURES
No. Page
1 Chesapeake Bay sampling stations
2 Southeast Atlantic stations sampled during R/V EASTWARD Cruise,
E—16B—74, November 1974
3 Survival of total viable bacteria (TVC) and E. coli in aquaria
water under PCB stress and non—stress conditions
4 Accumulation and elimination of E. coil by oysters following
PCB stress aitd non—stress conditions
5 Survival of total viable bacteria (TVC) and Salmonella
yphimurium in aquaria water under stress and non-stress
conditions
6 Accumulation of total viable bacteria (TVC) and Salmonella
typhimurium by the oyster, Crassostrea virginica , following
PCB stress
7 Comparative survival and release of bacteria, measured as total
viable bacterial counts (TVC), under control and PCB-stressed
conditions
8 Accumulation and retention by the clam, and survival o
Salmonella enteritidis under control conditions
9 Accumulation and retention by the clam, and survival of
Salmonella enteritidis under PCB—stressed conditions
10 Comparative survival of Salmonella enteritidis at various
salinities during control and PCB stress conditions
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TABLES
N o. Page
1 OUTLINE OF THE EXPERIMENTAL DESIGN FOR ASSESSMENT OF THE
PARTITIONING OF MERCURY AND HCB IN WATER, OIL AND SEDIMENT. . . 7
2 EXPERIMENTAL OUTLINE FOR ASSAY OF ENTERIC BACTERIA ACCU-
MULATED BY THE OYSTER, CRASSOSTREA VIRGINICA , FOLLOWING
ACUTE PCB STRESS 13
3 ELIMINATION OF TOTAL VIABLE BACTERIA (TVC) FROM OYSTERS
FOLLOWING PCB STRESS AND E. COLI DOSING 17
4 ACCUMULATION OF SALMONELLA TYPHIMURIUN IN OYSTER TISSUE
FOLLOWING PCB STRESS 21
5 ACCUMULATION OF TOTAL VIABLE BACTERIA (PVC) IN OYSTER
TISSUE FOLLOWING PCB STRESS AND SALMONELLA DOSING 23
6 ACCUMULATION OF SALMONELLA ENTERITIDIS IN CLAM TISSUE
FOLLOWING PCB STRESS 27
7 ELIMINATION OF SALMONELLA ENTERITIDIS FROM CLAM TISSUE
FOLLOWING PCB STRESS AND DEPURATION 28
8 FACTORIAL ANALYSIS OF VARIANCE (2x8x4) OF THE PARTITIONING
OF 203 HqC1 2 AND {u- - 4 c} HEXACHLOROBIPHEN L (HCB), BETWEEN
WATER, OIL AND SEDIMENT 30
9 RELATIVE PERCENT PARTITIONING OF 203 Hg AND 14 C-HCB RADIO-
ACTIVITY IN A THREE-PHASE WATER SYSTEM 31
10 RELATIVE PERCENT PARTITIONING OF 203 HgC1 2 AND 14 C-HCB
RADIOACTIVITY IN A THREE-PHASE SEAWATER SYSTEM 32
11 MEAN PERCENT LOSS OF 203 HgC1 2 AND 14 C-HCB RADIOACTIVITY
FROM THE WATER COLUMN TO THE OIL AND SUSPENDED SEDIMENT
PHASES . 32
12 CONCENTRATION OF HgC1 2 AND HCB IN OIL AND SUSPENDED SEDIMENT
FOLLOWING PARTITIONING FROM THE WATER PHASE 33
13 PHYSICAL AND CHEMICAL PARAMETERS FOR ALL STATIONS SAMPLED ON
R/V EASTWARD CRUISE E16B-74, NOVEMBER 16-21, 1974 35
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No. Page
14 PHYSICAL AND CHEMICAL PARAMETERS MEASURED AT THE CHESAPEAKE
BAY STATIONS INCLUDED IN THIS STUDY 36
15 ENUMERATION OF PCB-RESISTANT BACTERIA IN ATLANTIC OCEAN
SURFACE WATER AND SEDIMENT SAMPLES 37
16 ENUMERATION OF PCB-PESISTANT BACTERIA FROM CHESAPEAKE BAY
BOTI’OM WATER AND SEDIMENT SAMPLES 39
17 CORRELATION OF MICROBIAL POPULATIONS WITH PCB CONCENTRATIONS
IN ESTUARINE AND MARINE ENVIRONMENTS SAMPLED 40
viii
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ACKNO QLEDGMENTS
The technical assistance and contributions of the following individuals and
organizations are gratefully acknowledged: Ms. And! Hirsh and Mr. Mark Shon,
Department of Microbiology, University of Maryland; Dr. R. Thomas, Mr. B.
Olive and Mr. R. L. Marshall, U.S. EnvironnEntal Protection Agency, Chemical
and Biological Investigations Branch, Beltsville, Maryland; and the crews of
the R/V RIDGELY WARFIELD, Chesapeake Bay Institute, Johns Hopkins University,
and R/V EASTWARD, Duke University Marine Laboratory. Conjoint support for
this study was made available through Maryland Department of Natural Re-
sources Grant, Westinghouse Agency, No. 34-A-03427 and National Oceanographic
Atn spheric Administration Sea Grant No. 04—5-15811. Shiptirne was made
available through National Science Foundation Grant No. GD-31707. Con uter
tine and facilities were made available through the University of Maryland
Conputation Center, Grant No. 204104.
ix
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SECTION I
INTRODUCTION
The occurrence of polychiorinated biphenyls (PCB) in freshwater, estuarine,
and marine environments has been documented (5, 7, 13, 15, 34). These indus-
trial compounds are recognized as persistent pollutants of global importance
(24, 25). They have been shown to be toxic to aquatic invertebrates and ver-
tebrates (7, 11) and can be transferred and accumulated in food webs which
may include man’s (26). At the microbial level, PCB’s have been reported to
inhibit growth of phytoplankton populations (8, 22) and to interfere with
protozoan chemotaxis (39). They also can stimulate or inhibit bacterial
growth (4, 18).
Recent evidence has indicated a reduction in the concentration of PCB’s in
some marine environments (13). It was concluded that the decline in PCB
levels was due primarily to reduction in the use of PCB’s, mandated by the
federal government. Significant biodegradation was assun d to be nonexis-
tent. However, stimulatory and inhibitory effects of PCB formulations on
bacterial growth and activity have been reported (4, 18), as has microbial
degradation of PCB’s (1, 17, 40).
Hypothetically, microbial degradation of chlorinated biphenyls is a potential
mechanism for their removal from the aquatic environment. Therefore, a pri-
mary objective of this investigation was to assess the potential for degrada-
tion of PCB by estuarine bacteria of Chesapeake Bay. In addition, the
metabolic fate of PCN was determined in order to assess ecological effects of
PCB contamination on heterotrophic bacterial populations in the estuarine
environment.
Investigations in our laboratory have focussed on the biodegradation of three
ubiquitous pollutants: mercury compounds, petroleum hydrocarbons, and poly-
chlorinated biphenyls (PCB) (23, 29, 36). These pollutants commonly are lo-
calized in the sediments of the aquatic environment. Resultant concentra-
tions and interactions among these components are relatively unknown.
Available evidence indicates a propensity of petroleum hydrocarbons to con-
centrate chlorinated hydrocarbon pesticides (12, 30) and mercury compounds
(37), but the sequestering of both of these pollutants by petroleum in a
fresh or marine system has yet to be shown.
Little information is available concerning secondary levels of impact of PCB
contamination on estuarine and marine animals. A secondary level of impact
includes PCB-induced stress, altering the normal physiology of the animal,
and rendering it vulnerable to invading parasites or pathogens.
1
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n objective of this investigation was to determine whether PCB-induced
stress on the oyster, Crassostrea virginica , caused it to accumulate enteric
bacteria. A study was undertaken to test the hypothesis that PCB concentra-
tions commonly encountered by estuarine invertebrates may result in reduced
bacteriological quality of a commercially iu ortant shellfish. Other inves-
tigators have shown that the oyster can effectively filter pathogenic bac-
teria and viruses from overlying waters and accumulate significant quantities
of these microorganisms in tissue and on gill surfaces (9, 14). Retention of
enteric or pathogenic bacteria in stressed oysters could lead to serious eco-
nomic, as well as public health situations, if commercial oyster beds are
c-losed as a result of high coliform counts from PCB or other stress, exclud-
ing sewage contamination.
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SECTION II
CON LUS IONS
At low concentrations (<100 pg 11), the effect of PCB may be stimulatory to
heterotrophic bacterial growth. PCB stress on estuarine invertebrates is
such that an inproved bacteriological quality of comn rcially important
shellfish may be deceptive since, in fact, it may be the result of preferen-
tial effect of P B on enteric bacteria. Additional study should be under-
taken to evaluate the full inpact of PCB contamination on the ecology of
aquatic microorganisms.
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SECTION III
RECOMMENDATIONS
Results of this study, nairely that PCB contamination has a detectable impact
on the microbial activity of selected estuarine and marine bacteria, suggest
that environmsntal discharges of PCB, including incineration and other forms
of release, should be restricted and be subject to critical monitoring. The
impact of PCB on autochthonous, heterotrophic microorganisms should also be
monitored, both under in situ and laboratory conditions to describe accurate-
ly total impact of PCB. Effects of PCB in microbial ecology should receive
greater attention. Predation of higher trophic levels on bacteria, nutrient
cycling by bacteria, and changes in species diversity o microorganisn can
provide indices of environn ntal quality. These indices should be further
investigated so their potential can be developed. Secondary stress on higher
organisms as a consequence of PCB contanilnation, such as bacterial invasion
and pathogenesis, also should be investigated. Synergistic effects on hetero-
trophic processes, as co—contamination of PCB and heavy mstals, appear signif-
icant and should be investigated further.
4
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SECTION IV
MATERIALS AND METHODS
PCB EFFECT UPON ACCUMULATION OF ENTERIC BACTERIA BY S1 LLFISH
Culture Conditions
Laboratory investigations were conducted by using three bacterial strains;
one an indicator of contamination by don stic sewage and the other two, known
pathogens: Escherichia coli type I and Salmonella enteritidis , isolated from
Upper C1iesapeake Bay and a laboratQry stock culture of Salmonella typhi —
muri urn.
