FIELD VERIFICATION PROGRAM
(AQUATIC DISPOSAL)
TECHNICAL REPORT D-85-1
APPLICATION OF SISTER CHROMATID
EXCHANGE IN MARINE POLYCHAETES
TO BLACK ROCK HARBOR SEDIMENT
LABORATORY DOCUMENTATION PHASE
by
Gerald G. Pesch, Cornelia Mueller, Carol E. Pesch,
James Heltshe, Paul S. Schauer
Environmental Research Laboratory
US Environmental Protection Agency
Narragansett, Rhode Island 02882
January 1985
Final Report
Approved For Public Release: Distribution Unlimited
prepared for DEPARTMENT OF THE ARMY
US Army Corps of Engineers
Washington, DC 20314-1000
and US Environmental Protection Agency
Washington, DC 20460
Monitored 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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SUBJECT: Transmittal of Field Verification Program Technical Report Entitled
"Application of Sister Chromatid Exchange in Marine Polychaetes to
Black Rock Harbor Sediment; Laboratory Documentation Phase"
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.
A. 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: Transmittal of Field Verification Program Technical Report Entitled
"Application of Sister Chromatid Exchange in Marine Polychaetes to
Blatk Rock Harbor Sediment; Laboratory Documentation Phase"
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.
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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Unclassified
SECURITY CLASSIFICATION OF THIS PAGE (When Data Entered)
REPORT DOCUMENTATION PAGE
READ INSTRUCTIONS
BEFORE COMPLETING FORM
1. REPORT NUMBER
Technical Report D-85.-1
2. GOVT ACCESSION NO
3. RECIPIENT'S CATALOG NUMBER
4. TITLE (and Subtitle)
APPLICATION OF SISTER CHROMATID EXCHANGE IN
MARINE POLYCHAETES TO BLACK ROCK HARBOR SEDIMENT;
Laboratory Documentation Phase
5. TYPE OF REPORT 4 PERIOD COVERED
Final report
6. PERFORMING ORG. REPORT NUMBER
7. AUTHOR(»;
8. CONTRACT OR GRANT NUMBERfsJ
Gerald G. Pesch, Cornelia Mueller, Carol E. Pesch,
James Heltshe, Paul S. Schauer
9. PERFORMING ORGANIZATION NAME AND ADDRESS
US Environmental Protection Agency
Environmental Research Laboratory
Narragansett, Rhode Island 02882
10. PROGRAM ELEMENT, PROJECT, TASK
AREA 4 WORK UNIT NUMBERS
Field Verification Program
(Aquatic Disposal)
11. 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
January 1985
13. NUMBER OF PAGES
50
14. MONITORING AGENCY NAME « ADORESSf/f dlllerent Irom Controlling OHIce)
US Army Engineer Waterways Experiment Station
Environmental Laboratory
PO Box 631, Vicksburg, Mississippi 39180-0631
15. SECURITY CLASS, (of thli report)
Unclassified
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SCHEDULE
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17. DISTRIBUTION STATEMENT (of the abstract entered In Block 30, II dlllerent Irom Report)
IB. SUPPLEMENTARY NOTES
Available from National Technical Information Service, 5285 Port Royal Road,
Springfield, Virginia 22161.
19. KEY WORDS (Continue on reverse mtde II neceeiery and Identify by block number;
Marine pollution—Genetic effects (LC)
Dredged material (WES)
Sister Chromatid Exchange (SCE) (WES)
Black Rock Harbor (Conn.)—Environmental aspects (LC)
Polychaeta (LC)
Biological assay (LC)
20. ABSTRACT fConffau* en, r*WM
emty end Identity by btock number)
This report presents an evaluation of the applicability of the cytogenetic
technique of sister chromatid exchange (SCE) to measure genotoxic effects of
highly contaminated dredged material, and the degree of variability and repro-
ducibility inherent in the procedure. This project is part of the US Environ-
mental Protection Agency/Corps of Engineers Field Verification Program.
The SCE technique was applied to Nephtys incisa, an inf aunal polychaete
_ _ (Continued)
1473 EDITION OF • MOV 6S IS OBSOLETE
Unclassified
SECURITY CLASSIFICATION OF THIS PA1.E (When Del* Entered)
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Unclassified
SECURITY CLASSIFICATION OF THIS PAGEfH7l»n D*tm Enlfttd)
20 ABSTRACT (Continued).
dominant in the benthic community at the Central Long Island Sound disposal
site. The SCE response was measured in !N. incisa exposed to suspended particu-
late and bedded phases of Black Rock Harbor (BRH) sediment in the laboratory.
Neanthes arenaceodentata, a surrogate species, was tested in parallel to
II. incisa.
With the exception of one treatment in one experiment, the worm chromo-
somes were uniformly nonresponsive to BRH sediment. Replicate treatments
within an experiment did not differ significantly for Ifl. arenaceodentata.
Differences between experiments and between species within an experiment were
found. The reasons for the differences are not known. Differences in ability
to metabolize polynuclear aromatic hydrocarbons, found in high concentrations
in BRH sediments, is a likely but speculative reason. Clearly, additional
research is needed before SCE could be used for routine testing.
This investigation is the first phase in developing field-verified bio-
assessment evaluations for the Corps of Engineers and the US Environmental
Protection Agency regulatory program for dredged material disposal. This
report is not suitable for regulatory purposes; however, appropriate assessment
methodologies that are field verified will be available at the conclusion of
this program.
Unclassified
SECURITY CLASSIFICATION OF THIS PAGEflFJiBn D»t* Entttfd)
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PREFACE
This report describes work performed by the US Environmental Protection
Agency, Environmental Research Laboratory, Narragansett, Rhode Island (ERLN),
as part of the Interagency Field Verification of Testing and Predictive
Methodologies for Dredged Material Disposal Alternatives Program (Field
Verification Program (FVP)). This program is sponsored by the Office, Chief
of Engineers, and the US Army Engineer Waterways Experiment Station (WES),
Vicksburg, Miss. The program objective of this interagency agreement
is to verify existing predictive techniques for evaluating the environmental
consequences of dredged material disposal under aquatic, wetland, and upland
conditions. The aquatic portion of the FVP study is being conducted by ERLN,
with the wetland and upland portions conducted by WES.
The principal investigators for this aquatic study were Dr. Gerald G.
Pesch, Research Aquatic Biologist; Ms. Cornelia Mueller, Cytogenetic Technolo-
gist; Ms. Carol E. Pesch, Research Aquatic Biologist; Dr. Paul Schauer,
Nutritionist; and Dr. James Heltshe, Statistician. The laboratory exposure
system was designed by Dr. Paul Schauer. Mr. Michael Balboni, Dr. D. Michael
Johns, and Ms. Ruth Gutjahr-Gobell assisted with collecting worms and con-
ducting experiments. Data management and analysis were conducted by
Mr. Jeffrey Rosen.
The EPA Technical Director for the FVP was Dr. John H. Gentile; Technical
Coordinator was Mr. Walter Galloway; and Project Manager was Mr. Allan Beck.
Special thanks are due to Capt. Robert Alix and Dr. Anthony Calabrese
of the National Marine Fisheries Service Laboratory, Milford, Conn., for
field support and for use of their boat, the Shang Wheeler.
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This study was conducted under the supervision of Drs. Richard K.
Peddicord and Thomas M. Dillon, Environmental Laboratory (EL), Vicksburg,
Miss. Mr. Charles C. Calhoun was Manager, Environmental Effects of Dredging
Programs. Dr. John Harrison was Chief, EL. Commander and Director of WES
during the conduct of this study and the preparation of this report was
COL Tilford C. Creel, CE. Technical Director was Mr. F. R. Brown. The OCE
Technical Monitors were Drs. John Hall and William L. Klesch.
This report should be cited as follows:
Pesch, G. G., et al. 1985. "Application of Sister Chromatid
Exchange in Marine Polychaetes to Black Rock Harbor Sediment;
Laboratory Documentation Phase," Technical Report D-85-1,
prepared by US Environmental Protection Agency, Narragansett,
R. I., for the US Army Engineer Waterways Experiment Station,
Vicksburg, Miss.
