UPTAKE DEnillATn,
'
FIELD VERIFICATION PROGRAM
(AQUATIC DISPOSAL)
TECHNICAL REPORT D-85-2
BIOACCUMULATION OF CONTAMINANTS FROM
BLACK ROCK HARBOR DREDGED MATERIAL
BY MUSSELS AND POLYCHAETES
by
James Lake, Gerald L. Hoffman. Steven C. Schimmei
Environmental Research Laboratory
US Environmental Protection Agency
Narragansett. Rhode Island 02882
February 1985
Final Report
Approved For Pub'
prepared tor DEPARTMENT OF THE ARMY
US Army Corps of Engineers
Washington, DC 20314-1000
and US Environmental Protection Agency
Washington, DC 20460
r^ by Environmental Laboratory
US Army Engineer Waterways Experiment Station
PO Box 631, Vicksburg, Mississippi 39180-0631
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Destroy this report when no longer needed. Do not return
it to the originator.
The findings in this report are not to be construed as an official
Department of the Army position unless so designated
by other authorized documents.
The contents of this report are not to be used for
advertising, publication, or promotional purposes.
Citation of trade names does not constitute an
official endorsement or approval of the use of
such commercial products.
The D-series of reports includes publications of the
Environmental Effects of Dredging Programs:
Dredging Operations Technical Support
Long-Term Effects of Dredging Operations
Interagency Field Verification of Methodologies for
Evaluating Dredged Material Disposal Alternatives
(Field Verification Program)
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Unclassified
SECURITY CLASSIFICATION OF THIS PAGE (Wim r>nta Kutarnd)
REPORT DOCUMENTATION PAGE
READ INSTRUCTIONS
BKI'OKE COMPUKTINO KORM
1. REPORT NUMBER
Technical Report D-85-2
2. GOVT ACCESSION NO
3. RECIPIENT'S CATALOG NUMBER
4. TITLE (and Subtitle)
BIOACCUMULATION OF CONTAMINANTS FROM BLACK
ROCK HARBOR DREDGED MATERIAL BY MUSSELS AND
POLYCHAETES
5. TYPE OF REPORT a PERIOD COVERED
Final report
6. PERFORMING ORG. REPORT NUMBER
7. AUTHORf")
James Lake, Gerald L. Hoffman,
Steven C. Schimmel
8. CONTRACT OR GRANT NUMBER(«)
9. PERFORMING ORGANIZATION NAME AND ADDRESS
US Environmental Protection Agency
Environmental Research Laboratory
Narragansett, Rhode Island 02882
10. PROGRAM ELEMENT, PROJECT, TASK
AREA a WORK UNIT NUMBERS
Field Verification Program
(Aquatic Disposal)
II. CONTROLLING OFFICE NAME AND ADDRESS
DEPARTMENT OF THE ARMY, US Army Corps of
Engineers, Washington, DC 20314-1000 and
US Environmental Protection Agency,
Washington, DC 20460
12, REPORT DATE
February 1985
13. NUMBER OF PAGES
150
T4. MONITORING AGENCY NAME « ADORESSf// dlllerent from Controlling Olllce)
US Army Engineer Waterways Experiment Station
Environmental Laboratory
PO Box 631, Vicksburg, Mississippi 39180-0631
15. SECURITY CLASS, (at thle report)
Unclassified
ISa.
DECLASSIFI CATION/ DOWN GRADING
SCHEDULE
16. DISTRIBUTION STATEMEN T (at thle Report)
Approved for public release; distribution unlimited.
17. DISTRIBUTION STATEMENT (of the abstract entered In Block 30, It different from Report)
18. SUPPLEMENTARY NOTES
Available from National Technical Information Service, 5285 Port Royal Road,
Springfield, Virginia 22161. Appendices A and B are on microfiche and are
enclosed in a pocket attached to the back cover.
19. KEY WORDS (Continue on reveree title it neceateiy end Identify by block number)
Absorption (Physiology) (LC)
Dredged materials—research (LC)
Marine pollution—measurement (LC)
Polychlorinated biphenyls (LC)
20. ABSTRACT (Continue an rerere* aMb H n*c+eemr? fad Identity by block number)
Mussels (Mytilus edulis) and worms (Nereis virens) were exposed in
laboratory studies to dredged material from Black Rock Harbor (BRH),
Connecticut, to examine the bioaccumulation of organic and inorganic contam-
inants. Mussels were exposed in a dosing system designed to maintain a
constant concentration of suspended particulates and food (algae) in seawater.
Control mussels received only food (algae). Monitoring of concentrations
(Continued)
DO ,^1473
EDITION OF I MOV S» IS OBSOLETE
Unclassified
SECURITY CLASSIFICATION OF THIS PAriE (When Data Entered)
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Unclassified
SECURITY CLASSIFICATION OF THIS PAGE(H7l«n Dflf Entarfd)
20. ABSTRACT (Continued).
of organic and inorganic contaminants showed that the system maintained
constant concentrations during exposure. Exposed mussels accumulated organic
compounds and some inorganic elements, reaching steady-state values between the
first and second weeks of exposure. During the 28-day exposure period, mussels
showed increases in concentration of two to three orders of magnitude for
organic contaminants, but those metals accumulated showed increases of less
than a factor of 12.
In general, the depuration of organic contaminants was rapid during the
first week of depuration, and the depuration rate was inversely related to
the compound's n-octanol/water partition coefficient. After the first week
depuration rates decreased, and concentrations of most organic compounds re-
mained above control values to the end of the 5-week depuration period. Iron
and chromium depurated to control levels within a 2-week period.
The polychaete worm ^N. virens was exposed to BRH bedded sediment in glass
aquaria maintained under flowing seawater. Other worms were maintained in
reference sediments. Worms exposed for 28 days accumulated polychlorinated
biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) to concentrations
one to three orders of magnitude above those found in the reference organisms.
Of the metals determined, only Cr and Cu were found to accumulate to concen-
trations higher than those in the reference worms. Concentrations of PCBs
exposed to BRH sediment did not decrease during a 28-day depuration period in
reference sediments, but depuration of PAHs was apparent. Chromium and copper
depurated to control levels after 2 weeks.
Bioaccumulation factors for PCBs calculated for mussels and worms, when
total exposure concentrations were normalized to a gram dry weight sediment
basis, were generally within a factor of 1.5. This suggests that modeling
bioaccumulation of some organic compounds as a partitioning of contaminants
between sediments and organisms may have promise as a generalized predictive
technique.
Unclassified
SECURITY CLASSIFICATION OF THIS PAGEf»7i»n Dfta Entered)
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SUBJECT: Transmittal of Field Verification Program Technical Report Entitled
"Bioaccumulation of Contaminants from Black Rock Harbor Dredged
Material by Mussels and Polychaetes"
TO: All Report Recipients
1. This is one in a series of scientific reports documenting the findings of
studies conducted under the Interagency Field Verification of Testing and
Predictive Methodologies for Dredged Material Disposal Alternatives (referred
to as the Field Verification Program or FVP). This program is a comprehensive
evaluation of environmental effects of dredged material disposal under condi-
tions of upland and aquatic disposal and wetland creation.
2. The FVP originated out of the mutual need of both the Corps of Engineers
(Corps) and the Environmental Protection Agency (EPA) to continually improve
the technical basis for carrying out their shared regulatory missions. The
program is an expansion of studies proposed by EPA to the US Army Engineer
Division, New England (NED), in support of its regulatory and dredging mis-
sions related to dredged material disposal into Long Island Sound. Discus-
sions among the Corps' Waterways Experiment Station (WES), NED, and the EPA
Environmental Research Laboratory (ERLN) in Narragansett, RI, made it clear
that a dredging project at Black Rock Harbor in Bridgeport, CT, presented a
unique opportunity for simultaneous evaluation of aquatic disposal, upland
disposal, and wetland creation using the same dredged material. Evaluations
were to be based on technology existing within the two agencies or developed
during the six-year life of the program.
3. The program is generic in nature and will provide techniques and inter-
pretive approaches applicable to evaluation of many dredging and disposal
operations. Consequently, while the studies will provide detailed site-
specific information on disposal of material dredged from Black Rock Harbor,
they will also have great national significance for the Corps and EPA.
4. The FVP is designed to meet both Agencies' needs to document the effects
of disposal under various conditions, provide verification of the predictive
accuracy of evaluative techniques now in use, and provide a basis for deter-
mining the degree to which biological response is correlated with bioaccumula-
tion of key contaminants in the species under study. The latter is an
important aid in interpreting potential biological consequences of bioaccumu-
lation. The program also meets EPA mission needs by providing an opportunity
to document the application of a generic predictive hazard-assessment research
strategy applicable to all wastes disposed in the aquatic environment. There-
fore, the ERLN initiated exposure-assessment studies at the aquatic disposal
site. The Corps-sponsored studies on environmental consequences of aquatic
disposal will provide the effects assessment necessary to complement the EPA-
sponsored exposure assessment, thereby allowing ERLN to develop and apply a
hazard-assessment strategy. While not part of the Corps-funded FVP, the EPA
exposure assessment studies will complement the Corps' work, and together the
Corps and the EPA studies will satisfy the needs of both agencies.
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SUBJECT: Transmlttal of Field Verification Program Technical Report Entitled
"Bioaccumulation of Contaminants from Black Rock Harbor Dredged
Material by Mussels and Polychaetes"
5. In recognition of the potential national significance, the Office, Chief
of Engineers, approved and funded the studies in January 1982. The work is
managed through the Environmental Laboratory's Environmental Effects of
Dredging Programs at WES. Studies of the effects of upland disposal and
wetland creation are being conducted by WES and studies of aquatic disposal
are being carried out by the ERLN, applying techniques worked out at the
laboratory for evaluating sublethal effects of contaminants on aquatic organ-
isms. These studies are funded by the Corps while salary, support facilities,
etc., are provided by EPA. The EPA funding to support the exposure-assessment
studies followed in 1983; the exposure-assessment studies are managed and
conducted by ERLN.
6. The Corps and EPA are pleased at the opportunity to conduct cooperative
research and believe that the value in practical implementation and improve-
ment of environmental regulations of dredged material disposal will be con-
siderable. The studies conducted under this program are scientific in nature
and will be published in the scientific literature as appropriate and in a
series of Corps technical reports. The EPA will publish findings of the
exposure-assessment studies in the scientific literature and in EPA report
series. The FVP will provide the scientific basis upon which regulatory
recommendations will be made and upon which changes in regulatory implementa-
tion, and perhaps regulations themselves, will be based. However, the docu-
ments produced by the program do not in themselves constitute regulatory
guidance from either agency. Regulatory guidance will be provided under
separate authority after appropriate technical and administrative assessment
of the overall findings of the entire program.
ChoromokosV Jr77~?fc.D., P.E.
Director, Research and Development
U. S. Army Corps of Engineers
Bernard D. Goldstein, M.D.
Assistant Administrator for
Research and Development
U. S. Environmental Protection
Agency
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PREFACE
This report describes work performed by the U.S. Environmental Protec-
tion Agency (EPA), Environmental Research Laboratory, Narragansett, R.I.
(ERLN), as part of the Interagency Field Verification of Testing and Predic-
tive Methodologies for Dredged Material Disposal Alternatives Program or the
Field Verification Program (FVP). The FVP, sponsored by the Office, Chief of
Engineers (OCE), is assigned to the Environmental Laboratory (EL), U. S. Army
Engineer Waterways Experiment Station (WES), and is managed under the Environ-
mental Effects of Dredging Programs (EEDP). The OCE Technical Monitors for
FVP were Dr. John R. Hall and Dr. William L. Klesch.
The objective of the FVP is to verify existing predictive techniques for
evaluating the environmental consequence of dredged material disposal under
aquatic, wetland, and upland conditions. The aquatic portion of this study is
being conducted by ERLN, with the wetland and upland portions conducted by WES.
The principal ERLN investigators for this aquatic study were Drs. James
Lake and Gerald Hoffman, Analytical Chemists, and Mr. Steven Schimmel, Aquatic
Toxicologist. Laboratory exposure system design was coordinated by Mr. Jay
Sinnett and assisted by Ms. Dianne Black, Dr. Wayne Davis, and Mr. John Sewall.
Organic chemical sample preparation and analyses were conducted under the
supervision of Drs. Lake and Rogerson, and assisted by Mr. Curt Norwood,
Ms. Sharon Pavignano, Mr. Robert Bowen, Ms. Adria Elskus, and Mr. Lawrence
LeBlanc. Inorganic chemical preparation and analyses were conducted under the
supervision of Dr. Gerald Hoffman, and assisted by Mr. Frank Osterman,
Mr. Warren Boothman, and Mr. Dennis Migneault. Data management and data analy-
sis were conducted by Mr. Jerfrey Rosen and Dr. James Heltshe, respectively.
The EPA Technical Director for the FVP was Dr. John H. Gentile; the
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Technical Coordinator was Mr. Walter Galloway, and the Project Manager was
Mr. Allan Beck.
The study was conducted under the direct WES supervision of
Dr. Richard K. Peddicord and Dr. Thomas Dillon and under the general super-
vision of Dr. C. Richard Lee, Chief, Contaminant Mobility and Criteria Group;
Mr. Donald L. Robey, Chief, Ecosystem Research and Simulation Division;
Dr. John Harrison, Chief, EL. The EEDP Coordinator was Mr. Robert L. Lazor.
The EEDP Manager was Mr. Charles C. Calhoun.
Commanders and Directors of WES during preparation of the report were
COL Tilford C. Creel, CE, and COL Robert C. Lee, CE. Technical Director was
Mr. F. R. Brown.
This report should be cited as follows:
Lake, J., Hoffman, G., and Schimmel, S. 1985. "Bioaccumulation
of Contaminants From Black Rock Harbor Dredged Material by Mussels
and Polychaetes," Technical Report D-85-2, prepared by the US
Environmental Protection Agency, Environmental Research Laboratory,
Narragansett, R. I., for the US Army Engineer Waterways Experiment
Station, Vicksburg, Miss.
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CONTENTS
Page_
PREFACE 1
LIST OF FIGURES 4
LIST OF TABLES 7
PART I: INTRODUCTION 9
Background 9
Purpose and Scope « 9
PART II: MATERIALS AND METHODS 12
Sediment Collection and Preservation 12
Test Species 15
Mussel Bioaccumulation Study 16
Worm Bioaccumulation Study 23
Chemical Analysis 26
PART III: RESULTS AND DISCUSSION 38
Mussel Test 38
Worm Test 109
PART IV: SUMMARY 138
Mussel Bioaccumulation Study • 138
Worm Bioaccumulation Study 142
Bioaccumulation Mussels and Worms 143
PART V: RECOMMENDATIONS . 145
Mussels • 145
Worms 145
General ..... 146
REFERENCES « • 147
APPENDIX A: ORGANIC CHEMISTRY DATA *
APPENDIX B: INORGANIC CHEMISTRY DATA *
* Appendices A and B were reproduced on microfiche; they are
enclosed in an envelope attached inside the back cover of this report-
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LIST OF FIGURES
PAGE
Figure 1. Central Long Island Sound disposal site and south
reference Site (41°7.95' N and 72° 52.7'West) 13
Figure 2. Black Rock Harbor, Connecticut (73°13'W and 41° 90'N),
source of dredged material 14
Figure 3. Sediment dosing system with chilled water bath and argon
gas supply 17
Figure 4. Suspended sediment feedback control loop and strip chart
recorder 19
Figure 5. Blue mussel (Mytilus edulis) contaminant uptake system. * . 20
Figure 6. Capillary column electron capture gas chromatograms
of PF-50 (PCB) fractions from day 8 exposure,
a. unfiltered water, b. filtrate, c. filter, d. water
through continuous flow centrifuge (14,000 rpm) 45
Figure 7. Capillary column electron capture gas chromatograms
of PF-50 (PCB) fractions from day 8 exposure,
a. filtrate, b. water through continuous flow centrifuge
(14,000 rpm) 47
Figure 8. Capillary column electron capture gas chromatogram
of PF-50 (PCB) fraction from mussels, time 0 49
Figure 9. Capillary column electron capture gas chromatograms
of PF-50 (PCB) fractions from mussels, a. time 0,
b. day 28 exposure 52
Figure 10. Capillary column electron capture gas chromatograms
of PF-50 (PCB) fractions from exposure, a. unfiltered
water, day 8, b. mussel, day 28 53
Figure 11. Capillary column electron capture gas chromatograms
of PF-50 (PCB) fraction from exposure mussels, a. day
28, b. day 70 54
Figure 12. Concentration of total PCBs (as A-1254) in mussels
exposed to BRH sediment versus time. . . • 58
Figure 13. Capillary column electron capture gas chromatograms
of PF-50 (PCB) fraction from exposure, a. filter, day
8, b. filtrate, day 8, and c. mussels, day 28 62
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FIGURES (continued)
PAGE
Figure 14. Capillary column electron capture gas chromatograms
of PF-50 (PCB) fraction from exposure, a. filter, day
8, and b. mussels, day 28 65
Figure 15. Capillary column electron capture gas chromatograms
of PF-50 (PCB) fractions from mussels, a. time
0, b. control, day 28, and c. control, day 70 66
Figure 16. EICPs from GC/MS analysis of exposure tank
a. unfiltered water, b. filter, c. filtrate,
d. water through continuous flow centrifuge 71
Figure 17. EICPs from GC/MS analysis of a. BRH sediments,
b. mussels, day 0, c. exposure tank mussels,
day 1.8, d. exposure tank mussels, day 3.5 76
Figure 18. EICPs from GC/MS analysis of exposure tank mussels
from a. day 7, b. day 14, c. day 21, d. day
28 77
Figure 19. EICPs from GC/MS analysis of exposure tank mussels
from a. day 35, b. day 40, c. day 49, d. day
56 78
Figure 20. EICPs from GC/MS analysis of exposure tank mussels
from a. day 63, b. day 70 79
Figure 21. Concentration of sum of parent PAHs in mussels
exposed to BRH sediment versus time 85
Figure 22. Capillary column flame ionization detector gas
chromatograms of PF-50 fraction from a. exposure
tank water, day 0, b. BRH sediment, and
c. mussels, exposure day 28 87
Figure 23. Concentration of total petroleum hydrocarbons in
mussels exposed to BRH sediment versus time 89
Figure 24. Uptake and depuration of Fe, and Cr in mussels
exposed to BRH sediment 101
Figure 25. Uptake and depuration of Pb, and Cd in mussels
exposed to BRH sediment 102
Figure 26. Uptake and depuration of Cu, and As in mussels
exposed to BRH sediment 103
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FIGURES (continued)
PAGE
Figure 27. Uptake and depuration of Zn, and Mn in mussels
exposed to BRH sediment 104
Figure 28. Capillary column electron capture gas chromatograms
of PF-50 (PCB) fraction for BRH sediment, and
reference sediment 108
Figure 29. Capillary column electron capture gas chromatograms
of PF-50 (PCB) fraction for worms exposed to
BRH sediment a. day 0, b. day 14, and c. day
28 Ill
Figure 30. Capillary column electron capture gas chromato-
grams of PF-50 (PCB) fraction for worms exposed
to BRH sediment a. day 42, and b. day 56 112
Figure 31. Concentration of total PCBs (as A-1254) in worms
exposed to BRH sediment versus time 113
Figure 32. Concentration of sum of parent PAHs in worms
exposed to BRH sediment versus time 119
Figure 33. Capillary column flame ionization detector
gas chromatogram of PF-50 fraction from
worms exposed to BRH sediment for
28 days 122
Figure 34. Concentration of total petroleum hydrocarbons
in worms exposed to sediment verus time 123
Figure 35. Uptake and depuration of Fe, and Cr in worms
exposed to BRH sediment 133
Figure 36. Uptake and depuration of Cu, Zn and Cd in
worms exposed to BRH sediment 135
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LIST OF TABLES
PAGE
1. Summary of Experimental Conditions for Bioaccumulation
Study with Mytilus edulis ...................... 24
2. Mussel Dosing System; PCB Levels in Unfiltered Water ........ 39
3. PCB Blank Levels - Mussel Dosing System Samples ........... 40
4. Mussel Dosing System; PCB Levels in Water -Expo sure Tank ....... 42
5. Tentative Identifications of Compounds in BCD Gas
Chromatograms ............................ 43
6. Comparison of Experimental and Estimated Kp's ............ 48
7. Average Trace Metal Concentrations for Mussels Collected from
the Exposure Chamber on Day 28 ................... 56
8. Estimated and Measured Bioconcentration Factors (BCF) in Mussels
at Day 28 ....................... • ...... 59
9. Measured Log BAF for Each Separate PCB Peak in Exposed Mussels. . . 61
10. PAH and Ethylan Concentrations in Unfiltered Water Samples
(in Parts per Trillion) ....................... 70
11. PAH Compounds in Mussels ...................... 72
12. Comparison of Experimental and Estimated Sediment /Water
Partition Coefficients (Kp's) .............. ..... 75
13. Estimated and Measured Bioconcentration Factors (BCF) in Mussels
at Day 28 ............................. 80
14. PAH and Ethylan Concentrations in Exposed Mussels Expressed as
ng/g (dry) ......... .................... 82
15. Mussel Bioaccumulation Factors (Calculated for Day 28) ....... 83
16. Levels of PAH and Ethylan Compounds in Control Mussels During
Study ................................ 84
17. Average Trace Metal Concentrations for Black Rock Harbor
Sediment Samples .......................... 9°
18. Seawater Metal Concentrations Determined for the Black Rock
Harbor Sediment Exposure and Control Chambers ............ 91
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TABLES (continued)
PAGE
19. Average Fe/Metal Ratios for the Exposure Chamber
Seawater and Black Rock Harbor Sediments . . 93
20. Metal Bioaccumulation Factors for Mussels Exposed
to Black Rock Harbor Sediments 98
21. Average Fe/Metal Ratios for Control Mussels and
Black Rock Harbor Sediments 99
22. PCB Bioaccumulation Factors, Exposed Worms Day 28 116
23. PCB Bioaccumulation Factors of Worms in Reference
Sediment-Day 28 117
24. PAH and Ethylan Bioaccumulation Factors - Worms 120
25. Mussel Bioaccumulation Factors Calculated from Filters. . . . 125
26. Comparison of BAFs from Mussels and Worms 127
27. Average Trace Metal Concentration for Worms Collected
from the Exposure Chamber on Day 28 131
28. Metal Bioaccumulation Factors for Worms Exposed to
Black Rock Harbor Sediment 136
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BIOACCUMULATION OF CONTAMINANTS
FROM BLACK ROCK HARBOR DREDGED MATERIAL
BY MUSSELS AND POLYCHAETES
PART I: INTRODUCTION
Background
1. The U.S. Army Corps of Engineers (CE) and the U.S. Environmental
Protection Agency (EPA) are jointly conducting a comprehensive Field
Verification Program (FVP) to evaluate the potential environmental impact
associated with various disposal options for dredged material. The
approach being used in the FVP is to evaluate and field validate assessment
methodologies for predicting the environmental impacts of dredged material
disposal in aquatic, upland, and wetland environments. The research,
evaluation, and field verification of the upland and wetland disposal
options are being conducted by the Environmental Laboratory, U.S. Army
Engineer Waterways Experiment Station (WES), Vicksburg, Miss. The
application and field verification of predictive methodologies for the
aquatic disposal option will be conducted by the EPA Environmental
Research Laboratory (ERLN), Narragansett, R.I.