Bacterial cultures were harvested by centrifugation at 16,300 x g after
growth for 48 hr in nutrient broth. Pelleted cells were resuspended in ster—
ile salts broth. The resuspended cells were divided into equal portions and
used for inoculation of aquarium water in tanks containing oysters.
Analysis of shellfish tissue, following dosing with the bacteria, was accord-
ing to American Public Health Association (APHA) procedures (3). Oyster
shell surfaces were disinfected with 2.5% hypochiorite in an ice bath.
Oysters and clams were shucked, and their tissues were excised, rinsed with
phosphated buffered saline, weighed, and homogenized with 100 ml 0.5%
pep tone.
Total viable bacterial counts (TVC) of both the oyster homogenate and the
aquarium water were performed by using UBYE agar arid appropriate dilutions of
the samples. Quantitative E. coli determinations were made by ext loying Mac-
Cinkey agar. Since there was an absence of lactose-fermenting organisms
prior to E. coli dosing, all lactose-positive cultures growing on MacConkey
agar were recorded as E. coil . At high concentrations of E. coli , water or
tissue dilutions were plated directly on MacConkey agar. As the nuuber of
E. coil dropped, membrane filters (Millipore Corp., New Bedford, Mass.) were
used to concentrate the bacteria. The filters were placed on the surface of
MacConkey agar plates and incubated at 37°C for 24 to 48 hr.
A similar procedure was used for estimation of Salmonella typhimurium , except
that Bismuth Sulfate Agar (ESA) was used for enumeration. Green colonies on
BSA, after 24 hr incubation at 41°C, were recorded as S. typhimurium . Addi-
tional confirmatory tests were made on Kilegler iron agar, as warranted, to
determine if biochemical alteration of the Salmonella resulted from exposure
to PCB. Salmonella enteritidis was enumerated with similar methods and by
using Brilliant Green Agar (Difco Laboratories, Detroit, Mich.) as the selec-
tive differential plating medium.
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PCB stress was simulated by using Aroclor 1254 R, coated on diatomaceous earth
(Celite) ( J. T. Baker chemical Co., Phillipsburg, N.J.). PCB dosing was
maintained at 10 mg per liter (100 mg per liter Celite) for all experimental
work.
Oyster Maintenance
Oysters ( Crassostrea virginica ) used in this study were dredged from Tolly
Bar in the lower part of Upper chesapeake Bay, near Annapolis, Maryland.
This area of Chesapeake Bay, including water, sediment, and oysters harvested
in the area, has been found free of enteric pathogens and is judged fit for
shellfish harvesting (28). Each animal collected received a preliminary
cleaning aboard ship to remove mussels and associated animals from the shell.
All oysters were transported to the laboratory and stored at 6°C within 6 hr
of collection. Experimental work was initiated within 72 hr of collection.
Experiments using the soft shell clam, Mya arenaria , were similar to those
with oysters.
Oysters were maintained in 60 gallon, custom—designed, recirculating refrig-
erated aquaria (Sea Lake Systems, Inc., Euclid, Ohio). Operating ten erature
was maintained at 15°C. Each aquarium was sterilized by autoclaving in an
AMSCO steam autoclave (American Sterilizer Corp., Erie, Pa.). Two hundred
liters of steam—distilled water were filtered through 0.45 tim, 90 nun Milli-
pore membrane filters and added aseptically by gravity flow to each aquariu u.
Artificial sea salt (Sea Lake Systems) was autoclaved in the dry state and
was added to each aquarium, to a final salinity of 12 °/oo, equal approxi-
mately to the in situ salinity at Tolly Bar. Each aquarium was fitted with
glass covers to reduce, or eliminate, potential contamination. Refrigerant
coils and air lines were disinfected with 2.5% hypochiorite prior to each
experiment.
C ie hundred randomly sized oysters were selected from the total set of
oysters collected. Shell surfaces were thoroughly cleaned with a wire brush
and each animal was surface-disinfected in an ice bath, followed by an iced
2.5% hypochiorite bath for 3 to 5 mm. Icing insured that each animal re-
uiained tightly closed, hence preventing the disinfectant from reaching the
tissue of the animal. In each two aquaria were placed 50 cleaned and disin-
fected oysters. The oysters were retained in the aquaria for 48 hr so they
would become equilibrated to the system.
Following the equilibration period, one group of oysters received a dose of
10 mg per liter Aroclor 1254 coated on 100 mg per liter Celite. The dupli-
cate aquarium received a placebo of 100 mg per liter Celite and, therefore,
served as the control for the experiment. Both sets of oysters were held
under identical conditions except that stress was induced in one aquarium by
addition of PCB. Ninety-six hours after PCB dosing, five oysters were asep-
tically removed from each tank, disinfected, and assayed for bacterial
quality, according to APHA procedures (3). After removal of the five control
Aroclor 1254 R, Registered Tradenaze, Monsanto Industrial Chemicals, St. Louis,
6
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oysters, both tanks received a dose of a washed bacterial suspension, after
which five oysters were removed from each tank, disinfected, and assayed for
accumulated bacteria. Sampling of oysters and water from both aquaria pro-
ceeded at established time intervals for 12 days, after which the remaining
oysters from both tanks were removed, surface-disinfected, and placed in sep-
arate sterile aquaria. Elimination of the accumulated bacteria was monitored
in water of the aquaria to which the oysters had been transferred; purging of
bacteria from the animals was determined by periodic sampling of the oysters.
PARTITIONING OF PCB AND HG
chemicals
Isotopically labeled 203 HgC1 2 (An rsham Searle Corp., Arlington Heights,
Ill.) and {u- 14 c} 2, 4, 5, 2’, 4’, 5’ hexachiorobiphenyl (HCB) (New England
Nuclear Corp., Boston, Mass.), 98% purity as determined by thin layer chroma-
tography, were employed in all partitioning studies. Artificial seawater was
prepared with Tn Sea Salts (Sea Lake Systems, Inc.). Sediment was simu-
lated with the diatomaceous earth, Celite (J. T. Baker Chemical Co.).
Experimental Design
An experimental outline describing the partitioning of Hg and HCB between
three phases of an oil, water and sediment system is given in Table 1. In
order to assess the various partitioning of each phase in the presence of
HgC1 2 and HCB, separately or in con bination, experimental test systems were
established which included water, water and oil, water and sediment, and wa-
ter, oil and sediment for both freshwater and seawater.
TABLE 1. OUTLINE OF THE EXPERIMENTAL DESIGN FOR ASSESSMENT
OF THE PARTITIONING OF MERCURY AND HCB IN WATER,
OIL AND SEDIMENT
I. Water types
a. Fresh
b. Marine
II. Isotopic Assessment
a. HgC1 2
b. HgC1 2 in the presence of HCB
c. HCB
d. HCB in the presence of HgC1 2
III. Phase
a. Water
b. Water and oil
c. Water and sediment
d. Oil and water
e. Oil, water and sediment
f. Sediment and water
g. Sediment, water and oil
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The experimental test systems in the laboratory consisted of 130 mm x 15 nun
sterile screw-capped test tubes containing 10 ml of fresh (tap) water or
artificial seawater. To appropriate tubes were added 40.0 .ig (3.2 pCi)
203 Hgc1 2 , or 10.0 pg (0.14 pCi) (u— 14 c} HCB, or both. Control tubes received
no amendments. Additional test environments were established by adding
100 mg Celite, or 10% (v/v) Kuwait crude oil, or a combination of both to the
initial freshwater and seawater systems. The test tubes containing the com-
ponents were tightly capped and mixed for 5 sec in a vortex mixer. Following
mixing, the test tubes were placed in a 15°C chamber nd gently shaken at
100 rpm for 24 hr.
After incubation, the water, sediment, and oil phases were separated, collect-
ed, and assayed for radioactivity. Crude oil layers were separated and col—
lected by pipette, followed by centrifugation (2100 x g) of the remaining
water and sediment, to pellet any suspended sediments. The aqueous phase was
removed by pipetting and the remaining sediment was washed, centrifuged, re-
suspended, and harvested.
One ml of the aqueous phase or sediment resuspended in water was placed in
10 ml dioxane-based Omnifluor (New England Nuclear Corp.) cocktails; 14 C
radioactivity was measured with an Intertechnique liquid scintillation
counter Model SL-40 (Teledyne Corp., Westwood, N.J.), employing a standard
14 c window setting. Counting efficiency was 94%. Beta emission from 203 Hg
was also measured, with a standard 1 - 4 C window as a reference for total
radioactivity measured in the double label, 203 Hg + 14 C-HCB, test systems.
One ml of the 1/100 dilutions of oil was placed in toluene-based Onmifluor
(NE ) cocktails and counted in the same manner as the water samples. There
was no significant quenching effect observed in any of the liquid scintilla-
tion counting systems.
Gamma emission from the decay of 203 Hg (279.2 Key) was measured in a Packard
Tri—Carb scintillation counter (Packard Instrument Co., Inc., Downers Grove,
Ill.), equipped with an auto-gamma spectrometer. Harvested samples of sedi-
ment, oil, and water were placed directly into gamma tubes following appro-
priate dilution. Quantitative 203 Hg determinations were thus based on gamma
emission rather than beta emission. By comparing the ratio 203 Hg 8 : y to
8 emission in the double label experiments, it was possible to segregate
Hg and HCB partitioning in the various phases.
Sampling
Estuarine samples for enumeration of PCB—resistant bacteria were collected
over a 9-month sampling period, October 1974 to June 1975, aboard the P/V
RIDGELY WARFIELD. Estuarine samples analyzed for PCB content were collected
in June 1975. Marine samples for enumeration of PCB-resistant bacteria and
analysis for PCB were collected in November 1974 during P/V EASTWARD Cruise
E-l6B-74. The ocean sampling stations were located in the southeast Atlantic
outer continental shelf area, extending from Miami, Florida, to Cape
Hatteras, North Carolina (Fig. 2). Estuarine samples were collected at sta-
tions located along the entire length of Chesapeake Bay, from the Susquehanna
River to the Atlantic Ocean (Fig. 1).