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CONTENTS
Page
PREFACE 1
LIST OF TABLES 4
LIST OF FIGURES 5
PART I: INTRODUCTION 6
Background • •» 6
Objectives 11
PART II: METHODS AND MATERIALS 13
Overview 13
Sediment Collection and Preservation 14
Polychaete Collection, Culture, and Holding 16
Test System 17
Chromosome Labeling 20
Slide Preparation and Staining 22
Data Collection and Analysis 23
PART III: RESULTS AND DISCUSSION 24
Statistical Properties of SCE Data 24
Applicability of SCE Technique 27
Variability and Reproducibility of Data 31
PART IV: CONCLUSIONS 34
REFERENCES 35
APPENDIX A: KARYOTYPES A-l
APPENDIX B: DATA SHEETS B-l
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LIST OF TABLES
No. Page
1 Sister Chromatid Exchange/Chromosome Response for
Two Replicate Experiments, Solid Phase Dosing of
^ arenaceodentata 27
2 Sister Chromatid Exchange/Chromosome Response for
Replicate Experiments, Particulate Phase/Solid
Phase Dosing of N. incisa and !t._ arenaceodentata 28
3 Sister Chromatid Exchange/Chromosome Response for
Duplicate Treatments Within an Experiment, Particulate
Phase/Solid Phase Dosing of N. arenaceodentata 31
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LIST OF FIGURES
No. Page
1 Sister chromatic! exchange illustrated with the
chromosomes of the polychaete Nephtys incisa.,
Central Long Island Sound disposal site and
South reference site 15
Black Rock Harbor, Connecticut, source of dredged
material 16
Sediment dosing system with chilled water bath
and argon gas supply 17
Schematic of the distribution and dosing system
used to expose juvenile N^ incisa and IK_
arenaceodentata to reference and BRH sediment 19
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APPLICATION OF SISTER CHROMATID EXCHANGE IN
MARINE POLYCHAETES TO BLACK ROCK HARBOR SEDIMENT
Laboratory Documentation Phase
PART I: INTRODUCTION
Background
1. Pollutants in coastal marine environments may affect the
genetic constitution of exposed organisms by either causing shifts in
gene pool composition through selective mechanisms or by acting directly
on the genetic material to produce mutations. This report addresses the
latter problem. There is evidence that some areas of the marine environ-
ment are contaminated with mutagens and carcinogens. Longwell and Hughes
(1980) examined mackerel eggs sampled from the surface waters of the New
York Bight. They observed mitotic chromosome irregularities, variable
development rates as calculated by mitotic index, and differences in
viability as estimated by early indicators of cell death. These effects
were found in the more impacted areas of the Bight close to the coast and
disposal grounds. In addition to the New York Bight, mutagens have been
detected in other polluted marine environments (Parry et al. 1976; Payne
et al. 1979). These observations suggest that genetic toxicants may be
present in many polluted marine environments. Because the integrity of
an organism's genes is essential for its well being, and genetic damage
may accumulate from one generation to the next, genetic impairment may
represent a long-term threat to populations of marine organisms.
2. The importance of genetic effects in marine pollution studies
has been recognized only recently. The International Council for the
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Exploration of the Seas dedicated a section of their 1979 Workshop on
biological effects of marine pollution to genetics (Beardmore et al.
1980). They registered concern and recommended increased research efforts
in this area.
3. To determine whether mutagenic compounds pose real threats to
the marine environment, information is needed about the presence, distri-
bution, and abundance of such toxicants in the marine environment, the
degree of genetic damage suffered by marine organisms, and the ecological
consequences of such damage. No single test can address all of these
concerns. However, at present, no genetic tests are available for routine
use in managing waste disposal in estuarine, coastal, and oceanic environ-
ments. Short-term tests are needed to identify mutagens and to evaluate
complex mixtures. Long-term tests are needed also to investigate possible
effects of somatic and germinal mutations on populations.
4. Several approaches are possible to detect and study genetic
toxicants. Chemical analyses of environmental samples can be performed,
but provide no information on the bioavailability of sediment-sorbed com-
pounds. Futhermore, many classes of compounds are genetically active;
therefore, these analyses are time-consuming and expensive. A simpler
approach is to look for genetic damage in the exposed biota. Since
cytogenetic techniques are sensitive and reasonably simple, it is recom-
mended that genetic damage in marine organisms be determined by observing
their chromosomes directly (International Atomic Energy Agency 1979;
Kligerman 1980).
5. Polychaetes were selected for this study for several reasons.
First, they have suitable karyotypes, that is, relatively large chromosomes
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(Pesch and Peach 1980a, and Appendix A). Second, they are easily handled
and some species can be cultured to provide a continuous supply of experi-
mental material. Third, polychaetes are benthic and of particular interest
because sediments are often sinks for pollutants. Fourth, they are
important food web species for commercially important fishes, and, there-
fore, may contribute to the trophic transfer of toxicants. Fifth, they
are relatively sedentary so field-collected specimens would be represent-
ative of the area being sampled. Finally, some polychaetes are recognized
as pollution indicators, particularly pollution associated with organic
loading (Reish 1960; Wass 1967; Reish 1972).
6. The cytogenetic technique of choice is metaphase chromosome
analysis. As cells divide, the chromosomes are drawn together in a con-
densed form easily visible with a light microscope. This is the metaphase
stage of cell division. It is possible to arrest cells at this stage
using the chemical colchicine. This permits the observation of chromo-
somes in a wide variety of tissues containing metaphase-arrested cells.
Metaphase preparations allow accurate observation of chromosome structure
and permit sister chromatid exchange (SCE) analysis. A sister chromatid
exchange represents the breakage and reciprocol exchange of identical
DNA material between the two sister chromatids of a chromosome. This
was demonstrated originally by Taylor using tritium-labeled DNA (Taylor
et al. 1957). The methods for differential staining for light microscopy
were developed approximately 10 years ago (Figure 1) (Latt 1974; Perry
and Wolff 1974). These new techniques transformed SCE from a limited,
research tool to a tool which could be applied extensively to the study
of environmental mutagenesis.
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Figure 1. Sister chromatid exchange illustrated with
the chromosomes of the polychaete N. incisa
7. The mechanism of SCE is not known and is the subject of much
research. Several mechanisms have been postulated including altered
replication of DNA past damaged bases (Shafer 1977), asynchrony in DNA
synthesis between adjacent replicon clusters (Painter 1980), or a type
of recombinational repair (Bender et al. 1974). As yet, there is no
evidence to demonstrate conclusively a specific mechanism.
8. The usefulness of the SCE response as an indicator of DNA
damage is based on both empirical and biological grounds. There is a
wealth of SCE data showing dose responses to known mutagens in both in
vitro and in vivo test systems (Latt et al. 1981). The response itself
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indicates a direct effect on the DNA material; SCE is a visual conse-
quence of mutagens that affect changes in the DNA helix. Because of
our ignorance of the molecular mechanisms involved, we do not yet under-
stand the significance or consequences of these DNA changes. However,
SCE responses have been correlated with induced point mutations and may
be useful as a quantitative indicator of mutagenesis (Carrano et al.
1978).
9. The SCE response has been recommended for environmental appli-
cation by U.S. Environmental Protection Agency's Gene-Tox Program (Latt
et al. 1981). Several studies have shown that SCE is a more sensitive
method for detecting mutagens and carcinogens than the traditional chromo-
some and chromatid observations (Latt 1974; Perry and Evans 1975; Solomon
and Bobrow 1975; Bloom 1978). The application of SCE to polychaetes has
created a new tool that is both relevant and practical to study genetic
problems in marine environments (Pesch et al. 1981). The SCE technique
also makes it possible to measure the effect of toxicants on cell replica-
tion kinetics because differential staining of chromosomes permits easy
identification of first, second, third, and subsequent replication cycle
cells (Schneider et al. 1978). With the in vivo SCE assay, complex
wastes can be tested under conditions which simulate real world situations.
10. The study reported herein applies the SCE technique to the
polychaete Neanthes arenaceodentata. In earlier studies, N_. arenac eoden ta t a
exhibited a dose response to the known mutagen mitomycin C (MMC) at
concentrations comparable to those that elicited responses in in vivo
mammalian systems (Pesch & Pesch 1980b). Positive SCE responses in the
worm have also been demonstrated for other known, direct-acting mutagens
10
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such as 5-bromodeoxyuridine and methylmethanesulfonate, as well as for
compounds that need metabolic activation such as benzopyrene, dimethyl-
nit ros ami ne, and cyclophosphamide. These results imply that N.
arenaceodentata can metabolize promutagens and suggest that the worm may
be sensitive to a broad spectrum of genetic toxicants.
Objectives
11. This report is part of a comprehensive program sponsored by
the U.S. Army Corps of Engineers (CE) and the U.S. Environmental Protection
Agency (EPA) to evaluate the risk associated with various disposal options
for dredged material. The approach being used in this Field Verification
Program (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 EPA Environmental Research
Laboratory (ERLN), Narragansett, is responsible for the aquatic portion
of the FVP.
12. There are three primary objectives in the aquatic portion of
the FVP. The first objective is to demonstrate the applicability of the
SCE technique to measure effects of dredged material, and to determine
the degree of variability and reproducibility inherent in the procedure.
The SCE technique will be applied to Nephtys incisa, an infaunal poly-
chaete dominant in the benthic community at the Central Long Island
Sound disposal site. The SCE response will be measured in !N. incisa
exposed to particulate and solid phase Black Rock Harbor sediment in the
laboratory. N. arenaceodentata will be included in the laboratory
phases of the FVP study as a surrogate species and will be tested in
11
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parallel to N. incisa. This first objective is referred to as the Labo-
ratory Documentation Phase of the FVP and is the subject of this report.