Purpose and Scope
2. The aquatic disposal option of the FVP is to be used as a site-
specific case study for evaluating a hazard assessment research strategy.
Hazard assessment in terms of this study is a process by which data on
exposure and effects are assembled and interpreted to determine the
potential for harm to the aquatic environment that could result from the
ocean disposal of a particular material. To measure hazard, information
on the duration and intensity of exposure (exposure assessment) of organisms
to concentrations of materials disposed at the site (predicted environmental
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concentration) is coupled with concentrations of the material determined
from laboratory toxicity studies (effects assessment) on individual
species, populations, and communities. When properly synthesized, these
data provide an estimate of the probability of unacceptable adverse
impact on the aquatic environment as a result of the disposal of the
material. The verification of hazard assessment is comprised of two
components: (a) documentation and comparison of the accuracy and precision
of an individual method or protocol in the lab and field, and (b) verifi-
cation of the prediction of potential impact to the aquatic environment.
Within this context, hazard assessment contains parallel predictive
laboratory and field verification components. The achievement of the goal
of hazard assessment requires the development and verification of assessment
protocols for defining exposure and effects.
3. The second research component in the aquatic portion of the FVP
is an assessment of the bioaccumulation potential of available contaminants
within the dredged material by the blue mussel (Mytilus edulis) and the
polychaete worm Nereis virens. The focus of this study is twofold!
(a) determine the qualitative and quantitative aspects of the bioavailable
contaminants within BRH dredged material which are accumulated by the
mussel and the worm; and (b) examine the uptake and depuration kinetics
of the major contaminants within the material that constitute a potential
threat to man and the ecosystem. Results of this study will contribute
to the overall FVP by providing a predictive tool for predicting residues
of key contaminants in the fauna at the disposal site. The accuracy
of these predictive tools will be verified in the field and reported in
a future report.
10
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4. Chemicals of major environmental concern have three basic
characteristics: (a) they may be acutely or chronically toxic at low
concentrations; (b) they may bioaccumulate to concentrations in tissues
that cause adverse effects in the species contaminated with the
chemical, or otherwise make the species unsuitable for human consumption;
and (c) they may depurate slowly, causing a prolonged (chronic) adverse
effect or render the resource unsuitable for prolonged periods. The
latter two concerns are addressed in this report. The study of uptake
and depuration rates of the major bioavailable compounds and elements by
the organisms allows predictions to be made of the rate and extent of
chemical uptake and the time needed to depurate accumulated compounds
to an acceptable concentration.
5. Appendices A and B contain organic and inorganic chemistry
data, respectively. Because of the extent of the accumulated data, they
were reproduced on microfiche and are enclosed in an envelope attached
to the inside back cover of this report.
11
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PART II: MATERIALS AND METHODS
Sediment Collection and Preservation
fc
Reference Sediment
6. Reference sediment (REF) for the FVP studies was collected from
the South reference site (41°7.95'N and 72°52.7'W), which is approximately
700 m south of the southernmost perimeter of the central Long Island
Sound disposal site (Figure 1). Reference sediment was collected with a
Smith-MacIntyre grab sampler (0.1 m^) in both August and December 1982.
Sediment collected on each date was returned to the laboratory, press
sieved (wet) within 48 hr through a 2-mm mesh stainless steel screen,
homogenized, and stored at 4°C until used for experimental purposes.
Sediment was re-homogenized prior to use.
Black Rock Harbor Sediment
7. The source of the dredged material for the FVP was Black Rock
Harbor (BRH), located in Bridgeport, Connecticut (Figure 2), with
approximate coordinates of 73°13'W and 41°9'N. The study reach begins
400 m south of the fork in Cedar Creek and extends seaward for approximately
1700 m. Black Rock Harbor bottom sediments were collected at 25 locations
within the study area using a 0.1-m2 gravity box corer to a depth of
1.21 m and placed in 210-L barrels and transported in a refrigerated
truck (at 4°C) to WES. The contents of the 25 barrels were emptied
Into a nitrogen-purged cement mixer and homogenized. The homogenized
sediment was then redistributed to the 25 barrels and aliquots were
taken from each for sediment chemistry analysis. Twelve barrels were
kept at WES and thirteen barrels were transported to ERLN in a
refrigerated truck and stored at 4°C. Prior to use the contents of each
12
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NEW
HAVEN
BRIDGEPORT
BLACK ROCK
HARBOR
LONG ISLAND :
FVP
DISPOSAL
SITE
SOUTH REFERENCE
• SITE
Figure 1. Central Long Island Sound disposal site and south reference
site (41°7.95"N and 72°52.7"W)
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BRIDGEPORT
N
N
MAINTENANCE
DREDGING
FVP STUDY
REACH
BLACK
ROCK
HARBOR
\\
400m
Figure 2. Black Rock Harbor, Connecticut (73°13''W and 4l°90"N),
source of dredged material
14
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barrel were completely homogenized and wet seived through a 1-mm mesh
seive to remove large particles. Sediment was stored in glass bottles
at 4°C. To verify that the contents in the bottles were consistent,
400-ml samples were taken before the 1st, 25th, and 50th bottles for
moisture content and chemical analysis.
Test Species
8. Two species of marine invertebrates were used to conduct two
separate bioaccumulation studies, including depuration phases. A bivalve
mollusc, the blue mussel, Mytilus edulis, and the polychaete worm,
Nereis virens, were used in this study.
Mytilus edulis
9. The blue mussel is a filter-feeding bivalve mollusc that
ranges along the northern Atlantic coast of the United States and
Europe. In the United States, it ranges from Maine to North Carolina
and on the Pacific coast from Alaska to California (Bayne 1976). Mytilus
edulis was selected for this study because it is a filter-feeding mollusc,
capturing food as suspended particulates. Species of Mytilus have been used
extensively as a biological monitor worldwide (Farrington et al. 1983)
and its biology has been studied extensively.
10. One month prior to exposure, adult mussels were collected
from a well-characterized area of Narragansett Bay, Rhode Island, with
relatively low background concentrations of contaminants in the sediments
(Phelps et al. 1983; Phelps and Galloway 1980). Test organisms, 50 to 70 mm
shell length, were temperature acclimated from 5° to 10°C at the rate of
1°C per day, then held in unfiltered flowing seawater (28 to 30 °/oo
salinity) until initiation of the experiment.
15
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Nereis virens
11. Nereis virens is a marine polychaete worm that inhabits the
coastal United States from the Gulf of St. Lawrence to the Gulf of Mexico
on the east coast and the central California coast on the Pacific Ocean
(Pettibone 1963). They are raptorial and deposit feeders but generally
opportunistic in their feeding habits. This species was selected because
of its deposit-feeding habits (it will feed directly on sediment
constituents), its relatively large size, and its availability.
Approximately 600 adult worms were purchased from a bait dealer in Wiscasset,
Maine, packed in wet seaweed, and shipped to ERLN. Upon arrival at the
laboratory, they were immediately placed in sediment for testing.
Mussel Bioaccumulation Study
Sediment Dosing System
12. A sediment dosing system was constructed to provide BRH as sus-
pended sediment for the mussel bioaccumulation study (Figure 3). The
dosing system consisted of a conical-shaped slurry reservoir placed in a
chilled fiberglass chamber, a diaphragm pump, a 4-L separatory funnel,
and several return loops that directed the particulate slurry through a
dosing valve. The slurry reservoir (40 cm diameter x 55 cm high) contained
40-L of slurry comprised of 37.7 L of filtered seawater and 2.3 L of
BRH material. The slurry was changed every 2-3 days during exposure.
The fiberglass chamber (94 cm x 61 cm x 79 cm high), was maintained
between 4° and 10°C using an externally chilled water source. (The slurry
was chilled to minimize microbial degradation during the test.) A polypro-
pylene pipe (3.8 cm diameter) placed at the bottom of the reservoir
16
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ARGON
INJECTION
SEPARATORY
FUNNEL
DELIVERY
MANIFOLD
CHILLED
WATER BATH
DOSING
VALVE
TO EXPOSURE
SYSTEM
RETURN
MANIFOLD
SLURRY
RESERVOIR
Figure 3. Sediment dosing system with chilled water bath and argon
gas supply
17
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cone was connected to the diaphragm pump (16- to 40-L/mln capacity)
that had a Teflon® diaphragm. This pump was used to circulate the
slurry with minimal abrasion so that the physical properties and particle
sizes of the material remained as unchanged as possible. The separatory
funnel was connected to the pump and returned to the reservoir by
polypropylene pipes. The separatory funnel served two functions: (a)
to ensure that a constant head pressure was provided at the overflow,
and (b) to serve as a connection for the manifold located 4 cm below the
constant head level. The manifold served to distribute the slurry by
directing a portion of the flow from the funnel, through 6-mm-inside
diameter polypropylene tubes through the Teflon® dosing valves (Figures
3 and 4) and back to the reservoir. At the dosing valves, the slurry
was mixed with Narragansett Bay seawater which had been filtered (to 15 y)
through sand filters. The valves were controlled by a microprocessor that
was connected to a transmissometer (Figure 4). Under transmissometer
control, the microprocessor responds by modulating the pulse length to
achieve the desired setpoint of suspended sediment measured as turbidity
(Sinnett and Davis 1983).
Mussel Exposure System
13. The system used to expose blue mussels to BRH material in the
bioaccumulation test is shown in Figure 5. The exposure apparatus
consisted of a fiberglass, resin-coated plywood tank (123-L capacity)
partitioned into two components. Filtered seawater entered the mixing
chamber at 2 L/min where it was vigorously combined with the BRH material
and marine algae as a food source (a mixture of Phaeodactylum tricornutum
and T-Isochrysis galbana). The mixture cascaded over a partition into
18
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STRIP CHART
RECORDER
RETURN TO
RESERVOIR
MICRO-
PROCESSOR
CONTROL
BOX,
I
SLURRY
sssssssa
DOSING VALVE
SOLENOID
EXPOSURE SYSTEM
\
TRANSMISSOMETER
Figure 4. Suspended sediment feedback control loop and strip chart
recorder
19
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SEAWATER/SEDIMENT
SLURRY
ALGAE
TO MICROPROCESSOR
TRANSMISSOMETER
MIXING
CHAMBER
RECIRCULATING
PUMP
I
Figure 5. Blue mussel (Mytilus edulis) contaminant uptake system
20
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the exposure chamber containing the mussels and a transmissometer which
measured the amount of suspended particulates in the water. To ensure
that the particles were rapidly and evenly dispersed throughout the
tank, water was collected through a manifold near the transmissometer
and returned to the mixing chamber at a rate of 38 L/min. Polypropylene
or polyethylene plumbing materials were used throughout.
14. The sediment dosing system delivered BRH sediment directly into
the mussel exposure chamber via the dosing valve which was controlled by
the microprocessor and transmissometer. As the mussels removed the suspended
particles to a level below the desired concentration, the microprocessor
simultaneously opened the dosing valve to deliver the BRH suspension and
turned on a peristaltic pump to deliver algae to the chamber. Delivery
volumes by the valve and peristaltic pump were adjusted to maintain a
constant ratio of sediment and algae during a microprocessor pulse. In
response to a transmissometer signal every 5 min, the microprocessor
modulated the pulse length to achieve an exposure concentration in the
chamber of 9.5 mg/L of suspended particles, consisting of 9 mg/L
sediment and 0.5 mg/L algae (30 million cells/L). This concentration
of suspended sediments was estimated to be below the concentration
that would stress or adversely affect the organisms during the test
because a preliminary test demonstrated no appreciable mortality, histo-
pathological responses, or adverse changes in scope for growth (SFG) after
2 weeks of exposure to 20 mg/L.
15. The control for this experiment was designed to ensure that
contaminants observed in the mussels were accumulated from BRH material
rather than from the seawater or the algal cultures. The control exposure
21
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was conducted in an identical test apparatus, but no sediment was
delivered to the chamber. Instead, a suspended particulate concentration
of 0.5 mg/L consisting entirely of algae was maintained by the micro-
processor feedback system.
Experimental Conditions
16. Whenever possible, the general bioconcentration test methods
used were from Proposed Standard Practice for Conducting Bioconcentration
Tests with Fishes and Saltwater Bivalve Molluscs (American Society
for Testing and Materials (ASTM) 1982). Although not specifically
intended for suspended sediment testing, the general recommendations
defining test animal care, handling, acclimation procedures, seawater
quality, and acceptable exposure conditions were suitable for this test.
17. At the start of the bioaccumulation study, 300 mussels
were initially placed in each of the BRH and control chambers. Before
placing the animals in the test chamber, 20 animals were randomly
selected for organic and inorganic chemical analysis to determine the
baseline residues in the mussels before the exposures began. During
the test, 20 mussels were sampled for chemical analysis on days 1.8,
3.5, 7, 14, 21, and 28 during exposure and 35, 40, 49, 56, 63, and 70
during depuration in the BRH chamber, and 20 mussels were sampled on
days 28, 56, and 70 in the control chamber. To avoid excessive loading
of the tanks, the shorter exposures were conducted after some of the
mussels had been removed by sampling. Specifically, the mussels for days
1.8 and 3.5 exposures were placed in the tank on day 14 and removed on
days 16 and 18 respectively. Likewise, mussels for the 7 day exposure
were placed in the tank on day 21 and removed on day 28.
22
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Since this design assumes that the exposure system operates in a
consistent manner, two 14-day exposures were conducted to verify con-
sistency, one from day 0 to day 14 and a second from day 14 to day 28.
18. Twice each week suspended particulate concentrations from the
control exposure chambers were analyzed by dry weight determination and
by electronic particle counting (1 to 40 u particle range). The dry
weight determinations were conducted according to Standard Methods
(American Public Health Association (APHA) 1976) with the following
modifications. Before sample filtration, filters were washed with a 50-
ml aliquot of delonized water, then with three 10-ml aliquots of deionized
water. Following filtration, filters were rinsed with three 10-ml rinses
of 2.4 percent ammonium formate to remove salt. Measurements of dissolved
oxygen, salinity, temperature, and ammonia nitrogen were made to determine
water quality and are presented in Table 1.
Worm Bioaccumulation Study
19. The worm bioaccumulation study consisted of an exposure of
Nereis^virens to solid phase BRH or REF materials for as long as 40
days under flowing seawater conditions. Twenty-four hours prior to
introducing the animals to the exposure aquaria, approximately 9.5 L
of either sediment was placed in aquaria measuring 32 cm x 38 cm x 16
cm high. Ambient temperature (9° to 13°C) seawater was then provided
to each of 14 aquaria at the rate of 120 ml/min. Sediment depth in
each aquarium was approximately 8 cm; seawater depth (maintained by a
standpipe) was approximately 5 cm.
20. The test was initiated at time zero (TQ) by randomly placing
24 adult worms in each of 12 aquaria. Nine aquaria contained BRH
23
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Table 1
Summary of Experimental Conditions for Bioaccumulatlon
Study with Mytilus edulis*
Parameter
Control
Exposure
Suspended solids
dry wt, mg/L
Particle density,
No./L
Temperature, °C
Dissolved oxygen,
mg/L
Salinity, °/oo
Unionized ammonia,
Uptake Period
1.72 + 0.18
(1.45 - 2.02)
2.60 + 0.2 X 107
(2.00 - 3.1 X 10?
15.7 + 0.4
(15.0 - 16.4)
7.5 + 0.6
(7.0 - 8.5)
28.4 + 1.8
(24 - 30)
2.9 + 1.29
(0.64 + 5.40)
9.32 + 0.58
(8.19 - 10.33)
12.00 + 1.3 X 10?
(9.60 - 13.7 X 10?)
15.6 + 0.3
(15.4 - 16.4)
7.6 + 0.4
(7.1 - 8.4)
28,4 + 1.8
(24 - 30)
3.83 + 1.68
(1.04 - 6.40)
Suspended solids
dry wt, mg/L
Particle density,
No./L
Temperature, °C
Dissolved oxygen,
mg/L
Salinity, c/oo
Unionized ammonia,
ug/L
Depuration Period
23.5 + 1.17
(1.18 - 3.53)
2.8 + 0.3 X 10?
(2.5 - 3.2 X 10?)
15.2 + 0.3
(15.0 - 15.8)
8.0 + 0.2
(7.6 - 8.5)
27.9 + 1.9
(23 - 30)
1.30 + 0.26
(0.94 - 1.66)
2.48 + 1.64
(1.16 - 4.86)
2.9 + 0.2 X 107
(2.7 - 3.2 X 107)
15.2 + 0.3
(15.0 - 15.8)
8.0 + 0.3
(7.6 - 8.4)
27.9 + 1.9
(23 - 30)
1.33 + 0.22
(0.96 - 1.52)
* Tabular values are mean and standard deviation with range denoted in
parentheses.
24
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material and three REF sediment. A subset of 15 worms was randomly
selected for organic and inorganic chemical analysis to determine the
contaminants in worms at T0. Prior to chemical analysis, all worms
at all sampling times were placed in Petri dishes containing filtered
seawater and allowed to purge their gut contents for 14 hr.
21. For each sampling period, all the worms from a single aquarium
were removed for analysis. Sampling periods during the uptake portion
of the study were days 14 and 28. After 28 days, all worms in four of
the five remaining BRH aquaria were removed, the sediment emptied, and
the aquaria cleaned. The aquaria were then filled with REF sediment
and the worms placed back into the aquaria. Sampling of these worms
during the depuration phase was on days 42 and 56 (14 and 28 days of
depuration). The worms in the ninth BRH aquarium were allowed to
remain an additional 12 days (total of 40 days exposure) and archived
at -20°C.
22. Three aquaria were each provided with REF sediment and
24 worms. The worms were sampled on days 28, 40, and 56 to determine
what contaminants, if any, were obtained from the REF sediment.
