v>EPA
United States
Environmental Protection
Agency
Office Of Water
(WH-556)
EPA 823-R93-001
February 1993
Tiered Testing Issues
For Freshwater And
Marine Sediments
Proceedings Of A Workshop
Held In Washington, DC
September 16 -18,1992
Printed on Recycled Paper
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Proceedings
Tiered Testing Issues
for
Freshwater and Marine Sediments
Washington, D.C.
September 16-18, 1992
U.S. Environmental Protection Agency
Office of Water, Office of Science and Technology,
and Office of Research and Development
Washington, D.C.
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TABLE OF CONTENTS
Introduction .'....' ............>...
Workshop Summary .' ' ' ' ^
Day One ; -. :
Day Two v . . ..
Marine and Estuarine Sediment Testing Break-out Session . , ป
Freshwater Sediment Testing Break-Out Session 21
Day Three .
... 37
Workplans
Development of Sediment Toxicity Bioassay
for Marine and Estuarine Organisms
41
Development of Sediment Toxicity Bioassays
for Freshwater Organisms ..... ....... . ..... ....... ...... ......
Outlines of Speaker Presentations .................. ...... ........ .....
EPA's Contaminated Sediment Management Strategy 65
Elizabeth Southerland ....... ..... ....... ........ .......... 65
Standardized Sediment Testing:
Needs and Requirements of Key Agency Programs .
Thomas Armitage ... ..... . . . . . ......... ......... . . ...... 87
EPA Regional Sediment Needs ,
William Peltier .../:..... ...... ................ .......
Tiered Sediment Testing Conceptual Overview
Elizabeth Southerland ..... . . ...... ............. .........
Summary of ASTM Activities on Freshwater and Marine Sediment Test Methods
Chris Ingersoll ........... ..... ......... ..... ......
EPA Approaches for Biological Methods Standardization:
Historical Perspective and Present Guidance
Jim Lazorchak ........... ..... ..... .... ........ '. ' ' ' '
EMMC Methods Format . , 91 -a
William Telliard ...... ...... ............ ......... . ...... .4 ซo
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Freshwater Sediment Toxicity Assays: Necessary and Desirable Attributes
G. Allen Burton 227
Midge Whole Sediment Bioassays
John P. Giesy and Jody A. Kubitz 257
Desirable and Necessary Attributes for Freshwater Sediment Toxicity Tests:
Hvalella azteca
Chris Ingersoll 269
Discussion of Desirable and Necessary Attributes for Marine and Estuarine
Sediment Toxicity Tests, and the Use of Ampelisca abdita.
Rhepoxvnius abronius. Leptocheirus plumulosus. and Eohaustorius estuarius
in Marine and Estuarine Sediments
Richard C. Swartz \ ...... 281
Bioaccumulation of Sediment-Associated Contaminants:
Significance, Current Status, and Future
Peter Landrum , . . 295
Bioaccumulation of Sediment-Associated Contaminants:
Present Status, Laboratory Methods, and Related Research Needs
Henry Lee II 315
Discussion of the Use of Lumbriculus variegatus
in Freshwater Sediments
Gary Ankley '. -....-.' 347
Identification of Long Term Needs for Assessing Sediments
Norm Rubinstein ". 349
Break-Out Workgroup for Freshwater Sediment Issues: Overview of the Day
Gary Ankley . 357
Development of a Standard Protocol for Testing Hyalella azteca
Teresa Norberg-King 361
Development of a Standard Protocol for Chironomus tentans
Robert Hoke 387
Issues for Lumbriculus varieaatus1
Peter Landrum '. . 393
Development of a Standard Acute Amphipod Protocol
Richard C. Swartz, Michelle Redmond, Janet Lamberson
Beth McGee, Ray Alden, Richard Scroggins, Peter Chapman 403
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Development of a Standard Chronic Amphipod Protocol _
John Scott, Ted Dewitt, Michelle Redmond, Chris Schlekat ........ v; 439
'-' . . . . . 473
APPENDIX A: Workshop Agenda
APPENDIX B: Sediment Toxicity Tests Under Development by Environment Canada ... 483
...... 491
APPENDIX C: Freshwater Surveys ........
APPENDIX D: Bibliographies for Freshwater Test Species . . ','''
. . ........ 538
APPENDIX E: Workshop Participants
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introduction
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WORKSHOP SUMMARY
1
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Workshop Summary
The two and one half day workshop was designed to address general issues affecting both
marine and freshwater sediment testing on day one. Break-out sessions on day two focused on
specific details and requirements for freshwater and marine tests. Day three consisted of reports
back to the full workshop by break-out session leaders on issues discussed, conclusions, research
needs, and next steps followed by an overall summary and wrap-up.
Day One
During day one of the workshop, the following talks were presented:
Introduction and Description of EPA's Sediment Strategy
Elizabeth Southerland, U.S. EPA Office of Science and Technology
Dr. Southerland welcomed participants to the workshop and stated that EPA was
sponsoring this meeting to provide a forum for discussion of issues related to the standardization
of sediment bioassay test methods for cross-program use. She described EPA's Draft
Contaminated Sediment Management Strategy and noted that the development of consistent
assessment methods was one of the major goals of the Strategy. An outline of the draft strategy
was sent to more than 1000 representatives of industry, state, federal governments, and various
constituent group in March of 1992. Based on comments received from the mailing and from'
three national forums held during the spring and summer of 1992, a final strategy will be
developed and published in the Federal Register in 1993.
EPA Program Office Sediment Evaluation Needs
Thomas Armitage, U.S. EPA Office of Science and Technology
Dr. Armitage described the statutory authority and regulatory responsibility of EPA
Program Offices that could use the results of sediment toxicity tests. Based on differences hi
regulatory responsibilities, different programs may interpret test results differently. Interpretation
of individual test results may vary among programs, but standard bioassay protocols could be
used by all programs. . Once the method protocols have been developed, they may be used
immediately by Office of Water programs, the Superfund Contract Lab Program, and EPA's
Environmental Services Divisions. The Office of Pollution Prevention and Toxic Substances may
begin a test rule process leading to publication of test methods in the Federal Register, and the
Office of Pesticide Programs may begin their Science Advisory Panel review process. The
methods to be developed will also be submitted to ASTM to begin the balloting process leading
to completion of an ASTM standard method.
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EPA Regional Sediment Evaluation Needs
William Peltier, U.S. EPA Region IV, Environmental Services Division - Athens, GA
Mr. Peltier discussed the EPA Regions' sediment testing needs and reminded the
audience that the Regions are important clients for the methods under development. He
discussed the test methods that are currently available, and noted that there are significant
differences in test conditions among them. He emphasized that methods must be validated arid
must be run by Regional labs, state labs and consulting labs. He also noted that test conditions
such as feeding, age of organisms, sediment depth, and water renewal must be clearly specified
in the protocols. If pore water is to be tested, the pore extraction method must also be specified.
He also raised the issue of reference and control organism survival acceptability, and discussed
the possibility of using synthetic sediments for controls. Concerning species selection, he noted
that there should be criteria for using regional species instead of the species selected for
standardization, and that reference toxicant testing should be conducted. The question of
whether reference toxicant testing should be conducted each time a toxicity test is done, or
whether less frequent (weekly/monthly) testing would suffice was discussed. The importance
of a good QA/QC program was emphasized. This should include laboratory evaluation,
accreditation, and other checks on lab quality. Mr. Peltier also briefly discussed Regional needs
for bioaccumulation testing, especially data interpretation, and closed his presentation with the
reminder that Regional and state outreach was the key to success of this program.
Tiered Sediment Testing Conceptual Overview
Elizabeth Southerland, U.S. EPA Office of Science and Technology
Dr. Southerland presented an overview of tiered sediment testing. The Office of Water
(OW) the Office of Pesticide Programs (OPP), the Office of Pollution Prevention and Toxic
Substances (OPPTS), the Office of Solid Waste (OSW) and the Office of Emergency and
Remedial Response (OERR) are all committed to the principle of consistent tiered testing
outlined in the Agencywide Contaminated Sediment Strategy. Agencywide consistent testing is
desirable because all EPA programs would be able to agree on whether a sediment poses an
ecological or human health risk, and comparable data would be generated. It would also provide
the basis for uniform cross-program decision-making within EPA. Each program, however,
should retain the flexibility of deciding whether identified risks would trigger regulatory actions.
Tiered testing should include a hierarchy of tests with the tests in each successive tier becoming
progressively more rigorous, complex, and costly. Interpretative guidance must be developed
to explain how information generated within, each tier would trigger regulatory action. The
interpretative guidance could be program specific describing decisions based on a weight of
evidence approach, a pass/fail approach, or comparison to a reference depending on statutory
and regulatory requirements. There are currently two models of sediment tiered testing used by
EPA: 1) the Office of Water/US Army Corps of Engineers dredged material testing framework;
and, 2) the OPP ecological risk assessment tiered testing framework. Tier one of the dredged
material testing framework consists of a review of existing chemical and biological data and/or
an inventory of nearby sources. In tier two, chemical data are generated and compared to water
and sediment quality criteria. Tier three evaluation consists of acute toxicity and bioaccumulation
testing, and a comparison of the results to a reference area. A tier four evaluation consists of
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site-specific field studies. The OPP testing framework consists of acute toxicity testing in tier
one followed by chronic (early life stage) toxicity testing in tier two and further chronic toxicity
testing (full life cycle) in tier three. Tier four consists of field or mesocosm testing. A tiered
testing framework has not yet been chosen for Agency wide use, but some of the components that
have been identified will be standardized as a result of this workshop. These components ar?
acute and chronic toxicity bioassays, bioaccumulation tests, chemical criteria, and any others that
may have ecological significance including benthic community structure evaluation, colonization
rate, and in situ sediment testing within a mesocosm.
Summary of ASTM Activities
Chris Ingersoll, U.S. Fish and Wildlife Service, NFCR -Columbia, MO
Dr Ingersoll of the U.S. Fish and Wildlife Service presented a summary of ASTM
activities to standardize freshwater and marine sediment test methods. ASTM has not yet
developed any standard methods for sediment testing but has developed guides. The most recent
of thesTguides include: "Standard Guide for Collection, Storage Characterization aid
Manipulation of Sediment for Toxicologies! Testing." (ASTM 1991 Method E1391-90);
"Standard Guide for Conducting Static Acute Toxicity Tests Starting with Embryos of Four
Species of Saltwater Bivalve Molluscs." (ASTM 1991 Method E724-89); "Standard Guide for
Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians -(AbTM
1991 E 729-88)- "Standard Guide form Conducting Sediment Toxicity Tests with Marine,
Estuarine and Freshwater Invertebrates." (ASTM 1991 E-47); "Standard Guide ^Conducting
10-day Static Sediment Toxicity Tests with Marine and Estuarine Amphipods. (ASTM 19yi
Method E1367-90); and "Standard Guide for Conducting Sediment Toxicity Tests with
Freshwater Invertebrates." (ASTM 1991 Method E1383-90). Dr Ingersoll .discussed the
differences between ASTM guides and methods, and briefly described the guides listed above.
Approaches for Test Standardization;
Historical Perspective and Present Guidance . ;
Jim Lazorchak, U.S. EPA Environmental Monitoring and Systems Laboratory - Cincinnati, OH
William Telliard, U.S. EPA Office of Science and Technology
Dr Lazorchak described a 1987 ORD document, "Guidance on Methods Standardization"
which was never .finalized but may serve as a framework for the methods to be standardized as
a result of this workshop. There is no specific standardization process for biological methods
in the document, but the process developed for chemical testing may be applied to biological
testing methods development. As outlined in the document, method requirements and data
quality objectives must first be established. Method selection and development is men followed
by a single-laboratory evaluation involving a precision check and tests for sensitivity of method
variables. This is followed by confirmatory testing by a minimum of three labs. An mtenm
.
method description may then be prepared, although this has not yet been done for a^iological
test.. A formal collaborative or round robin testing procedure may then be conducted with a
.
minimum of six labs.
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Dr. TelJiard briefly described the activities of the EPA's Environmental Monitoring
Management Council (EMMC) whose charter is to: 1) coordinate the Agency's environmental
methods research and development activities; 2) foster consistency and simplicity in measurement
methodology across regulatory programs; 3) facilitate cooperative efforts with other federal
agencies, academia, industry, and other interested external parties on methods development; 4)
promote'and facilitate the adoption of new monitoring technology and instrumentation; and 5)
evaluate the feasibility of a national laboratory accreditation program. He described the
activities of the Methods Consolidation Workgroup. Their focus to date has been on methods
for water, solid waste, and air, although QA/QC and biological methods will be considered by
the group as well. A method validation process has not yet been developed, but a format for
EMMC approved methods has been finalized. /
Desirable and Necessary Attributes for Freshwater Sediment Toxicity Tests
G. Allen Burton, Wright State University - Dayton, OH
John Giesy, Michigan State University - East Lansing, MI
Chris Ingersott, U.S. Fish and Wildlife Service, NFCR - Columbia, MO
Dr. Burton noted that desirable attributes, for freshwater species and tests include:
species sensitivity; reproducibility of the test; discriminatory function of the test; and "do-
ability". Dr. Giesy remarked that compromises are often necessary when deciding which species
to standardize. Generally, a battery of tests with several species is required to explain most of
the variation in a test. Several disadvantages to the use of Chironomus tentans in sediment tests
were described: these insects are relatively insensitive for use in acute tbxicity tests (they are
especially tolerant to metals); they do not feed directly on sediments but eat resuspended
particles; genetic drift has been observed in lab-to-lab variation; there is unexplained sporadic
loss of vigor in culture; and there is the potential for loss (pupation or emergence) of adults.
Several other issues were identified: when to, start the test; test duration; endpoints; volume of
test sediments and water; food/culture medium. Similar questions about test conditions for
Hyalella azteca were posed. Many of the issues highlighted by these speakers, will be described
in the freshwater break-out session notes.
Desirable and Necessary Attributes for Marine and Estuarine
Sediment Toxicity Tests
Rick Swarfz, U.S. EPA Environmental Research Laboratory - Pacific Division ,
Several of the same attributes and issues identified for freshwater bioassays were
described for marine/estuarine tests. A major difference between marine and freshwater testing
is that many marine test species are field collected not cultured. Adult organisms are used! for
tests rather than the young cultured organisms used in freshwater testing. This difference aftects
feeding regime as well as the necessity for performing routine reference toxicant testing.
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Desirable and Necessary Attributes for Freshwater, Marine and Estuarine
Bioaccumulation Tests mn
Peter Landrum NOAA Great Lakes Environmental Research Laboratory - Ann Arbor, MI
Henry Lee II, U.S. EPA Environmental Research Laboratory - Pacific Division
The two required attributes of bioaccumulation test species are that they ingest sediment
--SMS?
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' residue levels, and the need for interpretative guidance on test results.
Attributes of Lumbnmte variegatus that make it a. good ^f f01*^
usiln long-term tests; standard culture and test methods have been developed; and some field
validation has been done.
Identification of Long Term Research Needs _
GaryAnkley, U.S. EPA Environmental Research Laboratory -Duluth, MN
No^n Rubimtein, U.S. EPA Environmental Research Laboratory - Narragansett, RI
Several long term research needs for sediment toxicity testing were ide ntifi ed. These
L Development Contaminated Sediment Research Strategy -is include in the end of the manne
break-out session summary.
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Day Two
Marine and Estuarine Sediment Testing Break-out Session
- ' . - > !
During this break-out session of the workshop, session leaders facilitated discussion of
issues to be resolved for marine and estuarine sediment testing.
Objectives of the Session - '" m'r''
Norm Rubinstein, U.S. EPA Environmental Research Laboratory - Narragansett, Rl
Dr Rubinstein identified three overall objectives for the workshop session dealing with
sediment tests for marine and estuarine organisms: 1) the workshop participants should agree
upon the definition of a standard method; 2) the workshop participants should reach^ agreement
onthe test species and protocols that can be standardized within the next year; and 3) workshop
participants should reach agreement on the process for standardizing protocols on sediment
spiking, handling, and storage.
Discussion of Marine and Estuarine Test Methods
Rick'Swartz, U.S. EPA Environmental Research Laboratory - Newport, OR
Standardization Process
Dr Swartz noted that, since exact guidance for standardizing test methods has not been
developed, the ASTM process for standardizing methods should be used to provide peer review
and more general participation in the process.
Identification of Uncertainties to be Addressed in Standardization Process ;
It was suggested that methods for sediment handling and manipulation should be
addressed separately from the toxicological methods .themselves. It was noted that Environment
Canada is evaluating sediment handling and manipulation as a separate issue, and will soon have
guidance available. There was general agreement that guidance is needed to determine how
sediment should be sampled and handled in the field, transported or shipped to the laboratory
and manipulated or otherwise treated in the laboratory. It was suggested tot handling;imd.
spiking could be addressed in guidance documents to accompany test method documents.; There
was also discussion about developing an additional standard method for experimental design that
could provide specific information on such issues as sampling replicates and selection, of held
reference sites.
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Dr. Swartz identified three categories of uncertainty that must be addressed in developing
guidance: 1) limitations of the method must be described (this is distinct from information
gaps); 2) issues that can be resolved on the basis of consensus should be identified (e.g. selection
of the temperature for conducting tests); and 3) critical research needs that must be met should
be identified (e.g. grain size tolerance). Workshop participants agreed to identify these issues
for the available test organisms.
Discussion of Rhepoxvnius dbronius
Rick Swartz, U.S. EPA Environmental Research Laboratory - Newport, OR
Dr. Swartz indicated that Rhepoxvnius has been well studied, there are 40-50 papers in
the literature, and there is already one interlaboratory comparison that has been completed. Dr.
Swartz noted that there are probably not many critical research needs to be rilled for
Rhepoxvnius tests. The following consensus issues were identified for Rhepoxynius:
1. A written protocol for the standard acute toxicity test is needed. Testing procedures
which must be followed should be identified.
2. Agreement must be reached on the data needed for reference toxicants. Dr. Swartz
already has data on cadmium.
3. Reference sediment quality assurance/quality control requirements must be identified.
Specifically, the question of the need to set a minimum desirable reference sediment
survival limit was raised. If one has intermediate levels of survival in reference sediment,
the difference between reference sediment and test site sediment, if detected, are difficult
to interpret, if not meaningless.