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Water samples for microbiological analysis were collected by using a Niskin
sterile bag sampler (General Oceanics Inc., Miami, Fla.). Estuarine and ma-
rine sediment samples were collected by means of non—aseptic Ponar and
Shipeck grabs, respectively. Sediment samples for bacteriological analysis
were taken aseptically from the subsurface of the grab sample. Surface water
samples for PCB analysis were collected by using shipboard submersible pumps.
Methods for the determination of physical and chemical parameters at the time
of sample collection are reported in detail elsewhere (27). Phosphate and
nitrate measurements of estuarine samples were measured according to the
methods of Strickland and Parsons (32).
9
Figure 1. Chesapeake Bay sampling stations.
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72°
700
800
1974.
76°
36°
340
32°
300
28°
26°
240
Figure 2. Southwest Atlantic stations sampled during
R/V EASTWARD Cruise, E-16B-74, November
Methods for determining salinity, phosphate, and productivity for marine
samples were according to Strickland and Parsons (32). Mimonia nitrogen con-
tent of the marine samples was determined using the method of Koroleff (20).
Transparency was measured by using a Secchi disc. Dissolved oxygen concen-
tration was determined by titration, employing the Alsterberg modification of
the Winkler method (32).
Bacterial Enumeration
Total ‘viable bacterial counts of samples containing less than 20 0/00 salinIty
were obtained by using Upper Bay yeast extract agar (UBYE) (15). Total
viable counts of samples of salinities greater than 20 were obtained by
using marine agar 2216 (Difco). Fungi and yeasts were enumerated for
78°
740
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selected samples by plating on Sabouraud dextrose agar (Difco) and Littman
Oxgall agar (Difco). Presumptive counts of fungi and yeasts were obtained by
examination of colonial morphology and microscopic observation. Indication
of PCB degradation, with the PCB present as a primary carbon source, was ob—
tamed by plating on 1254 agar (29), formulated as follows: NaC1, 23.4 g
MgSO 4 7H 2 O, 6.9 g i1, and KC1, 0.9 g i . The latter was used for marine
samples.
Samples were enumerated by spread plate count following appropriate dilution,
and inoculated media were incubated at 25°C. Counts were made at 2 and 4
weeks. .n enrichment broth containing marine salts ( vide supra ) or estuarine
strength salts (15) was supplemented with NH 4 NO 3 , 0.2 g 11 and Aroclor 1254,
1.0 g 1 coated on Celite, 1.0 g 1 (J. T. Baker chemical Co.) or 3 mm
glass beads, 10.0 g 1 . Each flask containing 100 ml enrichment broth was
inoculated with 1.0 ml of a 1/10 dilution of bottom sediment or 1.0 ml of
surface or bottom water and the inoculated flasks were incubated at 15°C for
4 weeks. Isolated colonies picked from count plates and streak plates pre-
pared from the enrichment broths were purified on 1254 agar or UBYE agar.
The pure cultures were presumptively identified to genus following the scheme
of Johnson and Colwell (16).
Extracts of Polychiorinated Biphenyls
Polychiorinated biphenyls were extracted with hexane (Burdick and Jackson
Laboratories Inc., Muskegon, Mich.) from 10-liter water samples, following
the method of Vieth and Lee (35). Hexane extracts were concentrated on board
ship by using a gentle stream of warm air. Extracts concentrated to 10 ml
were returned to the laboratory in acetone—washed, screw—capped tubes for
liquid column chromatographic clean-up prior to further chemical analysis.
Marine sediment samples were placed in acetone—washed jars and were frozen on
board ship. Later, the samples were thawed in the laboratory and 100 g sub-
samples were dried at 100 C for 12 hr to provide dry weight data. Each ma-
rine sediment sample was extracted for 12 hr with 200 ml of a (1:1) hexane
and acetone (Burdick and Jackson) mixture. Sediment extracts were evaporated
to dryness at 60°C in a rotary evaporator and were reconstituted in 30 ml of
hexane.
Estua.rine sediment samples were batch-extracted with 200 ml of hexane and
acetone mixture by shaking for 12 hr on an orbital shaker. These extracts
were handled like the marine sediment extracts.
Column Chromatography of Hexane Extracts
Each extract was mixed with 10 g anhydrous Na 2 SO 4 (Fischer Scientific Co.,
Fair Lawn, N.J.) and filtered through glass fiber filters. The extracts were
evaporated to dryness, resuspended in 10 ml of hexane, and applied to the top
of a 10 cm x 25 imn (19 g) activated fluorsil (J. T. Baker Chemical Co.)
column topped with 10 g (2.5 cm) of Na 2 SO 4 . Samples were eluted with 200 ml
hexane, followed by elution with 200 ml 20% ethyl ether (Fischer) in hexane.
Each fraction was concentrated to 10 ml in a rotary evaporator and stored in
the dark in acetone washed screw—capped tubes.
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Analytical Analysis
Concentrated sample extracts were analyzed for total PCB’ s by gas liquid
chromatography (GLC). GLC analysis was performed on a Shimadzu CG-4BM PF gas
chromatograph (American Instrument Co., Silver Spring, Md.), equipped with a
3% OV-l, 80-100 mesh Shimalite W column (1500 nun x 3 mm) and a 63 Ni electron
capture detector. Operating conditions were maintained as follows: injec-
tion and detector temperature, 285°C; column temperature, 240 0 C; and nitrogen
carrier gas flow rate, 50 ml per mm. Relative peak height, area, and reten-
tion times were measured with a Shimadzu R—201 recorder and a Hewlett Packard
digital integrator, Model 3373B (Hewlett Packard Analytical Instruments,
Avondale, Pa.).
Computerized gas chromatography-mass spectrometry (GC/MS) analysis of ex-
tracted san 1es was performed by using a Hewlett Packard Model 5930A GC/MS
data system. Initial separation of PCB components was obtained with a 5%
OV-l7 60—80 mesh AW chroixsorb W, 4 ft x 1/8 in pyrex column.
Individual PCB components were also identified with thin layer chromatog-
raphy (TLC), following the method described by the Environmental Protection
Agency (33) and GLC, employing a Perkin-Elmer Model 3920 gas chromatogra h
(Perkin-Elmer, Norwalk, Conn.). The instrument was equipped with dual 6 N!
(EC) electron capture detectors and 5% OV-17 and 20% SE3O, 60-80 mesh, AW
chron s orb W, 5 ft x 1/4 in pyrex columns.
All glassware used in the procedures, viz., extraction of PCB from the
sair les, extract concentration and clean—up, and analytical analysis, was
boiled in detergent, distilled water rinsed, and hexane and acetone washed
to eliminate P B contamination from outside sources. All reagents were of
spectrograde suitable for pesticide analysis. Saxr ples were extracted in
diffuse light or in the dark; extracts were stored in the dark to eliminate
potential photo-decomposition (6).
12
-------
SECTION V
RESULTS
EFFECT OF PCB ON ENTERIC ACCUMULATION IN THE OYSTER AND THE SOFT SHELL CLAM
Three groups of experiments investigated accumulation, retention, and sur-
vival of enteric bacteria. The first set examined the accumulation and
elimination of E. coli in the oyster, Crassostrea virginica , under PCB stress
and non-stress conditions. Additionally, the removal of E. coli from aquaria
water and survival of E. coli in aquaria wate; were investigated. 14n outline
of the experimental procedure is given in Table 2.
ThBLE 2. EXPERIMENT? L OUTLINE FOR ASSAY OF ENTERIC BACTERIA
ACCUMULATED BY THE OYSTER, CRASSOSTREA VIRGIN ICA ,
FOLL( ING ACUTE PCB STRESS
quaria
Days
Stress
No stress
—2
50 random oysters
48-hr equilibration
50 random oysters
48-hr equi. librati on
0
P B stress
No stress
4
Bacterial dose--under
stress
Bacterial dose
4—il
Survival and accumulation
——under stress
Survival and accumula—
tion
12
Transfer to fresh aquarium
——post—stress
Transfer to fresh
aquarium
12-19
Survival and elimination
——post— stress
Survival and elimination
Due to a faulty aquarium, the elimination of Salmonella typhimuriuxn by
Crassostrea virginica could not be assessed in a group of experiments simi-
lar to the E. coli accumulation experiments. However, the experimental
13
-------
procedure allowed for the study of accumulation and retention of Salmonella
and a partial study of the elimination of Salmonella by Crassostrea
virginica .
In the third group of experiments, the accumulation, retention, survival,
and elimination of Salmonella enteritidis by the soft shell clam, Mya
arenaria , were investigated. The experimental procedures were identical to
those used in the oyster studies, with minor modification in the initial
cleaning of the clams t exterior surfaces.
PCB Stress and the Accumulation of E. coli
The effect of Aroclor 1254 on the survival of E. coli in aquarium water is
demonstrated in Fig. 3. After E. coli addition (four days post PCB dosing),
an E. coli concentration of 106 cells per liter was reached in both the PCB-
dosing aquarium and control aquarium. This concentration was maintained for
24 hr in the control tank; however, there was ca. 99% reduction in E. coli
concentration in the PCB-dosed aquarium water. Within 48 hr, 90% of the E.
coli added to the control aquarium were no longer detectable. The bacteria
rapidly declined in the water column in both aquaria thereafter, although
the decline was slightly less pronounced in the control aquarium. Six days
following addition of E. coli to the PCB-stressed oyster aquarium, E. coli
concentrations dropped to undetectable levels (less than 1 per ml). E. coli
were detectable in the control aquarium for an additional four days, indi-
cating a slightly longer survival in the non-PCB—stressed environment. Com-
parison of the survival of E. coli with fluctuations in total viable counts
(TVC), as shown in Fig. 3, indicated trends similar to that demonstrated by
E. coli , except that there was no immediate marked loss of TVC from the
water column. Twelve days after dosing, oysters were removed from the
aquaria (Fig. 3). Absence of the oysters apparently had only a negligible
effect on E. coli . However, the TVC increased after the oysters were re-
moved from the aquaria, suggesting growth of heterotrophic bacteria intro-
duced into the aquaria with the oysters. It is impossible to eliminate all
bacteria from the oysters without killing the animals. Therefore, a back-
ground TVC, as indicated in counts at the outset, must be established for
experimental work of this kind.