23. For clarity, mussels exposed to BRH sediment and algae are
referred to as "exposed mussels," while mussels exposed to algae only
are referred to as "control mussels," For the worn study, worms exposed
to BRH sediment are "exposed worms," while those depurated in reference
sediment are referred to as "depurated worms." Worms exposed to reference
sediments are referred to as "reference worms."
25
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Chemical Analysis
Organic Sample Preparation
24. The analytical procedures described below represent the state
of the art in marine organic analysis and have been intercalibrated with
several oceanographic laboratories. EPA-recognized analytical methods,
while available for these classes of contaminants, have been developed
primarily for freshwater and wastewater systems. These methods require
extensive modification and intercalibration when applied to marine systems
for the types of matrices and levels of detection that are required in this
study.
25. Cleaning of Glassware and Equipment. All glassware used for
the collection, storage, extraction and analysis of samples was washed
with Alconox®, rinsed four times with hot tap water, four times with
deionized water, capped with aluminum foil, and muffled for 6 hr at 450°C.
Immediately prior to use glassware was rinsed three times with an appropriate
solvent.
26. Stainless steel centrifuge bottles were washed in the same
manner as glassware and then rinsed twice with methanol, twice with
methylene chloride and twice with hexane immediately prior to use.
27. Stainless steel tissue homogenizers were washed in the
same manner as glassware and then placed in an ultrasonic bath in
graduated cylinders filled first with methanol, then with methylene
chloride, and finally with hexane just prior to use.
28. Glass fiber filters were placed individually in aluminum foil
and muffled for 6 hr 450°C. The stainless steel filter housing was
washed and rinsed with acetone and hexane prior to use.
26
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29. Sediment. The methods that follow were used for the extraction
and analysis of BRH sediment and the reference sediment from the worm
dosing system. Approximately 10 gr of wet sediment was placed in a
stainless steel centrifuge tube, and 50 ml of acetone was added. The
mixture was homogenized for 40 sec using a brass-bearing-equipped
tissue homogenizer and then centrifuged at 10,000 RFM for 5 min. The
acetone was decanted into a 1-L separatory funnel containing 150 ml of
pre-extracted deionized water. The extraction and centrifugation steps
were repeated twice more and all extracts were combined in the separatory
funnel. The aqueous layer in the separatory funnel was extracted three
times with 50 ml of Freon 113 each time, and the extracts combined in
a 500-ml Erlenmeyer flask. Extracts were frozen to remove water. The
sample extract was then subjected to column chromatography (see Column
Chromatography, paragraphs 39 and 40).
30. Water. The following procedure was used for unfiltered water
samples (dissolved plus particle-bound contaminants), samples of filtered
water collected after the glass fiber filter, and water taken after
passage through a continuous flow centrifuge. Water samples were collected
in 6-L separatory funnels. Samples were extracted twice by the addition
of 100 ml Freon 113 followed by vigorous shaking. Extracts were combined
in a 500-ml Erlenmeyer flask, and sodium sulfate (previously muffled at
700°C for 4 hr) was added to remove water.
31. The Freon extract was poured off and volume reduced in a round
bottom flask fitted with a Kuderna-Danish evaporator, and the solvent was
changed to hexane. Extracts (5 ml) were fractionated using the second silicic
acid column (see Column Chromatography, paragraphs 39 and 40).
27
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32. Suspended Particulate Material. For the mussel study suspended
particulate material (SPM) was collected using a 273-mm glass fiber filter
(Gelman Type'AE, 0.1 micron) in a stainless steel housing (Millipore®
273 mm). Water from the exposure system tanks was allowed to gravity
feed into this filtering system through Teflon® tubing.
33. Each filter was carefully removed, placed in a stainless
steel centrifuge bottle, and frozen until preparation and analysis.
Acetone (50 ml) was added to the centrifuge bottles containing the
filter, and the filter was homogenized with a stainless steel tissue
homogenizer for 20 sec at 25,000 RIM. Samples were centrifuged at 10,000
RPM for 5 min, and the acetone water layer was decanted into a 1-L
separatory funnel containing 150 ml extracted deionized water. This
extraction procedure was repeated two more times using 50 ml of Freon
113. The Freon was added to the separatory funnel, which was then
shaken. The Freon layer was then drawn off and saved. The remaining
aqueous layer was extracted again with 50 ml of Freon, and the extracts
were combined. The sample extract was then subjected to column
chromatography (see Column Chromatography, paragraphs 39 and 40).
34. Organisms. Mussel samples were taken for background analysis
at day zero, and removed from the exposure tank at day 1.8, 3.5, 7,
14, 28, 35, 40, 49, 56, 63 and 70; control mussels were sampled on days
28, 56, and 70. At each sampling time, 20 mussels were removed using a
stratified random sampling plan and stored in muffled aluminum foil in a
freezer prior to analysis. From each group of 20 mussels, three replicates
consisting of four individuals each were shucked into pre-weighed glass
centrifuge tubes, homogenized with a tissue homogenizer for 20 sec, and
28
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centrifuged at 25,000 RPM for 5 min. The remaining 8 organisms were
archived at -20°C.
35. Dead mussels were removed daily when discovered and mortality
recorded. Mortality data were analyzed by calculating survivorship
functions for mussels from control and exposed treatment conditions.
These survivorship functions incorporated the effect of the periodic
removal of individuals for analyses other than mortality. A comparison
of these functions was made using the Mantel and Haenszel (1959) Chi
square test for comparing two survival distributions.
36. Worms were removed from exposure tanks for chemical analysis
on days 14, 28, 42, and 56 and from the control tanks on day 28. A
sample was also collected at day 0, prior to exposure. Following
collection from the experimental tanks and gut depuration (see Methods
paragraph 20), worms were frozen until analysis in muffled glass jars.
From these samples, three replicates of 1-2 individuals each were placed
into preweighed glass centrifuge tubes, homogenized with a tissue
homogenizer for 20 sec and centrifuged at 25,000 RPM for 5 min.
37. Approximately 2 g of the mussel and worm homogenates was
taken for inorganic analysis. A small portion (approximately 2 g) was
taken for wet:dry ratio determinations. The remaining homogenate was
weighed and used for organic analysis.
38. Each of the sample homogenates from above was treated as a
separate sample with appropriate blanks carried through the entire
procedure. To each sample was added 15 ml of acetone; the mixture was
then homogenized with a tissue homogenizer for 20 sec and centrifuged
at 1750 RPM for 5 min. The fluid layer was decanted into a separatory
29
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funnel containing 150 ml of pre-extracted deionized water. The acetone
extractions and centrlfugation were repeated once more and the extracts
were combined in the separatory funnel. The tissue homogenization,
extraction, and centrlfugation were repeated twice more using 25 ml of
Freon 113 as the solvent. Because of the density of the Freon, the
solvent was withdrawn from the bottom of the centrifuge tubes using a
syringe. The Freon extracts were combined in the separatory funnel,
which was then shaken and the Freon layer was drawn off and saved. The
remaining aqueous layer was extracted twice more with 50 ml of Freon
each time. The Freon extracts were combined and the aqueous layer was
discarded. The sample extract was then subjected to column chromatography
(see Column Chromatography, paragraphs 39 and 40).
39. Column Chromatography, Final Volume and Storage. To remove
interfering biogenlc material and some residual particulates, the combined
Freon extracts were passed through the first column (2 x 25 cm of 100%
activated 100-200 mesh silicic acid). For sediment samples, 2.5 cm of
activated copper powder was added to the bottom of the first column to remove
elemental sulfur. The column was then rinsed with 25 ml Freon followed
by 50 ml of methylene chloride. The eluate was collected and volume
reduced in a round bottom flask fitted with a Kuderna-Danish evaporator
and 3-ball Snyder column. The solvent was exchanged to hexane as the
sample approached 5 ml. Final volume reduction to 5 ml was accomplished
by placing the sample in a concentrator tube fitted with a mlcrosnyder column
and placing it into a tube heater.
40. The 5-ml sample extracts were then charged onto a 0.9 x 45 cm
second column of 5% water deactivated 100-200 mesh silicic acid. Three
30
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fractions were collected from the column. Fraction 1 (PF-50) consisted
of 50 ml of pentane, fraction 2 (F-2) consisted of 35 ml of 20% methylene
chloride in pentane, and fraction 3 (F-3) consisted of 35 ml of methylene
chloride. The PF-50 fraction is an expansion of a 1st fraction formally
used by this laboratory. The PF-50 fraction is designed to include PCBs
and related chlorinated pesticides of similar polarity in addition to a
large portion of the petroleum hydrocarbons. Petroleum hydrocarbons
(PHC) as referenced in this report include only those hydrocarbon compounds
in the PF-50 fraction. The polycyclic aromatic hydrocarbons (PAH) which
are collected in the F-2 fraction may also be of petroleum origin; however,
these PAH compounds and the small amount of unresolved material found in
the F-2 (as separated in the present study) represented only a small portion
(approximately 10%) of the total petroleum hydrocarbons and were not
included in petroleum hydrocarbon calculations. The F-3 fraction
collected more polar material. Each column fraction was reduced in volume
by a Kuderna-Danish evaporation as above, with the solvent changed to
hexane. The final sample volume of 1 ml was achieved by adding 1 ml
of heptane to the sample in a 10-ml concentrator tube. Glass ebullators,
microsnyder columns, and a tube heater were utilized to reduce the sample
to 1 ml. The extracts were then divided in half between sealed glass
ampules for archival storage and screw cap vials for gas chromatographic
and GC/MS analyses.
Organic Instrumental Analysis
41. Electron capture gas chromatographic analyses were conducted on
a Hewlett-Packard Model 5840 gas chromatograph equipped with a 30 meter DB-
5 fused silica capillary column from J & W. The chromatograph was
31
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temperature programmed from 80°C to 290°C at 10°C/mln with a 4-min
hold at 80°C. Flame ionization gas chromatographic analyses were conducted
on a Carlo Erba 4160 gas chromatograph equipped with an identical
column. The temperature was programmed from 60°C to 325°C at 10°C/min
with a 4-min hold at 60°C.
42. Gas chromatograph/mass spectrometric (GO/MS) analyses were
conducted on a Finnigan Model 4500 also equipped with J & W DB-5 30
meter fused silica capillary column. The tail of the capillary column
was positioned inside the mass spectrometer so that the effluent
from the column was directed into the ionization volume of the mass
spectrometer. The mass spectrometer was operated through a standard
Incos data system and was tuned at all times to meet EPA quality assurance
specifications using decafluorotriphenylphosphine. The ionizing current
was typically set at 300 milliamperes and 70 EV, and the instrument
operated such that 100 picograms of PAHs from naphthalene to benzopyrene
gave easily quantifiable signals on their molecular ions with signal-to-
noise ratios of 50:1 or better. The mass spectrometer's gas chromatograph
was typically programmed from 50°C to 330°C at 10°C/min with a 2-min
hold at 50°C, but was occasionally progammed at 4°C/min to permit higher
chromatographic resolution.
43. All instruments were calibrated with standards each day. The
concentrations of the standards used were chosen to be close to the
levels of the materials of interest, and periodic linearity checks were
made to ensure the proper performance of each system. When standards
were not available for some compounds, response factors were calculated
using mean responses of appropriate standards.
32
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jnorganic Sample Preparation
44. Seawater. Two sets of seawater samples were collected from
the mussel exposure system on day 25 of the exposure period. Approximately
1 hr before the BRH sediment slurry was renewed in the reservoir, duplicate
20-ml samples of seawater were taken from the control and exposure chambers.
Two 20-ml seawater samples were also taken 3 hr after renewing the slurry
from the exposure chamber only. The unfiltered samples were acidified
with 2.0 ml of ultra-pure concentrated nitric acid and placed in acid-
cleaned polyethylene bottles fitted with polyethylene screw caps. The
acidified samples were stored at room temperature for 1 week before
trace metal analysis.
45. Sediment. After the BRH sediment contained in a barrel
was thoroughly homogenized (see Sediment Collection and Preservation,
paragraphs 6 and 7), nine samples were taken for analysis. These samples
included three from the top, three from the middle, and three from the
bottom. The wet weight of all samples was determined. The samples
were frozen and then freeze dried in a Virtus® lypholyzer (Model #
10-145MR-BA) for 2 days. The dry weight of each sample was then
determined.
46. The dried BRH sediment samples were acidified with a total of
50-ml of concentrated HN03 (reagent grade). The acid was added in 10-ml
aliquots since BRH sediment is very reactive to acid. All reaction
was allowed to subside before the next addition of acid was made.
After several days the samples were heated at 60°C for several days.
The samples were subsequently evaporated down to approximately 10 ml
after which 30% H202 was added in 2-ml aliquots until 50 ml had been
33
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added. The H202 was added cautiously since BRH sediment reacts vigorously
with strong oxidizing agents. The samples were evaporated down to
approximately 25 ml and filtered through acid-rinsed (5% HNC^) Whatman
41 filter paper into 250-ml volumetric flasks. The beakers were rinsed
with 25-ml quantities of 5% HNC>3. The-rinse solution was also filtered
through the filter paper and added to the volumetric flask. The volumetric
flasks were brought up to volume with 5% HN03. This nitric acid-hydrogen
peroxide extraction procedure for sediment samples has been described by
Krishmanurty et al. (1976).
47. Organisms. From each sample homogenate, described in Organic
Sample Preparation, about 2 g of wet tissue was taken for inorganic
analysis and placed in a tared beaker and weighed. The samples were
oven dried at 110°C for 2 days, cooled in a desiccator, and weighed.
Ten milliliters of concentrated reagent grade nitric acid was added to
each sample, which was then allowed to digest at room temperature in a
hood for 24 hr. The samples were heated at 60°C for several days until
complete dissolution of the sample had occurred. The samples were then
evaporated to near dryness at 90-95°C, and cooled to room temperature.
Three milliliters of 30% hydrogen peroxide were slowly added in 1-ml
increments since the effervescent reaction was quite vigorous. The
solutions were then heated to 60°C for another day, evaporated to near
dryness, and cooled to room temperature. At this point the clear and
colorless solutions were transferred to 25-ml volumetric flasks with
several rinses of 5% nitric acid, and were diluted to the mark with 5%
nitric acid. The solutions were finally transferred to screw cap poly-
34
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ethylene bottles. This nitric acid-hydrogen peroxide dissolution procedure
has been reported by Knauer and Martin (1973).
Inorganic Instrumental Analysis
48. All flame atomization (FA) atomic absorption (AA) analysis
was conducted with a Perkin-Elmer (Model #603) atomic absorption instrument.
All Hg determinations were conducted by the method of Hatch and Ott (1968)
using a Perkin-Elmer (Model #MHS-1) mercury/hydride system adapted to
the 603 AA. The transient Hg signals were recorded with a Perkin-Elmer
(Model #56) strip chart recorder. All heated graphite atomization (HGA)
atomic absorption determinations were conducted with a Perkin-Elmer
(Model #500) HGA unit coupled to a Perkin-Elmer (Model #5000) atomic
absorption instrument retrofitted with a Zeeman HGA background correction
unit. The model 500 HGA unit was equipped with an auto injector (Model
# AS-40). The transient HGA-AA signals were recorded with a Perkin-Elmer
strip chart recorder (Model #56) and also sent automatically to a Perkin-
Elmer data station microcomputer (Model #3600). Software supplied with
the data station reduced the transient signals to a peak height and peak
area for each element determined. The instrument setup procedures for
the FA-AA, MHS-1, and HGA-AA determinations were in accordance with
procedures described in "Methods for Chemical Analysis of Water and
Wastes" (EPA 1979) and are also found in the manufacturer's reference
manuals.
49. The AA instruments were calibrated each time samples were
analyzed for a given element* Instrument calibrations were generally
checked after every five samples had been atomized into the flame unit*
injected into the HGA unit, or pipetted into the MHS-1 sample reaction
35
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flask. All samples were analyzed at least twice to determine signal
reproduclbility. Most were analyzed three times. Generally, for each
15 samples processed, one sample was determined by the method of standard
addition, and one procedural blank sample was analyzed.
50. All elements except Hg and As were determined in the sediment
samples by FA-AA. Mercury was determined only in the BRH sediment
samples by the MHS-l-AA technique. Arsenic could not be determined in
the sediment samples because of a chemical interference. At this time
the cause of the chemical interference is under investigation.
51. All seawater samples were analyzed by H6A-AA. No chemical
separation techniques were utilized to concentrate the elements of interest
from the seawater matrix. All samples were analyzed by direct injection
into the HGA unit (Ediger et al. 1974; Sturgeon et al. 1979; and Slavin
1980). The large non-atomic background signal was eliminated by the use
of the Zeeman background correction system (Fernandez et al. 1980, and
Fernandez and Giddlngs 1982). It was necessary to matrix match the
unknown samples with the standards since chemical interferences are not
corrected by the Zeeman effect. Therefore, all standards were prepared
in trace metal stripped seawater and acidified in the same manner as the
samples. The trace metal-free seawater was prepared by the methods of
Davey et al. (1970).
52. Due to the limited size of the mussel and worm samples (2 g wet
weight), only Fe and Zn could be determined by conventional FA-AA. All
other elements (i.e., Mn, Cu, Pb, Cd, Cr, and As) were determined by HGA-
AA. All mussel and worm samples determined by HGA-AA were matrix matched
before analysis. A matrix solution containing 10% seawater and 90% 0.16
36
-------
N nitric acid (V/V) was used as a diluent for both standards and samples.
Samples were diluted with this matrix modification solution so that the
sample extracts never exceeded 20% of the total volume of the solution
analyzed. Standards were made up in an identical manner to the samples.
53. It should be noted that, unlike the As determined in BRH
sediment samples, no chemical interference was detected for the As
determined in the mussel samples. There is a large difference in the
two sample matrixes with respect to the inorganic and organic composition
which could account for the absence or presence of a chemical interference
during the determination of As by HGA-AA or MHS-1 AA analysis.
37
-------
PART III: RESULTS AND DISCUSSION
Mussel Test
Organic Contaminants
54. PCBs - Unfiltered Seawater. The PCB concentrations (quantitated
as Aroclor-1254) in whole water samples (dissolved plus particle bound
PCB compounds) taken during the exposure and depuration phases of the
bioaccumulation study are shown in Table 2. The PCB concentrations found
in blanks processed through the analytical procedure averaged 0.21 ng/1
(Table 3). The average concentration of PCBs (as A-1254) found in control
tanks was 0.52 ng/1. PCBs found in unfiltered seawater samples from the
exposure tanks showed an average concentration of 112+29.3 ng/1 during
the exposure. During the exposure the RSD* of the measurement for
total PCBs was 20%, indicating that the exposure system was working well
and that it delivered a relatively constant concentration of PCB
contaminants to the mussels. During the depuration period the PCB
concentrations in the exposure tank decreased; however, they remained
elevated above those in the control tank during the depuration period
(Table 2). Since the fiberglass exposure tank was cleaned with soap and
water and thoroughly rinsed following the exposure period, the elevated
concentrations found in the tank during depuration may reflect the further
introduction of contaminants from a variety of possible sources (i.e., par-
ticulates associated with the mussels1 shells or byssal threads, feces and
pseudofeces, etc.).
* RSD - Relative standard deviation = standard deviation x 100
mean
38
-------
Table 2
Mussel Dosing System; PCS Levels in Unfiltered Water
Day
0
0
28
28
35
70
70
Day
0
0
8
8
14
14
16
16
21
21
*28
28
35
70
70
CONTROL TANK
Replicate
A
B
A
B
A
A
B
EXPOSURE TANK
Replicate
A
B
A
B
A
B
A
B
A
B
A
B
A
A
B
PCB (as A-1254)
ng/1 (not corrected for blank levels )
0.46 .50 + .05
0.53
0.23 .34 + .16
0.45
0.65 .65
0.66 .66 + .00
0.66
.52 + .16
PCB (as A-1254)
ng/1 (not corrected for blank levels)
95.3 115. + 27.2
134.
116. 123. + 9.2
129.
97.4 80.2 + 24.3
63.
133. 155. + 31.1
177.
80.3 91.7 + 16.1
103.
105. 107. + 2.8
109.
112. + 29.3
2.27
1.79 1.83 + 0.06
1.87
*At day 28 exposure ended and depuration period began.
39
-------
Table 3
PCS Blank Levels - Mussel Dosing System Samples*
PCB (as A-1254)
Day ng/1
24 February 83 .18
2 March 83 .16
4 March 83 .16
9 March 83 .34
16 March 83 .24
23 March 83 .20
.21 + .07
* For PCB levels given (Table 2), the blank levels have not
been subtracted.