4. Data are available on the seasonal sensitivity of Rhepoxynius. It should be possible to
understand seasonal variability of this test species.
5. Consensus must be reached on how to ship and handle Rhepoxynius.
The need for evaluating relative sensitivity of Rhepoxynius was discussed. Reference
toxicants were discussed, and it was agreed that the method guidance developed for Rhepoxynius
should include information describing how to interpret sensitivity to reference toxicant tests.
The utility of reference toxicants spiked in sediment was addressed. Workshop participants
stated that this is a generic issue to be addressed in all of the method guidance documents to be
developed.
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Discussion of Ampelisca dbdita
Michelle Redmond, U.S. EPA Environmental Research Laboratory,- Newport* OR
Ms Redmond described the test species and the, acute toxicity test. Ampelisca abdjta
occurs in the high salinity range of estuaries. It is a particle feeder. Research to support the
Ampelisca test is needed in a number of areas:
. 1. Research is needed on grain size tolerance for this species.
2. No data are available to test the sensitivity of ovigerous females. Males are not used in
the test.
3. A critical research question is how to interpret control survival. Frequently problems are
encountered when laboratories run the test for the first time.
Other issues were discussed. It was noted that problems have been encountered when
Ampelisca is shipped to laboratories to run the test. The shipping process must be standardized.
Test sensitivity has also been observed related to season. It was noted that EPA's Environmental
Monitoring and Assessment Program has adopted a control survival limit of 90 percent for this
species For Hyalella a lower rate of 80 percent has been established. Salinity ranges for
AWlisca were also discussed. Some workshop participants recommended using full strength
seawater for the test. The species was not recommended for tests at salinities below five parts
per thousand Workshop participants agreed that a standard method should be written for a high
salinity range, and additional research can be conducted to broaden the range. There was some
discussion of ammonia tolerance, and it was agreed that recent research has established the
ammonia tolerance limits of Ampelisca.
ntsni.ssinn nf T^ptocheirus plumulosus
Beth McGee, Maryland Department of the Environment - Baltimore, MD
Ms McGee described tests using L^tocheirus. She noted that it has a wide tolerance
range for both salinity (2-30 parts per thousand) and grain size (sand to silt). It was agreed that
test temperature is a consensus issue, not a research issue for this species. The test is run at
temperatures between 20-25 degrees C. The salinity at which, tests are run should depend upon
the objectives of the test. When testing the toxicity of in-place pollutants, the test site salinity
may be used for the test. A photoperiod of 16:8 hours light/dark is used for the test. Juvenile
and young adults (sized 3-5 mm) are tested and 20 animals are used per test chamber. Both
field collected and cultured organisms are used for the test. Cadmium chlonde is used as a
reference toxicant for the test (96-hr LC-50).
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A number of research issues were identified for Leptocheirus:
1. What are the effects of salinity on test results?
2. Must the animals be acclimated if culture or collection site salinity is different from test
salinity? Tests suggest tolerance limits for sudden and extreme (i.e. 5 to 32 parts per
thousand) salinity changes.
3. Are different test results obtained due to differences in culturing methods, acclimation,
and salinity? ' .
4. More data are needed to determine the sensitivity of this species to different chemicals,
particularly ammonia and PAHs.
5. More interspecies comparisons are needed. Data from tests on this species could be
compared to marine species, and to freshwater species capable of tolerating low salinity
(i.e. Hyalella azteca).
6. More data are needed to identify sensitivity differences between laboratory and field
collected animals.
7. ' Field validation is needed.
It was noted that there are some advantages to laboratory culture of this test species. In
culture it is available at all times of the year, and cultures have been reared under known
conditions. The disadvantages associated with laboratory culture include: the deleterious effects
of inbreeding and the influence of culture conditions on test sensitivity. Advantages associated
with field collection include: testing a natural population; the availability of large numbers of
animals at low cost; the seasonal availability of size classes; accounting for the seasonal effects
on sensitivity; and accounting for geographic differences in sensitivity.
A number of positive attributes of Leptocheirus as a test animal were discussed: 1) the
animal is tolerant of a wide range of environmental conditions; 2) the animal has sensitivity
which is comparable to other amphipods; 3) the animal is hardy and tolerant of handling, and
it can be well maintained in the laboratory; and, 4) a partial life cycle test using Leptocheirus
is now being developed. It was noted that investigators should be careful not to release animals
in non-native regions. Workshop participants agreed that indigenous species should be used in,
testing when this is possible.
Note: An interlaboratory comparison has been conducted. Four laboratories participated in the
acute I- plumulosus portion of a broader comparison of test methods for Chesapeake Bay
methods.
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ous T __
vnentvl Research Moratov - Ne^on, OK
A o^to tn-riHtv test using Eohaustorius were described. The species
in the spring. The young "*** eis nojrowe m g g ^
AW used with this
species is cadmium chloride.
The following research issues were identified Eohaustorius:
1. Research is needed to de^^^
2. The response to cadmium has varied. Additional data are needed.
3. /Additional'research is needed on factors affecting the sensitivity to other reference
toxicants.
4. The toxicity test has not been subjected to interlaboratory comparison.
5 The toxicity test has not been subjected to rigorous field validation.
to'the use of the species is that it cannot be cultured. ,
***ซ
ซ
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to toxicity testing in the fall, it seems to be sensitive to organic toxicants, and is tolerant of a
wide range of salinities. Sediment particle tolerance may be a problem. Lower survival in fine
sediment has been noted. Subadults are used for testing with 20 animals placed in each exposures
chamber.
This animal appears to be a good candidate test species for acute testing but not for
chronic testing, since it is slow in growing and reproducing. The amphipod is tolerant of
ammonia but more research is needed to define its tolerance levels. A 16:8 light to dark
photoperiod is used in most testing with this species. This has been used to simulate summer
conditions. The Chesapeake Bay Program has been running tests with this species at a salinity
of 15 parts per thousand, reference toxicant testing is conducted at 20 parts per thousand. The
appropriate photoperiod for testing was discussed. It was noted that when the lights are turned
off animals come out of their tubes and may be exposed to more fresh sediment. The
availability of the species in the field was discussed. They are a dominant amphipod in North
Carolina and South Carolina, and are also readily available in the Chesapeake Bay.
Discussion of Canadian Experience -with Test Methods
Richard Scroggins, Environment Canada - Quebec, Canada
The Canadian experience with toxicity testing for that country's ocean dumping program
was discussed. Seven amphipods, all found in Canada, were evaluated in a series of round robin
tests. The testing resulted in similar responses at all laboratories for a number of species. It
was mentioned that Environment Canada will recommend that organisms for testing for Atlantic
and Pacific sites will be coast specific. The preferred animals for testing on the Pacific coast
appear to be Rhepoxynius or Eohaustorius. For tests on the Atlantic Coast, Leptocheirus pjnouis
was selected.
General Discussion of Acute Amphipod Tests
A number of issues were discussed by workshop participants:
I.
Experimental Design
Guidance on experimental design should be provided to support the test method guidance.
A separate document could be developed that would address experimental design and
interpretation of data. Workshop participants noted that it is difficult to separate effects
observed in the lab from those that may or may not be observed in the field. Richard Scroggins
recommended the use of five field replicates for conducting sediment tests. It was noted that
appendices could be prepared for the test methods by program offices indicating how their
regulatory needs could be met by the tests.
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II. Commercial Availability of the Species
*>,
Workshop participants discussed the importance of commercial availability of test species.
Ampelisca is available from a commercial supplier on both the east and west coasts. There are
also suppliers for Eohaustorius and Rhepoxvnius. Some workshop participants indicated that a
directory of suppliers for test animals would be very useful. .
m. Sediment Handling and Storage
It was agreed that the sediment handling and storage issue is very important:
Geochemists should assist in the development of guidance in this area. Sediment handling
jnvolves a number of uncertainties. The Canadian government is trying to build on the 1990
ASTM guidelines and has formed a subcommittee of geocherhists and field collection
lexicologists to refine the document. The issue of storage is also important. The EPA Regional
people at the session indicated that the commercial aspects of sample handling and storage are
important as well.
IV. Other Issues Related to Acute Tests
Some participants noted that a separate guidance document addressing experimental
design and providing guidance on sampling would be useful. It was stated that the Regions and
Program Offices should provide input to address this issue. A number of comments on acute
test method development were received from the Regions and Program Offices.
1.. Superfund Enforcement indicated that, generally, they do hot have problems using
ASTM methods, and supported pursuing ASTM approval of methods developed.
2. OPPTS indicated that their real interest,is in spiked sediment tests. '
3. Region 1 indicated that methods developed should be subjected to interiaboratory
validation.
4. Region 9 (represented by Brian Melzian currently at the Narragansett ERL)
indicated that the development of methods should be accompanied, by a
technology transfer effort. '.,_ .' .
5 Region 4 indicated that they are looking for standard methods that can be
, ' customized to meet a particular program need. The Region will probably use a
set of reference toxicants to evaluate methods.
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Discussion of Chronic Test Methods
John Scott, Sciences Applications International Corporation - Narrdgansett, RI
A number of advantages to the use of chronic tests were identified. The tests are more
sensitive than acute tests, and they may be more relevant to ecological processes. They may
provide a better estimation of population level effects. A number of technical issues were
identified that must be addressed to complete development of the chronic tests. These included:
1. Standardization of the age of species to be used.
2. Understanding species sensitivity in chronic tests.
3. Understanding nutritional requirements of test species.
4. Developing feeding protocols. ,
5. Understanding effects of sediment aging.
6. Selecting the proper endpoints and biological responses for testing.
Ted Dewitt described four species indigenous to Chesapeake Bay that were evaluated for
chronic tests. He indicated that Leptocheirus appears to be the most promising one for chronic
tests at this time. The species is sensitive, it reproduces easily, and it can be handled easily.
The animals can be tested in "dishpans" using static renewal conditions. A water bath is used
to keep the test temperature constant, and an algal (a flagellated chrysophyte) and dry food
mixture is used to maintain the animals. ,
It was noted that in developing chronic tests, a procedure for minimizing the release of
nonindigenous species should also be developed. One of the differences between a chronic test
and a 10 day acute is the requirement for feeding of animals in the longer test. A number of
endpoints for the chronic test were discussed. These include:
1. Mortality of adults.
2. Size of F0 generation.
3. Fertility (number of offspring/female survivor).
4. Timing of brood emergence.
\
5. Reburial timing.
6. Growth.
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An advantage of using ^BafitetaS in chronic tests is that the animal can survive a range of
sediment grain sizes and salinities.
m. ^MpVat of the Maryland Department of the Environment discussed the
laboratory.
is developed: ,
1. Appropriate test temperature. . .;'.'"-
2. Appropriate age (U,< 24 hours xs < 1 week old) of animal to be used for fte^.
3. Effect of source of animal on the test.
4. Effects of using cultured versus field collected animals for the tests.
5. Effects of the feeding regime used for the test.
6. Development of consistent QA/QC guideUnes (reference ;toxi^^
survival).
>7. interaction between1'nutrition and toxicological sensitivity.
8. Effects of other variables on the test (e.g. salinity and grain size),
9; Simplification of the memcdolo^
10. Comparison of ^relative sensitivity of endpoints between species.
17
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Michelle Redmond discussed the use of Ampelisca in chronic testing. This species has
a growth curve similar to Leptocheirus. A number of experiments have been conducted to
improve the reproduction of this animal in the laboratory. The effects of differences in
population density, aeration, and other variables has been investigated. Success in maintaining
the ^cultures has been variable. Experiments indicate that aeration is necessary to conduct the
sediment test, and that survival of juvenile offspring obtained from cultured females is greater.
It will be necessary to look at shipping and handling to determine if this is related to decreased
survival. The following observations were made based on test results:
1.
2.
3.
4.
5.
6.
Short term and chronic sublethal tests can be conducted using Ampelisca.
Known age animals can be released from the females.
Newly released or 10 day old animals can be used for the test.
Problems of low reproduction and poor survival were encountered, perhaps related to
shipping and handling.
A flow through system and changes in photoperiod may be necessary to conduct the test.
Nutrition may be a factor affecting test results.
Discussion on Bioaccumulation Testing
Henry Lee, U.S. EPA Environmental Research Laboratory - Newport, RI
The development of a standard 28 day bioaccumulation method for two species, Neries
and Macoma, was discussed. A draft guidance manual on bedded sediment bioaccumulation
tests has been produced by the EPA Newport Laboratory. Workshop participants agreed that,
given the current state of knowledge, these test protocols can now be written. Some additional
research must be completed. Longer term tests and kinetic models will also provide tools to
evaluate bioaccumulation. It was recommended that a series of round robin tests be complete
using Neries and Macoma to provide some indication of the precision to be obtained with the
tests. The round robin experiments will not be cheap because of the requirement for tissue
analysis. It would probably be necessary to use spiked sediment with a high Kow compound.
Region 9 has expressed interest in using'their own species for the bioaccumulation test.
Workshop participants noted that it will be necessary to develop guidance on interpretation of
bioaccumulation results. This will depend upon whether human health or ecological health is
of concern. Human health may be the most significant endpoint to be addressed.
18
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It was agreed that the protocol will be developed as follows:
I/ Protocol will be for a 28 day solid phase test.
2. Test will use a flow through seawater system.
3 Test protocol will describe test organism acclimation, maintenance, introduction, data
recording, removal, gut depuration, and sample preservation.
With this protocol, 80 percent of the steady state level will be reached There was some
Discussion of Office of Research and Development Contaminated Sediment
EPA EnrirovneMol Research Laboraory - Narraganse,,, RI
The EPA Office of Research and Development (ORD) *ntamina^ฑd"S^
ซmfซv vL discussed ORD has restructured the research planning process Contaminated
STon STe "^Hbrium Phoning approach for the development of sediment quahty,
criteria Research ^ill also be J^conducted on evaluation of ecological nsk.
19
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20
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Day Two
Freshwater Sediment Testing Break-Out Session
During this break-out session of the workshop, session leaders facilitated discussion of
issues to be resolved for freshwater sediment testing.
Objectives of the Sessions t' ซ. ^ i * ป
Gary Ankley, U.S. EPA Environmental Research Lab - Duluth, MN
As a preliminary step for the second day of the workshop, a questionnaire was sent out
to worklop'SLSd o*er selected researchers on July 13, 1992. The pnm*y ^formation
reauested was about culturing and testing for three freshwater species OL azteca, C ten^s,
STrofeSS T'Hie survey was done in order to assemble as much information a^ possibly
exampLSe culturing issues ranked for IL azteca were: known age cu tiire systems; feeding
rSmt;' w^r for coring; flow-through versus static V^-.^'S^K
control e.g., reproduction levels, reference toxicants); and genetic dnft/^^ej^s -
testing issues for Hvalella were: test length/endpoint; organism age to stort the tes t water
TeS ^frequency ofc method); interpreting the effect of sediment vanable on test r^
rg!orgSc carbon, particle size); feeding levels/appropriateness in ^^^^
Ssurkncl/quality control (criteria for acceptable tests). These were generally the same issues
for C.. tentans and I- vanggatui. ,-.
This session was designed to: (1) identify freshwater sediment toxicity tests as candidates
for stan^dStion Sn th! next year; and (2) to explore specific: technical issues associated
w^^rjpurposes of reaching a general consensus or for ซf^*
future research. Each issue identified during the session was discussed and, where
consensus was reached for development of standard protocols.
Development of a Standard Testing Protocol for mS^em
Teresa Norberg-King, U.S. EPA Environmental Research Lab, - Duluth, MN
Twenty one responses to the survey were received, and eighteen laboratories (see
AppendbTS reported formation on HyaOeUa aztecaf The summary of the survey responses
are as follows: .
I c.^^rYfarffwzfeZfaoaeai.-- The most common procedural response is underlined and
when no item is underlined it indicates no single most common response.
21
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A. Culture Methods
Flow:
Temperature:
Light:
Chamber:
Age animals:
Freq. restart:
Water Quality:
Source of Strains:
Aeration:
Feeding:
Substrate:
Reference
Toxicants:
Static vs. renewal
19 to 25ฐC (23!Cy
16:8 photoperiod; 50 to 100 ft. candles
!Lto40L
Known age vs. mixed age
Monthly, every 2 months
Natural vs. reconstituted
ERL-Duluth, ERL-Corvalis. Burlington, Michigan State (most
cultured in moderately hard water or hard water)
Moderate
Leaves. Tetraminฎ. rabbit chow, diatoms, yeast, wheat grass,
Chlorella, alfalfa, Nutrafinฎ, YCT. paper towels, Selenastrum,
Ankistrodesmus, brine shrimp, aquatic plants, sediment. Feed 2
to 3 times/week typical.
Leaves, nylon mesh, cotton gauze, 3-M web plastic, paper towels
Cd. Cu, KC1, Zn, NaCl, Cr (water-only exposures)
B. Testing Procedures
Flow:
Aeration:
Temperature:
Light:
Chamber:
Sed. ratio:
Age animals:
No. animals:
No. reps:
Duration:
Feeding:
Endpoints:
Acceptability:
Static vs. renewal. .
None or moderate
20 to 25ฐC (2Q1Q
16:8 photoperiod; 25 to 50 ft. candles"
30 mL to 1 L (250 to 300 mL)
1:1 to 1:4 ratio sediment:water
Known age (0 to 7 d, 7 to 14 d) vs. mixed age (size about 7
to 14 d) (sieved) '
5 to 20/beaker (10/beakert
2 to 10/treatment (3 to 5/treatmenfl
2-1028^(10^)
None, Rabbit Chow, YCT, maple leaves, Tetraminฎ
Survival, length, weight, sexual maturation (males), young
production, bioaccumulation
Survival (80%). length, weight
22
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Development of a Standard Testing Protocol
for Chironomus tentans
Robert Hoke> AScl - Duluth, MN
Twenty one responses to the survey were received, and twelve laboratories (see Appendix
D) reported information on Chironomus tentans. The summary of the survey responses are as
follows:
Summary for Chironomus tentans. The most common procedure is underlined and when
no item is underlined it indicates no suigle most common response.
A. Culture Methods
Flow: Static vs. renewal
Temperature: 19 to 25ฐC (2310
Light: 16:8 photoperiod; 50 to 120 ft. candles
Chamber: 1 L to 40 L
.Age animals: Known age vs. mixed age
Freq. restart: 2x week to every 6 months
Age restart org: egg cases to <24 h old larvae
'Water Quality: Natural vs. reconstituted
Aeration: Moderate
Feeding: Tetraminฎ. Nutrafinฎ, YCT and algae, alfalfa and Tetramin
Feed daily to 3x/week .