Oyster tissues assayed for E. coli when oysters were removed to fresh
aquaria (day 12) were found to have accumulated large numbers of E. coli
(Fig. 4). There was no significant difference between accumulation of E.
coli , after exposure for 12 days, by the stressed and non—stressed oysters.
Both groups of oysters accumulated ca. 10 times more E. coli than the number
of E. coli added to the aquarium water (see Figs. 3 and 4). There was sig-
nificantly greater survival of E. coli in the non—stressed oysters, from day
12 through day 19, compared with stressed oysters, which eliminated all ac-
cumulated E. coli by day 19. The peak in E. coli accumulation, at day 14 in
the non—stressed oysters, was most likely due to experimental error; growth
of E. coli in the oyster tissue, is doubtful, but not an impossible situa-
tion.
14
-------
7
a
E
C
05
4-
a
4-
C
a
C
0
0
4
a
a,
4-
C.,
a
m
o3
-J
0
Figure 3.
Days
Survival of total viable bacteria (TVC) and
E. ccli in aquaria water under PCB stress
and non—stress conditions. (A E. coli - PCB
stress, A E. ccli — no stress, • TVC - PCB
stress, o TVC — no stress; oysters were re-
moved from the aquaria at day 12).
Elimination of E. ccli , as seen in Fig. 4, was interesting, in that E. coli
lost from stressed oysters were not recovered in the aquarium water. The
resulting conclusion is that these cells were no longer viable. However, in
non—stressed , E. coil was recovered in the water at day 14, corre-
sponding to the marked loss of E. coli from non-stressed oyster tissue. Al-
though E. coli recovered from aquarium water were insignificant in the total
accumulation of E. ccli by the oysters (<1.0%), it was significantly more
that that recovered from oysters dosed with Aroclor 1254.
0
0 I 2 3 4 5 6 7 8 9 10 I I 2 3 14 15 6 17 18
15
-------
4,
U,
U)
1— 4
‘I ,
0
0
c3
0
4-
a
4-
C
4,
a
C
0
0
2
a
I .-
4,
C D
0
-J
4,
E
C
0
a
L.
4-
C
a
C
0
0
a
4-
a
a
CD
0
‘0
-2
0 12 13 14 15 16 17 18 19
Days
Figure 4. Accumulation and elimination of E. coli
by oysters following PCB stress and non-
stress conditions. (A E. coli tissue
accumulation-—PCB stress, t E. coli tis-
sue accumulation——no stress, • E. coli
elimination——PCB stress, o E. coli elim-
ination——no stress).
The depressive effect of Aroclor 1254 on the elimination, or depuration, by
the oyster of total viable bacteria was clearly evident (Table 3). Elimina-
tion of the viable heterotrophic bacteria by PCB-stressed oysters amounted
to. a maximum of 3.8% of the total bacteria accumulated, conçared with 407%
for the control oysters, even though initial accumulation of TVC was approxi-
mately the same.
0
16
-------
I - .
- J
TABLE 3. ELIMINATIONa OF TOTAL VIABLE BACTERIA (TVC) FROM OYSTERS FOLLOWING PCB STRESS
D E. COLI DOSING
aEl.. . from
bpvc per ml.
CTVC per 100 g tissue.
dcii percent eliminated, Z water/E tissue x 100.
oysters to water, assuming no growth in water.
Stressed
Non-stressed
water
TissueC
Percent
Eliminatedd
Day
water’
TissueC
Percent
Eliminatedd
12
2.5 x io2
2.8 x 1O 5
.007
3.5 x io2
2.6 x 10
.01
13
5.3 x 1O 3
1.2 x
1.9
5.6 x 1O 3
3.6 x 1O
1.5
14
4.8 x
2.2 x 10
2.0
2.3 x
1.2 x 10
190.0
17
1.5 x io 6
x io6
3.8
2.2 x 1o 6
5.0 x 1O 4
407.0
-------
These data support two theories: 1) E. ccli is sensitive to Aroclor 1254 and
2) the ability of the oyster to accumulate bacteria is not inhibited by PCB
but depuration is diminished.
PCB Stress and the Accumulation of Salmonella typhimuriuxn
Accumulation of S. typhimuriuxn by stressed and non—stressed oysters revealed
patterns similar to those of E. coil , with some exceptions. It was immedi-
ately obvious that the quantitative rate of recovery of Salmonella by using
Bismuth Sulfite agar was much less than that of E. ccli . This was evident
from the discrepancy observed between TVC and numbers of recovered Salmo-
nella following addition of >106 cells per ml to the water of each aquarium
(Fig. 5 &. However, results for groups of oysters receiving the same treat-
ment would not be affected by the problem of quantitation.
As noted for E. coil (Fig. 3), the number of Salmonella in the aquarium water
decreased rapidly, starting with the addition of the bacteria four days after
PCB dosing (Fig. 5). There was a slight difference between decline in Salmo-
nella levels between 8 and 12 days, as the aquarium water without PCB showed
what could be interpreted as growth of the Salmonella , paralleled with a rise
in total viable bacteria. Both increases ceased at day 13, with a precipi-
tous drop in the number of S. typhimurium in the control aquaria. In gener-
al, the decline in the number of Salmonella was much less gradual than that
noted for E. ccli although the length of time during which a detectable
number of viable cells could be recovered was approximately the same, i.e.,
10 days. The total viable counts followed closely the trends observed for
Salmonella , with higher TVC concentrations detected in the non—stressed en-
vironment.
It was not possible to follow depuration of S. typhimurium because of a de-
fect that in one aquarium prohibited removal of the oysters to a fresh, ster-
ile environment for purging experiments. It was possible, however, to assay
accumulation of S. typhimurium in the presence of low levels of residual Sal-
monella in the initial dosing tanks.
At day 6, oysters in both environments accumulated approximately 1/10th the
concentration of cells as was present in the surrounding water (Figs. 5 and
6). Minor loss of Salmonella from control oysters, between days 6 and 12,
may have been responsible for the observed increase in concentration of Sal-
monella in the water, as shown in Fig. 5. As the concentration of Salmonella
in the water declined, following day 12 (Fig. 5), a dramatic reduction occur-
red in the concentration of Salmonella in the tissues (Fig. 6). The results
indicated depuration of Salmonella by the oyster.
Con arisons between accumulation of Salmonella by stressed and non—stressed
oysters are presented in Table 4. There was little difference noted, both
in absolute accumulation of Salmonella , or in relative percent accumulation
of Salmonella , between the groups. One difference, however, was the high
initial rate of accumulation of bacteria by non-stressed oysters at day 6.
Interestingly, only deaths of the oysters occurring throughout all the ex-
periments were between days 6 and 14 for the control oysters dosed with
18
-------
0
C.,
0
03
-J
0
o I 234567891011 1213141516
Days
Figure 5.
Survival of total viable bacteria (TVC)
and Salmonella typhimurium in aquarium
water under stress and non—stress con-
ditions. (A Salmonella - P B stress,
Salmonella - no stress, • TVC — PCB
stress, o TVC — no stress; oysters re-
moved at day 12).
Salmonella. Salmonella typhimurium was recovered from the gut of one of the
dead animals. The evidence is suggestive, but not conclusive, of Salmonella —
induced death.
7
6
5
4
a,
E
C
0
4-
0
4-
C
a,
C.)
C
0
0
1
Addition of Saimonelig
19
-------
6
5
a,
C o
I- .
3
C
0
4-
I-
4-
C
C)
C2
0
C-)
0123456
0
Figure 6.
12 3 14 15 16 17
Accumulation of total viable bacteria
(TVC) ai-id Salmonella typhimuriuiu by
the oyster, Crassostrea virginica ,
following P*8 stress. (. TVC - P B
stress, o TVC — no stress, A Salmo-
nella — PCB stress, Salmonella —
no stress).
0’
0
0
a
1
a,
4-
C,
0
m
0’
0
-J
7 8 9 10 II
Days
20
-------
TABLE 4. ACCUMULATIONa OF SALMONELLA TYPHIMURIUM IN OYSTER TISSUE FOLLOWING PCB STRESS
Stressed
Non-stressed
Day
b
Water
. c
Tissue
Accumulated
d
b
Water
. c
Tissue
Accumulated
d
6
4.4 x 1O 3
4.8 x 1O 3
109.0
1.7 x 1O 3
1.3 x
764.7
12
4.7 x 1O 3
1.9 x 1O 4
261.5
7.5 x 1O
5.5 x 1O 3
201.1
14
1.0 x 101
3.0 x i0 2
264.5
2.0 x io2
4.0 x io2
201.1
15
1.0 x 100
2.8 x 101
265.0
3.0 x 101
5.0 x 101
201.0
aAccumulation assuming no growth of Salmonella in tissue.
bsai neiia per ml aquarium water.
CSa1 nella per 100 g oyster tissue.
.dcumuiative percent accumulated, Z tissue/E water x 100.
-------
Accumulation of total heterotrophic bacteria in oyster tissue (Fig. 6) was
greater in non—stressed oysters. This observation was made for TVC in the
E. coli accumulation experiit nts. In terms of relative percent accumulation
of TVC, accumulation by the control oysters ranged from 26% to 11.4% of the
total number of Salmonella , compared with 10.0% to 8.5% for PCB-stressed
oysters (Table 5).
Janssen (14) reported oyster retention of 2.8 x 10 S. typhimurium per oyster
from water containing 2 x 10 cells per ml after 48 hr exposure. Although it
is difficult to compare the results reported by Janssen, on the basis of per
oyster accumulation, it does appear that the results of this investigation
are comparable in non—stressed oysters.