13. The second objective is to field verify the response observed
in the laboratory and determine the accuracy of the laboratory prediction.
Consequently, this portion of the study is referred to as the Field
Verification Phase.
14. The third objective is to determine the degree of correlation
of tissue residues resulting from the bioaccumulation of contaminants
from dredged material and the response in SCE as observed in both the
laboratory and the field. The second and third objectives will be com-
bined in a final report for the FVP due in 1986.
12
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PART II: METHODS AND MATERIALS
Overview
15. Two types of tests were conducted with polychaetes: short-term
solid phase tests and short-term tests with both suspended particulate
and solid (bedded) phase sediments. Both tests were 10-day flow-through
tests. The solid phase tests used a gradation of highly contaminated Black
Rock Harbor (BRH) sediment and Reference (REF) sediment from the reference
station just south of the disposal site. Only one species, N. arenaceodentata»
was tested in the short-term solid phase tests, whereas both species
were included in the short-term suspended particulate/solid phase tests.
N. incisa was not used in solid phase tests because the suspended partic-
ulate/solid phase tests included "worst" case solid phase exposure and
because the cytogenetic technique for N^ incisa was not perfected at
the time solid phase tests were conducted. In the suspended particulate/
solid phase tests, suspensions of either REF or BRH sediments were dosed
in combination with a solid phase of 100 percent REF or 100 percent BRH
sediment. This test, which combines the solid and particulate phase, is
representative of the type of condition at the disposal site; however,
the concentrations of suspended material used in the tests do not neces-
sarily simulate actual field concentrations. These higher concentrations
of suspended particulates were chosen to produce a dose response in the
endpoint measurements in the short-term tests.
16. The tests described below generally follow methods prescribed
in "Standard Practice for Conducting Acute Toxicity Tests with Fishes,
Macroinvertebrates, and Amphibians" (American Society for Testing and
13
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and Materials 1980). Although the ASTM test methods were not specifically
designed for polychaetes or sediment tests, they provide guidelines for
experimental designs, water quality parameters, statistical analyses,
and animal care, handling, and acclimation. The length of the tests, 10
days, was chosen based on the data provided in Pesch and Hoffman (1983).
The 10 day LC5Q values approached closely the incipient LC5Q values;
therefore, the cost of conducting 28-day tests was unwarranted unless
chronic exposures were desired. Chronic exposures were not needed for
purposes of laboratory documentation.
Sediment Collection and Preservation
17. Reference sediment for these studies was collected from
the South reference site (41°7.95'N and 72°52.7'W), which is approxi-
mately 700 m south of the southern perimeter of the Central Long Island
Sound disposal site (Figure 2). Reference sediment was collected with a
Smith-Mclntyre grab sampler (0.1 m^) in August and December 1982 and
May 1983 (collection I, II, and III, respectively). Sediment from each
collection was returned to the laboratory, press sieved (wet) through a
2-mm mesh stainless steel screen, homogenized, and stored in polypropylene
(collection I) or glass (collection II and III) containers at 4°C until
used in experiments. Each container of material was coded with collection
number and date, and jar number (Lake et al. 1984).
14
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BLACK ROCK
HARBOR-
SOUTH REFERENCE
• SITE
Figure 2. Central Long Island Sound disposal site
and South reference site
18. Black Rock Harbor sediment was collected from 25 locations
within the highly industrialized Black Rock Harbor (Bridgeport, Conn.) study
2
area with a 0.1-m gravity box corer to a depth of 1.21 m (Figure 3). The
sediment was homogenized, distributed to barrels, and stored at 4°C. The
contents of each barrel were homogenized, wet sieved through a 2-mm sieve,
distributed to glass jars, and stored at 4°C until used in experiments.
Samples of sediment were taken at various points in the collection, mixing,
and distribution procedure for moisture content and chemical analysis (Lake
et al. 1984).
15
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BLACK \
ROCK
HARBOR
MAINTENANCE
DREDGING
FVP STUDY
REACH
400m
Figure 3. Black Rock Harbor, Connecticut,
source of dredged material
Polychaete Collection, Culture, and Holding
19. Of the two species of polychaetes used, N. incisa and N.
arenaceodentata, N. incisa is indigenous to the disposal area in Central
Long Island Sound. They were collected with a Smith-Mclntyre grab sampler
(0.1 m^) from the South reference site at various times in 1983 prior
to the test periods and held in the laboratory for a short acclimation
period (Appendix B). Neanthes arenaceodentata used in this study were
from laboratory cultures at the same salinity and temperature used in
these tests. Details of culture methods and conditions have been published
16
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by Reish (1980). Nutritional requirements were determined by Schauer
and Pesch (In Press). For both species, all tests were conducted with
juvenile worms (Appendix B).
Test System
20. The suspended sediment experimental system consisted of three
modules: the controlled dosing system, the dilution and distribution
system, and the test chambers. Two identical dosing systems, one for REF
and one for BRH, provided a constantly recirculating source of concentrated
sediment slurry (in seawater) passing by a three-way valve that leads to
the dilution and distribution system (Figure 4). Argon gas was added to
the reservoir of the dosing system to minimize oxidation of the slurry.
The three-way valve was controlled by a microprocessor programmed to
deliver a pulse of slurry at periodic intervals. In the dilution and
ARGON
INJECTION
SEPARATORY
FUNNEL
DELIVERY
MANIFOLD
Figure 4. Sediment dosing system
with chilled water bath
and argon gas supply
DOSING
VALVE
TO EXPOSURE
SYSTEM
SLURRY
RESERVOIR
17
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distribution system, the concentrated slurry was mixed with seawater to
the proper concentration of suspended solids and distributed to the
individual test chambers. Actual concentration of suspended particulates
in the test chambers was determined (by dry weights) periodically (Lake
et al. 1984).
21. Two types of tests were conducted: a solid phase (bedded
sediment) test without suspended particulates and a solid phase test
with suspended particulates. The tests were conducted in glass crystal-
lizing dishes (150 by 75 mm). Each dish contained a smaller glass crys-
tallizing dish (60 by 35 mm) in the center of the larger dish. A Teflon®-
coated stir bar was placed in the small dish in the center, which received
the inflow water, to keep the particulate material in suspension (Figure
5). In the solid phase tests, the stir bar was omitted and the in-flow
water was sandfiltered seawater. The inflow water flowed out of the
central dish over the sediment surface, and overflowed the edge of the
crystallizing dish. Each dish contained 400 ml of sediment (2.5 to 3.5
cm deep). The exposure concentrations used in the solid phase tests
were: 100, 75, 50, 25, and 0 percent BRH (100 percent REF). The mixtures
of the two sediments were made volumetrically, mixed thoroughly, and
then distributed to the exposure chambers (Rogerson et al. 1984). Worms
were fed prawn flakes, 30 mg every other day, directly onto the sediment
surface during the tests. Two solid phase tests without suspended partic-
ulates were conducted with 19. arenaceodentata. All tests included a
mitomycin C treatment (5 by 10~*> M). Mitomycin C is a known mutagen
and is included as a test standard for the SCE response (Fesch et al.
1981). The worms for the mitomycin C (MMC) treatment were exposed to
18
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reference sediment for 9 days, then were sieved and exposed without
sediment to MMC in seawater for the last day of the 10-day test.
DOSING SYSTEM
Spigot-«_
fr='
•Suspended Particles
OOOQ
--•Distribution Jar
'Stir Bar
EXPOSURE SYSTEM
Stir Bar
Exposure Container
Figure 5. Schematic of the distribution and dosing system used
to expose juvenile jl^ incisa and _N. arenaceodentata
to reference and BRH sediment
19
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22. Exposure conditions for the solid phase portion of the sus-
pended particulate tests were 100 percent REF or 100 percent BRH. These
two solid phase exposure conditions in combination with the two suspended
sediment exposures, REF or BRH at about 200 mg/& (dry weight), gave a
total of 4 treatments. The worms were fed prawn flakes (ADT-Prime,
Aquatic Diet Technology, Brooklyn, N.Y.) in a suspension of seawater,
which was pumped by peristaltic pump into the distribution chamber of
the dosing system. The amount fed was 127 mg per test chamber per day.
This amount of food was determined in prior feeding studies with N_._
incisa. Three identical suspended particulate tests were conducted with
!i. incisa and two tests with N. arenaceodentata.
23. During the tests, all dishes were examined daily for the
appearance of any worms on the surface of the sediment. Stressed worms
will come to the surface of the sediment and remain there. On the last
day of the test, observations were made on the burrows visible through
the sides of the dishes and the depth of suspended material deposited on
top of the solid phase was measured. Then the sediment was sieved (0.335
mm mesh) and the worms retrieved and counted.