Substrate: paper towels (bleached or unbleached); sand
Reference
Toxicants: Cu, NaCl, Cd, KC1 (water-only exposures)
B. Testing Procedures
Flow: Static vs. renewal
Aeration: None or moderate
Temperature: 20 to 25ฐC (23ฐC) ;
Light: 16:8 photoperiod; 25 to 120 ft. candles
Chamber: 50 mL to 2 L
Sed. ratio: 1:1 to 111 ratio sediment:water
Age animals: Known age (0 to 16 d; 10 to 14 d)
No. animals: - 15 to 80/beaker (10 to 15/beaker)
No. reps: 2-15 (3 to 4) . .
Duration: 2 to 14-d (10-d)
Feeding:, trout chow, Tetrafinฎ, YCT
Endpoints: Survival, weight
Acceptability: Survival (70%). weight (dry weight)
23
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Development of a Standard Testing Protocol for
Lumbriculus variegatus
Peter Landrum, NOAA Great Lakes Environmental Research Laboratory - Ann Arbor, MI
Twenty one responses to the survey were received, and five laboratories (see Appendix
D) reported information on Lumbriculus variegatus. The summary of the survey responses are
as follows:
I. Summary for Lumbriculus varieeatus. The most common procedure is underlined and
when no item is underlined it indicates no single most common response.
A. Culture Methods
Flow:
Temperature:
Light:
Chamber:
Age animals:
Freq. restart:
Water Quality:
Aeration:
Feeding:
Substrate:
Reference
Toxicant:
Static vs. renewal
22to24ฐC
16:8 photoperiod; intensity unspecified
1 L to 40 L
mixed
Monthly, every 2 months
Natural vs. reconstituted
Moderate
Frozen silver cup trout chow, salmon starter, sediment, Tetraminฎ,
yeast, wheat grass, Chlorella, alfalfa, Nutrafinฎ, YCT, paper
towels food. Feed 2 to 3 times/week typical.
paper towels, sediment
no reference toxicants specified
B. Testing Procedure
Flow:
Aeration:
Temperature:
Light:
Sed. Ratio:
Age animals:
No. animals:
No. reps:
Duration:
Endpoints:
Feeding:
Acceptability:
Static vs. renewal
None or moderate
10to23ฐC
16:6 photoperiod
1:1 to 1:4 ratio sediment: water (sediment volumes should be,
adequate to allow feeding and .burrowing)
Adults, 3.8 cm.
Adequate number to provide tissue mass for analysis of
residue of concern
4 to 5/treatment
10 to 28 d
Bioaccumulation
None
Adequate tissue mass for residue analysis
24
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Workgroup Recommendations
I - i
Following the presentations of the culturing and testing survey results, there was an open
dialogue about the key issues. Generally for most of these issues, workgroup participants
offered suggestions about currently used versus preferred approaches. The workgroup arrived
at a consents on several culturing and testing specifics. Where it was not possible to make a
decision because of lack of information,, the group identified research items that need further
consideration before a specific decisions could be made. The workgroup's consensus and
research issues for the various species tests are summarized below.
In developing guidance for culturing freshwater species to be included in the EPA
methods manual for sediment tests, it was generally agreed that *ere^f/^tU^fofmt
that may be used to culture the three species. It was generally concluded that success of the
tests would rely heavily on the health of the culture from which the animals were taken for
testine That is having healthy animals of known quality and age for testing was deemed to be
Sfkey ^deration Relative to culture conditions. Therefore, a perforrnance-based cntena
approach was selected as the preferred method through which individual labฐrat^s^
evaluate their culture protocol rather than by a control-based cntena approach. This method
was chosen to allow each laboratory to optimize their own, perhaps umque, culture techniques
and meet certain quality control monitoring steps in cultures, while providing organisms that
would produce reliable, comparable test results.
Tfvalelia azteca:
Performance-based culturing criteria, for Hyalella azteca would .include:
1 Laboratories must perform .monthly water-lily reference toxicant tests to assess the
health of their culture organisms or the organisms they purchase from other laboratories.
The reference toxicant test should be performed as a 96 h water-only test. laboratories
should also evaluate the slope of their LC50 curve for each reference toxicant test
Results of these monthly tests then, would be entered into "control" charts; test results
greater than two standard deviations from the mean LC50 might indicate a cause for
concern that the cultures are unhealthy.
2 Laboratories should track the parental survival because it is important to know the
^ reproduction trends of reproduction for the cultures; it was, suggested that this; be
developed in a manner similar to that used as the control chart for reference toxicant
data. '
3 Laboratories should routinely measure and record the following culture water chemical
parameters: pH; D.O.; hardness; alkalinity; and ammonia. Again it was suggested_ tha
{Ssbe developed in a manner to that used as the control chart for reference toxicant
data.
25
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4. Laboratories should characterize and monitor the quality of the food they use in terms
of nutrient content and contamination.
5. Laboratories should keep records of their culture restart interval and keep accurate
records on the age of the brood animals and, as far as possible, track the source of test
animal to the age of the brood animals. Physiological parameters such as lipid content
also might be considered as a culture parameter to determine the health of the organisms.
6. Laboratories should develop standard operating procedures to ensure that their cultures
are renewed and monitored on a regular and standardized manner.
Given that the performance-based culture criteria will be part of the requirements for the
test performance, the major consensus items for the Hyalella azteca test protocol were as
follows:
1. The use of the performance-based culture criteria for cultures will be used to judge the
health of the animals in the toxicity test, along with the acceptability criteria. The test (
acceptability criteria were agreed upon as 80% control survival in a 10 d test as well as
acceptable water chemistry parameters over the course of the test.
2. " The renewal of the overlying water in the sediment test at a rate of 1.25 to 4
volumes/day was agreed upon. The specific procedures were not specified for the water
renewals; however, the intervals should be evenly spaced over 24 h.
3. The age of the animals for testing was 0 to 14 d. However, the workgroup recognized
that older animals (7 to 14 d) are easier to recover in the whole sediment compansd to
0-7 d old animals. This necessitates the need for known age culture systems.
4. The test length discussed ranged from 7-28 d, yet since the majority of the workgroup
participants were using 10-14 d for the survival endpoint, consensus was to use the 10
d test period. This test duration may allow the growth endpoint to be measured after 10
d, but more research "would be needed before this could be added as a criterion for test
acceptability.
5. The number of animals needed per concentration and the number of replicates needed was
discussed. The choice of each (replicates, number per concentration) will depend in part
on the probability level selected, and the type of statistical analysis. When variability
remains constant, the sensitivity of the test increases as the replicates increase. Further
evaluation of the appropriate alpha (a) (significance level) and beta (6) (power of the
test) for the Type I and Type n errors is needed along with consideration of the delta (5)
(minimum detectable difference). It was the group consensus'that this analysis could be
done with existing data.
26
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6 Although various size test chambers are used, it appeared that numerous researchers were
looking for smaller test volumes and test chambers and use of 250 to 300 ml beakers
with 50 to lop ml of sediment for 10 to 20 animals each was agreed upon. Larger
chambers have been used in the past but because space can be a limiting factor or
recovery of animals from larger volumes of sediment may be lower, it was the groups
consensus that smaller chambers would be acceptable. The minimum amount of sediment
needed to use in tests may need further consideration.
7 Feeding during the 10-d survival exposure was deemed necessary. Otherwise, acceptable
survival cannot be achieved wim R azteca. One food should be used for the standard
method. However, the decision of what the food should be and the quantity of it are yet
to be determined.
8. The test temperature was agreed to be 23 ฑ 2ฐC as most laboratories could accommodate
that now.
9 The analysis of abiotic factors that may affect test results is needed: this includes issues
such as the sediment grain size and/or organic carbon content which may affect organism
survival and/or growth. It was the group consensus that guidance on this issue could be
developed based upon existing data sets developed in IL azteca tests with a range of
clean .sediments. .
; - '-"./.'
The following research needs were identified for IL azteca.
1 Additional research is needed to develop approaches to produce known age animals for
testing. Alternatively, the manuals would state that if cultures were sieved to obtain test
organisms, the age of the animals retained by the sieve must have been previously
determined in that culture system. ,
2 Additional research is needed to evaluate the sensitivity of the various strains of IL
azteca to assess whether there is significant genetic drift depending on the waters that
animals were obtained from and/or the length of time they have been in monocultures.
3. Additional research is needed to develop the standard food for the tests that will provide
minimal organic carbon input while providing sufficient nutrition.
4 Additional research on the age of the test animals that can be used and still meet the
minimum required recovery is needed. Testing underway at Duluth may. help to decide
whether 0-7 d organisms are any different in sensitivity than the 7-14 d old organisms
which are easier to recover from whole sediment tests.
5 Additional research is needed on the abiotic factors that may affect test results (e.g.,
organic carbon, particle size). Data from Canada (Burlington), Duluth, the ARCS
program, and Mississippi should assist in making the decision.
6 Additional research is needed to determine the significance of various funding regimes
on the toxicity test. Feeding may alter the exposure through reduced uptake and
enhanced elimination of the contaminants.
v '27 "-. " : ,. .. . .
-------
nhirnnomv* tentans/riparius:
The workgroup consensus was that the a riparius could be included in the a tenitans.
protocols; these species are fairly similiar.
Most of the performance-based culture criteria listed for H, azteca are relevant to the
Chironomus spp. In addition to the chironomids, it is important that the following be considered
in fa& performance-based criteria:
1 Laboratories should measure and record the dry weight of at least one larval stage (e.g.,
. day 12) for the purpose of maintaining control charts for each culture. This is a similar
parameter to young production counts for 1L azteca.
2 Laboratories should record the time to first emergence for each culture and keep this data
' .in a control chart. Fluctuations in time to emergence often precede loss of vigor in
chironomid cultures.
3 Laboratories must perform monthly water-only reference toxicant tests to
health of their culture organisms or the organisms they purchase from other laboratories.
The reference toxicant test should be performed as a 96 h water-only test. Laboratories
should also evaluate the slope of their LC50 curve for each reference toxicant test.
' Result of thSe monthly tests then would be entered into "control" charts; test results
greater than two standard deviations from the mean LC50 might indicate a cause for
concern that the cultures are unhealthy.
4 Laboratories should routinely measure and record the following culture water chemical
parameters: pH; D.O. ; hardness; alkalinity; and ammonia. Again, it was suggested that
this be developed in manner similar to that used as the control chart for reference
toxicant data. . .
5. Laboratories should characterize and monitor the quality of the food they use in terms
of nutrient content and contamination.
6 Laboratories should keep records of their culture restart interval and keep accurate
records on the age of the brood animals and, as far as. possible, track the source of test
animals to the age of the brood animals.
7. Laboratories should develop standard operating procedures to ensure that
' are renewed and monitored on a regular and standardized manner.
parameters such as lipid content also might be considered as a culture parameter to
determine the health of the organisms.
28
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follows:
The age of the test animals should be: 8 to 12 d old far d tentans and 6-8 d old for
1.
. rioanus.
2.
course of the test.
af4?0 Hซ more research wouW be needed before.flus could be added as
'a criteria for test acceptability.
done with existing data.
6 Although various size test chambers are used ,it appearedI that numerous
would be acceptable.
"
to be determined. ,
8. The test temperature was agreed 'to be 23 ฑ 2ฐC as most laboratories could accommodate
that now.
29
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9. The analysis of abiotic factors that may affect test results is needed. This includes issues
such as the sediment grain size and/or organic carbon content which may affect organism
survival and/or growth. It was the group consensus that guidance on this issue could be
developed based upon existing data sets developed in C. tentans tests with a range of
clean sediments.
The following research needs were identified for Chironomus spp. toxicity tests as well as those
for the amphipod test above.
I. Additional research is needed on the abiotic factors that may affect test results (e.g.,
organic carbon, particle size). Data from Canada (Burlington), Duluth, the ARCS
program, and Mississippi should assist in making the decision.
2. Additional research is needed to develop approaches to produce known age animals for
testing. Alternatively, the manuals would state that if cultures were sieved to obtain test
organisms, the age of the animals retained by the sieve must have been previously
determined in that culture system.
3. Additional research is needed to develop the relative sensitivity data of midges and other
taxa.
4. , Additional research is needed to develop the standard food for the tests that will provide
minimal organic carbon input while providing sufficient nutrition.
5. Additional research on the age of the test animals that can be used and still meet the
minimum required recovery is needed.
30
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Tjgnhriculus vnrieeatus:
Many-of te performance-based criteria described above for H, งzteca are relevant to
variegatus. Additional culture considerations follow:
1
Laboratories must perform monthly water-only reference toxicant tests to assess the
^S to cultur^organisms or the organisms
preference toxicant test should be performed as a 9 h water-only test. *ซป*
.hould also evaluate the slope of their LC50 curve for each reference toxicant test
S* o the^tSy tes?then would be entered into "control" charts; test resuUs
gSte* San two standard deviations from the mean LC50 might indicate a cause for
concern that the cultures are unhealthy. '
2 Laboratories should monitor and record the frequency .wifc which the population is
' doubling. Again, this might be done using the control chart concept.
3 Physiological parameters such as lipid content also might be considered as a culture
parameter to determine the health of the organisms. ^
4 Laboratories should routinely measure and record the followin g-cultur e ^ter ch^cal
dieters- pH- D O.; hardness; alkalinity; and ammonia. Again it was suggested that
Ke devdopW in manner similar to that used as the control chart for reference
toxicant data.
5 Laboratories should characterize and monitor the quality of food i thq
nutrient content and contamination, particularly for the compounds to be evaluated for
bioaccumulation .
6. Laboratories should keep records of their culture restart interval.
7 Laboratories should develop standard operating procedures to ensure that their cultures
are renewed and monitored on a regular and standardized manner.
above:
1.
Given that ^performance-based culture criteria will be partof the requirements for Ae
hSTtest perfoVmice, the major consensus items for the test protoo* I ซ ซ ซ^
the vSous parameters of importance for the test method as discussed for H, azteca
For the time being, 28 d tests are recommended; however, ongoing research may indicate
SS shorter tests could be used. The use of the performance-based culture cntena for
SLres wUl be used to judge the health of the animals in the bioaccumulation test along
3 ttie acceptability criteria. The test acceptability criteria will include acceptable
water chemistry parameters over the course of the test.
31
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2. During tests, the organisms will not have any food added to the test chambers.
3. The desirable chamber size is more variable than for the previous two species and is
dependent upon the organism/sediment carbon ratios needed.
4. The number of organisms used per replicate will depend on obtaining an adequate tissue
mass for analysis of compounds of concern. Also, it was suggested that the ratio of
organism carbon/sediment organic carbon in the test systems should be on the order of
greater than 1/10 to 1/100.
5. The, renewal of the overlying water in the sediment test at a rate of 1.25 to 4
volumes/day was agreed upon. The specific procedures were not specified for the waiter
renewals; however, the intervals should be evenly spaced over 24 h.
6. The test temperature was agreed to be 23 ฑ 2ฐC as most laboratories could
accommodate that now. ,
7. The analysis of abiotic factors that may affect test results is needed. This includes issues
such as the sediment grain size and/or organic carbon content.
The following research needs were identified for L.. variegatus.
J. Additional research to determine the minimum and optimal time of exposure for
bioaccumulative compounds is needed. This would include tests that would evaluate
whether a shorter test (7-10 d) might be possible rather than the 28 d test.
2. Additional research to evaluate gut purging times in relation to elimination of
contaminants is needed. Evaluate the appropriate time to allow for gut purging and
whether this is contingent on chemical class.
5. Additional research to evaluate kinetics of the uptake/depuration for chemicals over a
wide range of Kow's.
fc
4. Additional research is needed to evaluate optimal organism carbon/sediment ratios for
tests.
5. Additional research is needed to determine the potential of "sediment avoidance" on
bioaccumulation of contaminants by L.. variegatus.
6. Additional research is needed on organism loading and doubling of populations during
kinetic studies.
32
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Dav Three
Conclusions and Next Steps
Dr Southland opened the session. She stated that, on the basis of discussion held at
this woShop EPA^ffl develop standard test protocols for acute toxicity and bioaccumulation
leading to completion of a standard method.
Dr Ankley described the results of the freshwater breakout session. He stated that the
bases for ^Dedb selection for standardization were: current and historical acceptance; logistical
%*%%ฃ** organisms are generally epibeathic and matin, testjฃP"ฃ^*g
o^re criteria for H azteca. Consensus from the breakout session was reached on what
criteria mTbe^orZde^d what criteria should be considered. Factors that mu^ be
cSerSlre: reference toxicants for short-term water ^.^^&^f^^^v
- describes the tasks to be performed in developing the final protocols.
33
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Dr. AnMey also summarized major issues discussed concerning the freshwater toxicity
tests. The following major issues related to the use of H. azteca were covered in the session:
1) Age of test animals.
i
There appears to be a range of ages most appropriate for testing. Organisms of age 0-14
days are most appropriate. It may be best to use animals of age 7-14 days, but difficulties in
recovering the animals may be encountered.
2) Length of test.
The length of the test agreed upon was 10 days with survival as the endpoint.
3) Feeding.
A minimal amount of food is necessary, but more data on feeding must be generated.
4) Water renewal.
Limited renewal of test water was recommended (1 volume/day).
i '
5) Sediment volumes.
Sediment volume in the 1/2 liter range was recommended but it seems acceptable to use
smaller test volumes.
6) Grain size.
There does not seem to be a grain size effect in the short term Hyalella test.
7) Strains of animals.
There are different strains of Hyalella used for testing. Reference toxicant comparisons
of the strains are needed.
In discussing the Chironomus test it was agreed that minimal feeding would be
appropriate (the acceptability of a change in total sediment organic carbon during the test of
.01% - 1.0% was discussed). Workshop participants did not believe that genetic diversity was
an important issue for Chironomus.
34
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In discussion of the Lum^a^ bioaccumulation test^a num
reached:
1) The protocol should recommend a 28 day test.
Ti No feeding is needed during the test. ..,
3 The ScL be conducted with water renewal or static conditions.
I
necessary.
Tpfkin^^^
are described in the breakout session notes.