40
-------
55. The distribution of PCB compounds between the dissolved**
and particle-bound form was examined in samples of the exposure water.
Both the filters and the filtrate were extracted and analyzed. Another
separation of dissolved and particulate phases was accomplished using a
continuous flow centrifuge. The results of these studies are shown in
Table 4. The mean PCB concentration for the filters (day 8) added to the
mean PCB concentration for the dissolved compounds (day 8) is close to
the value obtained for analysis of unfiltered water on day 8. These data
indicate that methylene chloride method for extracting PCB compounds from
the suspended particulate suspensions was as efficient as the sum of the
individual extractions of the filtrate and the particles (see Methods).
56. The electron capture detection gas chromatograms from the
analysis of unfiltered water, filters, filtrate, and centrifuged water
taken from the dosing system on day 8 of exposure are shown in Figure 6.
The chromatogram of the unfiltered water (dissolved and particle-bound
contaminants) shows a distribution of PCB compounds from Cl2 to Cl% with
the majority of material containing four, five, and six chlorine atoms.
Tentative identification of the compounds in electron capture chromatograms
are shown in Table 5. The same general patterns of peaks are shown in
the filter sample; however, there appears to be a relative decrease in
** Dissolved as used in this report refers to the compounds passing
through the O.!-(JL glass fiber filter and that material which passed
through the continuous flow centrifuge. These compounds may be
associated with surfactants or may be in colloidal forms and not
truly dissolved.
41
-------
Table 4
Mussel Dosing System; PCS Levels in Water-Exposure Tank
Day
Unfiltered Water
8
8
Replicate
A
B
PCB (as A-1254)
ng/1 (not corrected for blank levels^
116.
129.
123. + 9.2
Filtered Water
8
8
A
B
11.1
12.0
11.6 + .64
Water thru Centrifuge at 14,000 RPM
Filter
10.6
108.
42
-------
Table 5
Tentative Identifications of Compounds tn BCD Gas Chromatograms*
Peak It Tentative ID
1 2,3 - Dichlorobiphenyl
2 Dichloroblphenyl
3 Dichlorobiphenyl
4 2,2',5 - trichlorobiphenyl
5 Trichlorobiphenyl
6 Trichlorobiphenyl
7 Trichlorobiphenyl
8 Trichlorobiphenyl
9 2,4',5 - trichlorobiphenyl
10 2,4,4' - trichlorobiphenyl
11 2,3,4 - trichlorobiphenyl
12 Trichlorobiphenyl
13 Trichlorobiphenyl
14 Tetrachlorobiphenyl
15 2,2',4',5, - tetrachlorobiphenyl
16 2,2',4,4' - tetrachlorobiphenyl
17 2,2',3',5 - tetrachlorobiphenyl
18 Tetrachlorobiphenyl
19 Tetrachlorobiphenyl
20 Tetrachlorobiphenyl
21 2,3',4',5 - tetrachlorobiphenyl
22** 2,3',4,5',6 - pentachlorobiphenyl, 2,3',4,4' - tetrachlorobiphenyl
2,2',3,5,6 - pentachlorobiphenyl
23 Pentachlorobiphenyl, 2,3,8 - trichlorodibenzofuran,
tetrachlorodiphenyl ether
24 Tetrachlorobiphenyl, Pentachlorobiphenyl
*Since all PCB isomer standards were not available, the possibility exists that
other isomers nay elute with identical retention times as the PCB in this table.
Therefore we prefer the conservative approach by listing identifications as
tentative.
**More than one PCB isomer standard with this retention time eluted in this
position.
A3
-------
Table 5. (Cont'd)
Peak // Tentative ID
25 2,2',4,5,5'- pentachlorobiphenyl
26 Pentachlorobiphenyl
27 Pentachlorobiphenyl
28 Pentachlorobiphenyl, 1,1 - bis (p-chlorophenyl) - 2,2-dichloroethylene
29 Pentachlorobiphenyl
30 Pentachlorobiphenyl
31 Pentachlorobiphenyl, Hexachlorobiphenyl
32 Pentachlorobiphenyl, Hexachlorobiphenyl
33 Pentachlorobiphenyl
34 Pentachlorobiphenyl, Hexachlorobiphenyl
35 Hexachlorobiphenyl
36 2,2',4,4',5,5' - hexachlorobiphenyl
37 Pentachlorobiphenyl, Hexachlorobiphenyl
38 Hexachlorobiphenyl
39 Hexachlorobiphenyl,
40 2,2',3,3',4,5 -hexachlorobiphenyl
41 Heptachlorobiphenyl
42 2,2',3,4,4',5',6- heptachlorobiphenyl
43 2,2',3,3',4,4'- hexachlorobiphenyl
44 Hexachlorobiphenyl
45 Heptachlorobiphenyl
46 2,3,3•,4,4',5- hexachlorobiphenyl
47 2,2',3,3',4,5',6,6'- octachlorobiphenyl
48 Heptachlorobiphenyl
49 Heptachlorobiphenyl
50 Octachlorobiphenyl
51 Octachlorobiphenyl
52 2,2',3,3',4,4',5,5' -octachlorobiphenyl
53 2,2',3,3',4,4',5,5',6 - nonachlorobiphenyl
54 Decachlorobiphenyl
44
-------
Time
Time
a. Unfiltered water
b. Filtrate
•is
Time
d. Water through continuous flow
centrifuge (14,000 rpm)
Figure 6. Capillary column electron capture gas chromatograms of PF-50
(PCB) fractions from day 8 exposure
-------
the height of the lower molecular weight peaks in comparison with the
chromatogram of the unfiltered water. The chromatogram of the filtrate shows
a relative enhancement of the lower molecular weight PCB compounds when
compared with the unfiltered water. The distributions found are logically
consistent with the solubilities of the compounds. With lower molecular
weight, more water-soluble PCB compounds were found in the filtrate and
the higher molecular weight, less soluble compounds were found associated
with particles.
57. In order to determine whether the distributions found on the
filter and in the filtrate were artifacts of the filtration process (i.e.,
adsorption of less soluble PCB components on the filter while more soluble
components passed into the filtrate), continuous flow centrifugation at
14,000 RPM was utilized to remove particles. Analysis of the water
following passage through the centrifuge showed a distribution of PCB
compounds that was very similar to the distributions found in the filtrate
(Figure 7). While PCBs in the water passing though the centrifuge may
still be associated with extremely fine particles or exist in colloidal
form, this experiment showed that the separations were not artifacts of
the filtration process.
58. Data from the analysis of filtered material and filtrates were
utilized to calculate sediment-water partition coefficients, Kp, where
Kp = Cs_
Cw
Cs» concentration of compound in sediment
Cw» concentration of compound in water
Kps were estimated for PCB compounds where Log P (Log of the n-octanol/
water partition coefficient) were known (Table 6). The estimated
46
-------
t
c
o
Q.
cr
o
o
NUMBERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 5.
Time
a. Filtrate
15
30
Time
b. Water through continuous flow centrifuge (14,000 rpm)
Figure 7. Capillary colunm electron capture gas chromatograms of PF-50
(PCB) fractions from day 8 exposure
47
-------
Table 6
Comparison of Experimental and Estimated Kps
Peak No. PCS Compound Log P* Kp Experimental** Kp Estlmatedt
4 2,2,'5 - trichlorobiphenyl 4.7 .99 X 105 .02 X 105
10 2,4,4' - trichlorobiphenyl 5.0 2.1 X 105 .036 X 105
25 2,2',4,5,5'-pentachlorobiphenyl 6.3 12. X 105 .66 X 105
36 2,2l,4,4',5,5'-hexachlorobiphenyl 6.7 42. X 105 1.8 X 10$
43 2,2',3,3',4,4'-hexachlorobiphenyl 7.0 51. X 1Q5 3.2 X 105
* Solubility from Mackay et al.(1980b). Converted to Log
P using Log P • 5.00-.670 Log S where S is solubility in pmol/1 (Chlou
et al. 1977).
** Kp measured as means of 3 Kps determined on day 28 from mussel exposure tank,
t Kp estimated using Log Koc - Log Row - 0.21
Row - n-octanol/water partition coefficient (Log P)
Koc » organic carbon/water partition coefficient from (Karickhoff et
al. 1979)
and Kp - Koc (%OC) from Briggs (1973).
100
48
-------
NUMBERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 5.
35
t
0)
o
a.
m
OJ
a:
o
-------
results were considerably lower than the measured results for representative
PCB Isomers. This may indicate that equilibrium was not established with
respect to PCBs in the aqueous and particle bound phases during the
residence time of the suspensions in the dosing system.
59. PCBs - Mussels* An electron capture detection (BCD) gas
chromatogram from mussels taken at day 0 (Figure 8) shows a pattern of
electron capturing compounds that is typical of mussel samples from lower
Narragansett Bay (Lake et al. 1981). This chromatogram shows a peak
consisting of 2,4,8-trichlorodlbenzofuran and a tetrachlordiphenyl
ether (which co-elute under the gas chromatographic conditions employed).
The predominant peaks are Clg PCBs.
60. Following exposure to suspensions of BRH material (day 28),
more PCB peaks are evident in the BCD chromatograms of mussels and the
distributions are changed considerably (Figure 9). In particular, the
lower molecular weight PCB compounds consisting of PCBs with two, three,
and four chlorine atoms are significantly increased, and the maximum
peaks consist of Cl5 compounds. Relative increases in some Cl^ and
Cly peaks eluting in the later portions of chromatograms are also
evident.
61. Comparison of this chromatogram with that of the unfiltered
dosing water (Figure 10) shows that the mussels accumulated most of the
different PCB isomers present in the unfiltered water. The organisms appear-
ed to show a distribution which was very similar to that in the unfiltered
water; and, as was observed in other studies (Lake et al. 1983), PCBs with
seven or more chlorine atoms were not accumulated as effectively as
those with four, five, and six chlorine atoms.
50
-------
62. The chromatogram from mussels exposed for 28 days, and the
chromatogram of mussels exposed for 28 days followed by 42 days of
depuration, are shown In Figure 11. Comparison of these chromatograms
shows relative decreases in lower molecular weight PCB compounds (Cl2,
013, and 014 isomers) and in some Gig and Cly PCB isomers in the
depurated sample, as well as relative increases in other Cl$ isomers
(peaks No. 36 and 39). The peaks which are becoming more prominent are
the same PCB peaks that are predominant in the chromatograms from
control mussels and mussels from lower Narragansett Bay.
63. Since mussels were not gut purged in the present study, the
extracts of mussels include a PCB contribution from SPM in the gut of the
organisms. While the significance of this material to the total PCB
content of the organisms has not been determined in the present study by
examining gut-purging, two facts support the contention that it is not
dominant in determining the PCB distributions in mussels. First,
research examining the uptake of PCBs in similar dosing studies found no
differences in PCB concentrations between non gut-depurated mussels and
mussels depurated for 6 hr (Pruell et al. 1983). In addition, these
researchers found the SPM contained C18, Clg, and C110 P088 which
were not observed in the mussel extracts but which would have been present
if material in the gut had significantly influenced the PCB contaminants
in the extracts. Secondly, the amount of SPM present in the organisms
at day 28 can be calculated from the accumulation of Fe (which is not
highly bioaccumulated) and the concentration of Fe in the BRH sediment.
The amount of sediment accumulated multiplied by the concentration of
51
-------
t
U>
§
o.
I
NUMBERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 5
40
Time
a. Time 0
Figure 9,
Time >
b. Day 28 exposure
Capillary column electron capture gas chromatograms of PF-50
(PCB) fractions from mussels
52
-------
t
O)
§
Q.
«
0)
Q
NUMBERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 5.
50
JILj^-jJuJI
a.
Time >
Unfiltered water, day
Time >
b. Mussel, day 28
Figure 10. Capillary column electron capture gas chromatograms of PF-50
(PCB) tractions from exposure
53
-------
NUMBERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 5.
Time >
a. Day 28
Figure 11.
Time >
b. Day 70
Capillary column electron capture gas chromatograms of PF-50
(PCB) fraction from exposure mussels
-------
PCBs in the BRH material, then, gives the amount of PCBs in the SPM in
the organism's guts.
Fe concentration Day 28 Mussels Fe concentration Control Mussels
500 ug/g dry wt mussel - 193 ug/g dry wt mussel
(Table 7) (Table 7)
equals
Difference 307
ug/g dry wt mussel
If the assumption is made that all the Fe in the mussel at day 28 results
from Fe on the SPM in the gut of the organism,
(307 ug Fe/g dry wt mussel) (1000 mg dry wt BRH sed./29600 ug/Fe)
(Table 17)
equals
(10 mg dry wt BRH sed/g dry wt mussel)
(10 mg dry wt BRH sed/g dry wt mussel) (6800 ng A-1254/1000 mg dry
dry wt. BRH sed)*
equals
(71 ng A-1254/g dry wt mussel)
Since the increase observed in the concentration of PCBs in the mussels
is much larger than this, 28 day mussel - 2800 ng/g (Figure 12), the
contribution from PCBs on SPM in the gut of the organisms appears to be
almost inconsequential.
64. The concentrations of PCB in mussels exposed for 28 days were
divided by the concentrations of PCBs in filtered water samples to obtain
bioconcentration factors (BCFs).** These data expressed as Log BCF
* Value from Rogerson et al. (1983).
** Bioconcentration in this report refers to the process of uptake
of contaminants from water.
55
-------
Table 7
Average Trace Metal Concentrations for Mussels collected
Metal
Fe
Zn
Mn
Cu
Pb
Cd
Cr
from the Exposure
Chamber on Day 28*
Mussel
28 Day
500 +
333 +
11 +
55 +
13.9 +
7.0 +
25.1 +
191
84
5
18
4.7
2.0
10.7
Mussel
Control
193 + 24
178 + 53
12+5
12+5
5.0 + 1.5
2.6 + 0.4
2.2 + 1.0
*The control concentrations reported for the mussels are the
average of all the control samples and not just day 28. All
concentrations are in Ug/g dry weight.
56
-------
(Table 8) were converted from dry wt to wet wt using a common wet to dry
conversion factor to facilitate comparisons with estimates of Log BCFs
from Geyer et al.(1982). Due to variability in the amount of water in
the organism tissues, the authors prefer the use of dry weights from
individual samples for calculations, as is done in the remainder of the
report. The measured Log BCFs for representative PCB compounds increase
with increasing Log P (decreasing aqueous solubility) as observed for
Log BCFs with mussels (Ernst 1977) and fish (Veith et al. 1979). In
addition, the measured values are in close agreement with estimated Log
BCFs (Geyer et al. 1982).
65. The concentrations of compounds in the mussel samples at day
28 (dry weight) and the concentrations of compounds in the unfiltered
water samples at day 28 were used to calculate bioaccumulation factors
(BAFs).*
BAF =« concentration of individual PCB compound in mussel (dry weight)
concentration of individual PCB compound in unfiltered water
In order to facilitate comparisons of these large values, log BAFs were
calculated (Table 9). In spite of considerable differences in the n-
octanol/water partition coefficients (Log Ps) for these PCB compounds,
the Log BAFs appear to be quite constant. This is in contrast to BCFs
(accumulation from water only) from the literature for single compound
tests with dissolved components, which show increasing BCFs with
57
-------
1000
T3
O»
\
O>
CVJ
<
CO
o
0_
100
10
exposure
tank
control
tank
UPTAKE
DEPURATION
1.83.57 14 21 28 35 40 49 56 63 70
TIME (days)
Figure 12. Concentration of total PCBs (as A-1254) in mussels
exposed to BRH sediment versus time
58
-------
Table 8
Estimated and Measured Bioconcentration Factors (BCF) in
Mussels at Day 28
Log P* Estimated Log BCF**
Peak No. PCB Compound (Log Row) (wet wt.)
4 2, 2 ',5 - trichlorobiphenyl
10 2,4,4', -
25 2,2'4,5,5
36 2, 2', 4, 4'
43 2, 2', 3, 3'
PAHs
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz(a)anthracene
Chrysene
Benz(a)pyrene
Perylene
trichlorobiphenyl
'-pentachlorobiphenyl
,5,5' -hexachlorobiphenyl
,4,4' -hexachlorobiphenyl
4.7
5.0
6.3
6.7
7.0
4.4
5.3
4.9
5.1
5.8
6.4
6.2
6.9
3.2
3.5
4.6
4.9
5.2
3.0
3.7
3.4
3.6
4.2
4.7
4.5
5.1
Measured Logt BCF
(wet wt.)
3.2
3.6
4.2
4.7
4.8
2.2
2.3
2.9
3.1
4.0
3.9
4.2
3.8
* PCB solubilities from Mackay et al. (1980a). PAH solubilities from
Mackay et al. (1980b). Solubility converted to Log P using Log P -
5.00 - .67 Log S where S is solubility in umol/L (Chiou et al. 1977.)
** BCF estimated from log BF - 0.858 x Log Row - 0.808 from Geyer et al.(1982),
(BCF can be substituted for BF.)
t BCF measured from mean of concentrations in three 28-day exposed mussels
divided by mean of concentrations in three 28-day filtered water samples
from the exposed tank.
59
-------
decreasing water solubility (increasing Log P) for mussels (Ernst 1977;
Geyer et al. 1982). The uniform BAFs observed in the present study
probably resulted from the presence of SPM in the dosing system.
66. The constant BAFs observed may have resulted from two
processes competing for the dissolved phase contaminants. The first is
re-adsorption of dissolved PCB contaminants by the SPM including algae; the
second is the bioconcentration of dissolved PCB contaminants by the mussels.
If these two distributions vary to approximately the same extent over the
range of PCB contaminants, then constancy of BAFs could result. Another
possible explanation for the relatively constant BAFs observed in this
study is that the mussels accumulate individual PCB compounds by a similar
constant process (i.e., transfer from particles across the lining of the
gut). This method of accumulation could result in distributions which
were very similar to those in the unfiltered water and filter samples if
depuration rates for the individual compounds were approximately equal
during accumulation. A third possible explanation for the constant BAFs
observed in this study is that steady-state values were not reached for
all PCB compounds during the uptake period (see discussion of Kinetics).
67. The distributions of dissolved (filtrate) and particle-bound
(filter) PCBs were compared with those in mussels (Figure 13). The
distribution in the water (filtrate) is dominated by low molecular weight
compounds and the peak heights of the PCBs decrease with increasing
molecular weight. The distribution on the SPM (filter) closely matches
that found in mussels (Figure 14), suggesting that PCBs in the SPM
influence the distribution found in the mussels.
60
-------
Table 9
Measured Log BAF* for Each Separate PCB Peak in Exposed Mussels
Peak Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Log BAF
4.3
4.4
4.5
4.4
4.4
4.1
4.4
4.6
4.5
4.4
4.4
4.4
4.4
4.4
4.5
4.5
4.4
4.4
4.5
4.5
4.4
4.4
4.4
4.5
4.4
4.4
4.4
Peak Number
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Log BAF
4.5
4.6
4.5
4.5
4.5
4.6
4.5
4.5
4.5
4.5
3.9
4.4
4.2
4.4
4.4
4.4
3.4
4.3
4.2
4.4
4.1
3.0
3.3
3.8
*Method for calculation of Log BAF shown in text,
61
-------
1
Time >
a. Filter, day
Tim«
b. Filtrate, day
c. Mussels, day 28
Figure 13. Capillary column electron capture gas chromatograms of PF-50
(PCB) fraction from exposure
62
-------
68. Chromatograms from control mussels sampled at day 0,
day 28, and day 70 (Figure 15) showed only minor changes between day 0
and day 28. Between day 28 and day 70 an Increase In the height of the
2,4,8-trichlorodibenzofuran and tetrachlorodiphenyl ether peak was
evident in the control mussels. This increase probably reflects an
increased input of these industrial contaminants to the upper
Narragansett Bay followed by down Bay transport and entrance of small
amounts of these contaminants into our laboratory seawater supply. In
addition, a small relative increase in some lower molecular weight PCB
compounds was observed in the day 70 control. During the depuration
period, a late eluting peak appeared in chromatograms. GC/MS analysis
showed that it was not a chlorine or bromine-containing compound• It is
probably an electron capturing biological compound. It should be noted
that the PCB concentrations in control mussels remained low during the
experiment and that these organisms fulfilled their intended purpose as
chemical controls by accumulating background concentrations of pollutants
from the control seawater.
69. PCB Kinetics. The accumulation and depuration of PCB
contaminants (quantified as Aroclor®-1254) are shown in Figure 12. To
determine if steady-state was reached during the uptake period, the
A-1254 residue concentration in the mussels, and the time data (in days),
were entered into a computerized non-linear model in accordance with
proposed ASTM recommendations (ASTM 1982).