24. All tests were conducted with sand-filtered Narragansett Bay
seawater at 20°C and approximately 30 ppt salinity. Flow rates were
about 35 ml/min. The photoperiod was a 14:10 hr light-dark cycle.
Chromosome Labeling
25. In order to facilitate the SCE observations, the chromosomes
must be differentially stained. The differential staining is a consequence
of labeling the chromosomes with the base analog 5-bromodeoxyuridine (BrdU)
20
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for two cell cycles (Latt 1982). Because the labeling phase must include
two cell cycles, the time needed will vary according to growth rate.
Therefore, this time may differ by species and age of an organism. In
this study, these times were determined empirically.
26. The labeling phase was dorie subsequent to the exposure phase.
Because of different holding requirements, the labeling conditions were
different for the two species of worms. However, both species were exposed
to the same concentration of the label BrdU, 3 mg/jj,. Neanthes
arenaceodentata were labeled in 1£ of filtered seawater (30 ppt salinity)
in crystallizing dishes at 20°C in the dark. Each treatment had approx-
imately 15 worms and these were fed 30 mg of prawn flakes every other
day. The labeled seawater was renewed every other day. Labeling lasted
from 4 to 10 days depending on the size of worms. The larger worms were
labeled longer because of longer cell cycle times. Colchicine (0.05
percent) was added to the seawater for the last 5 hr of the labeling
period to arrest cell division.
27. To maintain healthy worms, N. incisa must be held in sediment;
therefore, they were placed in clean, fine-grained sand with 2jj, of
filtered, labeled seawater (30 ppt). They were held at 20°C in subdued
light. Each treatment had approximately 15 worms and these were fed 30
mg of prawn flakes every other day. Each time food was added, 2.3 g of
reference sediment was added also, because in prior feeding studies, the
presence of suspended sediment was found to enhance growth. The sediment
was maintained in suspension by gentle aeration of the seawater. The
labeled seawater was renewed.every other day. The worms were labeled
21
-------
for 10 days after exposure to sediments. Colchicine (0.05 percent) was
added to the seawater for the last 15 hr of the labeling period.
Slide Preparation and Staining
28. The following procedure for slide preparation was adapted
from Kligerman and Bloom (1977). The worms were removed from the labeling
treatments and placed in 100 ml of 0.075 M solution of potassium chloride
for 1 hr. They were then fixed in three changes of cold ethanol-acetic
acid 3:1) for 0.5 hr each. Fixed worms were placed individually in a
clean well slide and 1 ml of 60 percent acetic acid was added. The worm
was macerated for approximately 1 min or until the tissue appeared trans-
lucent. The material was then drawn up into Pasteur pipettes and applied
to clean, hot (45°C) slides. Excess acetic acid was immediately removed
from the slides. This procedure produced a monolayer of separated cells
on each slide.
29. Slides were stained according to a procedure recommended by
Bloom.* Slides with BrdU-labeled chromosomes were stained with 225 \ig
ml"* of 33258 Hoechst stain for 10 min (several drops placed on slide
and coverslip added), rinsed in distilled water, and air dried. The
slide was then wet mounted using an excess of Mcllvaine's buffer (0.1 M
citric acid, 0.2 M disodium phosphate) at pH 8.0 and placed between two
black lights for 60 min. The slide was then rinsed in distilled water,
air dried briefly, and stained with 2 percent Giemsa in delonized water
* Personal Communication, S.E. Bloom, 1981, Cornell University, Ithaca,
New York.
22
-------
for 7 min. The slide was rinsed in distilled water, air dried, soaked
in xylene, and mounted in Coverbond. Observations were made with a
research microscope at 1250X, oil immersion.
Data Collection and Analysis
30. The SCE observations were made on 25 second-division
(metaphase stage) cells for each treatment unless otherwise noted.
Cells were selected under low power, then counted under high power. For
each treatment, the individual worms were screened sequentially. Counting
continues until a total of 25 cells were counted regardless of the number
of individual worms. This assumes that organism-to-organism variance was
small compared to within organism variance. This has been tested for
these data and found to be true.
31. The data were examined to see whether criteria were met for
parametric statistical analysis or whether data transformation was neces-
sary. All of the SCE data in this report were transformed to log 10
(SCE/chromosome +0.1) prior to statistical analysis. The 0.1 is added
because log (0) is undefined. The means and standard error of the
untransformed data are included in Appendix B. Statistical procedures
used in this report are from Snedecor and Cochran (1980). Photographs
were taken at 1000X through a green filter using high contrast copy
film.
23
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PART III: RESULTS AND DISCUSSION
Statistical Properties of SCE Data
32. Parametric statistical procedures used to analyze data (e.g.,
analysis of variance) assume that the data are normally distributed and
that the variances are equal for all treatments being compared. These
fundamental assumptions have been examined for SCE data in two different
test systems. Dixon and Clarke (1982) reported that for adult blue
mussels, Mytilus edulis, SCE data follow a Poisson distribution for
individual animals. However, SCE counts for single cells from randomly
selected animals within each treatment indicated the presence of varia-
bility between animals. They corrected for these problems by summing
the SCE/cell counts for each animal and then applying a square root trans-
formation. This stabilized the variance for all treatments and permitted
parametric statistical analysis of the data. Carrano and Moore (1982)
have provided an extensive evaluation of SCE data from human lymphocytes.
They found that no single family of probability distributions could be
used to describe all of their data sets. Consequently, they applied a
nonparametric test for statistical comparisons.
33. The statistical attributes of SCE data for N^ arenaceodentata
were examined using results from the larval bioassay test (Pesch et al.
In Press). This section is based on these data. All baseline observation
data were pooled and the variable X = SCE's/chromosome was considered.
There were 447 metaphase cell observations in this data set and their
frequency distribution was skewed to the right and clearly did not follow
a normal distribution. Based upon these results, a transformation of
24
-------
the SCE/chromosome data prior to statistical analysis seemed appropriate.
To determine the most appropriate transformation, the mean SCE's/chromo-
some were plotted for all experimental treatments (63 treatments, 25
observations/treatment) against their standard deviation. There was a
very good relationship between standard deviation and mean, further
evidence that a transformation of the data was appropriate. Although
Latt et al. (1981) mention several possible transformations, it was
concluded that the log 10 transformation is appropriate because it removes
the relationship between mean and standard deviation; also, means based
upon sample sizes as small as 5 are normally distributed (Pesch et al.
In Press).
34. Another concern is the statistical sensitivity of the N.
arenaceodentata SCE assay to detect an increased SCE response over base-
line. The sensitivity of the method is evaluated by calculating the
associated statistical power of the test to detect an increase in SCE
frequency. The power of a statistical test is defined to be one minus
the probability of making a type II error (false negative). The proba-
bility of type I error (false positive) is the preselected level of
significance of a statistical test, typically P - 0.05. The probability
of type II error (and power) is a function of the differences to be
detected, the variance of the mean response, and sample size. A twofold
increase over baseline SCE frequencies is recommended to indicate a
positive response (Latt et al. 1981). To increase sample size is labor
intensive with the N. arenaceodentata SCE assay. Therefore, the simplest
approach to increase the power of the test is to decrease the variance
of the mean baseline response. Carrano and Moore (1982) examined this
25
-------
problem thoroughly and concluded that in the human lymphocyte system the
"major source of inherent variation in baseline SCE frequencies can be
attributed to the amount of BrdU present in the culture medium relative
to the number of lymphocytes initially added." They dealt with this
problem by selecting, as baseline, a BrdU concentration that was in the
middle of the plateau region of the dose response curve. The plateau of
the curve is by definition a region of minimum slope, so variations in
BrdU concentration or cell numbers have minimal impact on baseline SCE
frequencies.
35. BrdU elicits a dose response in N_. arenaceodendata as in other
test systems (Carrano et al. 1980; Wolff and Perry 1974). However, with
IJ. arenaceodentata the variance associated with the mean response (log
scale) increases by a factor of 2 in the plateau region of the curve.
Therefore, a baseline BrdU concentration selected from the plateau region
would reduce greatly the power of a statistical test to detect increases
above baseline. An examination of the data indicates that the sensitivity
of the assay is enhanced greatly using a baseline concentration of 3
mg/A BrdU vs. 15 mg/St, (Pesch et al. In Press). The accepted protocol
(Latt et al. 1981) of using 25 counts per replicate is sufficient to
detect a doubling of the mean response using 3 mg/Jl BrdU as a baseline
concentration. The power to detect this doubling is 0.9 using a two-sample
t-test with variance estimated from baseline data. A baseline concentra-
tion of 3 mg/Jl BrdU allows for a more sensitive statistical test because
of a lower variance of the mean.
-------
Applicability of SCE Technique
36. Neanthes arenaceodentata were exposed to a series of the test
sediments in solid phase only (Appendix B). This experiment was replicated
as a randomized block design. In no case were the SCE frequencies of
any treatment different from any other treatment (Table 1).