35
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Dr. Lee summarized the discussion of the marine bioaccumulation tests. Several
approaches to bioaccumulation testing were discussed. These included: use of equilibrium
partitioning models; kinetic modeling; and the direct approach of testing field collected sediment.
The development of tissue residue criteria for the protection of human health was identified as
a research need. It was agreed in the breakout session that the existing 28 day protocols for
Series and Macoma would be standardized. Field validation and round robin testing will be
required to complete development of this protocol. Over a longer period of time, it will be
necessary to develop kinetic models. It will also be necessary to develop guidance on the
ecological significance of tissue residue levels.
Dr. Southerland concluded the meeting by thanking all the participants for their hard
work and reminded everyone that methods protocols would be completed for the species
discussed by each workgroup by the end of fiscal year 1993 (October 1, 1993). The following
fiscal year, if more funds become available, the focus will be on standard chronic test methods
and sediment toxicity identification evaluations.
36
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WORKPLANS
37
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38
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The following workplans describe research that will.be undertaken to develop sediment
toxicity test protocols for marine, estuarine, and freshwater organisms. Bunding has been
proved to complete work on test protocols for species included in the.workplans dunng fiscd
year 1993 Work to develop additional test protocols will be completed if additional funding is
available. The workplans were developed on the basis of discussions held at this workshop.
39
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40
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Development of Sediment Toxicity Bioassay for
Marine and Estuarine Organisms
Technical Approach
This workplan was developed on the basis of discussions held at an EPA Office of
Science and Technology (OST)/Office of Research and Development (ORD) sponsored workshop
on the standardization of sediment toxicity tests. Meetings were also held with ORD scientists
from the Environmental Research Laboratories in Narragansett, Rhode Island, and Newport,
Oregon, to discuss and identify the objectives of the proposed work. In addition, some
preliminary analyses of the statistical characteristics of acute sediment toxicity data were
conducted.
This workplan describes research to be conducted to standardize acute, chronic, and
bioaccumulation tests for sediment toxicity. The standardization of these methods is critical
because of the need to instill consistency in their application in Federal, Regional ,and State
sediment characterization programs. This consistency is of particular importance to the
implementation of EPA's Contaminated Sediment Management Strategy.; The strategy calls for
completion of a national inventory of contaminated sites and continued monitoring to assess the,
extent and severity of the problem. These actions will require a nationally consistent means for
determining sediment quality.
.The general approach to standardization of sediment bioassays is to start with reasonably
well-defined procedures, such as the ten-day sediment bioassay with the amphipod Rhepoxynius
abroriius. and proceed to more complicated, less well-defined procedures, for example, chronic
.tests and bioaccumulation tests. Field validation of test procedures completes the cycle of
standardization prior to implementation of the methods.
The research described in this work plan supports the development of standard methods
for acute and chronic bioassays and bioaccumulation tests. This work will concentrate on
.development to the ASTM "Standard Test Method" phase of me ten-day acute bioassay
procedure with four species of amphipods. Other activities will support the development of the
chronic bioassay and the bioaccumulation test to the ASTM "Standard Guide" phase. The latter
documents can benefit from the ASTM review process, without the rigorous scrutiny necessary
for standard methods.
Standard protocols for acute sediment toxicity tests using the benthic marine and estuarine
amphipods Ampelisca abdita. Rhepoxvnius abronius. Leptocheirus plumulosus, and Eohaustorius
estuarius will be prepared. The protocols will include details on: culture and/or acquisition of
test organisms; test design; QA/QC requirements; effects of abiotic factors; contaminant
interactions; reference sediment requirements; relative sensitivity; biological significance of acute
toxicity; and interpretive guidance. Additionally, protocols for the conduct of the 28-day
bioaccumulation test, and for the collection, handling, and spiking of test sediments will be
prepared. ,
The approach to completion of each research task is presented below. Research will be
. conducted at the SAIC, Inc. in Narragansett, RI, testing center in consultation with EPA's
Environmental Research Laboratories in Narragansett, RI, and Newport, OR, the EPA
Headquarters Office of Science and Technology, and EPA's Tiered Testing Workgroup.
''..'. .- - ^ 41 ,' -.''_'
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Specific Research Tasks and Methodological Approach
Research to Support the Acute Standard Method:
There are a number of technical areas where data are needed to support an understanding
of the responses of the four amphipod species to sediment contaminants. Some of the factors that
affect an organism's response to contaminants are temperature, salinity, and grain size. Other
sediment characteristics not related to contaminants, e.g., ammonia, sulfides, and low dissolved
oxygen, may cause toxicity thus confounding the interpretation of contaminant effects. This
workplan focuses on elaborating test species responses to salinity, ammonia, and grain size
where existing data are lacking. Temperature effects are not important since a standard species-
specific temperature within the upper tolerance range is normally chosen. Similarly, low
dissolved oxygen or sulfide toxicity will not be addressed because the test chambers are well
aerated during sediment exposures.
There also are no data on the relative sensitivity among these species to dissolved and
sediment-associated contaminants. Field validation data also are lacking for three of the four
species. In addition to the collection of supplementary data through the conduct of laboratory
experiments, existing data on these elements of the tests"will be gathered and synthesized.
" The experimental design addressing each of the issues discussed below will be prepzired
and agreed upon in consultation with technical experts from the ORD's Environmental Research
Laboratories and EPA's Office of Science and Technology.
Grain Size Tolerance:
, \
Most marine and estuarine organisms prefer a particular range of sediment type that may
be related to feeding or burrowing habits. In contaminant effects studies, sensitivity to particle
size is important since unusual stress associated with extremes of the particle size range may
induce mortality. The effect of fine particle size as an artifact causing non-contaminant induced
mortality in Rhepoxvnius abronius is well understood, and algorithms describing that relationship
have been developed. It is possible that the opposite effect, i.e., coarse particle size-induced
mortality, may occur in Ampelisca abdita. While contaminants are generally associated with fine
sediments, contaminated coarse sediments can be extremely toxic also. Experiments will be
conducted to identify the potential for grain size effects in both Ampelisca and Leptocheinjs.
The hypothesis to be tested is that extremes in sediment particle size do not cause
excessive mortality in the test organisms. To test this hypothesis, a range of particle size
treatments (up to five) will be established by mixing,- by volume, non-toxic coarse sediments
with silt/clay sediments. Natural sediments with different particle sizes may also be tested: I&ch
species will be exposed to these sediment mixtures using the existing ASTM procedure for the
conduct of 10-day solid phase bioassays.
42
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not exist at present. . . - -
Ammonia Tolerance:
,0 the other three test species in 10-day *ttc .exposures.
sets of expend wu
sediment-spiked ammonia.
^r^ "^^
ammonia concentrations will be determined.
Salinity Tolerance:
ugss^m and An^.^^^-^m^SfaSL Detest salinity
^faSpCs^U rKCSry SwUl be evaluafcd in Ught of existing info^on
on the distribution of these two' species relative to salinity.
This research will not address the potential interactive affects of sublethal salinity stress
and contaminant toxicity within the non-lethal salinity range.
43
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Relative Sensitivity:
Typically, only one of the four amphipod test species will be used in a solid phase
assessment of sediment toxicity. The species of choice would be selected for a number of
reasons including: availability; regional interest; and particle size compatibility. The
interpretation of a toxic response will require an understanding of the sensitivity of the test
organism relative to that of "benchmark" species for which a large data base exists. Large data
bases do exist for Rhepoxynius and Ampelisca. however, direct comparisons of the two species
are rare. .
Experiments will be conducted to test the hypothesis that there are no differences in
sensitivity of the four amphipod species. The relative sensitivity to two inorganic and two
organic compounds will be determined through two sets of experiments with the four species:
four-day water-only and 10-day spiked sediment bioassays. LCSOs for each species and exposure
type will be determined. The relative sensitivity to field: sediments of known contaminant
concentrations and toxicity will also be determined in round robin testing described below.
Further information will be gained by analysis of existing data on the relative sensitivity of these
species.
Field Validation:
The ultimate utility of these acute bioassay methods will depend on the extent to which
they predict contaminant effects in natural populations and communities. The toxic responsie in
Rhepoxynius has been related to the absence of amphipods and alterations in benthic community
structure.
A field validation of the acute response is necessary ,for Ampelisca. Eohaustorius. and
Leptocheirus. Data are currently available from the EMAP and NOAA Status and Trends isites
on the benthic community composition and-abundance of amphipods. Concurrent data are also
available on Ampelisca toxicity for many of .these sites. These data will be reviewed and
analyzed to determine if sufficient data exist to field validate the acute test for this species. The
criterion for validation will be the co-occurrence of toxicity with decreased amphipod abundance
and/or significant benthic community effects.
Analysis of these data will identify sites that may be used to validate the test for
Leptocheirus. Additional samples, not to exceed 20, will be collected in cooperation with these
programs for toxicity tests with this species. Similarly, if the data on Ampelisca are insufficient,
this species will also be tested.
44
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Testing Summary:
ฐ?n tmDelisca - 10-day sediment - 5 treatment levels - 2
o .uSSSU-lMiy sediment -5 treatment levels -2 runs
- 10-day spiked sediment - 5 treatment levels
- 10-day spiked sediment - 5 treatment levels
^^ฃ%^-,*
- 10-day spiked sediment - 5 treatment levels
Field Validation '"..,.
AmEeUsca - 10-day - 20 sediments
Le^tocheirus - 10-day , 20 sediments
o
At least 5 replicates will be used per run.
-1-'
Research to Support the Chronic Standard Method:
Research wiU be initiated to dfel^
j^j^ and Anodto .MJJf^S ^vt'nlnS ResSrcrilboratory in Newport
development and validation by the E^e^nm^.h condubted to develop acute, test
Oregon (ERL-Newport) and others. Some of ftc ^,^A.g EnvirOnmental Research
methods wDl apply to the ^%^^^gisett) is also supporting research on
Laboratory in Narragansett, ^^^SSh with tbeฃ two species will be
chronic sediment bioassays ^^^^ S for the ERL-Newport and Narragansett
defined at a later date when ^.c^m^^s^ith up to five treatments each will be
^rtr*^^^^
of S work at & two EPA E8L laboratones.
45
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Since the ability to culture .test species will be a requirement of chronic methods,
additional research will address the culture requirements of Ampelisca. Short-term experiments
will investigate the effects of culture container size and configuration, and feeding procedures
and frequency on the size and fecundity of laboratory populations.
Research to Support the Bioaccumulation Standard Method:
The accuracy of the marine sediment bioaccumulation test is poorly quantified, and the
extent to which it predicts tissue residues under field conditions is unknown. The accuracy, as.
defined by a field validation of the laboratory results, is critical to the future development and
application of this test. A key to the laboratory to field comparison is the achievement of steady
state conditions in the laboratory exposures. Also, ideal circumstances would dictate that the site
chosen be inhabited by the same organisms being used in the bioaccumulation test.
Research described in this workplan will provide an initial field validation of the
bioaccumulation method using Macoma nasuta and Neries virens. This research entails a
minimum of a 60-day laboratory exposure to a naturally contaminated sediment with
measurements of tissue residues in each species at a minimum of two time intervals (28 and 60
days). These values will be compared to those measured in field collected organisms, preferably
the same species, inhabiting the same sediments.
Up to 50 tissue residue and eight sediment residue measurements will be required for this
research. Chemical analyses will target high and low molecular weight PAHs, metals, and
chlorinated compounds.
Criteria for the selection of an appropriate site include the presence of the test organisms,
or an adequate sediment-ingesting surrogate, and concentrations of target contaminants
sufficiently high to be bioaccumulated, but not high enough to cause mortality in test organisms.
Only one site validation will be conducted due to the limited availability of resources.
Therefore, the site will be carefully selected after consultation with ORD and OST experts. The
same consultation will be required to finalize the experimental design.
',''*
Statistical Analysis of Acute Bioassay Data:
The currently accepted criterion for the assignment of toxicity to a sediment sample relies
on the detection of a statistically significant decrease in survival in a test sediment relative to that
in a control (or reference) sediment. This research will provide a rigorous analysis of acute test
data for the four species to determine a level of statistical significance that relies on the
variability of the test performance among and between test runs. The goal of these analyses is
to describe procedures for examining the statistical nature of acute toxicity data bases relative
to variations in control mortality, test precision, and the ability to determine minimum detectable
differences. Preliminary analyses of selected Ampelisca data indicate that minimum detectable
differences may range from 20 to 30%. Ultimately, these statistical differences can be compared
to differences in mortality that are considered biologically significant.
46
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need, if any, for data transformation.
Acute Round Robin Tests:
-
K^sra
conducted to provide direct comparison with the other two species.
Four laboratories will be evaluated in this task. Up to four naturally contaminated and
Preparation of Draft Standard Methods:
Draft standard methods will be prepared for acute sediment, bioassays witii the four
The ASTM format will be followed for this and all other methods developed ^nderthis
workpla? ifm^foZt, the documents will be submitted for review and approval under the
ASTM Subcommittee E47.03 sediment toxicology balloting system.
A draft euide will be prepared for chronic sediment bioassays using T^ptocheirus. This
included in the draft guides.
Two draft guides for the 28-day bioaccumulation test with Macoma and Neries. will be
be baled on the existing EPA method for the former spec.es.
t/1. WL/u-L v*X* * i*"*-- - . ^
That'EPA method also is a draft ASTM guide.
- 47
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Additional standard methods for sediment collection, handling, and spiking will be
prepared in the ASTM format. The sediment spiking procedure will follow those developed by
the ERL-Newport laboratory for the sediment quality criteria program. The sediment collection
and handling document will be based on an existing guide prepared by ASTM. This document
will be revised in conjunction with ongoing efforts to develop standard methods for freshwater
sediment bioassays. Specifically, a committee of experts will be formed to come to consensus
on key issues. This group will meet at the fall 1992 and spring 1993 ASTM meetings to discuss
the proposed revisions to the existing document.
48
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Schedule
Acute Testing:
Complete ammonia tolerance tests' with^he^xynius abronius, Le^tocheirus plumulosus, and
Fohaustorius estuarius - data report (Jan/93)
Complete grain size tolerance tests with Ampdisca abdita and L^tocheirus r4umulosus - data
report (Jan/93)
Complete salinity tolerance tests - data report (Jan/93)
Complete comparative sensitivity tests with spiked sediments - data report (Mar/93)
Complete field validation data analysis and testing - data report (Sept/93)
Chronic Testing:
Complete development of culture methods for Ampelisca - draft protocol (Sept/93)
Bioaccumulation Tests:
Complete laboratory testing and chemical analysis for field validation - data report (Sept/93)
Statistical Analysis Procedures:
Completion of statistical analyses for mirdmum detectable difference for each of the fourspecies
- data report (Jun/93)
Acute Protocol Round Robin:
Completion of acute test round robin - data report (July/93)
49
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Document Preparation:
Draft Standard Method for Rhepoxynius abronius Acute Test (Apr/93)
Draft Standard Method for Ampelisca abdita Acute Test (Sept/93)
Draft Standard Method for Leptocheirus plumulosus Acute Test (Sept/93)
Draft Standard Method for Eohaustorius estuarius Acute Test (Sept/93)
Draft Guide for Macoma nasuta Bioaccumulation Test (Sept/93)
Draft Guide for Neries virens Bioaccumulation Test (Sept/93)
Draft Guide for Leptocheirus plumulosus Chronic Test (Sept/93)
Draft Standard Method for Sediment Spiking (Apr/93)
Draft Standard Method' for Sediment Collection, Handling, and Storage (Sept/93)
50
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Development of Sediment Toxicity Bioassays
for Freshwater Organisms
TM-hnical Approach
This workplan was <^op^
workshop on standardization of sediment cmaty ^^^^ ^ |nvironmental
contaminants in sediments to aquatic ?f"*ฃ^*SS Si areas where water quality
toxicology. Mounting evidence exists of ^^^^^^^^-^b^l^l^
criteria are not exceeded, yet orgamsmsare *^*^ J^ent ^ost assessments of
placed on evaluating ซPซ*^' f**^ considered a safe repository of
:Sn^
considering the fate of chemicals in sediment.
A variety of methods have "^
procedures range in complexity frฐt^ฐ^
Contaminants on single species "^S^iiictato whole sediment (often
Ideany, a sediment test should ^
study is to screen a large number of^ ซm a^y man ^^^ ^^^^
contaminated sediment can^be atxop^^^^S^ with the ability of an animal
of chemicals in sediments that are not fJ^STo^S chronic effects and long-term
to develop grow ซ''*^ J^SSS^^
bioaccumulation of chemicals from sed ^""f1 ^ coiiminants. Most estimations of
and to understand the enyironmen^ *&*%ฃ baled on T- to^-d exposures with midges,
. chronic sediment contaminant ^ effects have ^ ^^ .^ u^cyde exposures may not
from responses to natural sediment characteristics.
51
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Direct comparisons of animals exposed in the laboratory and in the field are required to
verity results from laboratory testing procedures. The assumption that laboratory results for a
specific sediment represent effects of similar sediments in the field needs to be evaluated.
Hazard evaluation of contaminated field sediments that integrate data from laboratory exposures,
chemical analyses, and in situ field assessments provide strong complementary evidence of the
degree of pollution-induced degradation to aquatic and benthic communities.
The goal of this research project is to develop state-of-the art, standardized protocols for
assessing the potential effects of contaminated sediments on aquatic ecosystems. These
laboratory tests are an essential component to the tiered testing approach currently being
developed by EPA. The general strategy behind the research is to start with the standardisation
of reasonably well-defined test procedures (10-d acute toxicity tests with benthic invertebrates),
proceeding to less well-defined protocols (bioaccumulation tests, food chain models, chronic
toxicity tests, toxicity identification evaluation), and ultimately culminating in field validation
of the tests. Because many contaminants of concern in sediments bioaccumulate, this research
will emphasize development and validation of toxicity and bioaccumulation tests to residue-effect
endpoints based on tissue concentrations. Part of this effort will involve developing toxicokinetic
and metabolism models for species exposed to different classes of representative sediment
contaminants. This information is needed in order to develop realistic models for predicting
exposure of organisms through the food chain, and will also provide a technical basis for
assessing the use of risk based residue-effect models.
The following objectives (elements) and associated timelines are based on the assumption
that support for this research effort will be available at comparable levels for 3 to 5 years.