Y - Ln Residue - Pl/(l + P2 ** (Time - P3))
63
-------
where Y « natural log of the residue
Pl= natural log of the maximum predicted residue concentration
P2= rising slope (0
-------
NUMBERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 5.
I
:.
in
-a
:•
o
n
v
r i
Time >
a. Filter, day 8
f1
Figure 14
Time >
b. Mussels, day 28
Capillary column electron capture gas chroraatograms of PF-50
(PCB) fraction from exposure
65
-------
NUMBERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 5
b. Control, day 28
c.
Time
Control, day 70
Figure 15. Capillary column electron capture gas chromatograms of PF-50
(PCB) fractions from mussels
66
-------
73. The kinetics of the depuration phase were examined in detail.
For most bioaccumulatlon studies, first-order kinetic expressions have
been applied (Niimi and Cho 1981; Ernst 1977; Veith et al. 1979). For
this process, the depuration rate is not dependent on the initial
concentration. Another study found that' second order kinetics were
followed for the elimination of pesticides from catfish (Ellgehausen et
al. 1980). In second-order processes the depuration rate is dependent
on the initial concentration. Plots of In C versus time (C=*
concentration) and 1/C versus time for all the peaks examined during the
depuration phase were made. If first-order kinetics were followed, then
the plot of In C versus time should be linear; if second-order kinetics
were followed, the plot of 1/C versus time should be linear (Glasstone
and Lewis 1960; Ellgehausen et al. 1980). No clear distinction of the
order of the kinetics was found in comparisons of the correlation co-
efficients (Table Al). In addition, scatter of the data during depuration
and the impact of slightly elevated levels of PCBs in the exposure tank
during depuration (Table 2) precluded a conclusive determination of the
order of kinetics.
74. If first-order kinetics are assumed, as was the case in other
studies on bioaccumulatlon (Niimi and Cho 1981; Ernst 1977; Vieth et al.
1979), differences in the depuration rates for the accumulated compounds
can be examined. The slopes of lines (the first-order depuration rates)
for the compounds examined in this study are shown on Table A2. A subset
of eight "representative" compounds was selected from the PCB dis-
tributions in mussels for more detailed study. Analysis of covariance
was used to test equality of the slopes for the eight compounds (ot. =»0.05)
67
-------
over different sections of the depuration period. This examination was
made over different depuration periods (1 week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, and 6 weeks) to determine if the depuration rates were
constant over the depuration period. The results (Table A2) show a
decrease in depuration rate (demonstrated as less negative slopes) with
increasing depuration time for most individual compounds. These results
may demonstrate the inapplicability of first-order kinetics to describe
depuration for some of these compounds. The results of the comparisons
of the equality of the slopes (depuration rates) during the depuration
time periods are shown in Table A2. The results show that statistical
differences between some lines exist (a =0.05) over some of the time
periods. Within each depuration period there are 28 possible compound
comparisons. Consequently, a very small a level (.05/28) was chosen for
each pairwise comparison. This was done to maintain a 5% level of
significance for all pairwise comparisons within each depuration period.
While differences between other lines were not significant at the same
concentration, an observed trend showed that the lower molecular weight
compounds were more rapidly depurated than the higher molecular weight
compounds. Some higher molecular weight compounds appear to depurate
faster than some mid range PCBs during the first weeks of depuration (up
to day 56 or 28 days depuration). The slopes of the depuration lines
for the different compounds converge as depuration time increases.
75. If all the depuration data are included, those compounds
which are resistant to transformation (Zell and Ballschmiter 1980) and
with higher chlorination have the slowest depuration rates (shown as
less negative slopes in Table A2).
68
-------
76. PAHs - Seawater. The concentrations of 11 polycyclic aromatic
hydrocarbons (PAHs) and one chlorinated pesticide, Ethylan (1,l-dichloro-2,
2-bis (p-ethylphenyl) ethane), in unfiltered water samples (dissolved plus
particle-bound compounds) taken during the exposure phase of the mussel
bioaccumulation study are shown in Table 10. The levels of these
contaminants in control water samples, water samples taken during
depuration, and blanks were below the detection limit «0.1 ng/L for the
methods used for extraction and analysis).
77. Extracted ion current profiles (EICPs) result from the GC/MS
analysis. These profiles display the concentrations of the major ion
for each compound as a function of retention time on the GC column.
By examining several of these plots corresponding to different times
during the course of the experiment it is possible to determine what
relative changes in the content of selected compounds occurred during
the experiment.
78. The EICPs for the PAH and Ethylan compounds (which are
reported together because they were all analyzed in the same GC/MS
analyses) in an unfiltered exposure water sample from the dosing system
day 8 are shown in Figure 16. The mass numbers (molecular weight/charge)
of fragments characteristic of the compounds (Figure 16) are shown on
the right axis.
79. Examination of the unfiltered water sample EICP (Figure 16)
and the data in Table 10 indicates that the relative distributions of PAH
compounds and the Ethylan were fairly consistent over the exposure studies,
but the total concentrations of these compounds changed (RSDs (S.D./mean
x 100) for PAH compounds were up to approximately 75%). The greater
69
-------
Table 10
PAH and Ethylan Concentrations in Unfiltered Water Samples*
Day
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz(a)anthracene
Chrysene
Benzo(b)f luoranthene and/or
Benzo(k)fluoranthene
Benzo (e )pyrene
Benzo(a)pyrene
Perylene
Sum of PAHs with MW of 276
Ethylan
SUM-PAHs
£
6.8
2.3
17.0
25.9
13.7
20.1
25.4
15.7
16.1
2.9
25.2
0.8
171.
8_
39.6
9.6
47.8
74.3
33.2
45.5
59.3
34.6
35.9
6.4
57.8
1.6
444.
J.4
8.2
2.1
11.5
18.5
8.7
13.1
16.8
9.4
9.7
1.9
16.8
0.6
117.
28
28.1
9.2
33.5
49.5
19.'
29.0
36.7
19.7
22.3
4.3
38.*
1.2,
290.
* (in Parts per Trillion)
70
-------
LETTERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 11.
A
-
B C
--
D
J
uu-~
L E
--
F G
1
H I J K
I I MASS
1 AL/L
1 -— 276
L (
— f^e.
\ A -
i- — . — ~ — . — _____ — . — . — — _ 223
•• —202
.
i [ i | • |~ i |- \FB
1200 1400 1600 1800 2000 2200 SCAN
2000 23*20 26*40 30*00 33*20 36*40 TIME
MAP:
a. Unfiltered water
A
B C
,1.
D L
-J
-*-*-
E
.._J
F G H
-
I J K
MASS
- - --276
CJi
— — .— — -V— _— ~- _ ^.jj
1 | 1 | l | l | 1 | l
1200 1400 !600 1800 2000 2200 SCAN
20*00 23*20 26*40 30*00 33*20 36*40 TIME
MAP*
c. Filtrate
A B
C D L
E F
GHIJ
._JJUL
— 223
-— 202
'2°°
20*00
I40°
23*20
I6O°
26*40
I80°
30*00
200°
36*20
2200 SCAN
36*40 TIME
MAP:
AB
b. Filter
COL
E F
G HIJ
1200
20*OO
1400
23*20
MAP:
1600
26*40
1800
30*00
2000
33*20
MASS
• - 302
•- 276
-- 252
•- 223
178
2200 SCAN
36*40 TIME
d. Water through continuous flow
centrifuge
Figure 16. EICPs from GC/MS analysis of exposure tank
-------
Table 11
PAH Compounds in Mussels
Peak Chemical ID
A Phenanthrene
B Anthracene
C Fluoranthene
D Pyrene
E Benz(a)anthracene
F Chrysene
G Benzo(b)fluoranthene and/or Benzo(k)fluoranthene
H Benzo(e)pyrene
I Benzo(a)pyrene
J Perylene
K Sum of PAHs with MW of 276
L Ethylan
72
-------
variability observed for the PAH compounds in water samples than for the
PCB compounds (paragraph 54) may reflect variability of the contaminants
in the BRH dredged material. It should be noted that soot particles
containing high concentrations of PAH compounds may be present in
contaminated sediments and that variability in the numbers of these
particles in samples may substantially contribute to concentration
variability.
80. The EICPs from the GC/MS analyses of unfiltered water, filters,
filtrate, and water passing through the continuous flow centrifuge
taken on day 8 of exposure are shown in Figure 16. The samples of un-
filtered water show a pattern of peaks for the compounds of interest
which is very similar to the patterns for the BRH sediment. A similar
distribution is observed in the sample from the filter. The EICPs from
the filtrate and the water passing through the continuous flow centrifuge
show a relative enhancement of the lower molecular weight PAH compounds.
As found with the PCB compounds, the PAH compounds appear to distribute
in accordance with their solubilities. With lower molecular weight, more
soluble PAH compounds are found in the filtrate, and with the higher
molecular weight, less soluble compounds are found associated with
particles.
81. Data from the analysis of filtered material and filtrates were
used to calculate sediment-water partition coefficients, Kp, where
Kp»Cs/Cw
Cs - concentration of compound in sediment (dry weight)
Cw » concentration of compound In water
Kps were estimated for compounds where the Log n-octanol/water partition
73
-------
coefficient (Log P) values were known. As observed for the PCB
compounds, the estimated results for the PAHs were considerably below
the experimental results (Table 12). This may indicate that the de-
sorption of PAH compounds from suspended sediment was not complete (i.e.,
equilibrium was not reached) during the residence time of the suspensions
in the dosing system.
82. PAHs - Mussels. Examination of the EICPs from mussels at
day 0 showed phenanthrene, fluoranthene and pyrene, benz[a]anthracene,
benzo[k] and/or benzo[b]fluoranthene, benzo(e]pyrene, and benzo[a]pyrene
and perylene (Figure 17). Ethylan was not found in these background
samples. At the first sampling period (day 1.8), the abundance of the
above compounds had increased and anthracene, Ethylan, and some PAHs
with MW 276 were apparent (Figure 17). Comparison of this EICP with one
from the BRH material shows that mussels had a relatively lower concen-
tration of peaks G, H, I, J, and K than was present in the sediment (Figure
17). This same general pattern of peaks is observed in all other mussel
samples taken during exposure (Figures 17 and 18). Following 7 days of
depuration, the lower molecular weight peaks A, B, C, and D had decreased
considerably while peaks E through K had become more prominent (Figure
19). The selective depuration of the lower molecular weight peaks may
result from the higher depuration rates associated with more water-soluble
compounds (Ernst 1977). As depuration continued, general decreases in
the concentrations of all compounds were observed (Figures 19 and 20).
-------
Table 12
Comparison of Experimental and Estimated Sediment/Water
"Partition Coefficients (KpsT
PAH Compound
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz(a)anthracene
Chrysene
Benz(a)pyrene
Perylene
Log P*
4.4
5.3
4.9
5.1
5.8
6.4
6.2
6.9
Kp Experimental**
.27 X 105
.23 X 105
.71 X 105
.79 X 105
4.7 X 105
4.1 X 105
24. X 105
17. X 105
Kp Estimatedt
.009 X 105
.066 X 105
.048 X 105
.031 X 105
.23 X 105
.87 X 105
.60 X 105
2.8 X 105
* Solubility from Mackay et al. (1980a) converted to Log P using
Log P = 5.00 .67 Log S where S is solubility in ymol/L (Chiou et al.
1977).
**Kp estimated as mean of 3 Kps determined on day 28 from mussel exposure
tank.
t Kp estimated as in Appendix Table A-l.
75
-------
~~J
A B
LETTERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 11.
C D L £F GH] J K
MA
A
_J
1
1200
20:00
P:
B C
JL
c
«.
.
,
--, i
1
k -
1 f 1 '
LJli
r
L .
' - i ii -
._j , .
i
2
) I
1
400
3^20
a.
E
--JL
1
1600
26:40
BRH 5
F G
I
1800
30:00
iedimer
HIJ K
1 i —
i -4_
L -
MASS
— 302
— 223
— 202
1 | I .10
2000 SCAN
33:20 TIME
its
MASS
— i
I ' ' 1 ' 1 '
1200 I40O I60O 1800
20:00 23:20 26:40 30:00
2OOO 22
33:20 36
— 252
— 202
00 SCAN
40 TIME
MAP:
c. Exposure tank mussels, day l.i
\B (
-
J-« — f.
:
4*
^
i.
D
E
-• ---•
,_
F
i
J
|
c
bd'j-
J,
|
H
"j
1
Ivl 1
J
1 — '
,.
;
— i
i
nn » .^i .
MASS
— 302
— 276
— 252
— 223
— 2O2
1200
20-00
I40O
23^20
1600
26^40
1800
30^00
2000
33^20
SCAN
TIME
MAP:
AS
Mussels, day 0
C D L
EF
G HIJ K
i
. .. LrfJ
l,__ -j .--M
fl
CC
__L
^
U
j
...
I
MASS
— i
•-— ri^" A, , , - -
r i |
i | i -
— OUi
— 276
— 252
-223
— 202
— 178
MAP:
1200 1400 1600 1800 200O 2200 SCAN
20:00 23:20 26:40 30:00 33:20 36:40 TIME
d. Exposure tank mussels, day 3.5
Figure 17. EICPs from GC/MS analysis
-------
A B
LETTERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 11.
CDL EF GHIJ K
CDL
EF
G HIJ
JL.."-« —
r '
J
t
*
L
\
V
' '
'I' ""i I"*" "l
223
1200 1400 1600 1800 2000
20>00 23*20 26*40 30^00 33*20
MAP
a. Day 7
AB CDL EF GHIJ K
2200
36-40
:
i
J
__L
L
*-.
*
-'
-T*""*T r 1
1200 1400
20*00 23*20
II 1
1600 1800 2000
26*40 30*00 33*20
276
252
223
202
178
SCAN
TIME
MAP:
t
f* —
12
JL-J.
*
^
-JL
L
p*""1" i
00 1400 16
I
LJ
i '
1
A— -. -
rfASS
- 302
— 276
252
- 202
— 178
1 1 ' 1 """
00 1800 2000 SCA
MAP:
b. Day 14
AB CDL
EF
GHIJ
MAP:
T1ME
__JLJV
\M~^*I~**-
,
A
DC
J
~V
-------
AB
LETTERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 11
C D L EF
G HIJ
*M
MB
c=A—
X
h
JU
J
«^A.
•TT ., i
bzs~
ttaM
k
***
\
_j.
r it
I BlMTII
1 '1 1
1200 1400 1600 1800
20=00 23=20 26=40 30=00
MASS
1 1
II
•BH^IMMAM
— 276
-252
— 223
- 202
— r— 3- 178
2000 SCAN
33=20 TIME
MAP
a. Day 35
MAP
b. Day 40
MASS
00
COL E F
G H IJ
c. Day 49
— h
1 ._.*
•f
Ik
«-
\M
Vv
***V*t
i
1200 1400
20=00 23=20
'
7
i —
MASS
1 1
-. *<..„.,
. .
L*-^1" ill i.i _._•>.. j
^L. _t.tu ^d**^''ft»**»««»»M
— 252
— 223
— 202
I7Q
1 1 . 1 ' 1 1
1600 1800 2000 2200 SCAN
26=40 30-00 33=20 36=40 TIME
MAP:
d. Day 56
Figure 19. EICPs from GC/MS analysis of exposure tank mussels
-------
LETTERS ABOVE PEAKS REFER TO IDENTITIES
OF COMPOUNDS LISTED IN TABLE 11
C D L E F GHIJ K
I200
20'00
I400
23^20
1600
26^40
1800
30-00
2000
3 3'20
MAP:
178
2200 SCAN
36'40 TIME
y- —r i i | , | 1 1 1 \~ 178
1200 1400 1600 1800 2000 2200 SCAN
20:00 23^20 26 = 40 30^00 33 = 20 36:40 TIME
MAP:
b. Day 70
Figure 20. EICPs from GC/MS analysis of exposure tank mussels
79
-------
Table 13
Estimated and Measured Bioconcentration Factors (BCF) in
Mussels at Day
Log P*
Peak No. PCB Compound (Log Kow)
4 2, 2', 5 -
10 2,4,4', -
25 2,2-4,5,5
36 2, 2', 4, 4'
43 2, 2', 3, 3'
PAHs
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz ( a ) anthrace ne
Chrysene
Benz(a)pyrene
Perylene
trichlorobiphenyl 4.7
trichloroblphenyl 5.0
'-pentachlorobiphenyl 6.3
,5,5'-hexachloroblphenyl 6.7
,4,4'-hexachloroblphenyl 7.0
4.4
5.3
4.9
5.1
5.8
6.4
6.2
6.9
28
Estimated Log BCF**
(wet wt.)
3.2
3.5
4.6
4.9
5.2
3.0
3.7
3.4
3.6
4.2
4.7
4.5
5.1
Measured Log! BCF
(wet wt.)
3.2
3.6
4.2
4.7
4.8
2.2
2.3
2.9
3.1
4.0
3.9
4.2
3.8
* PCB solubilities from Mackay et al. (1980b). PAH solubilities from
Mackay et al. (1980a). Solubility converted to Log P using Log P -
5.00 - .67 Log S where S is solubility in jjmol/L (Chiou et al., 1977)
** BCF estimated from log BF - 0.858 x Log Kow - 0.808 from Geyer et al.
(1982).
t BCF measured from mean of concentrations in three 28-day exposed mussels
divided by mean of concentrations in three 28-day filtered water samples
from the exposed tank.
80
-------
83. The mean concentrations of PAH compounds in mussels exposed
for 28 days were divided by the concentrations of PAHs in filtered
seawater samples to obtain bioconcentration factors (BCFs) (Table 13).
As with the PCBs (paragraph 64) these data are expressed in Log form on
a wet weight basis to facilitate comparisons with estimates of Log BCFs
from Geyer et al. (1982). The measured Log BCFs for the PAH compounds
increase with increasing Log P (decreasing aqueous solubility) as observed
for Log BCFs with mussels (Ernst 1977) and fish (Veith et al. 1979).
For PAHs the measured values are not as close to the estimated values
as were the PCBs (Table 13).
84. The mean concentration of PAH and Ethylan compounds in the
mussels at day 28 (Table 14) and the mean concentration of these compounds
in unfiltered water at day 28 (Table 10) were used to calculate bio-
accumulation factors (BAFs). The Log BAFs and the Log Ps are shown in
Table 15. The compounds with lower Log Ps showed lower BAFs. Benz(a)
anthracene and chrysene showed the highest BAF values in the mussels
while the higher molecular weight PAH compounds were accumulated less
effectively. The pesticide Ethylan showed a relatively high BAF compared
to the PAH compounds. Since organisms were not gut depurated prior to
analysis, the PAH content of the organisms included a contribution from
sediment in the gut (see discussion under PCBs, paragraph 63). The
levels of PAH contaminants found in control mussels were low and remained
relatively constant during the dosing-period. Ethylan was not found in
control samples (Table 16).
85. The uptake and depuration of total PAH compounds during the
mussel exposure study are shown in Figure 21. This plot was made using
81
-------
00
ro
Table 14
PAH and Ethylan Concentrations in Exposed Mussels Expressed as ng/g (dry)*
Day
Peak
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz ( a )anthracene
Chrysene
0
8.69
.488
17.4
15.8
2.72
6.63
Benzo(b)fluoranthene 9.82
and/or Benzo(k)fluoranthene
Benzo(e)pyrene
Benzo(a)pyrene
Perylene
Sum of PAHs with
MW of 276
Ethyl an
Sum of PAH Compounds
3.96
.532
1.12
3.66
0
70.8
7
162.
63.6
802
1359
754
1005
631
288
269
30.7
261
452.
5630
Expos ur
14
130.
49.6
444
811
448
650
408
234
216
25.5
170
177.
3590
e~
21
246.
73.0
475
1117
512
856
543
301
350
60.3
111
317.
4640
>_ „
™
28
130.
59.8
698
1228
864
1179
895
379
392
44.0
348
444.
6220
35
10.7
4.52
59.1
175
285
435
472
249
226
23.9
141
142.
2080
— — Tlav
itef
40
13.6
4.81
37.6
95.3
153
270
288
161
120
18.4
82.7
154.
1250
>uration
49
10.1
2.75
19.8
38.8
35.9
75.4
101
59.0
31.1
4.30
21.4
29.0
399
56
23.2
5.43
17.9
35.0
9.76
25.2
29.0
22.6
7.01
4.13
7.57
14.5
187
62
9.65
2.17
23.4
41.7
22.8
40.6
73.1
23.5
16.1
4.26
20.6
20.3
278
70
10.9
2.57
20.1
33.3
4.99
16.0
12.8
0
0
0
5.97
8.8
107
* Values not corrected for blank value.