Several preliminary conclusions can be drawn from the results of these inves-
tigations to date. As expected, the oyster, Crassostrea virginica , demon-
strated an ability to concentrate enteric bacteria. The effect of PCB stress
on oysters apparently is a more complex process than considered initially, in
terms of bacterial accumulation and depuration. A stress appears to be im-
posed on the bacterial population, as well as on the oyster, the end result
of which is that PCB stress artificially produced what superficially could
be considered, from bacteriological criteria, to be oysters of higher bacte-
riological quality than was, in fact, the case. This observation was totally
unexpected, as can be judged from the above hypoth sis that assuit d PCB
stress would result in poorer bacteriological quality. On the other hand,
indications are that PCB stress may result in a lessening of the ability of
the animal to purge itself of bacteria. These observations require further
study before they can be accepted as fact. In the interim, experimental work
was done with another estuarine invertebrate, the soft shell clam, to deter-
mine whether the effects observed for the oyster are specific or are appli-
cable to other estuarine animals.
PCB STRESS AND EFFECT ON SALMONELLA ENTERITIDIS ACCUMULATION IN THE CLAM
Total viable aerobic heterotrophic bacterial counts made of the control and
PCB-stressed clams showed a marked drop after dosing with Salmonella at day
2. The drop in the count was a sharp deflection in the TVC survival curve,
shown in Fig. 7. The number of total viable aerobic heterotrophic bacteria
stabilized at day 6 and 7, respectively, in the PCB-stressed and control
aquaria waters. However, the TVC associated with PCB—stressed clams were
found to be 10 tines higher than those in the control clams (Fig. 7). This
observation held true at day 12, two days after removal of the clams from the
dosing tanks to the purging tanks. Furthermore, release of TVC into the
aquarium water where the clams had been placed for purging was 10 to 100
tines greater in the control clams, compared with PCB—stressed clams.
Survival of Salmonella enteritidis in the dosing water was similar under both
control and PCB stress conditions (Figs. 8 and 9). However, PCB—stressed
clams initially accumulated greater than 10 tines as many Salmonella per
100 g of tissue than control clams. (See Figs. 8 and 9.) The accumulation
of Salmonella by stressed and control clams, relative to the concentration
of Salmonella in the aquaria water, was 20 to 50 times greater in the
stressed oysters during the following 2 through 6 days of Salmonella
22
-------
TABLE 5. ACCUMULATION OF TOTAL VIPIBLE BACTERIA (TVC) IN OYSTER TISSUE FOLLOWING PCB
STRESS ND SI LMONELLA DOSING
Stressed
Non—stressed
Watera
Tissueb
AccumulatedC
Day
Watera
pissueb
AccumulatedC
6
3.1 x 1O 5
3]. x 10
10.0
4.6 x 1O 5
1.2 x 1O 5
26.0
9
8.0 x 10
6.3 x 1O 4
8.5
8.0 x 10
1.6 x
22.2
12
4.8 x 1O 4
1.4 x 1O 4
9.3
1.6 x io6
5.6 x
11.7
13
1.6 x
1.6 x
9.4
6.8 x 10
6.7 x 1O 4
11.4
14
1.2x10 5
1.3x10 4
9.3
5.6x10 5
2.,0x10 5
14.7
a
¶ [ VC per ml.
bTVC per 100 g tissue.
CC u1ative percent accumulated, E tissue/Z water x 100.
-------
:
E
C 3
0
C
0
C
0
C-,
L
Days
Figure 7. Comparative survival and release of bacteria, measured
as total viable bacterial counts (TVC), under control
and PCB-stressed conditions. (Survival of the total
aerobic heterotrophic bacteria in the dosing tanks:
• = PcB stress, o = control, release of TVC: A = PcB
stress, = control).
dosing (Table 6). At day 10, at which time the clams were placed in the
purging aquaria, there was no significant difference in tissue accumulation
of Salmonella in PCB-stressed or control clams. Retention of accumulated
Salmonella by both stressed and control clams remained at high levels, i.e.,
105 to 106 Salmonella per 100 g throughout the period of purging. In com-
parison, the level of release of Salmonella , which was l0 ’ organism per ml,
was high in both experimental groups (Figs. .8 and 9). These results suggest
growth may occur in the clam tissue, as well as prolonged survival of Salmo-
nella under purging conditions. There was, however, no significant differ-
ence in the relative elimination of Salmonella from clam tiss during purg-
ing of both control or PCB-stressed oysters (Table 7).
As shown in Fig. 10, survival of pure cultures of Salmonella enteritidis
placed in flask cultures containing artificial seawater of various salinities
extended beyond the period of dosing and purging employed in these studies.
These data show little or no effect of PCB on the survival of S. enteritidis
at salinities of 10 0/00 or greater. Furthermore, the data suggest that, in
fact, there may be a potentially beneficial effect of PCB on S. enteritidis
survival at salinities of <10 0/oc — ___________
4 5 6 7 8 910 II 123415 1€ 1718192021 222324 25
24
-------
I.
— Purging——
4 5 6 7 8 9 10 I I 12 13 14 15 6 17 IS 19 20 21 22 23 24 25 26
Days
Figure 8.
Accumulation and retention by the clam, and survival
of Salmonella enteritidis under control conditions.
(0 = survival in water of the dosing tank, 0 = accu-
mulation and retention in oyster tissue, = release
of Salmonella into the purging aquarium water)
(Celite placebo added in place of PCB).
Some general observations can be made concerning results of studies on the
accumulation of enteric bacteria in oysters and clams. Accuinulat on and re-
tention of bacteria, measured by PVC counts, and of Salmonella in the clam
are significantly greater than in the oyster under both PCB stress and con-
trol conditions. However, the release of bacteria accumulated by the clam
was remarkably low. Physiological differences between clams and oysters are
sufficiently great that the effects of PCB stress on these animals should be
expected to be different.
The patterns observed for Salmonella enteritidis accumulation arid release by
soft shell clams and accumulation and release of E. coli by oysterswere very
similar (Figs. 5, 8 and 9). In both cases, the total bacterial accumulation
was approximately the same for each group of animals under both PCB stress
and control conditions. However, survival of E. coli and Salmonella enterit-
idis released from the animals during depuration appeared to be reduced in
the presence of PUB.
8
H—
E
C
26
0
C
U
C
0
C-)
0
A
25
-------
0,
0
0
0
E
C
0
0
C.)
C
0
C-)
0
C.)
a
3
0
3
Figure 9. Accumulation and retention by the clam, and survival
of Salmonella enteritidis under PCB—stressed condi-
tions. (. = survival in dosing aquarium water, *=
accumulation and retention in clam tissue, £ = re-
lease of Salmonella enteritidis into purging aquarium
water.)
• —q 1 L .. 11
—PCB Stress— — —
Purging— — — —
of Solmonello
Days
4 5 6 7 8 9 0 II 2 CS 14 IS 16 rr 18 19 20 21 22 23 24 25 26
26
-------
TABLE 6. ACCUMULATIONa OF SALMONELLA ENTERITIDIS IN CLAM TISSUE FOLLOWING PCB STRESS
Stressed
Non—stressed
Day
waterb
Tissue
- Accunlulatedd
waterb
Ti ss ueC
Accumulatedd
2
2.5 x 1O 4
6.0 x io6
24,000
1.5 , 1O
2.0 x 1O 5
1,333
3
5.6 x 1O 3
1.2 x i0 6
23,500
3.5 x 1O 4
2.6 x
452
6
1.5 x 1O 3
1.1 x io6
25,000
1.4 x
4.0 x 1O
1,217
aACcumulation assuming no growth of Salmonella in tissue.
b aimonei1a per ml aquarium water.
CSalmonella per 100 g oyster tissue.
dCumuiative percent accumulated, E tissue/E water x 100.
-------
TABLE 7. ELIMINATIONa OF SALMONELLA ENTERITIDIS FROM CLAN TISSUE FOLLOWING PCB STRESS
AND DEPURATION
Stressed
Non-stressed
Day
waterb
TissueC
Percent
Eliminatedd
waterb
TissueC
Percent
Eliminatedd
6
5.7x10 3
1.1x10 6
0.5
2.0x10 4
4.0x10 5
5.0
8
1.9x10 5
4.3x10 4
17.1
3.5x10 4
2.9x10 4
12.8
10
1.4 x 1O 5
5.2 x 1O 4
27.8
2.1 x 1O 4
5.6 x 1O 4
15.7
13
2.6 x
1.2 x io6
28.7
4.1 x 1O 7
2.7 x io6
2.5
17
1.2 x 1O 3
3.6 x 1O 5
12.0
7.8 x
4.9 x
2.4
22
4.2 x io 2
1.2 x io6
8.5
2.5 x 1O 3
2.3 x io6
1.5
aEl..U from clam to water.
b
SalnKnella per ml aquarium water.
C
Salmonella per 100 g clam tissue.
dcumuiati percent excreted, Z water/Z tissue x 100.
-------
C
0
24
C
a,
C -)
C
0
03
a
C-,
02
0’
0
-J
0
Days
Figure 10. Comparative survival of Salmonella enteritidis at
various salinities during control and PCB stress
conditions. (o = PCB stress, o = control).
PARTITIONING OF HG AND PCB BE’IWEEN OIL, WATER AND SUSPENDED SEDIMENT
Results of a factorial analysis of variance, assuming all major sources of
variation to be fixed treatment effects, showed signi ficant variation between
freshwater arid seawater and between the various oil, water, and sediment
phases (Table 8). No significant variation was attributable to the individu-
ally labeled compounds being partitioned. Variation attributed to all treat-
ment effect interactions, except that between water arid labeled compounds,
was found to be significant. These results refute the hypothesis that there
is no significant change in HCB or Hg concentration in the water column.
Furthermore, salinity and partitioning of the various phases were found to be
significant.
A comparison of the mean percent partitioning of 203 Hg and 14 C-HCB by the
various phases in the freshwater and seawater systems was revealing (Tables 9
and 10). From the data given in Tables 9 and 10, it is evident that oil is
an extremely effective partitioning agent for both HCB and HgC1 2 even in the
presence of sediment. There appears to be a significantly greater percent
partitioning of HCB by crude oil, compared with HgC1 2 , in both freshwater and
seawater. This effect was also observed in the significant variation found
for the Phase X Compound interaction (Table 8). In all cases, sediment was
2 3 4 5 6 7 B 9 0 II 2 13 14 5 6 IT 18 9 20 21 22
29
-------
TABLE 8. FACTORIAL ANALYSIS OF VARIANCEa (2x8x4) OF THE PARTITIONING
OF 203 HgC1 2 AND u- 14 c} HEXACHLOROBIPHENYL (I-IcB), BETWEEN
WATER, OIL AND SEDIMENT
Source of variation
d.f.