Available FY92 and FY93 funding will support only research activities dealing with Objectives
1, 2, and 3. Research will be conducted at the U.S. Fish and Wildlife Service National
Fisheries Contaminant Research Center in Columbia, Missouri (NFCR-C) and EPA's
Environmental Research Laboratory in Duluth, Minnesota (ERL-Duluth) in consultation with an
established workgroup of experts on freshwater sediment toxicity testing, EPA's Office of
Science and Technology, and an EPA Tiered Testing Workgroup.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Specific Research Objectives
Standard Protocol for an Acute Toxicity Test with Hyalella azteca (FY93)
Standard Protocol for an Acute Toxicity Test with Chironomus tentans (FY93)
Standard Protocol for a Bioaccumulation Test with Lumbriculus yariegatus (FY931)
Toxicity Identification Evaluation (TIE) Procedures for Contaminated Sediments (FY94)
Standard Protocol for a Chronic Toxicitv Test with Hvalella azteca rFY94)
Standard Protocol for a Chronic Toxicity Test with Chironomus tentans (FY95)
Develop a Generalized Model for-Predicting the Metabolism of Common Sediment-
Associated Contaminants in Benthos and Fish (FY95)
Develop an Effects-Based Tissue Residue Model for Assessing the Risk of Sediment-
Associated Contaminants (FY96)
Summarize Field Validation Studies for Standardized Toxicity and Bioaccumulation Tests
for Freshwater Sediments (FY96) .
52
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Fvperimentfll Design anf* Methodological Approiach
Investigations of sediment toxicity and bioaccumulation are limited by -k lack : .of
understanding of the factors controlling contaminant availability in sediment. Additionally, a
"act Turitide stendardized methods also Umits the use of sediment tests m ^ntammation
assessments. ASTM Subcommittee E47. 03 on Sediment Toxicology 'has *^&ฃ*ฃ
assessing the bioavailability of contaminants associated with sediments (e.g. , ASTM E 1383 92
^sSd Guide for Conducting Sediment Toxicity Tests witfi Freshwater f* ฃ
These guides are used to evaluate the toxicological hazard of contaminated sediment soil
sSe Sng fluids, and similar materials. The Subcommittee developed general guides and
no? sSndarftest methods or protocols, because most procedures for *M.coott^
sSinSnt have only been Decently developed. Definitive protocols are needed which describe
specific test methods. .
Objective 1: Standard Protocol for an Acute Toxicity Test with Hyjdella gzteca (FY93)
i ' ^. ' ,
' The protocol will include details for culturing and testing the amphipod, including test
svstem design Also covered "will be the development of a standard reference sediment,
Sures for'reference toxicants, procedures for interpreting the effects of abiotic factors (e.g
narticfe sSe)^ or ^results, use of cell lines for screening complex hydrophobic compounds and
deteS me^tency of these .compounds to aquatic organisms, the results of a relative
Lalys^Cfrom a series of single chemical tests) for K azteca, evaluation of genetic
Moratory cultures of the amphipod, and results of preliminary round-robm studxes.
, Hvalella azteca will be cultured to produce known-age or known-size animals. MFC^-C
of cukures wffl be compared using known-age or mixed-age methods and various diets.
A standard control sediment will be developed for use in determining
and
f\ dlxUlUAiU vWil U.WA tjw^*."*"-'*" ^ J. . . . *
nf the test and will facilitate inter-laboratory comparisons. A particle size
con* nuSoTof total organic carbon will be selected to be representative^ freshwater
SeSsNTCR-C will evaluate KC1 and ERL-Duluth wiU evaluate CuSO4 for use as a
reference toxicant.
NFCR-C wiU establish a culture of the ERL-Duluth strain of HyjMla a^a. Relative
sensitiv^of tMs^n will be compared to the NFCR-C strain. Taxonomy of both strains will
be confirmed by an identified expert.
53
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Preliminary round robin studies will be conducted by 8 laboratories using water only exposures
(Phase 1) and whole sediment exposures (Phase 2):
a.
b.
c.
Phase 1: Water only, 4-d exposure, 3 species (e.g., common strain), 1 reference
toxicant (KC1).
Phase 2: Whole sediment, 10-d toxicity exposures, 3 species (e.g., common
strain), 2 sediments (medium and high toxicity) + control. Versions of ERL-
Duluth methods would be used.
Timeline: .
1. Identify methods and laboratories for testing
2. Phase 1 testing: October, 1992
3. Phase 2 testing: February, 1993
Objective 2: Standard Protocol for an Acute Toxicity Test with Chironomus tentans (FYS>3)
The protocol will include details for culturing and testing ฃL tentans. including test system
design. Also covered will be the development of, a standard reference sediment, procedures for
.reference toxicants, procedures for interpreting the effects of abiotic factors (e.g., particle size) on test
results, use of cell lines for screening complex hydrophobic compounds and determining the potency
of these compounds to aquatic organisms, the results of a relative sensitivity analysis (from a series of
single chemical tests) for C. tentans. and results of preliminary round robin studies. Preliminary round
robin studies will be conducted by 8 laboratories using water only exposures (Phase 1) and whole
sediment exposures (Phase 2). , ,
Objective 3: Standard Protocol for a Bioaccumulation Test with Lumbriculus variegatus (FY93)
The oligochaete Lumbriculus variegatus is the most promising benthic test species available for
a standardized freshwater bioaccumulation test. Research will be focused on field validation of
bioaccumulation tests with Lumbriculus. as well as analysis of the kinetics of bioaccumulation of
different classes of chemicals of concern. Lumbriculus variegatus will be cultured at NFCR-C. ERL-
Duluth will supply their SOPs and animals. NFCR-C will determine performance of the cultures using
reference toxicants and estimates of population dynamics.
54
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Specific Research Tasks to Develop Acute Tnyicitv and Bioaccumulation Test Protocols
Specific research tasks that must be completed to develop the acute toxicity and
bioaccumulation test protocols are identified below. Each area of research is identified with a
group or groups having primary responsibility for conducting the work: (Columbia = NFCR-C,
WSU = Wright State University, Athens = NFCR-C Athens Georgia Field Research Station).
An area of research designated "round robin" will be evaluated by laboratories participating in
the round robin tests. An "*" indicates areas of research that will not be completely resolved
bv the end of fiscal year 1993. Best professional judgment will be used in some instances to
make decisions regarding areas of research indicated with an ''*V An area of research
designated with an "@" was identified as a high priority research need at EPA s test method
standardization workshop. NFCR-C will be responsible for writing the final document
describing the standard protocols. Chemical analyses will be done by the U.S. Fish and Wildlife
Service Patuxent Analytical Control Facility. This laboratory will meet QA/QC requirements
and anticipate a 30 to 90 day turn around time for samples.
1. CULTURING
a.1 amphipods: known-age vs. mixed cultures .
1. evaluate sensitivity to suite of compounds with different modes of action
(Columbia, Athens
2. consistency in size of organisms between known-age and mixed cultures
(Columbia, WSU, @)
ป-.' '
b. use of reconstituted water (*)
c. diet(*)
d. performance criteria (WSU, *, @)
2, WHOLE SEDIMENT TESTING
a. temperature: 20 to 25 ปC (*)
b. static renewal 1-4 volume additions/day (*)
c. methods for static renewal: evaluate water quality with various static renewal
exposure systems (All) ,
d chamber size: 30 mL to 1 L (typically 250 to 300 mL); volume of sediment: 100
mL (minimum for 300 mL chamber); sediment to water ratio between 1:1 and
, 1:4 (round robin, @)
' .-.'- .- 55 - - . . -;...'.
-------
e. known-age animals:
1. Hyalella azteca: 0 to 14 d old
a. evaluate using reference toxicants (All, @)
b. behavior in sediment by life stage (WSU, Columbia)
\
2. Chironomus tentans: 10 d old (*)
a. evaluate using reference toxicants (All, @)
3. . Lumbriculus yariegatus: adults (*)
a. evaluate using reference toxicants (All, @)
number of animals/chamber: minimum 10 (*)
number of replicates (chambers): power analyses are needed to determine desired
number of replicates (Athens, @)
h. feeding (All, round robin, *, @):
1. Hyalella azteca: 6 mg rabbit pellets/MWF/20 animals (Columbia); 0.8 mg
YTC/d (EPA); methods will be compared
2. Chironomus tentans: 4 mg Tetramin/d/10 animals
3. Lumbriculus variegatus: no feeding for bioaccumulation testing, 20 mg
trout starter every 5 d/10 animals in toxicity testing
i. water quality: evaluate use of reconstituted water (*) t
j. photoperiod: 16:8, minimum 25 foot candles (*)
k. endpoints: survival, growth (round robin, @) ,
1. test acceptability criterion (round robin, @).
f.
g-
56 '
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3. WATER-ONLY TESTING
a Reference toxicants: Water only, 4-d exposures, performed monthly Columbia
wm Sate KC1, WSU will evaluate CdCl2, and ERL-Duluth will evaluate
CuSO4
b. Use of reconstituted water (All, @)
c. Use of phenol or another non-ionic organic as a reference toxicant for water-only
or sediment testing (*) ' ,
4. STANDARD CONTROL SEDIMENT (@, Columbia, Athens)
5. PROCEDURES FOR EOTERPRETTNG EFFECTS OF ABIOTIC FACTORS
(Duluth, Columbia, WSU, .<ง>)
Reconstituted sediment will be used to evaluate particle size and organic carbon. . ERL-
ShT^veSS databases and regression equations with 50 sediment samples for
2 "dTEW.Dulซth is developing manuscripts dealing wi* interpreting the effects
of ^Sa. WSU is evaluating the influence of low dissolved oxygen.
6. RELATIVE SENSHTVITY ANALYSIS (Duluth, @)
ERL-Duluth is developing a database for 12 chemicals with all 3 species using 10-d .flow- ,
through water-only exposures. . '
7. GENETIC VARIABILITY OF Hyajella azteca (AU, @)
miumbia and WSU have started cultures of the ERL-Duluth strain of HyaMla aztecl.
"^ofsซ*ns to a suite of compounds with different modes of action will
be evaluated.
8. PRELIMINARY ROUND-ROBIN STUDIES (WSU, @)
a. Toxicity testing: 8 laboratories
^ 1 Phase 1: Water only, 4-d exposures,
next spring), KC1 reference toxicant. October
2 Phase 2: Whole sediment, 10-d toxicity exposures, 2 s^iments + control.
Versions of ERL-Duluth methods used. February 1993.
57
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9. EXPERIMENTAL DESIGN FOR FIELD VALIDATION OF BIOACCTJMULATION
TESTS WITH LTJMBRICULTJS
a. Objectives
1. Kinetics of bioaccumulation of different classes of chemicals of concern
with Lumbriculus
2. Field validation of laboratory bioaccumulation exposures with
Lumbriculus
b. Experimental design
i \ -
1. Field-collected sediments (2 sediments)
(Select sediments with broad, range of K^ compounds (up to K^ log 7-8);
organic carbon/lipid normalize.
Suggested location for sediment collection:
a. Little Scioto River in Ohio: high PAHs and metals, possibly PCBs
b. Huntsville Alabama: DDT and metabolites
2. Field-collected oligochaetes (5 samples/sediment)
3. Laboratory-exposed oligochaetes (56-day exposure sample over time)
c. Field collection '
1. Field-collected oligochaetes ,
5 samples x 2 sediments = 10 oligochaete samples
2. Field-collected sediment
A. 2 sediments: Little Scioto and 1 Huntsville
B. Collect multiple grabs of sediment (about 4 L/grab). Homogenize
and split into two subsamples-. one 2-L subsample for sediment
chemistry and a second 2-L subsample for field-collected
oligochaetes. Sieve oligochaetes in the field. Repeat 4 L grab
sampling until enough biomass and sediment is collected. Ship
oligochaetes and sediments to Columbia. No depuration of field-
collected oligochaetes (comparison to 28-D sample w/o
1 elimination).
58
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d. Chemistry samples (metals and organics sampled from the same replicate)
1. Chemical-specific analyses:
A. Little Scioto River: PAHs, metals, possibly PCBs
B. Huntsville: DDT and metabolites
2. Sediment
2 replicates/sampling period
! x 3 sample periods (day 0, 28, 56)
= 6 samples/sediment
,3. Field-collected oligochaetes '
5 replicates/sediment
4. Laboratory-exposed oligochaetes
A. Uptake: day 0, 2, 4, 7, 14, 28., 56
1. 3 replicates/sampling period .
x 6 sampling periods (days 0, 2, 4, 7, 14, 5t)
' ' , =18 samples/sediment
2. 5 replicates/sampling period
x 1 sampling period (day 28) ;
'.= 5 samples/sediment ,
/ .
> 3 Total: 23 oligochaete samples/each sediinent
B. Elimination: hour 12, 24, 48, 72, and day 7
1. 2 replicates/sampling period ,
x 5 sampling periods
x 2 treatments (with or without sediment)
= 20 samples/sediment
59
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Additional Research Proposed for FY94-96
Objective 4: Toxicity Identification Evaluation (TEE) Procedures for Contaminated
Sediments (FY94)
A draft document describing preliminary methods for sediment TEE has already been
completed; further resources will enable completion of research focused upon issues such as pore
water preparation, isolation and fractionation of high log K non-ionic organics, species
selection, TDB on whole sediments, and field validation.
Objective 5: Standard Protocol for a Chronic Toxicity Test with Hvalella azteca (FY94)
In addition to the research issues identified under Objective 1, a key factor to be
addressed in the development of a chronic test would focus on appropriate toxicity endpoints.
In addition, the length of the test will be evaluated; it may be, for example, that little additional
information is gained in long-term tests as compared to short-term tests.
Objective 6: Standard Protocol for a Chronic Toxicity Test with Chironomus tentans
(FY95)
See Objective 5.
Objective 7: Develop a Generalized Model for Predicting the Metabolism of Common
Sediment-Associated Contaminants in Benthos and Fish (FY95)
Residue-based risk assessments for hydrophobic sediment-associated contaminants are
highly dependent on accurate prediction and measurements of bioaccumulation. For super-
hydrophobic chemicals, bioaccumulation potential can be significantly overestimated by models,
which assume no metabolism, or by empirical exposure that are of insufficient length to account
for the kinetics of biotransformation. Procedures will be developed whereby computer-assisted
predictions of metabolic rates can be used to refine bioaccumulation estimates and identify
associated uncertainties.
Objective 8: Develop an Effects-Based Tissue Residue Model for Assessing the Risk of
Sediment-Associated Contaminants (FY96)
Current standard guides must be modified to provide a residue-effect based risk
assessment approach for very hydrophobic chemicals. Studies must be longer in length Jind
incorporate both dietary and water column routes of exposure to adequately quantify
bioaccumulation. Toxicity endpoints must also include effects on reproduction and the
probability of these effects must be related to residue levels. Research will be directed to
provide improved test protocols and predictive models for assessing risk.
60
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SrhPdnle for rnmpletion of Pmiliicts in Fiscal Year 1993
The final product of workifunded in FY93 will be standard protocols for acute^oxicity
The ASTM format will be followed for all methods developed under this ^; Jฐ
this fomat, ^d^ents>m be submitted for review and approval under the ASTM
Subcommittee E47.03 sediment toxicology balloting system.
61
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62
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OUTLINES
OF
SPEAKER PRESENTATIONS
63
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64
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EPAys Contaminated Sediment Management Strategy
Elizabeth Southerland, U. S: EPA Office of Science and Technology
In the 1980s EPA documented the extent and severity of contaminated sediment problems
at sites throughout the U.S. Concerned with the mounting evidence of ecological and human
health effects, EPA's Office of Water organized a Sediment Steering Committee chaired by the
Assistant Administrator of Water and composed of senior managers in all the EPA offices with
authority to handle contaminated sediments and EPA's ten Regional offices'.:
Over the past two years this committee has beenpreparing an Agencywide Contaminated
Sediment Management Strategy to coordinate and focus EPA's resources on contaminated
sediment problems. A draft outline of this strategy was released to the public this year to serve
as a proposal for discussion in three national forums scheduled for April, May, and June. The
draft strategy is designed around three major principles:
1. In-place sediment should be protected from contamination to ensure that the
beneficial uses of the nation's surface waters are maintained for future
generations;
2. Protection of in-place sediment should be achieved through pollution prevention
and source controls;
3. Natural recovery is the preferred remedial technique. In-place sediment
remediation, will be limited to high risk sites where natural recovery will not
occur in an acceptable time period and where the cleanup process will not cause
greater problems than leaving the site alone.
The draft strategy includes several component strategies: assessment, prevention,
remediation, dredged material management, research, and outreach. A brief summary of each
of these elements follows. -
In,the assessment strategy EPA is committing to develop a national inventory of
contaminated sediment sites and a pilot inventory of potential sources of sediment contamination,
based on existing data. The two types of inventories will be complementary because the source
database can be used to predict where sediments are contaminated in unsampled areas. The
inventories will be designed so that EPA's prevention and remediation programs can use them
to focus their resources on cleaning up the top priority sites and sources. Another key element
of the assessment strategy is the commitment to develop a consistent; tiered testing strategy that
will include a minimum set of sediment chemical criteria, bioassays, and bioaccumulation tests
that all programs will agree to use in determining if sediment are contaminated.
The prevention strategy includes a variety of pollution prevention measures and source
controls. The scale of contamination will guide the choice of a particular set of these measures.
If a sediment contaminant is causing harm or risk at numerous sites nationwide, it may be
65* - '' ' ' '.- '..'..,':/.
-------
relatively inefficient to deal with the problem on a site-by-site basis. Instead,-the strategy
discusses nationally applicable responses, such as prohibitions or use restrictions under TSCA
or EOFRA, technology-based effluent limitations for industrial dischargers, or a national initiative
to revise water-quality based limits in NPDES permits. If atmospheric deposition appears to be
a primary source of contamination, responses under the Clean Air Act will be considered.
Where sediment contamination is a concern at particular sites, but not on a national scale, case-
by-case assessments and response actions are recommended. Based on narrative and chemical-
specific criteria and standards, EPA or a State can develop NPDES permit limits for discharges
from'industrial sources, municipal sewage treatment plants, stormwater outfalls, and combined
sewer overflows. States that have ndnpoint source control programs can take actions to reduce
the contributions of these sources to sediment contamination.