-------
Table 15
Mussel Bioaccumulation Factors (Calculated for Day 28)
Peak
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz(a)anthracene
Chrysene
Benzo(b)fluoranthene and/or
Benzo(k)fluoranthene
Benzo(e)pyrene
Benzo(a)pyrene
Perylene
Sum of PAHs with MW of 276
Ethyl an
Log BAF
3.7
3.8
4.3
4.4
4.7
4.6
4.4
4.3
4.2
4.0
4.0
4.6
Log P*
4.4
5.3
4.9
5.1
5.8
6.4
6.2
6.9
7.0
* P » n-octanol/water partition coefficient obtained from solubility
data in (Mackay et al. 1980a). Converted to Log P using
Log P « 5.00-.67 log S where S is solubility in umol/1 (Chiou et
al. 1977).
83
-------
Table 16
Levels of PAH and Ethylan Compounds in Control Mussels
During
Peak
Phenanthrene
Anthracene
Fluor anthene
Pyrene
Benz ( a )anthracene
Chrysene
Benzo(b)£luoranthene and/or
Benzo(k)f luoranthene
Benzo(e)pyrene
Benzo(a)pyrene
Perylene
Sum of PAHs with MW of 276
Ethylan
Study (PPb;
0
8.69
.488
17.4
15.8
2.72
6.63
9.82
3.96
.532
1.12
3.66
0
(ng/g(dry))
Day
ire — ~ —x Depuration
28
6.36 5
.697 1
6.12 11
9.31 13
1.13
3.12 7
3.84 1
2.07 4
.621
.355
2.28 2
0
56
.70
.20
.7
.6
.852
.95
.62
.56
.399
.221
.01
0
- _ _ _S
70
6.22
.383
7.78
13.4
.911
5.80
2.14
3.92
.075
.075
.524
0
Sum-PAHs
70.8
35.9
49.8
41.2
84
-------
CO
Q.
Q.
EXPOSED
REFERENCE
DEPURATION
f UPTAKE
| 1 1 1 1 1 1 1 I I I 1" 1 1 I
05 10 15 20 25 30 35 40 45 50 55 60 65 70
DAYS
Figure 21. Concentration of sum of parent PAHs in mussels exposed
to BRH sediment versus time
85
-------
the sums of the concentrations of the eleven PAH compounds listed in
Table 14 and shows a rapid uptake of PAH compounds to day 7 of the exposure,
Following day 7 concentrations fluctuated until the end of the exposure
at day 28. Depuration of the PAH compounds was rapid during the first
week followed by several weeks of slower depuration. By day 70 the
concentrations of most compounds had decreased to approximately two to
three times their day 0 levels indicating that depuration was not
complete.
86. Figures A18 to A20 show the uptake and depuration of the
PAHs and the Ethylan. Most of the curves showed maximums at day 7 or
day 28. The curves for fluoranthene and pyrene showed maximum
concentrations at day 7, but in general larger PAH compounds showed
maximum concentrations at day 28.
87. During the depuration phase, the lower molecular weight PAH
compounds appeared to be depurated more rapidly that the higher molecular
weight PAH compounds. The rapid depuration of more soluble (lower Log
P) compounds by mussels has been observed in another study (Ernst 1977).
In general, the higher molecular weight PAH compounds (higher Log P)
took longer to reach their maximum level and were depurated more slowly
than the lower molecular weight (lower Log P) compounds. Ethylan which
was not detected in control organisms was accumulated and depurated similar
to chrysene.
88. Petroleum Hydrocarbons - Mussels. The petroleum hydrocarbons
from samples were detected as a large mound in flame ionization detection
gas chromatograms (Figure 22). This mound of material, usually referred
to as an unresolved complex mixture (UCM), consists of numerous petroleum
86
-------
a. Exposure tank water, day 0
b. BRH sediment
c. Mussels, exposute day 28
Figure 22. Capillary column flame ionization detector gas
chromatograms of PF-50 (contains mostly straight chain, branched,
and cyclic saturated hydrocarbons)
-------
hydrocarbons (i.e., alkanes, cycloalkanes). The petroleum hydrocarbons
found In unfiltered water samples from the dosing system showed a
distribution as an unresolved complex mixture (UCM) which was slightly
lower in molecular weight, but otherwise similar to the distribution
found in the BRH sediments (Figure 22). During the uptake phase the
mussels accumulated a UCM which was slightly lower in molecular weight
than the distribution found in the unfiltered water.
89. The petroleum hydrocarbons followed the same general pattern
of concentration changes observed during the uptake and depuration for
the other organic contaminants in mussels (Figure 23). By day 7 the
contaminants were near their maximum values. At day 14 and day 21 lower
concentrations were observed with the highest concentration found at day
28. The first week of depuration showed a rapid loss of total petroleum
hydrocarbon contaminants followed by a plateau phase of decreased loss
rates. This behavior for the depuration of petroleum hydrocarbons has
been observed in other studies with bivalve molluscs (Lee et al. 1972;
Clark and Finley 1975; Fossato 1975; Lake and Hershner 1977). Chromato-
grams from control mussels showed a low level of petroleum hydrocarbons
(as a UCM). Petroleum hydrocarbons in mussels from the control tank
showed slight concentration decreases during the study period (Figure
23).
Inorganic Contaminants
90. Sediment. The trace metal composition for the barrel of BRH
sediment analyzed is given in Table 17. The wet-to-dry-weight
88
-------
500 r
£
O
10
exposure
tank
control
tank
Figure 23.
UPTAKE h
DEPURATION
10 20 30 40
TIME (days)
50
60
70
Concentration of total petroleum hydrocarbons in mussels
exposed to BRH sediment versus time
89
-------
Table 17
Average Trace Metal Concentrations for Black Rock Harbor
Sediment Samples Expressed as yg/g Dry Weight
Metal pg/g Std. Dev. % Std. Dev.
Fe 29600 809 2
Zn 1200 59 4
Mn 359 37 10
Cu 2380 112 4
Pb 378 16 4
Cd 23.4 0.9 4
Cr 1430 77 5
Ni 139 4 3
Hg 1.7 0.1 4
Wet/Dry 3.22 0.02 0.6
90
-------
Table 18
Seavater Metal Concentrations Determined for the Black Rock Harbor
Sediment Exposure and Control Chambers Expressed as ug/liter
Metal
Fe
Std. Dev.
Zn
Std. Dev.
Mn
Std. Dev.
Cu
Std. Dev.
Pb
Std. Dev.
Cd
Std. Dev.
Cr
Std. Dev.
Exposure
Pre renewal
of BRH Slurry
288
4.2
25.5
0.4
4.8
0.2
34.8
0.2
4.5
0.5
0.82
0.1
16.6
0.2
Chamber
Post renewal
of BRH Slurry
343
9.9
22.0
0.4
4.8
0.5
34.6
0.4
4.5
0.5
0.7
0.2
17.8
0.2
Control
Chamber
5.2
0.8
5.6
0.7
0.7
0.5
1.4
0.3
<'
<0.2
<0.2
* Seawater samples were collected 2 hr before renewal of the Black
Rock Harbor sediment slurry and 3 hr post-renewal of the slurry.
91
-------
ratio is also given for the BRH sediment samples. No values for As are
listed in this table since a chemical interference was detected during
the analysis (for both HGA-AA and MHS-1 hydride generation techniques)
of these sediment samples. The results indicate that BRH sediment samples
are reasonably homogeneous for a given barrel if precautions are taken to
re-mix each barrel before sampling.
91. Seawater. The results for the monitoring of the mussel
exposure system are given in Table 18. Only the total acid leachable
concentrations present in the unfiltered seawater are reported.
Concentrations for the elements of interest in the control chamber were
generally undetectable by direct injection of seawater samples into the
HGA unit; however, seawater samples from the BRH exposure chamber could
be analyzed by this method* The results show that the concentrations
for most of the metals are reasonably consistent for the two sets of
samples collected prerenewal and post-renewal of the BRH sediment slurry.
92. It is important to know if the concentrations of the various
metals determined in the exposure chamber agree with the measured
particulate concentrations that were delivered by the dosing system.
The total concentration of BRH sediment delivered into the exposure
chamber can be calculated from the Fe concentrations presented in Table
18 assuming Fe is conservative. Using these data, the calculated
concentrations for BRH sediment are 10.8 and 12.8 mg/L for the prerenewal
samples and postrenewal samples, respectively. The values actually
measured by filtration of seawater samples from the exposure chamber
92
-------
Table 19
Average Fe/Metal Ratios (± Standard Deviation) for the Exposure
Chamber Seawater and Black Rock Harbor Sediments
Exposure Chamber
Fe/Metal BRH Sediment Seawater
Fe/Zn 24.8 + 0.9 17.7 + 2.4
Fe/Mn 80.9 + 7.9 77 + 17
Fe/Cu 12.4+0.5 9.1 + 1.0
Fe/Pb 78.4 + 2.6 70 +8
Fe/Cd 1270 + 45 420 + 156
Fe/Cr 20.8 + 0.98 18.4 + 1.9
93
-------
during the course of the exposure period range from 8.19 to 10.33 mg/L.
The values determined for the control chamber during this same period
range from 1.45 to 2.02 mg/L. The calculated concentration of BRH
sediments in the exposure chamber are, therefore, only 10 to 20 percent
higher than the actual range of concentrations determined over the entire
time course of the experiment. Iron was used to make these calculations
because Fe has the highest concentration of any metal measured in BRH
sediment and should be affected less by contamination during seawater
sample analysis.
93. The inter-elemental ratios of the metals (i.e. Fe/metal) are
given in Table 19. This table also contains the Fe/metal ratios of the
BRH sediment samples. Theoretically the Fe/metal ratios determined in
the seawater samples should agree with the Fe/metal ratios for the BRH
sediment samples. There are, however, some difficulties with this
concept. The seawater metal concentrations determined were analyzed at
concentrations 1000 times lower than were determined in the bulk BRH
sediment samples. Therefore, detection limits and contamination of
seawater samples could affect the metal concentrations and their sub-
sequent Fe/metal ratios. This is important since the measured metal
concentrations are used to determine bioaccumulation factors of metals
for the exposed mussel samples. The Fe/metal ratios for the exposure
chamber seawater samples presented in Table 19 are the average values
for the prerenewal and postrenewal of the BRH slurry. The Fe/metal
ratios for the BRH sediment samples were calculated from the data presented
in Table Bl. The BRH sediment Fe/metal ratios are the average of the
individual ratios for the nine samples. The Fe/metal ratios for the
94
-------
seawater samples compare favorably with the BRH sediment ratios for all
elements listed except Cd. The Fe/Cd ratio is different from the sediment
ratio by a factor of 3. This indicates that a secondary source of Cd
was present in the exposure tank or that the seawater samples were con-
taminated with Cd during collection. The detection limit for Cd in
seawater using the present analytical techniques is about 0.1 to 0.3 ug/L.
The concentration of Cd determined in the exposure chambers was 0.8 and
0.7 ug/L for the two sets of samples collected. Therefore, the concentration
of Cd determined in the exposure chamber was very close to the analytical
detection limit and this probably accounts for the factor of 3 difference
in the ratio compared to the BRH Fe/Cd sediment ratio. Also, the ratio
of Fe/Zn was corrected for the Zn concentration determined in the control
tank (i.e., 5.4 ug/L). This Zn concentration is probably due to the
large amount of PVC piping that is used in this facility to carry seawater
from Narragansett Bay to the various laboratory seawater exposure
experiments. Zinc is used as a catalyst in the production of PVC plastic.
The concentration of Zn for Narragansett Bay at a site near the ERL-N is
about 1 to 2 yg/L.
94. The concentration of Hg could not be detected In either the
control chamber or the exposure chamber. The exposure chamber should
have a Hg concentration of approximately 0.02 ug/L. This Hg concentration
was calculated by dividing the average Fe concentration in the exposure
chamber seawater samples by the Fe/Hg average ratio for the BRH sediment
samples. The detection limit for Hg with our present analytical
equipment is 0.05 ug/L.
95
-------
95. Arsenic was not determined in the control or exposure chamber
seawater samples. The As concentration for BRH sediments (provided by
New England Division, Corps of Engineers) is 6.1 mg/kg. The calculated
ratio of Fe/As for BRH sediments using the average Fe concentration from
Table Bl is 4850. The theoretical As seawater exposure chamber con-
centration can be calculated by dividing the Fe seawater concentration
with this calculated Fe/As ratio value. The calculated As concentration
due to the addition of 10 mg/L of BRH sediment to the exposure chamber
is 0.07 ug/L. The natural concentration of As in seawater is
approximately 1 to 2 ug/L. Therefore, the natural seawater concentration
of As is about 15 times greater than the BRH sediment As added to the
exposure chamber.
96. Mussels. The average metal concentrations and standard
deviations for the mussel samples collected from the BRH exposure chamber
on day 28 are given in Table 7. All of the inorganic data used to
calculate these averages are given in Tables B2, B3, and B4. The averages
reported for the control mussel samples in this Table are the averages
for all the control mussel samples and not just day 28. There is
a statistically significant (a « 0.05) difference between the means for
the Cu concentrations of the control mussel samples collected at the
different times during the experiment. However, for all the other metals
determined, there is no statistical difference in their means for the
control mussel samples collected during the course of the experiment.
There is no significant difference between the mean Mn, Zn, and As in
the control and exposed mussel samples for the 28-day sampling period
(Student t-test, P<0.05). However, there is a significant difference
96
-------
between the means for the 28-day control mussel samples and the 28-day
exposed mussel samples for all of the other elements determined (lie.,
Fe, Cr, Cu, Pb, and Cd). If the average of all of the control mussel
samples (days 0, 28, 56, and 70) are compared to the 28-day exposed
mussel samples, then only the mean Mn concentration for the 28-day BRH
exposed mussels is not significantly different from the mean Mn
concentration for all the control mussel samples.
97. During the uptake period (excluding time 0) there is no
significant difference (one way analysis of variance, OC »0.05) of the
means for Fe, Cu, Fb, Cr, and Zn for the BRH exposed mussel samples over
time. This would indicate that equilibrium was reached for these
metals by the time the first set of exposed mussel samples was collected
(i.e., 1.8 days). This might suggest that the mussels simply had BRH
sediment in their gut at the time of collection and that the mussels
depurated the BRH sediment at a constant rate during the uptake period*
However, this is refuted by the following. The Fe, Cr, Cu, Pb,
and Cd concentrations for the mussel samples collected from the exposure
chamber during the first week of depuration (day 35) were still elevated
relative to their mean concentrations in the control mussel samples.
Gut depuration of BRH sediment from the exposed mussel samples should
take place faster than 7 days. Also, Pb did not depurate readily
from the BRH exposed mussels between day 35 and day 70.
98. The bioaccumulation factors (BAFs) for the metals determined
in the 28-day exposed mussels are given in Table 20. To determine the BAF
values, the following calculations are made: (a) the average metal concen-
trations for the control mussels are subtracted from their respective
97
-------
Table 20
Metal Bioaccumulation Factors for Mussels Exposed^jio
Black Rock Harbor Sediment*
Metal
Fe
Zn
Mn
Cu
Pb
Cd
Cr
Mussel
BAF
972
6513
-208
1243
1977
6567
1331
Mussel
28 Day /Control
2.6
1.9
0.9
4.6
2.8
2.7
11.4
* The ratios reported are the day 28 mussel sample averages
divided by their respective average control concentrations
98
-------
Table 21
Average Fe/Metal Ratios (± Standard Deviation) for Control
Mussels and Black Rock Harbor Sediment
Fe/Metal BRH Sediments Mussel Control
Fe/Zn 24.8 + 0.9 1.1 ± 0.2
Fe/Mn 80.9 + 7.9 18.2 + 5.A
Fe/Cu 12.4 + 0.5 17.9 + 4.2
Fe/Pb 78.4 + 2.6 40.7 + 10.2
Fe/Cd 1270 + 45 73.8 ± 9.8
Fe/Cr 20.8 + 0.98 100 + 33
99
-------
metal concentrations for day 28 BRH exposed mussels; and (b) the resultant
metal concentrations are divided by their respective BRH exposure chamber
seawater metal concentrations. The units of measure for the mussels are
in micrograms per gram dry weight and the seawater concentration units
are in micrograms per milliliter. The ratios of the average metal
concentrations for the day 28 BRH exposed mussels to the average
metal concentrations for the control mussel samples are also reported
in Table 21. The ratios are a different method of representing the
metal accumulation in the day 28 BRH-exposed mussels. The BAF values
tend to give an impression of a large uptake by the mussels for the
metal concentrations determined. However, the metal ratios give only
the relative metal concentration increases for the BRH-exposed mussels
versus the control mussel samples in this study. For example, the BAF
values for Zn and Cr are 6513 and 1331, respectively. However, the day
28/control ratios are 1.9 and 11.4 for Zn and Cr, respectively. Using
only the BAF values one might conclude that Zn would have a larger
percent increase than Cr in exposed mussels.
99. There was no increase for the Mn concentration in the day 28
BRH-exposed mussels compared to the control mussels. However, the
increase for Cr for these same mussel samples was a factor of 11. The
increases for the other metal concentrations determined for the day 28
BRH-exposed mussels compared to the control mussels are generally greater
by a factor of 2 to 5.
100. The uptake and depuration curves for the metals determined
in the samples are given in Figures 24 to 27. There are two day 14
sets of mussel samples collected from the BRH exposure chamber (day 0
100
-------
600
1
3400
-------
~ 15
E
Q.
O.
- 10
JO
Q.
THE STANDARD DEVIATIONS OF THE AVERAGE
METAL CONCENTRATIONS ARE DEPICTED AS
A VERTICAL LINE. ALL Pb AND Cd CONCEN-
TRATIONS ARE IN G/G DRY WEIGHT.
o EXPOSED
• CONTROL
—UPTAKE—h- DEPURATION —
10 20 30 40 50 60 70
TIME (days)
a. Pb
8
E
Q.
Q.
°- 6
3 4
o EXPOSED
• CONTROL
—UPTAKE
DEPURATION-
Figure 25,
0 10 20 30 40 50 60 70
TIME (days)
b. Cd
Uptake and depuration in mussels exposed
to BRH sediment
102
-------
EIOO
ex
50
E 8
a
a.
THE STANDARD DEVIATIONS OF THE AVERAGE
METAL CONCENTRATIONS ARE DEPICTED AS
A VERTICAL LINE. ALL Cu AND As CONCEN-
TRATIONS ARE IN nG/G DRY WEIGHT.
o EXPOSED
• CONTROL
0 10 20 30 40 50 60 70
— UPTAKE-H-DEPURATION-
TIME (days)
a. Cu
° EXPOSED
• CONTROL
— UPTAKE—I—DEPURATION
I 1 1 1 i—
0 10 20 30 40 50 60 70
TIME (days)
b. As
Figure 26. Uptake and depuration in mussels exposed
to BRH sediment
103
-------
400
9- 300
200
100
40
E
o.
o.
c
a30
20
10
THE STANDARD DEVIATIONS OF THE AVERAGE
METAL CONCENTRATIONS ARE DEPICTED AS
A VERTICAL LINE. ALL Zn AND Mn CONCEN-
TRATIONS ARE IN pG/G DRY WEIGHT.
o EXPOSED
• CONTROL
-UPTAKE 1 DEPURATION —
10 30 30 40 50 60 70
TIME (days)
a. Zn
o EXPOSED
• CONTROL
—UPTAKE
DEPURATION
0 10 20 30 40 50 60 70
TIME (days)
b. Mn
Figure 27. Uptake and depuration in mussels exposed
to BRH sediment
104
-------
to 14 and day 14 to 28). These two sets have been combined to create
only one average concentration for day 14 of the uptake period for the
exposed mussel samples.
101. To determine if any relationships exist between any of the
metals determined for the homogenized mussel samples, correlation
coefficients (r) were calculated for all metals compared to the Fe
concentration for each sample. Iron was chosen as the element to
compare to the other metals for three reasons: (a) Fe has the highest
concentration of all the metals determined in BRH sediment; (b) Fe has
the smallest percent standard deviation of the means of the control
mussel samples for the entire experiment; and (c) Fe should be less
subject to contamination during analysis compared to any of the other
metals determined.
102. The number of sample pairs (i.e., mussel concentrations of X
and Y) must be considered in order to evaluate the probability of the
correlation coefficient being significant due to random sampling from
an uncorrelated population. All the correlation coefficients used
in the following discussion are based on P values of 0.05. For example,
24 data pairs require a correlation coefficient greater than 0.381 to
be at the 0.05 level of significance (Fisher 1985).