88
MS
Fs
Water (W)
1
879
879
198 kb
Phase (P)
7
151769
21681
488.0*
Coi ound (C)
3
21
7
0.2
WXP
7
1025
146
3•3*
WXC
3
1
0.6
0.0
PxC
21
16765
798
18.0*
WXPXC
21
4740
226
5.8*
Within subgroups
64
2839
44
Total
127
178040
aANOVA Model 1 with replication (c = .05).
b
* indicates variability significantly greater than within subgroup
va.ri ation.
much less effective in partitioning mercury and HCB from the water coluiwi,
conpared with the crude oil. The partitioning of HCB and mercury by sediment,
in the presence of oil, was from the oil rather than the water, which was sur-
prIsing, considering the distance between the sediment pellet and the floating
oil layer (approx. 7 cm).
The difference between the freshwater and seawater was less apparent than that
observed for the various phases. Thus, the ability of seawater to retain both
HCB and mercury is greater than freshwater. This was not an unexpected obser-
vation, in view of the ionic nature of HgCl 2 and the number of chlorine resi-
dues of the hexachlorobiphenyl.
Data on mean percent loss of radioactive label for both freshwater and sea-
water were pooled to determine potential loss of both HCB and mercury from
the water columu to the oil and sediment phases (Table 11). Eighty-five per-
cent of the HCB and mercury was removed from the water column by both the oil
phase, alone, or the oil in conjunction with sediment. However, sediment re-
moved only 28.4% of the HCB and mercury. The remainder was found in the
water. Greater selective partitioning of HCB by crude oil, con ared with
30
-------
TABLE 9. RELATIVE pERCENTa PARTITIONING OF 203 Hg ND 14 C-HCB
RADIOACTIVITY IN A THREE-PHASE WATER SYSTEM
Percent radioactivity partitioned
Water Oil Sediment
203 HgC 1 2 19.0 72.8 8.2
23.5 85.5 ——
75.7 —— 24.4
100.0 ——
203 HgC1 2 (HCB) 28.4 63.1 8.5
32.9 67.1 ——
85.7 —— 14.3
100.0 _.1
C—HCB 0.9 92.4 6.8
0.4 99.5 -—
74.8 —— 25.2
100.0 ——
14 C—HcB (HgC1 2 ) 2.7 93.7 2.6
2.5 97.5 ——
71.2 —— 28.8
100.0 -—
a an of duplicate observations relative to the total radio-
activity in the water column.
mercury, (ca. 97% removal vs. 74%) was an expected result, considering the
affinity of H B for non-polar solvents.
The relative percent partitioning, or removal, of mercury and HCB by sediment
was less than for crude oil (Tables 9, 10, and 11). However, when the data are
expressed in terms of net concentration of mercury or HCB in the oil layer,
coipared with the sediment, sediment was highly efficient in concentrating
both mercury and HCB (Table 12). Efficiency of sediment partitioning was
slightly reduced in the presence of oil. Concentrations of HCB in sediment
increased 13.6 fold and 28.1 fold, respectively, for sediment in the presence
of oil and sediment alone. About a ninefold increase was noted for the oil.
Less dramatic results were obtained for concentration of mercury in oil or
sediment. Separately, oil and sediment were found to be nearly equal in
ability to accumulate mercury. However, in a three-phase system, oil was a
better conpetitor in the partitioning of mercury, with a sixfold increase,
conpared with a twofold increase in mercury in suspended sediment.
31
-------
TABLE 10. RELATIVE PERCENTa PAR ITI0NING OF 203 HgC1 2 AND 14 C-HCB
RADIOACTIVITY IN A THREE-PHASE SEAWATER SYSTEM
Percent
radioactivity
partitioned
Water
Oil
Sediment
203 HgC1 2
28.3
17.2
61.5
100.0
54.9
82.4
——
——
16.8
——
38.5
—-
203 HgC1 2
(HCB)
30.0
30.9
76.4
100.0
61.9
69.1
——
——
8.1
——
23.6
—-
14 CHCE
10.7
0.9
69.3
100.0
81.1
99.1
——
——
8.3
——
30.7
—-
14 CHCE
(HgCl 2 )
4.1
1.2
95.9
100.0
85.4
98.8
——
——
10.5
—-
4.1
——
of duplicate observations relative to the total radio-
activity in the water column.
TABLE 11. MEAN PERCENT LOSS OF 203 HgC1 AND 14 C-HCB RADIO-
ACTIVITY FROM THE WATER COLUM TO THE OIL AND
SUSPENDED SEDIMENT PHASES
Phases
Radioactive
label
203 HgC1 2
14 c-HCB
Composite
Y s
Oil + sediment
73.6
95.4
84.5 12.4
Oil
73.9
98.7
85.4 12.9
Sediment
23.2
22.2
28.4 18.8
Water
0.0
0.0
0.0
a .
Relative to the total radioactivity in the water for both
the freshwater and seawater systems.
32
-------
TABLE 12. CONCENTRATION OF HgC1 2 AND HCB IN OIL AND SUSPENDED SEDIMENT
FOLLOWING PARTITIONING FROM THE WATER pI.1ASEa
Phase
HgC1 2
HCB
mg 1
s
Percent
increase
mg i1
s
Percent
increase
Oil
30.4
0.4
2640
9.5
0.001
857
Oil (sediment)C
25.3
0.4
2030
8.8
0.07
782
Sediment
24.8
3.9
2080
28.1
1.2
2710
Sediment ( 0 1 )d
10.4
1.8
640
13.6
0.3
1260
a an of eight observations. s, standard deviation.
bpercent increase relative to the initial concentration of HgC1 2
(4 mg or hexachiorobiphenyl (1 mg 11) in the water column.
C 0 fl partitioning in the presence of sediment.
d diment partitioning in the presence of oil.
Differences in partitioning efficiency, between sediment and oil, arise from
the highly non-polar nature of crude oil and the resulting partitioning on
the basis of surface adsorption occurring on the relatively large surface
area of the Celite.
Kenega (19) reported that adsorption of chlorinated pesticides in some en-
vironmental systems is 50% cort 1ete within a few hours. Thus, the findings
of the short—term (24—hr) laboratory experiments in this study can be extrap-
olated to the natural environment. Maximum partitioning of DDT (structurally
similar to HCB) has been reported to occur at ca. 10 days (12). Greater par-
titioning of PCB and mercury would be expected to occur as the time of incu-
bation increased. Hartung (12) reported steady-state oil partition coeffi-
cients for DDT that exceed 106, indicating an even greater potential for
chlorinated residues, such as PCB, to accumulate in areas continually receiv-
ing oil and PCB.
Since no significant difference was noted between mercury and PCB partition-
ing, it is doubtful that organic forms of mercury would be accumulated to any
less extent than the HgC1 2 and HCB employed in this study. However, Hg 0 vola-
tilization is a microbial metabolic pathway shown to occur in the aquatic en-
vironment and is a mechanism of escape of mercury from a combined Fig—oil
environment (21). Also, water-extractable material in oil (31) can be a
potential mercury volatilizing mechanism (2).
33
-------
It is evident from the data presented here that concentration of heavy metals
and chlorinated hydrocarbons in sediment and/or oil can result in a highly
toxic environment, i.e., inhibitory to microorganisms capable of degrading
each of the components separately but inhibited by the high concentrations in
combination. The relatively exotic substrates thereby occurring in sediment
must eventually be mineralized by microorganisms. Thus, spilled, dumped, or
seeping oil reaching marine or estuarine sediments may be rendered impervious
to microbiological attack (38). Furthermore, application of degradation ki-
netics, established for any one of the individual pollutants discussed here, to
a multiple—contaminated environment will not be valid. The consequences of
impeded or inhibited microbial degradation of the components of a mixed-
pollutant system are serious and should be investigated.
DISTRIBUTION OF PCB-RESISTANT BACTERIA AND PCB IN ESTUARINE AND MARINE ENVI-
RONMENTS
Physical and chemical parameters measured at the time of collection of the
samples from Chesapeake Bay and the Southeast Atlantic Coast are given in
Tables 13 and 14. The marine surface water samples collected in Miami Beach
Harbor and in the shallow water of Cape Hatteras, North Carolina, revealed
the highest concentrations of NH 4 -N and P0 4 -P (Table 13). Coincident with
the values for these nutrients was the relatively high level of heterotrophic
activity ( 14 C uptake) and chlorophyl-a content observed at Miami Beach and,
to a lesser extent, off Cape Hatteras. In general, nutrient concentrations
and net activity of the surface waters was lower off the continental shelf
extending to the deep station, #8, farthest from shore, i.e., ca. 200 mi off
Cape Hatteras.
Fewer nutrient data were available for samples collected in Chesapeake Bay,
but the data obtained indicated a high level of nutrient input in the Upper
Bay, from nnapo1is, Maryland to the Susquehanna River. The nutrient concen-
trations observed were most likely due to the influence of the Baltimore
Harbor area and the Susquehanna River (Table 14). Previously reported data
indicated cbnes tic sewage point source contamination at Chesapeake Bay sta-
tions CBSO1 and CBBO9 (15, 16). In general, stations located in the open bay
yield lower levels of contamination relative to both Baltimore Harbor and the
Susquehanna River areas. Salinities measured at the stations in Chesapeake
Bay included in this sutdy ranged from essentially freshwater to Ca. 70% sea-
water.