Table 1
Sister Chromatid Exchange/Chromosome Response for Two Replicate
Experiments, Solid Phase Dosing of N. arenaceodentata
Treatments
% BRH* Exp. 1 Exp. 2 Pooled Data
0 -0.803 ± 0.049**(25)t -0.802 ± 0.041(25) -0.802 ± 0.032(50)
25 -0.783 ± 0.047(25) -0.806 ± 0.043(25) -0.794 ± 0.031(50)
50 -0.714 ± 0.049(25) -0.826 ± 0.043(25) -0.769 ± 0.033(50)
75 -0.716 ± 0.040(25) -0.870 ± 0.031(25) -0.793 ± 0.028(50)
100 -0.807 ± 0.045(25) -0.789 ± 0.041(25) -0.798 ± 0.030(50)
MMCtt -0.534 ± 0.065(25) ND*
Note: Data expressed as log 10 of means.
* Percentage Black Rock Harbor sediment mixed volume for volume with
Reference sediment.
** Standard error of the mean.
t Number in parentheses is sample size (N).
tt MMC is a known mutagen and is included as a test standard for the
SCE response.
$ ND = no data.
37. The pattern of no SCE response to BRH sediment continued for
the particulate phase/solid phase experiments with N^ arenaceodentata
(Table 2). Again, in replicate experiments (ramdomized block design),
the SCE frequencies of any treatment were not different from any
other treatment.
27
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Table 2
Sister Chromatic! Exchange/Chromosome Response for Replicate Experiments,
Particulate Phase/Solid Phase Dosing of N. incisa and N. arenaceodentata
N>
00
Treatment
Exp. 1
B/R*
B/B
R/R
R/B
-0
-0
-0
-0
.450
,543
.494
.608
±
t
+
+
0.050**(2
0.050(26)
0.145 (7)
0.056(25)
MMCt 0.109 ± 0.275 (3)
R/B -0.644 ± 0.058(25)
B/R -0.656 ± 0.053(25)
R/R -0.689 ± 0.053(25)
B/B -0.719 ± 0.048(25)
MMC* -0.348 ± 0.058(25)
Exp. 2
Exp. 3
incisa
-0.414 ± 0.055(12)
-0.513 ± 0.054(15)
-0.512 ± 0.049(25)
NDTt
-0.194 ± 0.056(19)
-0.449 ± 0.053(25)
-0.482 ± 0.123 (9)
-0.403 ± 0.055(24)
-0.095 ± 0.051(20) 0.144 ± 0.246 (3)
N. arenaceodentata
-0.710 ± 0.060(25)
-0.705 ± 0.040(25)
-0.683 ± 0.048(25)
-0.655 ± 0.056(25)
-0.358 ± 0.054(25)
Pooled Data
-0.355 ± 0.035(56)
-0.500 ± 0.031(66)
-0.502 ± 0.046(41)
-0.508 ± 0.041(49)
-0.044 ± 0.056(26)
-0.676 ± 0.042(50)
-0.680 ± 0.033(50)
-0.685 ± 0.036(50)
-0.686 ± 0.037(50)
-0.353 ± 0.039(50)
Note: Data expressed as log 10 of means
* R = reference sediment, B = Black Rock Harbor sediment, numerator = particulate phase,
denominator = solid phase.
** Standard error of the mean.
t Number in parentheses is sample size (N).
tt ND - no data.
$ MMC is a known mutagen and is included as a test standard for the SCE response.
-------
38. The particulate phase/solid phase experiments were replicated
three times with 14. incisa using a randomized block design. Worms used
in these replicate experiments were from different collection trips to
the South reference site. In all three experiments, the B/R treatment
had the highest SCE frequencies (Table 2). In the pooled data, the SCE
frequencies for the B/R treatment were significantly higher (P < 0.01,
~ 50 percent higher) than any other treatment. There were no significant
differences among the other treatments.
39. These findings raise several questions. Would we expect to
see an increase in SCE frequencies due to Black Rock Harbor sediment?
If so, why only in the B/R treatment? Why not in the presumably worst
case treatment, B/B? Also, why would there be a difference in the response
for the two species tested?
40. Black Rock Harbor sediment contains high levels of polynuclear
aromatic hydrocarbons (Rogerson et al. 1984). Such compounds are known
to be mutagenic; however, they require metabolic activation to become so
(Bresnick 1976). The metabolic activation of hydrocarbons is mediated
by a mixed-function oxygenase (MFO) system. Polychaetes have been
shown to have active, inducible MFO systems (Lee and Singer 1980; Lee
et al. 1979). Pesch et al. (1981) demonstrated that the polynuclear
aromatic hydrocarbon, benzo(a)pyrene, caused increased SCE frequencies
in the polychaete _N. arenaceodentata.
41. The required induction of an MFO system provides a speculative
answer for the remaining questions. Johns and Gutjahr-Gobell (in prepa-
ration) have studied the impact of BRH sediment on the bioenergetics of
N. incisa. These studies were conducted simultaneously in the same
29
-------
system as the chromosome studies. They found that the presumably worst
case treatment (B/B) caused N. incisa to lose weight during the experi-
ments, whereas the B/R treatment was not significantly different from
R/R treatment for any of the measures of energetic effects. Because
metabolic activation of the contaminants is needed, the worst case worms
may be protected by their reduced metabolic activity. The worms in the
B/R treatment did well metabolically and thus may have been susceptible.
Since no data are available on MFO activity in these worms, this explana-
tion of the observed SCE response is speculative. The ability to metab-
olize polynuclear aromatic hydrocarbons is centrally important to the
toxicity of such compounds. Clearly, this is an area where additional
research is needed.
42. The difference between species has several possible explana-
tions. Pesch et al. (1981) demonstrated that IJ. arenaceodentata has an
active MFO system. It may be that 1J. incisa has a more effective MFO
system. More likely, the N\ incisa (field collected vs. laboratory
cultured) had been exposed previously to polynuclear aromatic hydrocarbons
(PAH). These worms (N. incisa) were collected from the reference site.
There are measurable levels of PAH (Rogerson et al. 1984) at the reference
site. This low level exposure may induce increased MFO activity. Thus,
the two species may have had considerably different levels of MFO
activity during the experiments. Such a difference could explain the
difference in SCE response.
30.
-------
Variability and Reproducibility of Data
43. Table 3 presents results from a separate experiment comparing
duplicate treatments within a particulate phase/solid phase dosing exper-
iment of N^ arenaceodentata. The particulate phase concentration was
25 mg/jt (Appendix B).
Table 3
Sister Chromatid Exchange/Chromosome Response for Duplicate Treatments
Within an Experiment, Particulate Phase/Solid Phase Dosing of
N. arenaceodentata
Treatment log Mean
R/R* -0.753 ± 0.052**(25)t
R/R -0.789 ± 0.044(25)
B/B -0.723 ± 0.052(25)
B/B -0.809 ± 0.042(23)
Note: Data expressed as log 10 of means. ~~~ ~
* R = reference sediment, B =• Black Rock Harbor sediment,
numerator = particulate phase, denominator - solid phase.
** Standard error of the mean.
t Number in parentheses is sample size (N).
44. The assumed worst case, B/B. was duplicated and compared to
duplicates of R/R. There were no statistically significant differences
among any of the four treatments. Replicates of the same conditions
were not significantly different from one another. These results indi-
cate that between-treatment variance is low.
45. With N^ arenaceodentata, replicate experiments were conducted
with solid phase dosing and with particulate phase/solid phase dosing.
31
-------
The particulate phase/solid phase replicates were not statistically dif-
ferent from each other; however, the overall means of the solid phase
replicates differed at the P - 0.05 level with N^_ arenaceodentata.
46. The particulate phase/solid phase dosing experiment was rep-
licated three times with N. incisa (Table 2). Two of the experiments
(Exps. 1 and 2) were not significantly different from each other, nor
were there any significant differences among treatments. The overall
mean for the third experiment (Exp. 3) was significantly higher than the
overall means for the other two experiments. Also, the B/R treatment
values were significantly higher than the other treatments within the
third experiment. These results indicate a lack of reproducibility
among experiments for 1*. incisa. However, the R/R (control) treatment
was not different among the three experiments. The most likely explana-
tion of the experiment-to-experiment differences is a biological differ-
ence among the three collections of worms. The collection site (South
reference site) has low levels of PAH's (Rogerson et al. 1984) and has
old patches of dredged material (personal observation). Prior exposure
to PAH's could influence the inducible MFO system in the worms. The
level of MFO activity in the worms could influence their SCE response to
BRH sediment. Since no MFO data are available, this explanation of the
observed SCE responses is speculative.