103. During the uptake period (24 sample pairs) only Mn was not
significantly correlated with Fe. All the other metals showed varying
degrees of correlation. The calculated correlation coefficients for Fe
versus Cr, Pb, As, Zn, Cd, Cu, and Mn are 0.957, 0.827, 0.635, 0.534,
105
-------
0.490, 0.461, and 0.263, respectively. None of the metals correlated
with Fe for the control mussel samples or for the mussel samples collected
during the depuration phase of the experiment.
104. It Is easily seen that the Fe and Cr concentrations In the
mussel samples covary linearily (r-0.957) during the uptake period (see
Figure 24). A comparison of the Fe/Cr ratio (see Table 21) for the
control samples versus the BRH sediment samples shows that the control
mussels have a ratio five times that of the sediment samples. The
only other metal determined that has a Fe/metal ratio larger for the
control mussels versus the BRH sediment is Cu, and this ratio difference
is a factor of 2. The Fe/Cr ratio difference in the controls and BRH
sediment may be an advantage in determining uptake of BRH sediment in
mussels during the field verification portion of this study. The
relatively low concentration of Cr in the control mussels versus the
BRH sediment makes Cr an ideal choice for a metal tracer of BRH sediment.
105. The uptake/depuration plots of Pb, Cd, and Cu in the mussels
from the BRH exposure chamber are given in Figures 25 and 26. All three
of these metals are elevated during the uptake period compared to the
control mussel samples, but there is no clear uptake pattern for any of
them. The correlation coefficients of Pb, Cd, and Cu versus Fe during
the uptake period are 0.827, 0.490 and 0.465, respectively. All three
of these correlation coefficients are significant (P=0.05) for a
population of 24 sample pairs. The Pb uptake curve also resembles many
of the same features of the Cr and Fe curves during the exposure phase
of the experiment.
106
-------
106. The maximum amount of uptake for Cu occurs early (day 3.5)
into the exposure period compared to all the other metal concentrations
determined and then tissue concentrations decline after 21 days
into the exposure. The depuration of Cu appears to be steady in
that a slow release of Cu occurs over a 3-week period. The Cu
concentration declines to the average control mussel concentration
on day 56 and then remains constant until the end of the experiment.
107. The metals Pb and Cd do not show the same type of depuration
curve. Neither of these elements shows a steady decline in concentration
during the depuration period. At day 70 (day 42 of depuration), the
average Cd concentration for the exposed mussels declined to the control
mussel concentration, but the average Fb concentration for the exposed
mussels did not decline to the control mussel concentration.
108. The last three metals determined in the mussels, As, Zn, and
Mn, have one common feature. During the uptake and depuration periods
the concentrations of these metals in the exposed mussels vary around
their respective concentrations in the control mussels. Several of
the average concentrations for these metals in the exposed mussels are
lower than the average for the controls during the uptake period. The
uptake and depuration plots for these three metals are given in Figures
26 and 27. The correlation coefficients for As, Zn, and Mn versus
Fe are 0.635, 0.534, and 0.263, respectively, for the uptake portion of
the exposure period. Of these three metals, only As and Zn have
significant correlation coefficients (P-0.05) for a population of 24
sample pairs.
107
-------
30
I
O
Q.
in
UJ
cc
O
o
UJ
H
LJ
Q
TIME
a. BRH sediment
t
UJ
o
a.
cc.
o
UJ
O
35
Figure 28,
TIME
b. Reference sediment
Capillary column electron capture gas chromatograms
of PF-50 (PCB) fraction
108
-------
Worm Test
Organic Contaminants
109. PCBs - Sediment. The electron capture detection (BCD)
chromatograms of extracts from BRH sediments taken from the exposure tank
show a distribution of PCB compounds with from two to ten chlorine atoms
with the predominant peaks containing five and six chlorine atoms (Figure
28). Tentative identifications of the peaks are shown in Table 5.
Chromatograms from day 0 and day 40 of the exposure sediment show almost
Identical distributions of compounds.
110. The ECD chromatograms of reference sediment from the reference
tanks show a distribution of PCBs with from two to ten chlorine atoms.
The distribution of PCBs in the reference sediment shows a relatively
greater abundance of higher molecular weight PCBs than is found in the
exposure sediment. Chromatograms from day 0 and day 40 of the reference
sediment show almost identical distributions of compounds.
111. Sediments from the exposure and reference tanks were sampled
throughout the study. Samples taken on day 0 and day 40 were analyzed
and the results show only small differences in the concentrations of
PCBs over the study period.
112. PCBs - Worms. The polychaete worms Nereis virens on
arrival at ERLN were large but visually appeared to lose weight
during exposure to BRH dredged material. Worms from the reference
tank did not appear to lose weight over the duration of the experiment.
Observations of worms after removal from the tanks and during the gut
depuration phase (see Methods, paragraph 20) showed that the exposed
109
-------
organisms appeared to process only small amounts of BRH material during
the uptake phase (some worms from the exposure tank had no sediment in
their guts). When the exposed worms were placed in reference sediment
for depuration, they processed only small amounts of sediment. Reference
worms, however, were always full of sediment prior to gut depuration.
113. Chromatograms of worms from the exposure and depuration
study are shown in Figures 29 and 30. The chromatograms from the day 0
worms show three predominant peaks (36, 39, and 48) which represent
compounds with structures that are resistant to degradation (Zell and
Ballschmiter 1980). The distribution of these peaks maximizes at the
Cl, PCBs. Only trace amounts of peaks No. 1 to 13 are evident.
Following exposure for 14 days the organisms had accumulated a
range of PCB compounds from C\2 to c^10* However, comparison of the
chromatogram from day 14 worms with the chromatogram from BRH sediments
shows that earlier eluting compounds appear to be preferentially
accumulated. A peak distribution similar to that observed in the day 14
worms is found in day 28 and day 42 organisms, but by day 56 (28 days of
depuration) peaks 36 and 39 are beginning to predominate. Reference
worms showed only minor changes in peak patterns during the experiment.
114. During the exposure to the Black Rock Harbor sediments, N^
virens accumulated the PCB A-1254 (Figure 31). It is not known if the
worms reached steady-state during this 28-day uptake. Some researchers
have found no indication of equilibrium concentrations being approached
during 32 days of exposure of worms (N. virens) to sandy sediment spiked
with A-1254 (McLeese et al. 1980). Other researchers found that (a)
110
-------
a. Day 0
I-
UJ
O
TIME
b. Day 14
Figure 29. Capillary column electron capture gas
chromatograms of PF-50 (PCB) fraction for worms
exposed to BRH sediment
111
-------
UJ
en
a
c/j
LJ
a
D
; >
' >
Figure 30,
TIME >
b. Day 56
Capillary column electron capture gas chromatograms of PF-50
fraction for worms exposed to BRH sediment
112
-------
1000-
in
CM
CD
u
a.
100
exposure
tank
control
tank
10
UPTAKE
10
DEPURATION-
20 30 40
TIME (days)
50
60
'Figure 31.
Concentration of total PCBs (as A-1254) in worms
exposed to BRH sediment versus time
113
-------
steady-state for N. virens exposed to A-1254 In naturally contaminated
sediments was reached by day 30 and day 40 depending upon the sediment
(Rubinstein et al. 1983); and (b) steady-state was reached between 35
days and 100 days (Interpolation of graphical data) for JJ. diversicolor
exposed to sediments spiked with different levels of PCBs (Fowler et al.
1978).
115. The bioaccumulation factors (BAFs) » PCS (worm dry wt.)
PCB (sediment dry wt.)
for A-1254 in the present study were 0.20 for the exposed worms and
1.33 for the reference organisms at day 28. Other researchers have
reported BAF values of approximately 8 to 10 (dry wt.) for 14. dlverslcolor
exposed to sediments spiked with A-1254 (Fowler et al. 1978), and
concentration factors of 3.8 and 10.8 for large and small N. virens
exposed to A-1254 in sandy sediment after 32 days exposure, although
steady-state conditions were not attained (McLeese et al. 1980). BAFs
ranged from 0.157 to 1.59 for II. virens exposed to sediments naturally
contaminanted with A-1254 (Rubinstein et al. 1983).
116. The present study found no depuration of A-1254 contaminants
during the 28-day depuration period (Figure 31). This is in agreement
with findings that there was no obvious excretion of PCB by N. virens
during 21 days post-exposure (McLeese et al. 1980). In contrast, other
researchers reported that 14. diversicolor eliminated PCB during post-
exposure depuration periods (Fowler et al. 1978).
117. The uptake and depuration of representative individual PCB
peaks (identified in Table 5) are shown in Figures A14 through A17. An
examination of these curves suggests that the lower molecular weight, more
soluble compounds are accumulated more rapidly than the higher molecular
114
-------
weight compounds, but no significant depurations were apparent.
Bioaccumulation data for the individual peaks are shown in Tables 22 and
23. While the data are not conclusive, there appears to be a slight
increase in BAF in the range of the C15 and Clg PCB peaks. The reason
for the lower BAF values observed in th6 exposed versus the reference
worms may be the result of the organic matter content of the sediments.
The reference sediment contained 1.8% organic matter while the exposure
sediment contained 5.9%. Other researchers have suggested that the
organic content of sediments play a key role in the availability of
organic pollutants to benthic organisms, with higher organic content
decreasing the bioavailability of contaminants (Rubinstein et al. 1983).
118. Another reason for the relatively low exposure BAF values
found in the present study may result from the low feeding rates observed
for the worms during the study period. Since the organisms fed little,
if at all, and appeared to lose weight during this study, a significant
portion of the accumulation of PCBs that occurred may have resulted
from bioconcentration of contaminants from interstitial water. This
route of uptake has contributed significantly to the PCB body residues
of Arenicola marina and Vt_. diversicolor in laboratory exposures (Courtney
and Langston 1978).
119. PAH - Sediment. Sediment samples collected from the exposure
and reference tanks during the worm exposure study showed distributions of
PAH compounds which are typical of distributions found in heavily
contaminated and lightly contaminated sediments, respectively. A much
more detailed analysis of these sediments can be found in Rogerson et al.
(1983).
115
-------
Table 22
PCB Bioaccumulation Factors. Exposed Worms Day 28
Peak No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
BAF
.10
.57
.27
.16
.30
.20
.15
.07
.20
.16
.19
.17
.39
.39
.20
.29
.13
.22
.19
.09
.07
.30
.38
.25
.28
.22
.36
.09
.22
.26
Peak No.
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
BAF
.21
.24
.04
.21
.35
.33
.32
.16
.26
.19
.29
.30
.21
.16
.23
.23
.23
.20
.19
.17
.19
.12
.15
.06
116
-------
Table 23
PCB Bioaccumulation Factors of Worms in Reference Sediment - Day 28
Peak No.
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
BAF
.70
.96
1.6
.57
.43
.84
2.6
1,5
1.4
.97
1.3
.38
2.7
1.0
1.6
1.5
.57
1.2
2.9
1.8
1.1
2.7
1.6
4.0
1.8
2.3
1.5
.96
1.6
1.3
.90
1.9
1.4
.94
1.5
.85
1.5
1.1
117
-------
120. PAH - Worms. During the exposure to BRH dredged material,
the polychaete worm N. virens accumulated PAH contaminants and the
pesticide Ethylan. Reference worms also contained PAH compounds; however,
the concentrations of PAH compounds in the reference worms following
exposure were similar to pre-exposure concentrations. Ethylan was not
detected in the reference worms. The uptake and depuration of the sum
of the PAH compounds identified in Table 11 are shown in Figure 32.
This curve is dominated by the lower molecular weight PAH compounds.
121. Figures A21 to A23 show the uptake and depuration of the
individual PAH compounds and the Ethylan during the worm exposure study.
The lower molecular weight compounds (i.e., phenanthrene, anthracene,
fluoranthrene, and pyrene) showed maximum uptakes at day 14, while the
higher molecular weight PAHs and the Ethylan reached their maximum at
day 28. Due to the limited sampling times and the fluctuating nature of
the data, it cannot be determined if steady-state was reached for the
accumulation of PAH and Ethylan by the worms.
122. While steady-state may not have been reached for the PAHs and
Ethylan, bioaccumulation factors (concentration in worm (dry weight)/
concentration in sediment (dry weight)) were determined for comparison
purposes. The data are shown in Table 24. With the exception of an
unexplained increase in BAF for pyrene in the reference worms, the BAFs
appear to range from almost zero to a few percent.
123. In the depuration phase the lower molecular weight PAH compounds
appeared to be depurated more rapidly from worms than the higher molecular
weight PAH compounds. Ethylan was accumulated by the worms from the
118
-------
10,000
1000-
a.
a
«^
CO
X
100-1
EXPOSURE
CONTROL
UPTAKE
•4-
DEPURATION
T
T
10
20
30
40
50
—T~
60
TIME (days)
Figure 32,
Concentration of sum of parent PAHs in worms exposed
to BRH sediment versus time
119
-------
Table 24
PAH and Ethylan Bioaccumulation Factors - Worms
Compound
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz (a )anthracene
Chrysene
Benzo(b)fluoranthene and/or
Benzo(k)fluoranthene
Benzo(e)pyrene
Benzo (a)pyrene
Perylene
Sum of PAHs with MW of 276
Ethylan
Exposed Worms*
.04
.04
.05
.04
.03
.06
.02
.02
.02
.07
.004
.09
Reference Worms**
.14
.008
.04
.28
.004
.01
.005
.01
.005
.01
NDt
NDt
* Calculated using mean (n=2) concentrations of PAH and Ethylan compounds
in exposed worm samples at day 28 divided by mean (n=3) concentrations
of compounds in exposure sediments (dry weight).
**Calculated using concentration of PAH and Ethylan compounds in reference
worms at day 28 (n-1) divided by concentration of compounds in reference
sediments (n=l)(dry weight.
t ND - not determined in reference worms.
120
-------
exposure sediments and depurated similar to the higher molecular weight
PAHs.
124, jetroleum Hydrocarbons - Worms. The UCM patterns found in
the worms following exposure to BRH sediments, when compared to those
of the sediment, were shifted toward lower molecular weight compounds
(Figure 33). Only small changes were observed in these patterns during
the depuration phases. The concentrations of petroleum hydrocarbons in
the exposed worms during the uptake and depuration phases of the
experiment are shown in Figure 34. The maximum concentration observed
was at day 28. Concentrations of total petroleum hydrocarbons appeared
to decrease during depuration. Only small changes in the patterns and
concentrations of total petroleum hydrocarbons in the worms from the
reference tank were observed.
125. Comparisons of Bioaccumulation - Mussels and Worms.
The determination of bioaccumulation or bioconcentration factors for
the organic compounds found in the exposed mussels and worms in this
study depends upon whether the accumulation is considered to have come
from the dissolved phase, the particulate phase, or both. If the mechanism
of accumulation is direct uptake from the aqueous phase, then bio-
concentration factors (BCFs) may be utilized. These factors are usually
determined in experiments where organisms (usually fish) are exposed to
known concentrations of the compound in the water, and where SPM is
usually not present. BCFs are determined by dividing the concentration
of contaminant in the organism at steady-state by the dissolved con-
centration in the exposure water. Since many organic pollutants have
low aqueous solubilities and high lipid solubilities, BCFs are usually
121
-------
t
LJ
O
0.
cn
UJ
cc
§
o
LJ
TIME
Figure 33. Capillary column flame ionization detector gas chromatogram of PF-50 fraction from worms
exposed to BRH sediment for 28 days. This fraction contains mostly straight chain, branched, and
cyclic saturated hydrocarbons
-------
500r
10
5L
exposure
tank
o control
tank
UPTAKE
DEPURATION
10 20 30 40
TIME (days)
50
60
70
Figure 34. Concentration of total petroleum hydrocarbons in worms
exposed to sediment versus time
123
-------
high. BCFs calculated utilizing the concentration of contaminants in
the mussels (dry wt) at day 28 divided by the concentration of contaminant
in the filtrate from the exposure water show the expected high values
(Table 13). In addition, a direct relationship between Log P and the
Log BCF of the compound is observed, as has been found in other studies
(Ernst 1977; Geyer et al. 1982).
126. With the exposure and possible accumulation from both dissolved
and particulate phases, bioaccumulation factors (BAFs) defined as the
concentration of contaminant in the mussels at steady-state divided by
the concentration of contaminant in the unfiltered water are appropriate.
In the present study, these calculations show Log BAF for PCBs and PAHs
which are constant at approximately 4.2 over a range of solubilities or
n-octanol/water partition coefficients (Tables 9 and 15) (see paragraph
65 for possible explanations for this constancy).
127. If the mechanism of accumulation is only direct uptake from
SPM, then BAFs calculated using the concentration of contaminant in the
mussels at day 28 (dry wt) divided by the concentration of contaminant in
the SPM (dry wt) are appropriate. BAFs calculated this way are less than
0.3 for PCB compounds. Greater variability was found for the PAH calculations
(Table 25).
128. One simplified view of bioaccumulation uses the concept of
organisms as lipids and other adsorbing materials in a semi-permeable
membrane. If the assumptions are made that membrane transport and the
quantity and adsorption efficiences of the adsorbing materials in different
organism types are approximately equal, then exposure of these "organisms"
to equivalent exposure environments, until attainment of steady-state,
124
-------
Table 25
Mussel Bioaccumulation Factors Calculated from Filters
PCBs
Peak No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
BAP*
.18
.23
.17
.18
.19
.10
.18
.18
.19
.19
.19
.20
.15
.17
.17
.17
.17
.19
.19
.16
.16
.15
.16
.17
.15
.16
.16
.18
.23
.16
.15
.15
.16
.15
PAHs
Compound
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo(a)anthracene
Chrysene
Benzo(b)f luoranthene and/or
Benzo(k)f luoranthene
Benzo(e)pyrene
Benzo(a)pyrene
Perylene
Sum of PAHs with MW of 276
(continued)
BAF1
.07
.12
.15
.20
.25
.22
.13
.11
.11
.05
.06
* BAFs calculated using mean mussel concentration of day 28 (n-3)/mean
concentration of compounds on day 28 filter (n«3) assuming .009 g
BRH sediment (dry weight)/liter.
125
-------
Table 25 (Cont'd)
PCBs
Peak No. BAF*
35 .16
36 .15
37 .15
38 .06
39 .14
40 .09
41 .15
42 .15
43 .14
44 .02
45 .12
46 .10
47 .17
48 .08
126
-------
Table 26
Comparison of BAFs from Mussels and Worms
PCB Peak No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
. 19
20
21
22
23
24
25
26
27
28
29
30
31
32
BAF - Exposed Mussels*
.71
.22
.28
.21
.24
,11
.24
.33
.26
.24
.24
.25
.23
.20
.27
.27
.25
,25
.27
.25
.24
.24
.24
.25
.24
.24
.25
.27
.35
.24
.24
.24
(Continued)
BAF - Exposed Worms**
.10
.57
.27
.16
.30
.20
.15
.07
.20
.16
.19
.17
.39
.39
.20
.29
.13
.22
.19
.09
.07
.30
.38
.25
.28
.22
.36
.09
.22
.26
.21
.24
* Bioaccumulation factors calculated using mean (n=3) concentrations of
PCBs, PAHs, and Ethylan in exposed mussels at day 28 divided by whole
unfiltered water concentration at day 28 (nsO) converted to concentration/
gram dry wt. BRH SPM using a .009 g (dry wt.)/liter.
** Calculated using mean (n=>3) concentrations of PCBs in exposed worms at day
28 divided by mean (n»3) exposure sediment concentration (dry wts).
127
-------
Table 26 (Cont'd)
PCS Peak. No.
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
BAF - Exposed Mussels
.26
.22
.24
.22
.22
.07
.22
.14
.22
.25
.23
.02
.18
.16
.22
.12
.09
.02
.05
BAF - Exposed Worms
.04
.21
.35
.33
.32
.16
.26
.19
.29
.30
.21
.16
.23
.23
.23
.20
.19
.17
.19
.12
.15
.06
PAH Compounds BAF - Exposed Mussels BAF - Exposed Worms*
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo ( a ) anthracene
Chrysene
Benzo (b)fluoranthene and/or
Benzo (k)fluoranthene
Benzo (e)pyrene
Benzo(a)pyrene
Perylene
Sum of PAHs with MW of 276
Ethyl an
.04
.06
.18
.22
.40
.37
.22
.18
.16
.09
.08
.33
.04
.04
.05
.04
.03
.06
.02
.02
.02
.007
.004
.09
* Calculated using mean (n=2) concentration of PAH and Ethylan compounds
in exposed worm samples at day 28 divided by mean (n=3) concentrations
of compounds in exposed sediments (dry wts).