1 covery of PCB and PCB-Resistant Bacteria
Polychlorinated biphenyls were recovered from all of the marine water and the
sediment samples collected during the study. However, in 9 of the 14 samples
analyzed, the concentrations of PCB detected in the samples were below the
sensitivity limits of the methods employed, i.e., ca. 10 jig/kg sediment and
0.1 pg/l000 ml H 2 0. Samples collected in Miami Beach Harbor, at stations 5
and 7, and off Cape Hatteras were all found to contain significant levels of
PCB. Qualitatively, the presence of Aroclor 1254 was detected at station 10
off Cape Hatteras, North Carolina. However, sediment samples collected in
Miami Beach Harbor contained significant levels of polychiorinated biphenyl.
34
-------
TABLE 13. PHYSICAL AND CHEMICAL PARAMETERS FOR ALL STATIONS SAMPLED ON R/V EASTWARD CRUISE E16B-74,
NOVEMBER 16—21, 1974
U I
Station
Location
Depth
(m)
Salinity
(°/oo)
Temp.
(C)
DO
(mg/i)
P0 4 -P
(ugat/i)
NH 4 -N
(ugat/i)
14 C
(mg/m 3 /day)
Ch l-a
(mg/rn)
1
Miami Beach Harbor
0
ii
34.1
34.2
23.4
——
3.0
3.1
0.02
0.06
2.10
2.25
32.0
92.8
25.6
30.8
2
26° 30.O’N, 79° 20.O’W
0
460
36.1
35.7
26.7
13.3
3.0
2.5
0.0
0.24
0.01
0.03
0.09
——
0.18
——
3
29° 05.6’N, 800 05.O’W
0
528
36.1
36.4
25.5
19.5
2.9
2.1
0.03
0.13
0.49
0.15
0.12
——
0.17
——
4
300 05.6’N, 80° 15.O’W
0
160
36.2
36.0
25.8
14.4
5.5
2.0
0.0
0.22
0.35
0.06
4.50
——
0.09
——
5
31° 00.O’N, 80° 00.O’W
0
160
36.1
35.7
24.2
15.8
3.5
2.0
0.0
0.26
0.68
0.37
5.4
——
0.13
——
6
32° 05.O’N, 790 05.4’W
0
200
36.2
35.0
24.6
8.3
2.9
1.9
0.0
0.44
0.27
0.17
0.2
——
0.27
——
7
33° 00.4’N, 770 40.O’W
0
152
36.2
36.1
25.7
13.3
2.9
2.1
0.0
0.17
0.17
0.12
0.4
——
0.25
——
8
330 53.5’N, 740 55.O’W
0
3199
36.3
35.0
24.0
2.5
2.9
3.7
0.01
0.01
0.17
0.24
1.5
——
0.18
——
9
340 19.8’N, 76° 04.7’W
0
36.3
22.4
3.0
0.0
0.05
0.03
0.34
50 36.2 20.1
3. 1
0.3
0.001
10 340 37.3’N, 76° 33.1’W
0 35.5 15.6 3.6 0.Q4
0.57 26.3 0.19
-------
TABLE 14. PHYSICAL AND CHEMICAL PARAMETERS MEASURED AT THE CHESAPEAKE BAY STATIONS IN-
CLUDED IN THIS STUDY
Stationa
Location
Depth
Cm)
Salinity
(°/oo)
DO
(mg/i)
Thans—
parency
(m)
Temp.
(C)
P0 4 —P
(mg/i)
N0 3 -N
(mg/i)
CBSO1
Havre de Grace
12.0
0.0
8.8
1.5
22.8
.028
3.10
CBS27
CBBO7
CBBO8
cBBO9
Conowingo Dam
Colgate Creek 1
Fort McHenryb
Jones Fallsb
2.0
9.1
6.5
2.6
0.0
3.9
3.9
1.4
——
8.5
10.3
6.9
0.3
0.8
0.7
0.6
——
22.5
21.].
22.6
.066
.048
.076
——
2.02
1.71
3.72
—
9180S
Pooles Island
6.7
4.3
10.7
2.0
19.5
.048
2.20
9110F
Baltimore Harbor
4.5
3.2
6.8
2.0
18.6
.048
2.50
9040N
chester River
10.0
8.6
9.5
2.0
14.5
——
——
8580E
Tolly Bar
6.5
7.0
6.2
1.8
15.1
.038
1.17
818A
Solomons
9.7
10.9
8.5
1.7
23.0
——
——
7480U
Tangier Island
6.1
14.0
3.6
2.3
23.0
.19
——
7140S
Cape Charles
28.0
25.8
7.4
3.3
——
——
——
6560H
Little CreekC
8.2
20.3
5.8
1.7
25.4
-—
a
Dates of sample collection: September and October 1974; June 18 and 19, 1975.
bBl timore Harbor.
CNorfQ lk, Virginia.
-------
Bacteria capable of growth on media containing Aroclor 1254 as the primary
carbon source were recovered from water and sediment sazples collected at all
stations sanpled (Table 15). In general, the number of PCB-degrading/resis-
tant bacteria was higher in the sediment than in the water coluzm . However,
the relative proportions of PCB degraders making up the TVC were higher in
the water than in the sediment. Total numbers of PCB—resistant bacteria were
highest at shallow stations and appeared to decrease with increasing depth
and distance from shore.
TABLE 15. ENUMERATION OF PCB-RES ISTANT BACTERIA IN ATLANTIC OCEAN
SURFACE WATER AND SEDIMENT SAMPLES
Station
San le
t a
TVC
PCB
resistant
Percent
composition
Total PCB
g i (kg 1 )
1
W
S
5.7
2.1
x
x
10
10
1.3
9.5
x
x
l0
10
22.8
4.5
0.3
12.0
3
W
S
2.0
1.2
x
x
10
10
1.0
3.5
x
x
10
10
0.5
2.9
<0.1
<10.0
5
W
S
1.6
2.6
x
x
l0
10
1.0
4.5
x
x
102
l0
6.2
17.3
0.5
<10.0
7
W
S
3.8
2.6
x
x
10
10
1.0
1.2
x
x
l0
10
2.7
4.6
0.5
<10.0
8
W
S
7.2
3.6
x
x
1O
10
7.2
1.2
x
x
10
10
100.0
33.3
<0.1
<10.0
9
W
S
3.0
4.0
x
x
10
10
3.0
5.4
x
x
lO
10
100.0
13.5
<0.1
<10.0
10
W
S
7.7
1.2
x
x
io2
1O 5
7.0
3.9
x
x
101
lO
9.0
3.2
0.7
<10.0
a = water; S = sediment.
An apparent inconsistency in the data was observed concerning the levels of
PcB-resistant bacteria at station 8 and station 9. The recovered PCB-
degrading bacteria accounted for 100% of the total viable heterotrophic bac-
terial population. This result could be interpreted as indicating a higher
level of PCB contamination, thereby inducing larger populations of bacteria
to metabolize PCB. However, the concentration of PCB at these stations did
not support such a hypothesis (Table 15). It is more likely that the micro-
organisme recovered on the low nutrient-containing PCB medium were adapted
to the low nutrient concentration prevalent in their environment and, con-
sequently, gave a better growth response on the 1254 medium than on the
richer 2216 marine agar used to enumerate the TVC.
In chesapeake Bay, the PCB-resistant bacteria and TVC were observed to be
present at exponentially higher levels than in the marine bacterial
37
-------
populations (Table 16). s in the case of the seawater and deep ocean sedi-
ment bacterial populations, there was a marked variability in the numbers of
PCB-resistant bacteria and in the proportion of the TVC observed for the
chesapeake Bay samples. Nine samples were assayed for PCB in June 1975.
These samples were found to contain low concentrations of PCB in the water
column, ranging from 0.01 to 0.14 pg per liter. However, the PCB concentra-
tions in the sediment ranged from 4.0 to 400 pg per kg. Sediment samp].es
collected in Baltin re Harbor, station CBBO9, and near Norfolk, Virginia,
station 6560H, both industrialized sites, contained 400 and 125 pg per kg
sediment, respectively, i . e., up to 100—fold greater concentrations than
those observed for samples collected at stations in the open bay. Suspended
sediment samples collected in the Upper Chesapeake Bay have been reported to
contain PCB in concentrations as high as 2 mg per kg (personal communication,
T. 0. Munson, Westinghouse Ocean Research Laboratory, Annapolis, Nd.). Such
data indicate that large influxes of polychlorinated biphenyls into chesa-
peake Bay may occur sporadically, with subsequent contamination of the
Atlantic Ocean via tidal outflow.
Levels of polychlorinated biphenyl contamination of the aquatic environment
can be traced directly to domestic and industrial pollution. Duke et al. (7)
reported PCB concentrations in water and sediment reaching 275 pg/i and
486 pg/kg, respectively, in areas directly polluted by PCB leaks from indus-
trial heat exchangers into waters of Escambia Bay, Florida. Elimination of
the source of pollutant resulted in an immediate decline in PCB concentra-
tions in the water column and sediment. It can be concluded that significant
concentrations of the contaminant were subsequently carried to the ocean.
Raicrow et al. (10) reported that PCB content of marine sediment samples col-
lected off the coast of Scotland ranged from 26 pg/kg to 1000 pg/kg, mainly
from sewage and garbage dumping. Recently data were published by Harvey et
al. (13), indicating lower PCB concentrations in the North Atlantic, with
surface water pcB concentrations showing highest levels, i.e., 4.3 pg/i, and
decreasing concentrations with depth.
Contamination of the freshwater environment is no less severe that that of
the marine and estuarine environment. Vieth and Lee (35) reported PCB con-
centrations in Milwaukee River water in the range, .05 pg/i to .1 pg/i.
Waste effluents of the same region carried PCB concentrations from .04 pg/i
to 2.5 pg/l. Crump—Wiesner et al. (5) published results of surveys of PCB
contamination undertaken throughout the United States. Freshwater samples
were found to contain PCB at concentrations of 0.3 pg/i to 4.0 pg/i, whereas
sediment samples were found to contain PCB at concentrations from non-detect-
able levels to 2400 pg/kg.