47. All statistical comparisons involving replicate experiments
were made using a randomized block design. In this way, differences among
experiments (blocks) were accounted for in determining differences among
treatments. Using this method, the B/R treatment was significantly higher
32
-------
than all other treatments with N_. incisa. The other treatments were not
significantly different from each other.
33
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PART IV: CONCLUSIONS
48. The objective of the Laboratory Documentation Phase of the
Field Verification Program for cytogenetic studies was to determine the
applicability of sister chromatid exchange to measuring contaminated dredged
material effects, and to determine the degree of variability and reproducibil-
ity inherent in the procedure. A major challenge was to apply the SCE tech-
nique to Nephtys incisa. This was done successfully. Nephtys incisa
and the surrogate species, Neanthes arenaceodentata, were then used in
parallel to test the effect of contaminated Black Rock Harbor sediment on the
sister chromatid exchange response. With the exception of one treatment in one
experiment (B/R, Exp. 3, Table 2, N. incisa), the worm chromosomes were
uniformly nonresponsive to BRH sediment.
49. Replicate treatments within an experiment did not differ
significantly for N. arenaceodentata (Table 3). Differences between
experiments and between species within an experiment were found (Tables 1
and 2). The reasons for these differences are not known. Differences in
ability to metabolize polynuclear aromatic hydrocarbons, found in high
concentrations in BRH sediments, is a likely but speculative reason.
Clearly, additional research is needed before SCE could be used for
routine testing.
34
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Aquatic Toxicology and Hazard Assessment; Sixth Symposium, W.E. Bishop,
R.D. Cardwell,and B.B. Heidolf, eds., ASTM STP802, American Society
for Testing and Materials, Philadelphia, pp 482-493.
Pesch, G., Heltshe, J., and Mueller, C. In Press. "A Statistical Analysis
of Neanthes arenaceodentata, Sister Chromatid Exchange Data," International
Symposium on Sister Chromatid Exchange, R. Tice, ed., Plenum Press,
New Xork.
Pesch, G., Pesch, C.E., and Malcolm, A.R. 1981. "Neanthes arenaceodentata,
a Cytogenetic Model for Marine Genetic Toxicology," Aquatic Toxicology,
Vol 1, pp 301-311.
Pesch, G., and Pesch, C.E. 1980a. "Chromosome Complement of the Marine
Worm Neanthes arenaceodentata (Polychaeta:Annelida)," Canadian Journal
of Fisheries and Aquatic Sciences, Vol 37, pp 286-288.
Pesch G., and Pesch, C.E. 1980b. "Neanthes arenaceodentata (Polychaeta:
Annelida), a Proposed Cytogenetic Model for Marine Genetic Toxicology,"
Canadian Journal of Fisheries and Aquatic Sciences, Vol 7, pp 1225-1228.
Reish, D. 1960. "The Uses of Marine Invertebrates as Indicators of Water
Quality," Waste Disposal in the Marine Environment, E. Pearson, ed.,
Proceedings of the First International Conference, Pergamon, New York,
pp 92-103.
Reish, D. 1972. "The Use of Marine Invertebrates as Indicators of
Varying Degrees of Marine Pollution," Marine Pollution and Sea Life,
M. Ruivo, ed., FAO of the United Nations, Fishing News (Books) Ltd.,
London, England, pp 203-207.
37
-------
Reish, D. 1980. "The Effect of Different Pollutants on Ecologically
Important Polychaete Worms," Ecological Research Report, EPA 600/3-80-053.
U.S. Environmental Protection Agency, Washington, D.C., 138 pp.
Rogerson, P., Schimmel, S., and Hoffman, G. 1984. "Chemical and Biolog-
ical Characterization of Black Rock Harbor Dredged Material," Technical
Report D-84- , prepared by U.S. Environmental Protection Agency,
Narragansett, R.I., for the U.S. Army Engineer Waterways Experi-
ment Station, CE, Vicksburg, Miss.
Schauer, P.S,,and Pesch, C.E. Submitted for Publication. "Influence of
Diet on Growth, Survival and Tube Production of the Laboratory Cultured
Polychaete Neanthes arenaceodentata."
Schneider, E.L., Tice, R.R., and Kram, D. 1978. "Bromodeoxyuridine Dif-
ferential Chromatid Staining Technique: a New Approach to Examining
Sister Chromatid Exchange and Cell Replication Kinetics," Methods in
Cell Biology, Vol 20, pp 379-409.
Shafer, D.A. 1977. "Replication Bypass Model of Sister Chromatid Exchanges
and Implications for Bloom's Syndrome and Fanconi's Anemia," Human Genetics,
Vol 39, pp 177-199.
Snedecor, G.W., and Cochran, W.G. 1980. Statistical Methods. 7th Edition.
The Iowa State University Press, Ames, Iowa, pp 102-105.
Solomon, E., and Bobrow, M. 1975. "Sister Chromatid Exchanges: a Sensi-
tive Assay of Agents Damaging Human Chromosomes," Mutation Research, Vol
30, pp 273-278.
Taylor, J.H., Woods, P.S., and Hughes, W.L. 1957. "The Organization and
Duplication of Chromosomes as Revealed by Autoradiographic Studies Using
Tritium-Labeled Thymidine," Proceedings of the National Academy of Sciences,
U.S. Vol 43, pp 122-128. ———
Wass, M.L. 1967. "Biological and Physiological Basis of Indicator
Organisms and Communities," Section II: Indicators of pollution,
Pollution and Marine Ecology, T.A. Olson and F.J. Burgess, eds.,
Interscience Publishers, New York, pp 271-283.
Wolff, S., and Perry, P. 1974. "Differential Giemsa Staining of Sister
Chromatids and the Study of Sister Chromatid Exchanges Without Auto-
radiography," Chromosoma, Vol 48, pp 341-353.
38
-------
APPENDIX A: KARYOTYPES
Chromosome complements (karyotypes) of Neanthes arenaceodentata
and Nephtys incisa are shown in Figures Al and A2, respectively.
A-l
-------
n H
if H H ri
6789
Figure Al. Chromosomes of Neanthes arenaceodentata
A-2
-------
U
U U U
it H it «l II
7 8 9 10 11
II It «» «» I*
12 13 14
M II
17 18 19
Figure A2. Chromosomes of Nephtys incisa
A-3
-------
APPENDIX B: DATA SHEETS
Tables Bl through B8 are the laboratory data sheets for the
experiments included in this report. The data sheets are identified by
table number and experiment number as these numbers appear in the text
of the report for each species tested.
B-l
-------
TABLE Bl
LABORATORY MORN DATA SHEET
COE/ERLN FVP
STUDY PLAN: 4
EXPERIMENI DESCRIPTION: SOLID
EXP. NUMBER:
INVESTIGATOR: PESCH EI.AL.
DATE OF TtST:
83O1O4
, , ... SPECIES: NEANTHfeS ARENACEODENTATA
UabiW;EXPERIMeNTAL CONDITIONS »»
RANOE: 19.OO - 21. OO
RANGE: 3O. OO - 32. OO
TfcMPERATUKE: 20. OO DEGREES CEN11GRADE
SALINITY: 31. OO PARTS PER THOUSAND
EXPOSURE DURATION: 1O DAYS
PHOTOPERIOD: 9 HOURS
FLOW RATE: 63 HLS/HIN VOLUME ADDITIONS/DAY 13O
NUMBER OF ANIMALS/REPLICATE- 29 NUMBER OF REPLICATES/TREATMENT:
ANIMAL'S LIFE STACE: JUVENILF AGE: 93 DAYS
SIZE: O. 7 +/- 0. 36 MG DRY WT RANGE:
CONTROLS: 1OOX REF
FOOD: PRAWN FLAKF DIRECTLY TO DISH
ANIMAL SOURCE: CULTURE
COLLECTION TEMPEHATURE: DEGREES C COLLECTION SATINITY:
ACCLIMATION:
SEDIMENT SGUKCE: BARREL OR COL» ECTION/JAR NUMBER
SOLID REFERENCE: I/- SOLID BRH: LL/19
SUSPENDED REFERENCE: SUSPENDED BRH:
PPT
• :•* smm a
SAMPLE
NUMBER
4OOO39
40OO4O
40O041
400042
400043
EXPOSURE CON<
NOMINAL
1OOX RE*
29% BRH
90% BRH
79% BRH
1OOX BRH
400133 IMilC
1
:ENTRATIONS
MEASURED
1
OXYGEN 1 I»;£AN
JLOOCSCE/
MG/L ICHROM-K 1>
-O. SO
-O. 78
O. 71
-O. 71
•O. 80
-0.93
e mmmvmmmmm
1 MEAN
SCE/CHROMQ
mtfm-mmmmm
O. O9 ± 0.03*
O. O9 ± 0.02
0. 12 ± 0.02
0. 11 + 0.01
O. O7± 0.02
0. 28 + 0.06
* Standard error of the mean.