128
-------
should result In equivalent "organism" concentrations. In the present
study, the exposure environments for the mussels and worms included
contaminants in both dissolved and particle bound form. For worms, the
contaminants in the sediment pore water represent the dissolved phase;
the particle bound phase is the sediment concentration minus the pore
water concentration. Since the extraction of sediment utilized in this
study included the contaminants in the pore water with the particle
bound contaminants, BAFs may be calculated utilizing the measured sediment
concentration as a representation of a total exposure concentration.*
In a similar way the total exposure concentration for the mussels is
represented by the unfiltered water samples. For comparison purposes
the total content of contaminants for both worm and mussel exposures is
assumed to reside on the particles. BAFs calculated as concentration in
organism (dry wt) divided by the total exposure concentration (dry wt)
of sediment (worms) or SPM (mussels) are shown in Table 26. The close
correspondence of BAFs for PCBs for both mussels and worms suggests that
bioaccumulation of relatively unreactive FCB molecules may be modeled as
a partitioning of these contaminants between the organisms and either
the sediment or SFM. Whether the actual bioaccumulation process occurs
through direct transfer of hydrophobic organics from sediment to organism
through the lining of the gut or through a dissolved intermediate phase
is unknown.
129. The correspondence between measured and estimated BCF values
found in the mussel studies (paragraph 64) is suggestive of a dissolved
* Not including the concentration of contaminants in overlying water
which are thought to be very small.
129
-------
phase intermediate between the contaminants on the SPM and those in the
mussels. However, the increase in measured BCF with increasing Log P
may result in another way. The concentrations of PCB compounds in the
aqueous phase decrease by orders of magnitude over the range of PCB
compounds. Since organisms may accumulate particle-bound contaminants
to constant concentrations (I.e., gut transfer), the division of
organism concentrations by measured aqueous concentrations will give
spreads of BCF values covering several orders of magnitude with BCFs
increasing with decreasing compound solubility (increasing Log P).
Similar relationships are possible in the pore water of the sediments
and in the worms.
130. For the more reactive PAH compounds greater differences
were observed in the BAFs for the worms and mussels. The worms showed
smaller BAFs than the mussels (Table 26). The reason(s) for these
differences are unclear, but may reflect metabolic differences of the
organisms.
131. For modeling bioaccumulation, researchers have suggested
utilizing normalization of sediment concentration to the organic
carbon content of the sediment (site of most of adsorption of hydrophobic
organic compounds) (Karickoff et al. 1979) and normalization of organism
concentrations to the lipid content of the organism (most important site
of storage of organic pollutants). These concentrations are utilized in
thermodynamic arguments with fugacity concepts and will be the subject
of future publications.
130
-------
Inorganic Contaminants—Worms
132. The average concentrations and standard deviations
for the day 28 worm samples collected from the BRH sediment exposure
chamber are given in Table 27. All of the inorganic data used to
calculate these averages are given in Table B5. Data for the time zero
worm samples are not used in the following discussion since much of the
Fe, Cr, and Cu data are higher than all of the other samples collected.
The large concentrations of Fe, Cu, and Cr in the time zero samples are
probably due to the fact that the worms were not allowed to acclimate in
the reference sediment prior to the start of the experiment so that a
firm baseline could have been established. The time zero worms probably
represent the sediment of the Maine coastline from which they were
collected since these worms had never been in BRH or REF sediment.
Several data points were eliminated from the uptake, depuration, and
reference worm samples. These results were rejected by application of
the "Q" test for rejection of an experimental observation (Dean and
Dixon 1951). Two Cd results were discarded: one from the day 28 uptake
samples and one from the day 40 reference sample. Also, one value for
Cr was rejected from the day 56 depuration phase of the experiment.
These data points are marked with an asterisk in Table B5.
133. The means for the two control mussel Fe concentration are
significantly different (Student t-test, P»0.05) for the two collection
times. However, there are no significant differences in the control worm
mean concentrations for Cu, Cr, Cd, and Zn for the two collection times.
134. The Fe uptake and depuration plot for the worms is given in
Figure 35. Only the average and standard deviation of the average of
131
-------
Table 27
Average Trace Metal Concentration for Worms
Collected from the Exposure Chamber on Day 28*
Metal
Fe
Zn
Cu
Cd
Cr
Worm
28 day
404 + 68
139 + 15
31.3 + 9.9
0.73 + 0.10
5.8 + 2.4
Worm
Control
316 + 75
109 + 16
12.2 + 1.2
0.60 + 0.08
2.3 + 0.6
The control concentrations reported for the worms are the average
of all the control samples and not just day 28. All concentrations
are in ug/g dry weight. The standard deviations of the means are also
reported.
132
-------
THE STANDARD DEVIATIONS OF THE AVERAGE
METAL CONCENTRATIONS ARE DEPICTED AS
VERTICAL LINES ASSOCIATED WITH EACH
DATA POINT
1400
o.
o.
4>
u.
200
° EXPOSED
• REFERENCE
O.
O.
— UPTAKE-
DEPURATION
10 20 30 40 50 60
TIME (days)
Fe
o EXPOSED
• REFERENCE
UPTAKE-
DEPURATION
10 20 30 40 50 60
TIME (days)
b. Cr
Figure 35. Uptake and depuration in worms exposed
to BRH sediment
133
-------
each set of exposed and control worm samples are shown in this figure.
There is no significant difference in the Fe concentration of the
day 28 BRH-exposed worms and the day 28 reference sediment-exposed
control worms. There is no significant difference in the Fe
concentrations for any of the worm samples collected from the BRH exposure
chamber during uptake or depuration (one way analysis variance °^ = 0.05).
135. The Cr uptake and depuration plot for the worms is given in
Figure 35. There is a large standard deviation of the concentration
of Cr for the day 28 BRH-exposed worms compared to the standard deviation
of the Cr concentration for the day 28 reference sediment worms.
However, there is a significant difference (Student t-test, P - 0.05)
between these two concentrations. Also, the difference between the
concentrations is significant for all the worm samples collected during
uptake and depuration.
136. The Cu uptake and depuration plot for the worms is given in
Figure 36. Like the Cr data the Cu data have a large standard deviation
of the mean for the day 28 BRH-exposed worms. Also, like Cr, the worm
Cu concentration means for the uptake portion of the study from the BRH
exposure chamber are significantly different from the worm Cu
concentration means for the depuration portion of the study. The two
control worm sample means for Cu are not significantly different from
each other. The Cu concentration in BRH-exposed worms declines to the
control worm concentration for the day 42 samples (14 days of depuration).
137. The Zn uptake and depuration plot for the worms is given in
Figure 36. There is no significant difference between the Zn concentrations
for the day 28 BRH exposed worms and the day 28 reference worms. There
134
-------
THE STANDARD DEVIATIONS OF THE AVERAGE
METAL CONCENTRATIONS ARE DEPICTED AS
VERTICAL LINES ASSOCIATED WITH EACH
DATA POINT
40
a 30
a
<3 20
10
o EXPOSED
• REFERENCE
UPTAKE-
•DEPURATION-
10 20 30 40 50
TIME (days)
a. Cu
60
140
_ 120
lioo
* 80
N 60
40
20
o EXPOSED
• REFERENCE
• UPTAKE—! DEPURATION-
10 20 30 40 50 60
TIME (days)
b. Zn
E
a.
a
•o
O
.0
0.5
o EXPOSED
• REFERENCE
UPTAKE-
• DEPURATION-
10 20 30 40 50 60
TIME (days)
c.
Cd
Figure 36. Uptake and depuration in worms
exposed to BRH sediment
135
-------
Table 28
Metal Bioaccumulation Factors for Worms
Metal
Fe
Zn
Cu
Cd
Cr
Exposed to Black Rock Harbor
Worm
BAF
0.0030
0.025
0.0080
0.0058
0.0025
Sediment*
Worm
28 day/control
1.3
1.3
2.6
1.2
2.5
The ratios reported are the 28 day worm sample average concentration
divided by their average controls, respectively.
136
-------
is also no significant difference between any of the Zn concentrations
for any of the samples collected from the BRH exposure chamber or the
b
reference sediment control chamber.
138. The Cd uptake and depuration plot for the worms is given in
Figure 36. There is no significant difference between the Cd concentration
for the day 28 BRH-exposed worms and the day 28 reference sediment
control worms. The two Cd concentrations for the control samples are
also not significantly different.
139. The BAF values for the metals determined in the day 28
BRH-exposed worms are given in Table 28. These BAF values were calculated
for all the metals even though the Zn, Cd, and Fe data show no significant
difference between the means of these elements for the BRH-exposed and the
reference-exposed control worms. The BAF values were calculated as follows:
(a)'the average control worm metal concentration was subtracted from
the average day 28 worm concentration; and (b) the corrected concentrations
were then divided by the concentration of the metals determined in BRH
sediment. The ratios of the average metal concentrations for the day
28 BRH-exposed worms to the average metal concentrations for the control
worm samples are also reported in Table 28. These ratios are probably
a better representation for the metal accumulation in the day 28 BRH-
exposed worms. The ratios for Cu and Cr for the day 28 exposed worms
and the reference sediment control worms are 2.6 and 2.5, respectively.
These increases in Cu and Cr, while not large, are significant. The
calculated ratios for Fe, Zn, and Cd are small and are not significant.
137
-------
PART IV: SUMMARY
Mussel Bioaccumulation Study
Organics
140. The system utilized to dose mussels with suspensions of
BRH dredged material worked well. The dose of PCB contaminants monitored
in the exposure tank was quite constant over the study period, and while
the concentration of total PAH compounds was found to show more variability,
this variability appeared to be in total levels of PAH compounds rather
than on a compound-to-compound basis. It seemed likely that the PAH
variability may have resulted from the unhomogeneous distribution of
soot particles (containing high concentrations of PAH compounds) in the
BRH sediment. The concentrations of PAH and PCB contaminants in the
control tanks were orders of magnitude below those in the exposure tanks.
141. Separation of dissolved and particle-bound PCB and PAH
contaminants resulted in distributions which were logically consistent
with the solubilities of the compounds. The more water-soluble compounds
were found in the dissolved form while the less water-soluble compounds
were found associated with the particles.
142. Based on comparisons of measured and estimated Kps for the PCB
and PAH contaminants in the exposure tanks, it appeared that equilibrium
conditions were not reached in the residence time of the suspensions in
the dosing system.
143. During the first 7 days of exposure, mussels in the exposure
tank showed a rapid uptake of PAH and PCB compounds. There was an un-
explained decrease in the concentration in mussels during the next 2 weeks
for most compounds, and the highest concentrations were found at day 28.
138
-------
144. PCB compounds with molecular weights above Cly PCB were not
effectively accumulated by the mussels, and, similarly, PAHs of higher
molecular weight were not accumulated as much as some lower molecular
weight compounds.
145. Application of a non-linear model to the mussel data indicated
that steady-state concentrations of PCBs had been reached during the 28-
day exposure period. From the shapes of the uptake curves for the PAH
compounds, it appeared that steady-state concentrations of PAHs also had
been reached during the 28-day exposure.
146. While the dominant method of accumulation of PAHs and PCBs
(i.e., uptake from water or uptake from the particles) could not be
determined from these studies, measured BCFs (assuming uptake from aqueous
phase only) showed increasing BCFs with increasing Log n-octanol/water
partition coefficients (decreasing aqueous solubilities) and reasonable
agreement with BCFs estimated from a correlation (BCF vs. Log P) in the
literature. In contrast, bioaccumulation factors (BAFs; calculated using
unfiltered water concentrations) for PCBs and PAHs were relatively
constant over the range of PAH and PCB contaminants examined. The
constancy of these BAFs suggests that similar processes determined the
distributions of these compounds. In this regard, bioaccumulation in the
exposure tanks may be viewed as the result of two processes competing for
the dissolved phase contaminants:, readsorption by the SPM, and bio-
concentration of dissolved contaminants by the mussels. Alternatively,
the constant bioaccumulations observed may have resulted from similar
constant processes like direct transfer of contaminants through the gut.
139
-------
147. In general, depuration rate in mussels appeared to be Inversely
related to Log P for PCB compounds; however, some higher molecular weight
PCB compounds were lost at higher rates than lower molecular weight
PCBs. Those compounds with recalcitrant structures appeared to be
depurated most slowly. The depuration of PAH compounds also appeared to
be inversely related to Log P. The concentrations of petroleum hydrocarbons
measured in the mussels over the exposure and depuration period generally
followed the patterns observed for the other organic contaminants.
148. Control mussels, which contained only low concentrations of
contaminants, showed only small fluctuations in concentrations of organic
contaminants during this study.
Inorganics
149. The acid-soluble trace metal concentrations in the seawater
were generally consistent with the quantity of BRH sediment added to the
exposure chamber. The total concentration of BRH sediment added to the
seawater could be calculated from the Fe concentration determined in the
seawater exposure chamber. The interelemental ratios determined in the
exposure chamber also compared favorably with the interelemental ratios
determined for BRH sediments.
150. Statistically, there was no difference for the respective means
of Fe, Cr, Mn, Pb, Cd, Zn, and As concentrations over time for the control
mussels collected from the control chamber. There was, however, a significant
difference in the mean Cu concentrations for the control mussel samples
collected over time from the control chamber. The means of Fe, Cu, Cr,
Zn, Pb, Cd, and As for the day 28 BRH-exposed mussels were significantly
different from their respective means for the control mussels.
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151. Typically, metal BAFs over 1000 were calculated for the day
28 mussels collected from the BRH exposure chamber. A different
impression of the uptake is observed in the ratios of metal concentrations
in day 28 exposed mussels divided by the metal concentrations in the
control mussels. The ratios which indicate only the relative metal
concentration increases for BRH-exposed mussels versus the control mussel
samples ranged from 0.9 to 11.
152. The uptake patterns of Fe and Cr for the exposed mussels were
almost identical. The correlation coefficient for Fe versus Cr in the
BRH-exposed mussels was 0.957. The relatively low concentration of Cr
in the control mussels versus that in the BRH sediment makes Cr an
ideal choice for a metal tracer of BRH sediment. The mussel con-
centrations of Fe, Cr, Cu, and Pb were all elevated during the uptake
period compared to the control mussel samples; however, only Pb and Cr
correlated well with Fe in the mussels during the uptake period. The
concentrations of Zn, As, and Mn in the exposed mussels varied around
their respective concentrations in the control mussels.
153. The depuration patterns of Fe and Cr from the mussels
exposed to BRH sediment appeared to be identical. Both elements declined
to control concentrations after 2 weeks of depuration. The depuration
of Cu appeared to begin before the end of the exposure period. The Cu
concentration in the BRH-exposed mussels fell to the control mussel
concentrations after 3 weeks of depuration. Neither Pb or Cd showed
a steady decline in concentration in the exposed mussels during the
depuration period. The concentrations of Mn and As in the exposed
mussels were generally below the concentrations of Mn and As concentrations
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of the control mussels during depuration. The average Zn concentrations
in the exposed mussels were elevated compared to the average control
Zn concentration during the depuration period. However, the standard
deviations of the average concentrations for Zn in the exposed mussels
overlapped with those for the control mussels during the depuration
period.
Worm Bioaccumulation Study
Organics
154. The exposure of worms Nereis virens to BRH sediment resulted
in accumulation of organic compounds even though the organisms showed
little evidence of feeding during the experiment. The PCBs accumulated
by the worms showed a pattern which was similar to the pattern observed
in the sediment. The PCB contaminants accumulated showed no apparent
decreases in total concentrations over the depuration period. Exposed
worms accumulated PAHs to concentrations which were orders of magnitude
greater than those in the reference worms. In contrast to the PCBs,
which showed no concentration decreases during the depuration period,
the concentrations of PAHs in the worms fell rapidly during depuration.
155. The petroleum hydrocarbons found in worms (measured as an
unresolved complex mixture) increased during the uptake phase and
decreased during the depuration phase.
156. Reference worms showed relatively constant low levels of
organic pollutants over the exposure and depuration study.
157. The bloaccumulation factors observed for the PCBs in the
exposed worms were lower than those found for worms in the reference
sediment. This may have resulted from the apparent poor health of the
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exposed organisms or from decreased bioavailability of PCB contaminants
in sediments with high organic carbon (i.e., Black Rock Harbor sediments).
Alternatively, the lower bioaccumulation factors found for worms in
Black Rock Harbor sediment may result from steady-state values not
being attained during the 28-day exposu're period. BAFs for PAH compounds
in the worms were lower than BAFs for the PCBs, and no consistent
differences between exposed and reference BAFs were found for PAHs. The
differences observed in the BAFs for PCBs and PAH may reflect metabolic
or bioavailability differences, or may result from steady-state values
not being attained during the exposure period.
Inorganics
158. There was no significant difference between the mean Fe, Zn,
and Cd concentrations in the day 28 BRH-exposed worms compared to the
reference sediment exposed worms. There was a significant uptake of Cu
and Cr for the day 28 BRH-exposed worms. The calculated ratio of the mean
Cu and Cr concentrations for the day 28 BRH-exposed mussels and the
reference sediment exposed mussel were 2.6 and 2.5, respectively. The
depuration of Cu and Cr was complete 2 weeks after the exposure period.
Bioaccumulation Mussels and Worms—Organic^
159. Exposure to organic contaminants in dissolved form and on SPM
(mussels) and in pore water and sediments (worms) can be simplified by
assuming that the total exposure concentration of PCBs resides on the
particles or the sediments. When this is done the calculated BAFs for
both mussels and worms are quite similar. Similar calculations with
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the more reactive and possibly less available (due to incorporation in
soot particles) PAHs show greater differences. Similarities in worm
and mussel BAFs suggest that modeling bioaccumulation of some organics
(at least less reactive compounds like PCBs) as a partitioning of
contaminants between the organisms (worms and mussels) and the sediment
or suspended sediment shows promise as a predictive technique for
assessing the accumulation of organic contaminants from dredged material
and other mixed wastes.
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PART V: RECOMMENDATIONS
Mussels
160. Due to the apparent discrepancy between estimated and
measured partition coefficients for organic compounds, it is recommended
that partitioning studies be conducted to determine the time to equilibrium
and the soluble/particulate distributions of organic and inorganic
compounds under both aerobic and anaerobic conditions.
161. Due to the variability in concentrations of organics and
inorganics observed in mussels during the uptake phase, the following
studies are recommended:
_a. Studies should be conducted to broaden our understanding
of the nutritional requirements of these organisms and
the potential nutritional value of the sediments.
_b. Future studies should be conducted to examine the
contribution of contaminants on sediment in the gut of the
exposed mussels to the concentrations found in extracts of
whole organisms.
162. It is recommended that a range of concentrations be used
in mussel exposures to establish the constancy of bioaccumulation
factors at different exposure concentrations.
Worms
163. Due to the poor feeding behavior of the worms in this
exposure study, it is recommended that further studies be conducted
to ensure that worms remain healthy and have adequate nutrition during
exposure and depuration studies with sediments which are heavily contamin-
ated with organic and inorganic compounds. This research should consider
the utilization of standardized control sediment in exposure studies.
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These standard sediments could be used to dilute toxic sediments and
to serve as carriers for the nutritional needs of the organisms.
164. It is recommended that worms be held in reference sediment
prior to initiation of exposure studies for a time sufficient to allow
worms to adjust their contaminant levels to those of the reference
sediment.
165. As adequate exposure conditions (i.e., no adverse effects)
become available, it is recommended that longer term bioaccumulation
studies be conducted to ensure that steady-state levels are reached
for both the organic and inorganic contaminants in these organisms.
General
166. In order to determine the potential for bioaccumulation of
sediment-bound contaminants, it is recommended that research be undertaken
to develop a short-term abiotic test to enable prediction of the bio-
availability and bioaccumulation of contaminants.
167. In order to link the laboratory bioaccumulations with
potential bioaccumulations in the field at the disposal site for BRH
dredged material (under the Field Verification Program), it is recom-
mended that:
£. Field bioaccumulation samples be analyzed for the same contaminants
that were accumulated in laboratory studies.
b_. Where possible, the same organisms (or similar surrogate organisms)
be used in field and laboratory studies.
£. If the same organisms cannot be deployed or are not indigenous
~~ at the disposal site, studies be undertaken to compare
bioaccumulation between indigenous and surrogate organisms.
d. Exposure concentrations at the disposal and reference site should
~ be determined at least seasonally to enable estimation of mean
annual exposure levels in water and sediments.
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