EPA may, remediate sediments under CERCLA, RCRA, CWA, and TSCA. The
remediation programs will use the national inventory to assist in selecting sites for cleanup and
the consistent tiered testing to assist in identifying contaminated areas and establishing cleanup
goals. The remediation strategy emphasizes that sources of contamination should be controlled
prior to remediation efforts unless the contaminated sediments pose a sufficiently great
environmental hazard. In making remediation decisions, the strategy also points out that it is
important to consider whether contaminated sediments at a site can be transported to downstream
or offshore areas if left in place, thereby increasing the size of the contaminated area and making
future remediation efforts much more difficult. Other factors to consider include the timefraiwe
for natural recovery, the potential for contaminant mobilization during remediation, and the
feasibility and cost of various treatment and removal options.
The maintenance of our nation's waterways for navigation requires the dredging and
disposal of 250 to 450 million cubic yards of material each year. Dredged material testing
manuals prepared jointly by EPA and the Corps of Engineers recommend the chemical and
biological tests that should be conducted to determine if the material is contaminated and must
be disposed of using special procedures. The tests selected for the Agencywide contaminated
sediment strategy will be included in these dredged material testing manuals. The strategy also
outlines additional guidance that will be developed by EPA and the Corps to improve the
management of these materials. . , >
The research strategy outlines all the work that EPA's Office of Research and
Development (ORD) has planned on sediment chemical criteria, sediment bioassay and
bioaccumulation test, fate and transport models, and remedial techniques. ORD is establishing
a Resource Center to provide EPA offices with centralized technical assistance in evaluating
sediment contamination and will also sponsor workshops and training sessions throughout the
country.
The outreach strategy describes how EPA will work with other Federal agencies and State
agencies to coordinate EPA's contaminated sediment activities with their efforts. EPA will
strive to ensure that these agencies share sediment related research findings and innovative
technologies. In addition, EPA is proposing a two-way public awareness program that will
disseminate contaminated sediment information to the public and also incorporate information
from the public into EPA activities. ' '
66
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Standardized Sediment Testing:
Needs and Requirements of Key Agency Programs
Thomas Armitage, U.S. EPA Office of Science and Technology
I. Office of Wetlands, Oceans, and Watersheds
A. Oceans arid Coastal Protection Division
1. Regulatory Responsibilities:
a Determine the acceptability of materials, including dredged
materials, for ocean dumping under the Ocean Dumping
Regulations.
B. Statutory Authority
1. Marine Protection, Research, and Sanctuaries Act (MPRS A);
2. Ocean Dumping Regulations (40 CFR 227 and 228) ,
C. Major Needs and Requirements for Standard Sediment Testing Methods:
1. Primary interest lies in testing dredged material to determine possible
effects of disposal. . .
2 Methods for the following tests should be standardized: chemical
analytical testing, toxicity bioassays, and bioaccumulation.
3 A sufficient number of test organism taxa must be standardized in order
. to provide an adequate range of test species and be applicable to a wide,
range of sites and contaminants.
it is recommended that standardized organisms to be used in acute
toxicity tests be from the taxonomic order Amphipoda, and include
at lea;t th~ ปrฃ*ซ Ampelisca abdita and RhPT>oxvnms abromus.
a
b Standard organisms for bioaccumulation tests should include a
' number of species from the following taxonomic groups:
polychaetes (particularly Neanthes sp, Neries sp, and Nepjhys sp),
moUuscs (particularly Macoma sp), and crustaceans. Because of
problems with availability and relative sensitivities, we nee
-------
5. Standard methods for use in marine sediment chemistry analysis have been
developed for tiie Clean Water Act 301(h) program (monitoring ocean
outfalls of sewage treatment plants), and are the methods specified for use
in testing sediment for ocean disposal. The Oceans and Coastal Protection
Division suggests that these methods be adopted for marine sediments
unless other methods can be standardized and used routinely.
D. Existing Program Guidance and Tests Used to Assess Sediment Contamination:
1. The testing manual, "Evaluation of Dredged Material Proposed for Ocean
Disposal" outlines procedures for water column and benthic effects tests.
a. The testing manual lists suggested and recommended species to be
used in specified tests. The decision concerning which species are
required for tests currently rests with the EPA Regions and Corps
of Engineers Districts, which must consider local factors in
determining species appropriateness. This is done through the
development of local testing manuals.
b. Guidance for performing tests includes discussion of: species
selection, apparatus, experimental conditions, sample preparation,
test design, QA/QC, and data presentation and interpretation.
c. Water column tests
i. Elutriate chemical concentrations are ' used to assess
compliance with water quality criteria; the water quality
criteria are considered the "Limiting Permissible
Concentration" not to be exceeded after consideration of
initial mixing. . .
ii. Elutriate bioassays are static 96 hour LC50 studies using
three concentrations of elutriate; 1% of the LC50
concentration is considered the "Limiting Concentration"
not to be exceeded after consideration of initial mixing.
iii. The regulations require at least one species from
phytoplankton or zooplankton, crustacean or mollusc, and
fish. A range of test species are suggested, and a subset is
recommended, in the testing manual for water column
bioassays. However, decisions concerning the choice of
test organisms currently rest with local decision makers.
-------
d. Whole Sediment Bioassays
i whole sediment bioassays, both acute 10 day bioassays and
10 or 28 day bioaccumulation, are described in the testing
manual. . ' .
u For both acute 10 day bioassays and 10 or 28 day
'' bioaccumulation, the dredged material results are compared
to reference site sediment results. This is done to ensure
that no unreasonable degradation will occur because of
disposal. ...-'.
iii
'
The regulations require that test species comprise filter-
feeding, deposit-feeding, and burrowing organisms. A
range of test species are suggested, and a subset is
recommended, in the testing manual, for whole sediment
bioassays. However, the decision concerning choice of test
organisms currently rests with local decision makers.
'' For acute toxicity bioassays, infaunal amphipods,
burrowing polychaetes, molluscs, and crustaceans
, . are suggested.
. For bioaccumulation tests, polychaetes, molluscs,
and crustaceans are suggested.
2 301(h) monitoring program guidance documents include a number of
volumes that may be relevant to contaminated sediment testing:
a. 301(h) toxic effects of sewage discharge, on coral 'reef
i communities. ,
b Summary of U.S. EPA approved methods to demonstrate
" compliance with applicable water quality standards.
c. QA/QC for 301(h) monitoring program. Guidance on field and lab
methods. -"
d Bioaccumulation series (5 volumes). Addresses target species,
detection limits, analytical methods, and sample replication.
Wetlands Division
A. Regulatory Responsibilities:
1 Develop guidelines to evaluate proposed discharges of dredged or fill
material into waters of the United States.
89
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B. Statutory arid Regulatory Authority:
1. Clean Water Act, Section 404
2. Section 404(b) (1) guidelines (40 CFR 230)
C. Major Needs and Requirements for Standard Sediment Testing Methods:
1. Interest lies in testing dredged material to determine possible effects of
discharge.
2. The program would like a tiered testing approach to evaluation of dredged
material proposed for discharge. Needs and requirements are similar to
the ocean disposal program. However, freshwater and estuarine species
should also be selected for test development.
i - ' !
D. Existing Program Guidance and Tests Used to Assess Sediment Contamination:
1. Currently there is no broadly applied testing manual for dredged material
evaluation. Evaluations are handled on a case-by-case basis using 404 (b)
(1) Guidelines. However, EPA and the Corps of Engineers are in the
process of developing the Inland Testing Manual to evaluate proposed
discharges of dredged material into waters inside the baseline of the
Territorial Sea. The manual will be completed in 1993. It incorporates
a tiered testing strategy.
2. The 404 (b) (1) Guidelines provide a general framework under which
testing is to be performed. The general framework includes: evaluation
of existing information, chemical and biological testing (chemical,
characterization of material, elutriate testing, and benthic effects testing),
and evaluation of physical effects of disposal.
3. Regional guidance for testing dredged material has been developed by:
Regions 1, 5, 9, and 10.
a. Region 1 protocols have been developed for: selection of sampling
sites; physical and bulk chemical analysis of sediments; tiered
evaluation testing for liquid phase assay, suspended paniculate
assay, whole sediment assay, and bioaccumulation analysis;
- elutriate testing procedures; and QA/QC measures.
b. Region 9 has developed guidance similar to Region 1 guidance.
90
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c Region 5 has produced guidance for sampling and testing efforts
related to navigational dredging, and also uses the IJC Guidelines
and Register for Evaluation of Great Lakes Dredging Projects.
d. Region 10 has developed the Puget Sound Dredged Disposal
Analysis, which includes a tiered battery of tests.
e Other Regions apply variations of the ocean disposal testing
manual to dredged material assessment programs in waters of the
United States.
4 It should be noted that standardized testing in the ocean dumping, 404,
'and other programs must address the interfering effects of sediment grain
size, ammonia, and hydrogen sulfide. These are important issues for
standardization.
Office of Pollution Prevention and Toxic Substances
A. Regulatory Responsibilities:
1 Assessment of risks resulting from possible releases of existing and new
chemicals that are manufactured, distributed, or disposed.
2. Decisions to regulate the use of new and existing chemicals.
B. Statutory Authority: .
1. Toxic Substances Control Act, Section 4, 5, 6, 8
C. Major Needs and Requirements for Standard Sediment Testing Methods:
1 OPPT is interested in fate, transport and effects of potential sediment
contaminants. Spiked sediment testing is the kind of standardized test that
would be of the greatest usd in these evaluations.
2 The PCB program is also interested in developing toxicity assays for use
' 'with sediments taken from sites contaminated with PCBs. If a disposal
method such as bioremediation is used to remove PCBs, toxicity tests are
necessary to ensure that PCBs have been destroyed, and that intermediates
more toxic than the original PCBs have not been formed.
91
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3. Since spiked sediment tests would be of greatest use to the program,
standardization of the test protocol elements described below -would be
most useful.
4. Sediment testing for one species is described in the program guidelines.
Standardization of methods for additional species would offer a greater
range of testing options.
D. Existing Program Guidance and Tests Used to Assess Sediment Contamination:
1. TSCA Guidelines used to develop data on the toxicity and bioavailability
of chemical substances and mixtures.
2. Section 795.135 of the Guidelines describes the chironomid Sediment
Toxicity Test,
3. Chironomid sediment test guideline describes three tests:
a. 14-day chironpmid aqueous exposure test with minimal sediments,
foods, and test substance added to the water.
b. 14-day exposure with test substance added to the sediment.
c. 14-day interstitial exposure with the test substance addecl to the
water.
4. The guidelines include the following key protocol elements:
a. Conducting range finding tests;
b. Conducting definitive tests (number of test organisms, specification
for controls, test duration, endpoints, water quality measurements);
c. Measurement of test substance;
d. Test conditions and selection of organisms
i. Acquisition, feeding, loading, care and handling,
accEmation, facilities;
e. Test substance delivery system;
f. Dilution water;
92
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g. Cleaning test system;
h, Sediments used for test, determination of contaminant partitioning and
. bioconcentration;
i. Additional test parameters and measurements;
' j. Reporting test results.
IV. Office of Pesticide Programs
A. Regulatory Responsibilities: ,
1 Review me uses of new and existing chemicals to be registered as pesticides
in order to determine effects on nontarget organisms.
' ' 2 Make decisions to: 1) label pesticides in order to control or restrict their use;
ratioofne^^
or
ban the use of existing pesticides.
B. Statutory Authority: . ,
1. Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)
C. Major Needs and Requirements for Standard Sediment Testing Methods:
1 The tiesticide program is primarily interested in spiked sediment toxicity tests
anlS?^
S^S of Chemicals would affect exposed species. Standardization of
spiking methods would therefore be very useful.
9 The program requires standardized tests to discriminate among exposure
SLaysTorS to determine the bioavailability of contaminants resulting
from different levels and methods of pesticide application.
3 OPP is also interested in evaluating contaminated sediment in the field in
order to trace runoff of pesticides applied in agricultural practices. .
4. Currently, no standardized sediment toxicity tests have been developed by the
program for evaluation of pesticides. '.-"'
5 The program would like to see standardization of marine and fr^terte^
for boA the acute and chronic studies. Representative species of appropriate
Snsiti^ty from a range of sites would have to be available for testing. Acute
SSSS testing currentiy requires an invertebrate, -da warm water ^d
probably be similar to those mentioned above.
93
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6. Mesocosm tests have also been developed by OPP for dose-response
studies of pesticides.
7. A major concern of the program is that standardized sediment studies
would have to be legally defensible if Agency decisions were challenged
by the agricultural or chemical industries.
8. A major issue for the program in sediment testing is the kind of sediment
to use in spiked testing, how the sediment should be handled, and how the
contaminant should be introduced to the test system.
9. The pesticide testing guidelines and standard evaluation procedures
developed by OPP provide the level of detail that would be required in
standardization. This kind of guidance is generally of the same level of
detail as an ASTM standard method.
10. The level of detail in a standardized test that is likely to be most useful to
the pesticide program would be that of the ASTM standard practice. A
standard guide may not offer enough structure, and a standard method
may not be flexible enough.
D. Existing Program Guidance and Tests Used to Assess Sediment Contamination:
1. The pesticide program currently has no existing guidance for sediment
testing. No standard tests or species have been developed. The program
has authority, to ask for such a study under special test requirements, but
generally has not required sediment bioassays. If they are required, the
pesticide registrant is asked to submit a protocol for evaluation by the
program.
2. The Office of Pesticide Programs Standard Evaluation Procedure for
Ecological Risk Assessment describes how the results of contaminated
sediment studies would be used to complete a risk assessment.
V. Office of Emergency and Remedial Response (Superfund)
A. Regulatory Responsibilities:
1. Cleanup of hazardous waste sites to protect human health, welfare, and
the environment.
2. Sediment assessment methods are identified and applied both for site
assessments and remedy selection.
94
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3 Once contaminants are identified, existing state and federal standards are
evaluated for applicability to the site. When standards are not available,
other evaluation methods are used to determine if the environment is
endangered and to select cleanup goals.
B. Statutory Authority:
1 Comprehensive Environmental Response, Compensation, and Liability Act
' (CERCLA), as amended by the Superfund Amendments and
Reauthorizatiori Act (SARA).
2 CERCLA mandates that sampling be conducted to characterize the release
of hazardous substances from a site and to determine if these releases
present a threat to human health, welfare, and the environment.
C. Major Needs and Requirements for Standard Sediment Testing Methods:
1 The program is looking for a battery of test methods that have been peer
reviewed and validated. These would be used with standard chemical
analytical methods. A list of standardized biological methods that could
be included in the Superfund Contract Laboratory Program would be very
useful.
i
2 The Superfund Program handles a wide variety of sites, requiring a wide
spectrum of testing options to meet site specific goals. More emphasis
; and resources are being placed on ecological assessment, and the program
will be doing more quantitative assessment. Any standard methods that
can be of use in these assessments will be important to the program.
3. The following standardized tests would be useful for the program:
a. " Chemical Testing - .
' , i. Sediment analysis to determine bioavailability of
contaminants.
, ii: Total Organic Carbon (TOC).
iii. Residue Analysis
b. Biological Testing
i. Bioaccumulation Tests
ii. Solid Phase Toxicity Tests
95
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D. Existing Program Guidance and Tests Used to Assess Sediment Contamination:
1. Superfund RI/FS Guidance and the Environmental Evaluation Manual
provide general guidance on methodologies used to determine the nature
and extent of sediment contamination at sites. Ecological Assessment of
Hazardous Waste Sites. EPA/600/3-89/013.
2. The Data Quality Objectives Guidance and Compendium outline a detailed
description of the investigation process. A Quality Action Plan (QAP) is
developed on each site, and may be carried out by the EPA established
contract laboratory program (CLP) or a non CLP laboratory that meets the
data quality objectives of the investigation.
3. Currently, the Superfund Program draws upon several sources for testing
methods. These include, but are not limited to ASTM, OECD, the U.S.
Army Corps of Engineers, EPA Methods for Measuring Acute and
Chronic Toxicity of Effluent Waters, and the Canadian ministry of the
Environment. A list of those available for sediment tests is attached. The
ASTM sediment methods are standard guides.
4. Region 4 Standard Operating Procedures for Toxicity Testing/Hazardous
Waste Assessments. April 15, 1990. Written by Todd Harris, Jay
Glover, Jim Maudsley, with foreword by Bill Peltier. U.S. EPA Region
4 Environmental Services Division and NSI Technology Services Corp. '
VI. Office of Solid Waste
A. Regulatory Responsibilities:
1. Under the Resource Conservation and Recovery Act (RCRA),
contaminated sediments exhibiting RCRA toxicity must be managed in a
RCRA permitted or interim status facility.
2. Authority to enforce cleanup of contaminated sediments is used under two
. conditions: 1) if the sediment is dredged and exhibits a hazardous waste
characteristic under RCRA, or if the sediment is mixed with a RCRA
listed hazardous waste; and 2) if the sediment contamination can be shown
to have resulted from a release form a specified solid waste management
unit at a RCRA permitted or interim status hazardous waste facility.
3. Contaminated sediments may be toxic under the toxicity characteristic
leaching procedure test, a test that compares the concentrations of various
chemicals in the leachate .from the dredged materials with levels
established to protect the environment.
96
-------
1.
B. Statutory Authority: ,
1. Resource Conservation and Recovery Act (RCRA), Sections 3004 (u) and
(v), and Section 7003.
C. Major Needs and Requirements for Standard Sediment Testing Methods:
If new sediment quality assessment methods receive adequate scientific
review, OSW would incorporate them in making RGRA permit decisions,
and in evaluation of remedial site restoration plans.
2 The program would be interested in tests that can link toxicity to a source
'of contamination. Toxicity identification evaluation would be useful.
D. Existing Program Guidance and Tests Used to Assess Sediment Contamination:
1 The program relies on the Superfund guidance described above No
' standard program approaches to evaluating or testing sediment
contamination have been developed. Testing decisions are made on a
case-by-case basis. The program would be interested in a range of testing
options that could provide the appropriate choice for a particular site.
Vri. Office of Wastewater Enforcement and Compliance
A. ' Regulatory Responsibilities:
1 NPDES permits issued under the Clean Water Act can be written to
' protect against sediment contamination. Sediment testing and monitoring
can be required as a condition of a discharge permit.
2 At present, water quality based effluent limits protect against sediment
contamination only to the extent that such contamination would cause
violations of water quality criteria.