B-2
-------
TABLE B2
LABORATORY WORM DATA SHEET
COE/ERLN FVP
STUDY PLAN: 4
EXPERIMENT DESCRIPTION; SOLID
EXP. NUMBER:
INVESTIGATOR: PESCH ET. AL.
DATE OF TEST:
83O931
2 (Table 1) SPECIES: NEANTHeS ARENACEODENTATA
»* EXPERIMENTAL CONDITIONS »*
RANGE: 21. OO - 22. OO
RANGE: 28. OO - 3O. 00
TEMPERATURE: 21. OO DEGREES CENTIGRADE
SALINITY: 3O. 00 PARTS PER THOUSAND
EXPOSURE DURATION: 1O DAYS
PHOTOPERIOD: 19 HOURS
FLOW RATE: 64 ML8/MIN VOLUME ADDITIONS/DAY 122
NUMBER OF ANIMALS/REPLICATE: 13 NUMBER OF REPLICATES/TREATMENT:
ANIMAL'S LIFE STAGE: JUVENILE AGE: 62 DAYS
SIZE: +/- MO DRY WT RANGE: NOT AVAILABLE
CONTROLS: 1OOX REF
FOOD: PRAWN FLAKE DIRECTLY TO DISH
ANIMAL SOURCE: CULTURE
COLLECTION TEMPERATURE: DEGREES C COLLECTION SALINITY:
ACCLIMATION:
SEDIMENT SOURCE: BARREL OR COLLECTION/JAR NUMBER
SOLID REFERENCE: 11/92 SOLID BRH: LL/39
SUSPENDED REFERENCE: SUSPENDED BRH:
SAMPLE I
PPT
NUMBER I NOMINAL
m -mwmmmm | mmmim—mm*imrmm
4OOO49 11OOX REF
1
4OOO90 129% BRH
I
400091 I90X BRH
t
400032 I79X BRH
I
400093 1100X BRH
I
* Standard error of the mean.
MM *mmmmm-*imm ai
AT IONS (1)
MEASURED
l» .)««••»•»»
OXYGEN
MG/L
xmmimm^mmmmt
MEAN
LOGCSCE/
CHROm. 1 )
-O. SO
-0.80
-O. 82
-O. 87
-0. 78
• •nmmMmmmmm
MEAN
8CE/CHROMO
O. O7 ± 0.01*
0. O7 ± 0.01
0. 06 ± 0 . 01
O. O4 ± 0.01
0. O8 ± 0.01
B-3
-------
TABLE B3
LABORATORY WORM DATA SHEET
COE/ERLN FVP
STUDY PLAN: 4
INVESTIGATOR: PESCH ET.AL.
EXPERIMENT DESCRIPTION: SUSPENDED
DATE OF TEST:
83O816
EXP. NtJMBER: 1 (Table 2) SPECIES: NEPHTYS INC ISA
** EXPERIMENTAL CONDITIONS **
TEMPERATURE: 21. OO DEGREES CENTIGRADE RANGE: 21. OO - 22. GO
SALINITY: 3O. OO PARTS PER THOUSAND RANGE: 3O. GO - 31. GO
EXPOSURE DURATION: 13 DAYS
PHOTOPERIOD: 14 HOURS
FLOW RATE: 33 MLS/MIN VOLUME ADDITIONS/DAY 67
NUMBER OF ANIMALS/REPLICATE: 14 NUMBER OF REPLICATES/TREATMENT: 1
ANIMAL'S LIFE STAGE: JUVENILE* AGE: DAYS
SIZE: +/- MG DRY WT RANGE: 438-4. 662 MO
CONTROLS: 200 KG/L REF/REF
FOOD: PRAMI FLAKE SUSPENSION
ANIMAL SOURCE: SOUTH REFERENCE SITEr LONG ISLAND SOUND
COLLECTION TEMPERATURE: 19.30 DEGREES C COLLECTION SALINITY: 28. 2G PPT
ACCLIMATION: 14 DAYS AT 20 C.
SEDIMENT SOURCE: BARREL OR COLLECTION/JAR NUMBER
SOLID REFERENCE: 111/13,14 SOLID BRH: EE/1.2
SUSPENDED REFERENCE: II1/13-17 SUSPENDED BRH: EE/1-9
I. .|B»**MB
SAMPLE
NUMBER
l^ HflUWfl
400122
4OO123
400124
4O0123
400190
> EXPOSURE CONC
t
i NOMINAL
I2OOMG/L REF/REF
1
:ENTRATIONS
MEASURED
217. 3± 85.9
I200MG/L BRH/REFI 19O. 1 + 60.7
1
I2OOMG/L REF/BRHt 217.3+ 85.9
t
J2OOMG/L BRH/BRHi 19O. 1+ 60.7
1
IMMC
1
OXYGEN ! MEAN
ILOOCSCE/
MG/L ICHROM-K 1)
-O. 49
-0.44
-0. 60
-O. 34
0. 1O
MEAN
SCE/CHROMO
O. 32 ± 0.10*
0. 3O ± 0.03
0. 20 ± 0 • 03
0.24 ± 0.05
1. 73 f 0.98
* Standard error of the mean.
B-4
-------
TABLE B4
LABORATORY WORM DATA SHEET
COE/ERLN FVP
STUDY PLAN: 4
EXPERIMENT DESCRIPTION: SUSPENDED
INVESTIGATOR: PESCH ET. AL.
DATE OF TEST:
83O920
EXP. NUMBER: 2 (Table 2) SPECIES: NEPHTYS INC ISA
** EXPERIMENTAL CONDITIONS *»
TEMPERATURE: 20. 60 DEGREES CENTIGRADE RANGE: 19. 8O - 22. OO
SALINITY: 30. 70 PARTS PER THOUSAND RANGE: 3O. OO - 31. SO
EXPOSURE DURATION: 10 DAYS
PHOTOPERIOD: 12 HOURS
FLOW RATE: 32 MLS/MIN VOLUME ADDITIONS/DAY 61
NUMBER OF ANIMALS/REPLICATE: 21 NUMBER OF REPLICATES/TREATMENT: 1
ANIMAL'S LIFE STAGE: JUVENILE AGE: DAYS
SIZE: +/- MG DRY WT RANGE: 1. 184-1. 799 MG
CONTROLS: 2OOMO/L REF/REF
FOOD: PRAWN FLAKE SUSPENSION
ANIMAL SOURCE: SOUTH REFERENCE SITE. LONG ISLAND SOUND
COLLECTION TEMPERATURE: 21. 6O DEGREES C COLLECTION SALINITY: 29. 2O PPT
ACCLIMATION: 9 DAYS AT 2O C.
SEDIMENT SOURCE: BARREL OR COLLECTION/JAR NUMBER
SOLID REFERENCE: 111/29 SOLID BRH: EE/17
SUSPENDED REFERENCE: II1/6.7,36 SUSPENDED BRH: EE/8, 1O, 14* 23, »
SAMPLE 1 EXPOSURE CONCENT*ATIONS (1)
I !
NUMBER i NOMINAL I MEASURED
-*•»•»«« * ] •••••*•» asBB -.mm^m J ••= mmmmmmmximmmi
400134 J200MG/L REF/REF1 198.7 +73.3
I i
4O0139 I2OOMG/L BRH/REF! 229.6 +47.5
I J
4O0137 I200MO/L BRH/BRHi 225.6 +47.5
I I
400192 IMMC I
I I
OXYGEN I MEAN I MEAN
ILOGCSCE/ I8CE/CHROMO
MG/L ICHROM+. 1)
-O. 91
6. 6O
6. 3O
6.00
-O. 41
-0. 91
-0.09
0.29 + 0.03*
O. 32 + 0.05
0. 23 + 0.03
0.82 + 0.12
* Standard error of the mean.
B-5
-------
TABLE B5
LABORATORY WORM DATA SHEET
COE/ERLN FVP
STUDY PLAN: 4 INVESTIGATOR: PESCH ET. AL.
EXPERIMENT DESCRIPTION: SUSPENDED DATE OF TEST: 83O9O2
EXP. NUMBER: 3 (Table 2) SPECIES: NEPHTYS INC ISA
»* EXPERIMENTAL CONDITIONS *«
RANGE: 2O. SO - 22. 9O
RANGE: 3O. OO - 31. OO
TEMPERATURE: 21. 20 DEGREES CENTIGRADE
SALINITY: 30. SO PARTS PER THOUSAND
EXPOSURE DURATION: 10 DAYS
PHOTOPERIOD: 13 HOURS
FLOW RATE: 39 MLS/MIN VOLUME ADDITIONS/DAY 67
NUMBER OF ANIMALS/REPLICATE: 19 NUMBER OF REPLICATES/TREATMENT: 1
ANIMAL'S LIFE STAOE: JUVENILE AGE: DAYS
SIZE: +/- MO DRY WT RANGE: 1.491-4.488 MG
CONTROLS: 20OMO/L REF/REF
FOOD: PRAWN FLAKE SUSPENSION
ANIMAL SOURCE: SOUTH REFERENCE SITE. LONG ISLAND SOUND
COLLECTION TEMPERATURE: 2O. 9O DEGREES C COLLECTION SALINITY: 28.SO PPT
ACCLIMATION: 7 DAYS AT 2O C.