The published data on PCB occurrence and concentration indicate the ubiqui-
tous nature of P08 contamination in the aquatic environment. Presumably much
of this material is deposited in the sediments, is lost to the atixsphere
through co—distillation at the air—water interface, or accumulates in plant
and/or animal life. Recent evidence suggests that bacterial decomposition
and natural weathering can be a significant factor in the elimination of PCB
from the environment (17, 29). Wong and Kaiser (40) found significant bac-
terial populations resistant to chlorinated biphenyls. hmed and Focht (1)
38
-------
TABLE 16. ENUMERATION OF PCB-RESISTANT BACTERIA FROM CHESAPEA1 BAY
BOP OM WATER AND SEDIMENT SAMPLES
Saxiple PCB
Station typea TVC n tabolisin
Percent
coxrçosition
Total
g
PCB
i -i
BSO1 W -— 6 ——
S 1.0 x 10 i0 2 .01
BS27 w 1.0 x i0 2 10.0
S
BB07 W 1.4 x 10 1.7 x 1O 3 0.2
S 1.7x10 1.2x10 6 7.1
CBBO8 W 1.0 x 107 2.2 x 1O 0.2
S 1.lxlO 1.7x10 16.3
CBBO9 W 4.0 x 10 8.0 x 10 2.0 0.14
S 3.3 x 10 1.0 x 10 0.3 400.0
9180S W 5.5 x 10 3.5 x 1O 6.3
S 1.2x10 4.2x10 3.6
9110F w 1.6 x 1O 1.1 x 10 7.2
S 2.2 x 10 4.5 x 10 20.2
9040N W 6.0 x 10 4.8 x 7.8
S 3.lxlO 6.0x10 5 19.0
8580E W 2.3 x 10 1.6 x 10 0.8
S 6.2 x 10 1.2 x 10 28.0
8180E W 1.8 x 7.3 x 10 38.8 0.01
S 4.1 x io6 8.0 x 10 1.9 ——
7480U W 5.0 x 10 0.0 0.0 0.01
S 2.9 x 10 0.0 0.0 4.0
7140S W 2.2 x 10 5.5 x 10 2.5 0.06
S 2.3 x 10 6.5 x 10 2.8 6.0
6560H W 3.3 x 10 1.2 x 10 3.6 0.01
S 1.5 x 10 1.2 x 10 0.7 125.0
= water; S = sediment.
39
-------
reported the isolation of an Achromobacter sp. from sewage that was capable
of degrading PCB.
In the study reported here, significant numbers of PCB—metabolizing bacteria
were recovered from all of the Chesapeake Bay stations sampled. All of the
samples collected at the southeastern coastal Atlantic Ocean stations also
contained PCB-metabolizing bacteria, although in lower numbers than in the
Bay (Table 17). In the marine environment, PCB resistant bacterial popula—
tions were found to be greater than both the fungal and yeast populations of
Atlantic Ocean surface waters and sediment.
TABLE 17. CORRELATION OF MICROBIAL POPULATIONS WITH PCB CONCENTRATIONS
IN ESTUARINE AND MARINE ENVIRONMENTS SAMPLEDa
Environment
sampled
Correlation coefficient
(r)
TVC
PCB
degraders
PCB
degraders/10 6
TVC
Marine
.54
—.24
—.38
Estuarine
98 C
.62
-.20
surface and/or bottom water.
bSignificant correlation, critical r = .53; a = .05 ..
Csiif. correlation, critical r = .67; a = .05.
To determine whether the -PCB—resistant bacteria were representative of only
one or a few microbial groups, predominantly allochthonous organisms capable
of PCB degradation, or were typical of the larger microbial populations in
the estuary and ocean, pure cultures were identified to genus. A significant
difference was noted in the composition of the marine and estuarine bacterial
flora capable of degrading PCB. Pure cultures of PCB-resistant bacteria iso-
lated from seawater and ocean sediment were predominantly Pseudomonas and
Vibrio spp. Of 44 pure cultures presumptively identified to genus, approxi-
mately 50% were Pseudomonas spp. and the remaining were Vibrio spp. There
was no immediate discernible difference, based on the limited number of iso-
lates examined, in the distribution of these two genera from station to sta-
tion. However, Pseudomonas spp. representing Groups 1 and 2 were isolated
less frequently in samples collected northward along the outer continental
shelf.
A greater diversity of bacterial genera was observed for the strains isolated
from chesapeake Bay samples. Seven bacterial genera were represented amng
the 25 PCB—resistant bacterial strains exandrzed. The small sample size,
multiple sample types arid enrichment methods, and the limited geographical
areas examined prevented further extrapolations concerning generic distribu-
tion. However, it should be noted that Gram positive bacteria were isolated
40
-------
from samples collected at the lower end of the Upper Chesapeake Bay. The oc-
currence of the Gram—positive PCB-resistant bacteria may be related to salin-
ity or nutrient and pollutant influx. However, further study will be
required to resolve this point.
It is apparent from the data that resistance to PCB is not restricted to
strains of bacteria representing a single genus. In addition, all of the
samples collected in this study were found to contain microorganisms capable
of growth on PCB medium. Thus, the bacterial populations of waters may con-
tain a small, but persistent fraction of bacteria capable of PCB degradation.
Even more important, the possibility may exist that a low level of PCB con-
tamination in the environment results in a corresponding level of induced
PGB-resistant bacteria.
In conclusion, PCB contamination was found to be higher in areas of urbaniza-
tion and industrialization. Such contamination was found to decrease with
distance from probable sources of contamination. Although not statistically
significant because of small sample sizes, correlations of PCB levels and
PcB—degrading bacteria suggest that the latter occur ubiquitously, along with
PCB residues. More extensive sampling of areas receiving high levels of PCB
contamination should be undertaken, if the hypothesis that increased numbers
of PcB—degrading bacteria occur as a direct response to increased PCB contam-
ination is to be established. If such a hypothesis is proven, it will then
be possible to develop a PCB-degrading bacterial index of PCB contamination
in the estuary and ocean. The concentrations of PCB observed for samples
examined in the study reported here were relatively low. However, certain
of the sediment samples did contain PCB levels reported by other workers to
be toxic to selected estuarine invertebrates (7). It should be emphasized
that very low concentrations of PCB in the aquatic environment can provide a
reservoir for bioaccumulation via bacteria, zooplankton, and higher life
forms. Experiments in progress in our laboratory suggest that, indeed, such
bioaccumulation can occur.
41
-------
SECTION VI
SUMMARY
Polychiorinated biphenyls (PCB) were found to be present in samples of chesa-
peake Bay and southeastern Atlantic surface water and sediment. PCB concen-
trations ranged from 0.01 pg i to 0.3 pg 11, for surface water, and
4.0 pg g 1 to 400 pg g 1 for sediments. Although detectable in all samples
analyzed in this study, PCB was observed to be correlated with urbanized
areas.
Partitioning of PCB residues in suspended sediments, oil-contaminated sedi-
ments, or surface films may result in elevated PCB levels at some localities.
Under laboratory conditions, both PCB residues and mercury compounds were
effectively partitioned and concentrated by suspended sediments and by petro-
leum hydrocarbons. Under the experimental conditions employed in this study,
up to 99.5% of the PCB in the water column was partitioned into an oil phase
during 24 hr incubation.
Bacteria capable of growth on, or in the presence of, high (500 ppm) concen-
trations of Aroclor 1254 were recovered from all but one sampling area ex-
amined, the latter a station in Chesapeake Bay. The total viable bacterial
population capable of growth on PCB ranged from <.1% to 100%. The variabil-
ity observed in this study was attributed to localized environmental and
nutrient conditions. In Chesapeake Bay, the niunber of bacteria grown in the
presence of PCB was found to be positively correlated with presence of PCB
in the water or sediment.
Acute PCB stress was reflected in the bacteriological quality of the oyster,
Crassostrea virginica . A decrease in depuration of fecal coliforms (E. coli )
and the human enteric bacterial pathogen ( Salmonella typhimuriuxn ) was ob-
served. However, net accumulation of these organisms was not affected by
PCB stress. PCB stress did effect a reduced long-term viability of enteric
bacteria accumulated by the oyster.
PcB—stressed soft shell clams, Mya arenaria , accumulated >10—fold more Salmo-
nella enteritidis , relative to the water column concentration, than did con-
trol clams. Depuration rates, however, for both stressed and control animals
remained approximately the same. As in the case of the oyster, long-term in
vivo survival of Salmonella was reduced in stressed clams, resulting in a
superficially improved bacteriological quality of the shellfish.
42
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45
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. 2.
EPA—600/3—77—070
3. RECIPIENT’S ACCESSIO +NO.
4. TITLE AND SUBTITLE
Effects and Interactions of Polychiorinated
Biphenyls (PCB) with Estuarine Microorganisms and
Shellfish
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTI-IOR(S)
Rita R. Coiwell and Gary S. Sayler
8. PERFORMING ORGANIZATION REPORT NO.
ERLI GB 324
9. PERFORMING ORGANIZATION NAME AND ADDRESS
University of Maryland
Department of Biology
College Park, Maryland 20742
10. PROGRAM ELEMENT NO.
1EA615
11.CONTRACT/GRANTNO.
R—803300—Ol- -0
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
final
14.SPONSORINGAGENCYCODE
EPA/ORD
15. SUPPLEMENTARY NOTES
16. p rc c
The role of estuarine bacteria in the mobilization, transport, and removal of poly—
chlorinated biphenyls (PCB) was investigated in estuarine environments. A main ob-
jective of this investigation was to determine a secondary impact of PCB contamina-
tion of estuarine systems. The specific secondary effect was the PCB—stress—induced
accumulation and depuration of enteric bacteria by shellfish, i.e., the Chesapeake
Bay oyster, Crassostrea virginica .
For this report, bacteria uninhibited by’PCB, but capable of growth in the presence of
PCB, are defined as PCB—resistant. In this regard, PCB—resistant bacteria were found
to be distributed ubiquitously throughout estuarine and marine environments sampled
in this study. The residence time of PCB in estuarine and marine environments Is
concluded to be sufficiently long to induce stress upon estuarine animals.
17. KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
C. COSATI Field/Group
PCB contamination
Effects of Bacteria
Marine Bacteria—PCB Interactions
Shellfish—Microorganisin—PCB Interactions
PCB—resistant Bacteria
Estuarine Bacteria
Shellfish Depuration
and Accumulation
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