B. .. Statutory Authority:
1. Clean Water Act, Sections 101(4) (3), 301 (b) (1) (C), 402, and 304 (1).
C. Major Needs and Requirements for Standard Sediment Testing Methods:
1. Acute and chronic sediment bioassays and bioaccumulation studies could
be used in the NPDES permitting process.
97
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D. Existing Program Guidance and Tests Used to Assess Sediment Contamination:
1. Sediment quality criteria are being developed to be used in NPDES
permitting. . - ' '
2. The guidance document, "Assessment and Control of Bioconcentratable
Contaminants in Surface Waters," March 1991, describes methods for
sampling and measuring bioconcentratable chemicals in sediment and biota
at point source discharge sites.
98
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PROGRAM OFFICES
RESPONSIBLE FOR
SEDIMENT TOXICITY TESTING
. OFFICE OF WETLANDS, OCEANS, AND WATERSHEDS
- OCEANS AND COASTAL PROTECTION DIVISION
- WETLANDS DIVISION
i
OFFICE OF WASTEWATER ENFORCEMENT AND
COMPLIANCE
OFFICE OF POLLUTION PREVENTION AND TOXIC
SUBSTANCES
. OFFICE OF PESTICIDE PROGRAMS
OFFICE OF EMERGENCY AND REMEDIAL RESPONSE
'ซ
. OFFICE OF SOLID WASTE
99
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OFFICE OF WETLANDS, OCEANS , AND
WATERSHEDS - OCEANS AND COASTAL
PROTECTION DIVISION
STATUTORY AUTHORITY AND REGULATORY
RESPONSIBILITY
DETERMINE ACCEPTABILITY OF MATERIALS
(INCLUDING DREDGED MATERIALS) FOR
OCEAN DUMPING
MARINE PROTECTION RESEARCH AND
SANCTUARIES ACT (MPRSA)
OCEAN DUMPING REGULATIONS (40 CFR 220-229)
100
-------
OCEANS AND COASTAL
PROTECTION DIVISION REQUIREMENTS
FOR STANDARD SEDIMENT TESTING
METHODS
v CHEMICAL ANALYTICAL TESTING, TOXICITY
BIOASSAYS, AND BIOACCUMULATION TESTS
v ORGANISM TAXA MUST BE APPLICABLE
TO WIDE RANGE OF SITES AND CONTAMINANTS
RECOMMEND USE OF AMPHIPODS FOR ACUTE
SEDIMENT TOXICITY TESTS
- AMPELISCA ABDITA. RHEPQXYNIUS ABRQNIUS
RECOMMEND SEVERAL TAXA FOR
BIOACCUMULATION TESTS
-- POLYCHAETES (NERJES SPECIES)
- MOLLUSCS (MACOMA SPECIES)
WHOLE SEDIMENT CHRONIC BIOASSAYS
NEEDED FOR OCEAN DUMPING PROGRAM
SEDIMENT CHEMISTRY METHODS DEVELOPED
FOR CWA 301 (H) PROGRAM
101
-------
OCEANS AND COASTAL PROTECTION
DIVISION EXISTING TESTING GUIDANCE
TESTING MANUAL, "EVALUATION OF DREDGED
MATERIAL PROPOSED FOR OCEAN DISPOSAL" -
"THE GREEN BOOK"
LISTS SUGGESTED AND RECOMMENDED SPECIES
FOR TESTS
PROVIDES GUIDANCE FOR PERFORMING TESTS
- SPECIES SELECTION
-- APPARATUS
-- EXPERIMENTAL CONDITIONS
-- SAMPLE PREPARATION
-- TEST DESIGN
-- QA/QC REQUIREMENTS
-- DATA PRESENTATION AND INTERPRETATION
102
-------
"GREEN BOOK" TESTS
ELUTRIATE BIOASSAYS
1 .
- STATIC 96 HR LC50 STUDIES
WHOLE SEDIMENT BIOASSAYS
-- 10 DAY BIOASSAYS
- 10 OR 28 DAY BIOACCUMULATION TESTS
..' ."' .. ' . ' \
DREDGED MATERIAL RESULTS ARE
COMPARED TO REFERENCE SITE SEDIMENT
TEST RESULTS
103
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OFFICE OF WETLANDS, OCEANS, AND
WATERSHEDS -- WETLANDS
DIVISION
STATUTORY AUTHORITY AND REGULATORY
RESPONSIBILITY
TESTING DREDGED MATERIAL TO DETERMINE
EFFECTS OF DISCHARGE
CLEAN WATER ACT SECTION 404
SECTION 404(B)(1) GUIDELINES
(40 CFR 230)
104
-------
WETLANDS DIVISION EXISTING
PROGRAM GUIDANCE
. CURRENTLY NO BROADLY APPLIED TESTING
MANUAL
EVALUATE PROPOSED DISCHARGES OF
DREDGED MATERIAL
. 404(BX1) GUIDELINES PROVIDE GENERAL
TESTING FRAMEWORK
MATERIAL TESTING
105
-------
OFFICE OF POLLUTION PREVENTION
AND TOXIC SUBSTANCES
STATUTORY AUTHORITY AND
REGULATORY RESPONSIBILITY
ASSESSMENT OF RISKS FROM RELEASE
OF EXISTING AND NEW CHEMICALS
THAT ARE MANUFACTURED, DISTRIBUTED,
OR DISPOSED
TOXIC SUBSTANCES CONTROL ACT, SECTIONS
4, 5, 6, AND 8
106
-------
OFFICE OF POLLUTION PREVENTION. ^
METHODS
INTERESTED IN FATE, TRANSPORT, AND EFFECTS
OF SEDIMENT CONTAMINANTS
SPIKED SEDIMENT TESTING IS OF GREATEST USE
PCB PROGRAM INTERESTED IN TOXICITY ASSAYS
FOR PCBS
107 .
-------
OFFICE OF POLLUTION PREVENTION
AND TOXIC SUBSTANCES EXISTING
TESTING GUIDANCE
SECTION 795.135 OF TSCA GUIDELINES
DESCRIBE CHIRONOMID SEDIMENT TOXICITY
TEST
TEST GUIDELINES DESCRIBE THREE TESTS
- 14 DAY CHIRONOMID AQUEOUS EXPOSURE
TEST WITH MINIMAL SEDIMENTS AND FOOD
ADDED TO THE WATER
-14 DAY CHIRONOMID TEST WITH TEST
SUBSTANCE ADDED TO SEDIMENT
-- 14 DAY INTERSTITIAL EXPOSURE WITH
TEST SUBSTANCE ADDED TO THE WATER
GUIDELINES INCLUDE A NUMBER OF KEY
PROTOCOL ELEMENTS
108
-------
OFFICE OF PESTICIDE PROGRAMS
STATUTORY AUTHORITY AND
REGULATORY RESPONSIBILITY
^ _ " ' - '
REVIEW USE OF NEW AND EXISTING
CHEMICALS REGISTERED AS PESTICIDES
TO DETERMINE EFFECTS ON
NONTARGET ORGANISMS
MAKE DECISIONS TO LABEL PESTICIDES
TO RESTmCT USE, PROHIBIT REGISTRATION
OR USES OR CANCEL/BAN USE OF EXISTING
PESTICIDES
FEDERAL INSECTICIDE, FUNGICIDE, AND
RODENTICIDE ACT (FIFRA)
109
-------
OFFICE OF PESTICIDE PROGRAMS
REQUIREMENTS FOR STANDARD
SEDIMENT TESTING METHODS
PRIMARILY BNTERESTED IN SPIKED SEDIMENT
TOXICITY TESTS TO DEVELOP DOSE/RESPONSE
RELATIONSHIPS
REQUIRES TESTS TO DISCRIMINATE AMONG
EXPOSURE PATHWAYS
INTERESTED IN EVALUATING CONTAMINATED
SEDIMENT IN THE FIELD TO TRACE
PESTICIDE RUNOFF
PROGRAM NEEDS ACUTE AND CHRONIC MARINE
AND FRESHWATER TESTS
REPRESENTATIVE SPECIES FROM A RANGE
OF SITES ARE NEEDED
MAJOR ISSUE FOR PROGRAM IS KIND
OF SEDIMENT TO USE IN SPIKED TESTING
AND HOW TO INTRODUCE CONTAMINANT
INTO TEST SYSTEM
no
-------
OFFICE OF PESTICIDE PROGRAMS
EXISTING TESTING GUIDANCE
OPP CURRENTLY HAS NO EXISTING GUIDANCE FOR
SEDIMENT TESTING
PROGRAM HAS AUTHORITY TO ASK FOR SEDIMENT
STUDY UNDER THE SPECIAL TEST
REQUIREMENTS
OPP STANDARD EVALUATION PROCEDURE
FOR ECOLOGICAL RISK ASSESSMENT _
npQPRlRES HOW RESULTS OF SEDIMENT
STUDIES WOULD BE USED IN RISK ASSESSMENT
111
-------
OFFICE OF EMERGENCY AND
REMEDIAL RESPONSE
STATUTORY AUTHORITY AND
REGULATORY RESPONSIBILITY
CLEANUP OF HAZARDOUS WASTE SITES
TO PROTECT HUMAN HEALTH, WELFARE,
AND THE ENVIRONMENT
SEDIMENT ASSESSMENT METHODS
APPLIED FOR SITE ASSESSMENT AND
REMEDY SELECTION
EXISTING STATE AND FEDERAL STANDARDS
USED TO SET CLEANUP GOALS, IN
ABSENCE OF STANDARDS, OTHER ASSESSMENT
METHODS USED
COMPREHENSIVE ENVIRONMENTAL RESPONSE,
COMPENSATION, AND LIABILITY ACT
(CERCLA) AS AMENDED BY SUPERFUND
AMENDMENTS AND REAUTHORIZATION ACT
(SARA)
CERCLA MANDATES SAMPLING TO
CHARACTERIZE RELEASE OF HAZARDOUS
SUBSTANCES FROM A SITE
112
-------
OFFICE OF EMERGENCY AND REMEDIAL
RESPONSE REQUIREMENTS FOR ^
STANDARD SEDIMENT TESTING METHODS
AND VALIDATED
MORE EMPHASIS IN PROGRAM IS BEING
PLACED^ ECOLOGICAL ASSESSMENT
113
-------
OFFICE OF EMERGENCY AND REMEDIAL
RESPONSE EXISTING TESTING GUIDANCE
SUPERFUND Rl/FS GUIDANCE AND ENVIRONMENTAL
EVALUATION MANUAL
DATA QUALITY OBJECTIVES GUIDANCE AND
COMPENDIUM
OTHER SOURCES OF TESTING METHODS INCLUDE
- ASTM, ARMY COE METHODS, EPA METHODS
FOR MEASURING EFFLUENT TOXICITY,
CANADIAN MINISTRY OF ENVIRONMENT
EPA REGION 4 STANDARD OPERATING .^
PROCEDURES FOR TOXICITY TESTING/ HAZARDOUS
WASTE ASSESSMENTS
114
-------
OFFICE OF SOLID WASTE
STATUTORY AUTHORITY AND
REGULATORY RESPONSIBILITY
RESOURCE CONSERVATION AND
RECOVERY ACT (RCRA) SECTIONS 3004 (U)
AND (V) AND SECTION 7003
PROGRAM DETERMINES WHETHER
CONTAMINATED SEDIMENT SHOULD BE^
MANAGED IN RCRA PERMITTED FACILITY
PROGRAM HAS AUTHORITY TO ENFORCED _
CLEANUP OF SEDIMENT IF IT IS HAZARDOUS
WASTE UNDER RCRA OR IF IT HAS BEEN
RELEASED FROM A RCRA PERMITTED FACILITY
115
-------
OFFICE OF SOLID WASTE REQUIREMENTS
FOR STANDARD SEDIMENT TESTING
METHODS
PROGRAM IS INTERESTED IN TESTS LINKING
TOXICITY TO A SOURCE OF CONTAMINATION
IF NEW SEDIMENT ASSESSMENT METHODS
ARE DEVELOPED THEY WOULD BE USED
IN MAKING RCRA PERMIT DECISIONS, AND
IN EVALUATING REMEDIAL SITE RESTORATION
PLANS
116
-------
OFFICE OF SOLID WASTE EXISTING
TESTING GUIDANCE
_ : ;
NO STANDARD PROGRAM APPROACHES
TO EVALUATING SEDIMENT
PROGRAM RELIES ON SUPERFUND GUIDANCE
TESTING DECISIONS ARE MADE ON A CASE-BY-
CASE BASIS
A RANGE OF TESTS WOULD BE NEEDED TO
PROVIDE AN APPROPRIATE TEST FOR
AN INDIVIDUAL SITE
117
-------
OFFICE OF WASTEWATER ENFORCEMENT
AND COMPLIANCE
STATUTORY AUTHORITY AND REGULATORY
RESPONSIBILTY
ISSUES NPDES PERMITS UNDER CLEAN WATER
ACT
PERMITS CAN BE WRITTEN TO PROTECT AGAINST
SEDIMENT CONTAMINATION
SEDIMENT TESTING AND MONITORING CAN
BE REQUIRED AS CONDITION OF A DISCHARGE
PERMIT
CLEAN WATER ACT SECTIONS 101 (A)(3),
301(B)(1)(C),AND304(L)
118
-------
OFFICE OF WASTEWATER ENFORCEMENT
AND COMPLIANCE REQUIREMENTS
FOR STANDARD SEDIMENT TESTING
METHODS
ACUTE AND CHRONIC SEDIMENT BIOASSAYS
AND B10ACCUMULATION STUDIES COULD
BE USED IN NPDES PERMITTING PROCESS
SEDIMENT QUALITY CRITERIA NOW
BEING DEVELOPED TO BE USED IN NPDES
PERMITTING
119
-------
OFFICE OF WASTEWATER ENFORCEMENT
AND COMPLIANCE EXISTING TESTING
GUIDANCE
"ASSESSMENT AND CONTROL OF
BIOCONCENTRATABLE CONTAMINANTS IN
SURFACE WATERS"
DESCRIBES METHODS FOR SAMPLING AND
MEASURING BIOCONCENTRATABLE
CHEMICALS IN SEDIMENT AND BIOTA AT
POINT SOURCE DISCHARGES
SEDIMENT CRITERIA METHODOLOGY
(EQUILIBRIUM PARTITIONING METHOD)
MODELS TO BACK CALCULATE PERMIT LIMITS
FROM SEDIMENT CRITERIA
120
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EPA Regional Sediment Needs .
William Peltier, U.S. EPA Region TV, Environmental Services Division - Athens, GA
I. Present Regional Activities
A. Superfund
B. MPDES
C. Dredge material
D. Special investigations
Standardized Test Methods
A. ASTM
B. EPA
C. COE/EPA
D. Modification of existing methods
HL Reference Sediment
A. Regional periodic reference area
B. Synthetic reference sediment
IV. Control Sediment
A. Synthetic sediment
B. Site sediment
C. Regional site sediment
V. Species Selection
A. Standard test species
B. Regional test species
C. Criteria for alternate test species*
VL Reference Toxicant Testing
A. Selection of chemicals for reference testing
B, Required series of chemicals used in reference testing for Regional or alternate
test species ,
121
-------
VH. Test Conditions
' A. Summary of test conditions and test acceptability for each selected test species
. QA/AC Program
A. Contract laboratory evaluations
B. Accreditation of laboratories'
IX. Sample Collection, Preservation and Holding
A. Consistency in collection methods
B. Preservation and holding times of sediment
X. Bioaccumulation >
A. Standardized sampling protocols
B. Minimum detection levels with available analytical methods
XI. Technical Transfer
A. Regional and State program assistance
XH. Regional Resources
A. Present Regional and State staffing
B. Facilities and future initiatives
122
-------
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Tiered Sediment Testing Conceptual Overview
Elizabeth Southland, 17.5. EPA Office of Science and Technology
ThP office of Water (OW), the Office of Pesticide Programs (OPP), the Office of
gSSwoufd be developed to explain how a decision could be made at the end of each tier
on whether a sediment poses a risk that would trigger a regulatory action.
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146
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Summary of ASTM Activities on
Freshwater and Marine Sediment Test Methods
Objectives
A. Overview ASTM subcommittee E47.03 on sediment toxicology
B. Standardization of sediment testing
Advantages of Standardization
A. ' Useof uniform testing procedures (e.g., dilution water, duration)
B. Increase data accuracy and precision
C. Facilitate test replication
D. increase comparative value of results
E. Greater regulatory and legal impact
Disadvantages of Standardization
A Sediment testing in infancy relative to aquatic testing
?: : SSSSSiSSSi - -*ป * ,,*ป
IV. American Society for Testing and Materials
A Goal- "Develop standards on characteristics and performance"of products,
svstems and s^s^promotion of related knowledge -voluntary
conSsus/ Publication: Annual Book of ASTM Standards, Volume ,
1L04
V. ASTM Definitions
A. Standard: Document development within principals of the society =
consensus '',."* ^nn
B Guide: Series of options, no recommended course of action
. , C. Test Method: Definitive procedure for measuring characteristics
f . ' .
147
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VI. Balloting a Document by Voting Members
A. Consensus at 4 levels
1. Task group (1 to 10 individuals ;
2. Subcommittee (100 individuals + 500)
3. Main committee (250 individuals)
4. Society (33,000 individuals)
B. Re-ballot every 3 years (at minimum)
VH. ASTM E47.03 SedJment Toxicology Subcommittee
A. Inception: May 1987
B. Goal: "Develop guides for assessing the bioavailability of contaminants
associated with sediments ... evaluate hazard of contaminated sediment,
soil, sludge, drilling fluids, and similar materials."
C. Meeting schedule: Spring - during annual ASTM symposium
Fall - weekend before SETAC
Approved ASTM Standards
vm.
A. E 1367-92:
Amphipods
B. E 1383-92: Standard Guide .
C. E 1391-90: Standard Guide
Manipulation
Standard Guide ... 10-d Toxicity with Marine & Estuarine
Toxicity with Freshwater Invertebrates
Collection. Storage. Characterization.