SEDIMENT SOURCE: BARREL OR COLLECTION/JAR NUMBER
SOLID REFERENCE: I11/19 SOLID BRH: EE/3,8
SUSPENDED REFERENCE: 111/19,21.22 SUSPENDED BRH: EE/7,11.12
i- -immmmm
SAMPLE
NUMBER
400130
4OO131
400132
400133
40O191
i EXPOSURE CONCENTRATIONS (1)
1 1
1 NOMINAL i MEASURED
J2OOMO/L REF/REF 1
1 i
S20OMO/L BRH/REFi
1 1
ISOOMO/L REF/BRHt
1 1
I2OOMG/L BRH/BRHI
1 1
JMMC 1
f 1
211. O ± 87
171.3 + 52
211.0 + 87
171. 3+52
.2
.9
.2
.9
OXYGEN 1 MEAN
iLOOCSCE/
MG/L 1CHROM+. 1)
,
-0.48
-O. 19
-0. 4O
-O. 44
O. 14
MEAN
SCE/CHROMO
O. 34 ± 0
0. 62 ± 0
0. 37 ± 0
0. 31 ± 0
1.83 + 1
Standard error of the mean.
B-6
-------
TABLE B6
LABORATORY WORM DATA SHEET
COE/ERLN FVP
STUDY PLAN: 4
EXPERIMENT DESCRIPTION: SUSPENDED
INVESTIGATOR: PESCH Ef. AL.
DATE OF TEST:
830816
EXP, NUMBER: 1 {Table 2) SPECIES: NEANTHES ARENACEODENTATA
«* EXPERIMENTAL CONDITIONS **
RANGE: 21. OO - 22. OO
RANGE: 30. OO - 31. OO
TEMPERATURE: 21. OO DEGREES CENTIGRADE
SALINITY: 3O. OO PARTS PER THOUSAND
EXPOSURE DURATION: 13 DAYS
PHOTOPERIOD: 14 HOURS
FLOW RATE: 39 MLS/MIN VOLUME ADDITIONS/DAY 67
NUMBER OF ANIMALS/REPLICATE: 10 NUMBER OF REPLICATES/TREATMENT:
ANIMAL'S LIFE STAGE: JUVENILE AGE: 97 DAYS
SIZE: +/- MG DRY WT RANGE: 7.394-12.012 MG
CONTROLS: 200MO/L REF/REF
FOOD: PRAWN FLAKE DIRECTLY TO DISH
ANIMAL SOURCE: CULTURE
COLLECTION TEMPERATURE: DEGREES C COLLECTION SALINITY:
ACCLIMATION:
SEDIMENT SOURCE: BARREL OR COLLECTION/JAR NUMBER
SOLID REFERENCE: 111/13.14 SOLID BRH: EE/1,2
SUSPENDED REFERENCE: II1/13-17 SUSPENDED BRH: EE/1-9
PPT
SAMPLE
NUMBER
400126
400127
400128
400129
400134
1 EXPOSURE CONC
1
I NOMINAL
I200MG/L
1
I200MO/L
1
J20OMG/L
|
I200MO/L
1
IMMC
1
REF/REF
:ENTRATIONS u>
MEASURED
217.3
BRH/REFI 190. I
REF/BRH! 217.3
BftH/BRHI 190. 1
± 85
+ 60
+ 85
+ 60
.9
.7
.9
.7
OXYGEN t MEAN
SLOG
-------
TABLE B7
LABORATORY WORM DATA SHEET
COE/ERLN FVP
STUDY PLAN: 4 INVESTIGATOR: PE8CH ET. AL.
EXPERIMENT DESCRIPTION: SUSPENDED DATE OF TEST:
EXP. NUMBER:
830922
2 (Table 2} SPECIES: NEANTHES ARENACEODENTATA
** EXPERIMENTAL CONDITIONS **
RANOE: 19. SO - 22. OO
RANGE: 30. OO - 31.80
TEMPERATURE: 2O. 60 DEGREES CENTIGRADE
SALINITY: 3O. 70 PARTS PER THOUSAND
EXPOSURE DURATION: 1O DAYS
PHOTOPERIOD: 12 HOURS
FLOW RATE: 33 MLS/MIN VOLUME ADDITIONS/DAY 63
NUMBER OF ANIMALS/REPLICATE: 19 NUMBER OF REPLICATES/TREATMENT: 1
ANIMAL'S LIFE STAGE: JUVENILE AGE: 42 DAYS
SIZE: +/- MG DRY WT RANGE: 1.496-5.124 MG
CONTROLS: 20OMC/L REF/REF
FOOD: PRAWN FLAKE SUSPENSION
ANIMAL SOURCE: CULTURE
COLLECTION TEMPERATURE: DEGREES C COLLECTION SALINITY: PPT
ACCLIMATION:
SEDIMENT SOUHCE: BARREL OR COLLECTION/JAR NUMBER
SOLID REFERENCE: 111/29.26 SOLID BRH: EE/17.18
SUSPENDED REFERENCE: HI/6. 7. 36 SUSPENDED BRH: EE/8, 10, 14, 23, *
SAMPLE ! EXPOSURE CONCENTRATIONS (1)
I I
NUMBER I NOMINAL I MEASURED
m-immmmss*\mmm*amm .•m-mmmm:-tm\ mrn-mmmtsmmujmm^fm
4OO138 J2OOMO/L REF/REFJ 199. 2±73.2
I !
400139 I2OOMQ/L BRH/REFi 227.4+43.5
1 I
400140 I2OOKO/L REF/BRHl 199. 2±73.2
I I
4OO141 I2OOMG/L BRH/BRHi 222.4+43.5
I 1
400159 IMMC I
I I
OXYGEN 1 MEAN i MEAN
ILOGCSCE/ I8CE/CHROMO
MG/L ICHROM+. 1>
-0.68
6.60
6.30
6.40
6. OO
-0. 7O
-0.70
-O. 63
-0.39
O. 13 + 0.02*
0. 11 + 0.01
O. 19 ± 0.04
O. 17 ± 0.05
0. 42 ± 0.06
* Standard error of the mean.
B-8
-------
TABLE B8
LABORATORY WORM DATA SHKET
COE/ERLN FVP
STUDY FLAN: 4 INVESTIGATOR: PE8CH El. AL.
EXPERIMENT DESCRIPTION: SUSPENDED DATE OF TEST:
EXP. NUMBER:
83O316
1 (Table 3) SPECIES: NEANTHFS ARENACEODENTATA
** EXPERIMfcNFAL CONDITIONS »*
RANGE: 20.00 - 21. 3O
RANGE: 28. OO - 29. 00
TtKPERATURE: 21.00 DEGREES CENIIGRADE
SALINITY: 28. 00 PARTS PER THOUSAND
EXPOSURE DURATION: 1O DAYS
PHOTOPERIOD: 13 HOURS
FLOW RATE: 1OO KLS/MIN VOLUME ADDITIONS/DAY 172
UUM8ER OF ANIMAf.S/REPLICATE: 3O NUMBER OF REPLICATES/TREATMENT: 2
ANIMAL'S LIFE STAGE: JUVtNILF AGE: 41 DAYS
SIZE: 2. 1 +/- 0. 42 MO DRY WT RANGE:
CONTROLS: 23MG/L REF/REF
FOOD: PRAWN FLAKE DIRECTLY TO DISH
ANIMAL SOURCE: CULTURE
COLLECTION TEMPERATURE: DEGREES C COLLECTION 8AI INITY: PPT
ACCLIMATION:
SEDIMENT SOURCE: BARREL OR COLLECTION/JAR NUMBER
SOLID REFERENCE: 11/30 SOLID BRH: LL/23
SUSPENDED REFERENCE: 11/49.32 SUSPENDED BRH: LL/23.28
SAMPLE
NUMBER
4OO118
4OO119
400120
4O0121
I EXPOSURE CONC
1
J NOMINAL.
I29MC/L REF/REF
J
I29MG/L REF/REF
J
I29MO/L BRH/BRH
I
I29MO/L BRH/BRH
1
:ENTRATIONS
MEASURED
27. 7+0.9
27.7 + 0.9
24.9 + 1.5
24.9 + 1.5
OXYGEN 1 MEAN 1
ILOG(SCE/ 1
MG/L ICHROM+. 1)
-0.73
-0. 78
-0. 72
-0.80 .
MEAN
SCE/CHROMO
0. 11 ± 0
0. OS ± 0
O. 12 ± 0
O. 07 ± 0
.03*
.02
Standard error of the mean.
B-9
------- |