K. Documents in Balloting Process (task Groups)
A. Designing Biological Tests (Dwyer; Main/Subcommittee)
B. Toxicity Tests with Polychaetes (Reish; Main/Subcommittee)
C. Toxicity Tests with Mayflies (Bedard; Main/Subcommittee)
D. Terminology (Ingersoll; Main/Subcommittee)
E. Bioaccumulation by Benthic Invertebrates (Lee; Subcommittee)
F. Bioaccumulation by Fish (Mac; Subcommittee)
X. Documents Proposed
A. Toxicity Tests with Oysters (Dinnel)
B. Toxicity Tests with Echinoderms (Dinnel)
C. Toxicity Tests with Earthworms (Callahan)
D. Toxicity Tests with Microtox (Evereklian)
E. Toxicity Tests with Tubifex tubifex (Day)
F. Sediment Resusoension (Burton)
G. Statistical Guidance (Schlekat)
H. Toxicitv Identification and Evaluation (??)
148
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XI. E 1367: Marine and Estuarine Amphipods
A. Scope, significance, and use
B. Interference
C Hazards
D. Test water, test and control sediments, test organisms
E. Experimental design, procedure, analyses
F. Acceptability and interpretation .
G. Species-specific 'annexes
XII. E 1367: Marine and Estuarine Amphipods
A. Species-specific annexes
Annex!. Rhepoxvnius abronius
Annex 2. Eohaustorius spp.
Annex 3. Ampelisca abdjta
Annex 4. flrandidierella japonica
Annex 5. T^ptocheirus plumulosus
Annex 6. Hyalella azteca (proposed)
XII E1367: Marine and Estuarine Amphipods
A' ^ To-d whole sediment, static, field contaminated/spiked sediment
2. Endpoints ^survival, reburial
B. Status: approved standard
G. Future plans: ;
1. Chronic endpoints (growth, reproduction)
2. "Rhepoxynius abronius test method?
Xin. E 1383: Freshwater Invertebrates
A. Species-specific annexes
Annex!. ; HyjMla azteca ,
Annex 2. Chironomus tentans :
Annex 3. Chironomus riparius . .
Annex 4. Daphina sp. and Ceriodaphnia sp.
Annex 5. Hexagenia sp. (Main/Subcommittee) _
Annex 6. Diporeia SP. (proposed; formerly Pontoporeia hoyj)
Annex 7. T.umbriculus sp. (proposed)
Annex 8. Oligochaeta (proposed, Jubifex tubifex)
Annex 9. MoUusks (proposed)
149
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XV, E 1383: Freshwater Invertebrates
A. Procedures:
1. Partial life cycle, static/flow-through, field contaminated/spiked
sediment
2. Endpoints = survival, growth, reproduction
B. Status: approved standard
C. Future plans:
1. Additional species-specific annexes
2. Hyalella azteca test method?
XVI. E1391: Collection, Storage, Characterization, Manipulation
A. Procedures:
1. Sediment collection, transport, storage, characterization, spiking,
and dilution
2. Recommendations and limitations
B. Status: approved standard
C. Future plans: test methods?
XVH. Designing Biological Tests with Sediment
A. Procedures:
1. Test type (e.g., whole sediment, pore water,.elutriate)
2. Sample collection, handling, and manipulation
3. Test organisms and endpoints
4. Experimental design
5. Statistics, data interpretation, QA/QC
B. Status: Main/subcommittee ballot
XVm Sediment Toxicity Tests with Polychaetes
A. Procedures: ,
1. 4 to 20-d test, juvenile/adult, field contaminated/spiked sediment
2. Endpoints = survival, growth
B. Status: Main/subcommittee ballot
XIX. Bioaccumulation Bv Benthic Invertebrates and Bioaccumulation By Fish
A. Procedures: .
1. 10 28-d, static/flow-through, field contaminated/spiked sediment
2. Bioaccumulation potential vs. steady state ,
B. Status: Subcommittee ballots
150
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XX. Research Needs for Standard Development
A; Multi-'species comparisons
B. Inter-laboratory comparisons
C. - Abiotic factors
D. Life history and chronic indicators of toxicity
E. Spiking methods and positive controls
F. Dilution studies and mixtures
G. Laboratory to injitu comparisons
151
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ADVANTAGES OF STANDARDIZATION
Use of uniform testing procedures (e.g.; dilution water, duration)
Increase data accuracy and precision
Facilitate test replication
Increase comparative value of results
Greater regulatory and legal impact
154
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DISADVANTAGES OF STANDARDIZATION
Sediment testing in infancy relative to aquatic testing
Inhibit creative approaches
inadequate characterization of effects because of optimized conditions
155
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AMERICAN SOCIETY FOR TESTING AND MATERIALS
GOAL:
"Develop standards on characteristics and performance of
products, systems, and services; promotion of related
knowledge...voluntary consensus."
PUBLICATION: Annual Book of ASTM Standards, Volume 11.04
156
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ASTM DEFINITIONS
STANDARD:
GUIDE:
TEST METHOD:
DOCUMENT DEVELOPED WITHIN PRINCIPALS OF THE
SOCIETY = CONSENSUS
SERIES OF OPTIONS, NO RECOMMENDED COURSE OF
ACTION
DEFINITIVE PROCEDURE FOR MEASURING
/ CHARACTERISTICS
157
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BALLOTING A DOCUMENT BY VOTING MEMBERS
CONSENSUS AT 4 LEVELS
1. Task Group (1 to 10 individuals)
2. Subcommittee (100 individuals+500)
3. Main Committee (250 individuals)
4. Society (33,000 individuals)
RE-BALLOT EVERY 3 YEARS (at minimum)
158
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ASTM E47.03 SEDIMENT TOXICOLOGY SUBCOMMITTEE
INCEPTION: May 1987
GOAL: "Develop guides for assessing the bioavailability of contaminants
associated with sediments... evaluate hazard of contaminated
sediment, soil, sludge, drilling fluids, and similar materials."
MEETING SCHEDULE:
Spring-During Annual ASTM Symposium
Fall-Weekend before SETAC
159
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APPROVED ASTM STANDARDS
ป
E 1367-92: Standard Guide ... 10-d Toxicity with Marine & Estuarine Amohipods .
E 1383-92: Standard Guide ... Toxicity with Freshwater Invertebrates
E 1391-90: Standard Guide ... Collection. Storage. Characterization. Manipulation
160
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DOCUMENTS IN BALLOTING PROCESS (Task Groups)
- reigning Biological Tests (Dwyer; Main/Subcommittee)
Toxicity Tests with Polvchaetes (Reish; Main/Subcommittee)
Toxicity Tests with Mayflies (Bedard; Main/Subcommittee)
Terminology (Ingersoll; Main/Subcommittee)
Bioaccumuiation *y Ronthin invertebrates (Lee; Subcommittee)
Bioaccumuiation by Fish (Mac; Subcommittee)
161
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DOCUMENTS PROPOSED
Toxicity Tests with Oysters (Dinnel)
Toxicity Tests with Echinoderms (Dinnel)
Toxicitv Tests with earthworms (Callahan)
Toxicity Tests with Microtox (Evereklian)
Toxicity Tests with Tubifex tubifex (Day)
Sediment Resuspension (Burton)
Statistical Guidance (Schlekat)
Toxicitv Identification and Evaluation (??)
162
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E 1367: MARINE AND ESTUARINE AMPHIPODS
A. Scope, Significance, and Use
B. Interferences
C. Hazards
p Test Water, Test and Control Sediments, Test Organisms
.E..' Experimental-Design, Procedure, Analyses
F. Acceptability and Interpretation
G. Species-specific Annexes
. 163
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E 1367: MARINE AND ESTUARINE AMPHIPODS
SPECIES-SPECIFIC ANNEXES
Annex 1. Rhepoxynius abronius
Annex 2. Eohaustorius spp.
Annex 3. Amoelisca abdita
Annex 4. Grandidierella iaponica
Annex 5. Leotocheirus plumulosus
6. Hvaiella azte'ca (proposed)
164
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E 1367: MARINE AND ESTUARINE AMPHIPODS
PROCEDURES:
-10-d whole sediment, static, field contaminated/spiked sediment
Endpoints = survival, reburial
STATUS: approved standard
FUTURE PLANS:
-Chronic endpoints (growth, reproduction)
-Rhepoxvnius abronius test method?
165
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E 1383: FRESHWATER INVERTEBRATES
SPECIES-SPECIFIC ANNEXES
Annex 1. Hvalella azteca
Annex 2. Chironomus tentans
Annex 3. Chironomus riparius
Annex 4. Daphnia sp. and Ceriodaphnia sp.
Annex 5. Hexaaenia sp. (Main/Subcommittee)
166
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E 1383: FRESHWATER INVERTEBRATES
SPECIES-SPECIFIC ANNEXES (cont.)
6. Diporeia sp. (proposed; formerly Pontoporeia hoyi)
7. Lumbricyius sp. (proposed)
8. Oligochaeta (proposed;
9. Mollusks (proposed)
167
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E 1383: FRESHWATER INVERTEBRATES
PROCEDURES:
-Partial life cycle, static/flow-through, field contaminated/spiked sediment
-Endpoints = survival, growth, reproduction
STATUS: approved standard
FUTURE PLANS:
-Additional species-specific annexes
-Hvalella azteca test method?
168
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E1391: COLLECTION, STORAGE, CHARACTERIZATION, MANIPULATION
. i '-.' " . ' '. .
PROCEDURES:
-Sediment collection, transport, storage, characterization, spiking, and dilution
-Recommendations and Limitations
STATUS: approved standard
FUTURE PLANS: Test methods?
169
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DESIGNING BIOLOGICAL TESTS WITH SEDIMENT
PROCEDURES:
-Test type (e.g., whole sediment, pore water, elutriate)
-Sample collection, handling, and manipulation
-Test organisms and endpoints
-Experimental design
-Statistics, data interpretation, QA/QC
STATUS: Main/Subcommittee ballot
170
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SEDIMENT TOXICITY TESTS WITH POLYCHAETES
PROCEDURES:
-4 to 20-d test, juvenile/adult, field contaminated/spiked sediment
-Endpoints = survival, growth
STATUS: Main/Subcommittee ballot
171
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BIOACCUMULATION BY BENTHIC INVERTEBRATES and
BIOACCUMULATION BY FISH
PROCEDURES:
-10 to 28-d, static/flow-through, field contaminated/spiked sediment
-Bioaccumulation potential vs. steady state
STATUS: Subcommittee ballots
172
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RESEARCH NEEDS FOR STANDARD DEVELOPMENT
MULTI-SPECIES COMPARISONS
INTER-LABORATORY COMPARISONS
ABIOTIC FACTORS
LIFE HISTORY AND CHRONIC INDICATORS OF TOXICITY
SPIKING METHODS AND POSITIVE CONTROLS
DILUTION STUDIES AND MIXTURES
LABORATORY TO ]N STTU COMPARISONS
173
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EPA Approaches for Biological Methods Standardization:
- perspective and Present Guidance
ok, U.S. EPA Environmental Monitoring and Systems Laboratory -
' ฐ
I Background - Standardization, of Biological Methods 1960 - 1989
A National Water Quality Network (NWQN)
B' Methods Development and Quality Assurance Research Lab
C Environmental Monitoring and Support Laboratory - Cincinnati
' D'. Environmental Monitoring Systems Laboratory - Cincinnati
E. Biological Advisory Committee
F! Biological Methods Manual (1973)
H. Whole Effluent Toxicity Testing Manuals
A. Research Method to 304(H) CWA Approved Method (1981-1992)
m. Current Status of Biological Methods Standardization
A. EPA Biological Advisory Committee Charter
IV. Existing Agency (ORD, 1987) Guidance on Methods Standardization/Validation
A. Six Steps: N
1. Determination of Method Requirements and DQO's
, 2. Method Selection/Development
3. Single-Laboratory Evaluation
4. Confirmatory Testing
5. Interim Methods Description
6. 'Formal Collaborative (interlaboratory study)
V. New EMMC Workgroup on Biological Methods Integration
175
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OUTLINE OF ACTION TAKEN (1989-1991)
MAY 20, 1992
1.
2.
3.
4.
5.
6.
12/4/89
2/4/90
3/90
4/90
1/91
5/7/91
7. 5/27/91
8. 6/91
PUBLICATION OF PROPOSED RULE
PUBLIC COMMENT PERIOD CLOSED
PUBLIC COMMENTS SUMMARIZED
FOUR TECHNICAL SUBGROUPS ORGANIZED
(AQUA.TOX., STAT, AMES TEST, VIRUSES)
RESPONSES TO PUBLIC COMMENTS COMPLETED
FINAL RULE TO RED BORDER REVIEW (OPPE,
OPTS, OGC, OW, REG. 3 & 65) (NEW LAWYER,
REGAS, ASSIGNED)
RED BORDER REVIEW COMPLETE (OW AND
OGC NON-CONCUR)
FURTHER ACTION ON RULE PLACED ON HOLD
UNTIL TOXICiry TEST MANUALS ARE REVISED
176
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BIOLOGICAL METHODS TO BE INCLUDED IN 304 (H)
MAY 20, 1992
PROPOSED ACTION:
1. TOXICITY TEST METHODS
A. ACUTE AND CHRONIC TOXICITY TESTS FOR EFFLUENTS
AND RECEIVING WATERS
B. ACUTE TOXICITY TESTS FOR DRILLING MUDS
C. AMES (MUTAGENICITY) TEST FOR EFFLUENTS, RECEIVING
WATERS, AND SLUDGES.
2. METHODSFORCOLLECTION^ONCENTRATION^NUMERATION,
AND IDENTIFICATION OF VIRUSES IN SURFACE WATERS,
WASTEWATERS, AND SLUDGES.
3. UPDATED REFERENCES FOR BACTERIOLOGICAL TESTS
177
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INCLUSION OF TOXICITY TESTS IN 304 (H)
MAY 20, 1992 .
RESULTS OF 1991 RED BORDER REVIEW:
t ' , - . ' -
1. OPPE & OPTS* CONCURRED WITHOUT COMMENT
2. REGIONS 3 & 5 CONCURRED WITH MINOR COMMENTS
3. . OW CONCURRED CONTINGENT ON COMPLETION OF REVISED
TOXICITY TEST MANUALS , BEFORE THE FINAL RULE IS
PUBLISHED
4. OGC NON-CONCURRED. CONCURRENCE DEPENDS ON:
A. REVISION/APPROVAL OF FINAL RULE
B. REVISION/APPROVAL OF RESPONSES TO PUBLIC
COMMENTS
C REVISION/APPROVAL/PUBLICATION OF MANUALS
* OPTS is .now the Off. Poll. Prevention & Toxics
178
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STATUS OF INCLUSION OF TOXICITY TESTS IN 304 (H)
MAY 20, 1992
ACTION STNCE 1991 RED BORDER REVIEW;
1. 9/91 - REVISED ACUTE MANUAL SENT TO PRINTER - .
V
2. 9/91 - NEW OGC LAWYER (SWEENEY) ASSIGNED
3. 1/92 - CHRONIC MANUALS NEAR COMPLETION
4. 2/92 - ACTION ON RULE RESTARTED (MEETING IN DC WITH OGC,
, - i |
OWEC)
- REVISION OF COMMENTS AND RULE
HARMONIZATION OF POLICY IN MANUALS, RESPONSE TO
... COMMENTS, AND FINAL RULE ,
TARGET DATES FOR COMPLETION AND PUBLICATION OF RULE
5. 5/92 - COMPLETION OF REVISED RESPONSE TO COMMENTS (RTC)
AND FINAL RULE (FR)
6. 6/92 - REVIEW OF RTC AND FR BY 304H WKGP
7. 7/92 - RED BORDER REVIEW
8. 8/92 - SUBMISSION OF RULE TO ADMINISTRATOR ,
/ > - - ' . ' '' '.
9. 12/92 - PUBLICATION OF RULE IN FED REG
, 179 .
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BIOLOGICAT ADVISORY COMMITTEE
The vision of the Biological Advisory Committee is to provide technical advice to the agency
on aU biological methods and related ecological issues.
MISSION OF THE COMMITTEE
1. Review, comment, and assist Regions, ORD and Program Offices in standardizing and
evaluating EPA biological methods and indicators to be used by Regional and State programs.
2. Ensure that states develop logically consistent and ecologically meaningful biological criteria
that facilitate interstate, interregion and Environmental Monitoring Assessment Programs
(EMAP) national comparisons.
3. Exchange technical information and experience in the collection, analyses, and use of
biological methods and indicators in assessing the effects of impacts on biological integrity.
4. Review, comment, and assist Regions and Program offices in developing agency biological
monitoring or biocriteria policy. ,
5. Represent agency for ecological methods and other'related issues on National EPA and other
Federal committees (e.g., EPA Environmental Monitoring Management Council (EMMC) and
OMB Intergovernmental Task Force on Monitoring Water Quality).
Biological Advisory Committee (BAG) will be headed by a Chairperson from EMSL-Cincinnati,,
Deputy Chairperson from ORD or a Program Office.
Each subcommittee will have a chairperson and vice-chairperson, one of which is:
1. an ORD or Program Office Representative, and
2. a Regional Representative
The following subcommittees were agreed upon by the BAG during the FY91 meeting:
Toxicitv Assessment Subcommittee;
This subcommittee's mission is to address methods and other issues related to assessing acute
toxicity, chronic toxicity, bioaccumulation, and organism physiological (biomarker level)
dysfunction due to contaminant in water, sediment and soil.
Ecological Assessment Subcommittee;
This subcommittee's mission will be to address methods and issues that measure ecosystem
health in aquatic and terrestrial systems.
The following addition of steering committees to the BAG Committee and its subcommittees was
recommended by the BAG Chairperson and Subcommittee Chairpersons during a November,
1992 Society for Environmental Toxicology and Chemistry meeting:
180
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RA.C Steering Committee;
Committee.
Tmricitv Assessment Su*ปittee Steering Committee;
The mission of the steering committee will be to organize, prioritize, and expedite subcommittee
activities.
Ffnlogical Aซpssnient S"*ปmniitteซ' Steering Committee;
The mission of the steering committee will be to organize, prioritize, and expedite subcommittee
activities.
REPRESENTED ON COMMITTEE
1 A minimum of 1-2 Biologists from each Regional Environmental Services Division ^upon
JecommTS from each BSD and Biologist from Water Management and/or Waste
Management).
2. A minimum of 1-2 Biologists from each ORD Laboratory.
3. A minimum of 1-2 Biologists from each of the following ProgramOffices:
Office of Wastewater Enforcement and Permits
Office of Science and Technology
Office of Wetlands, Oceans and Watersheds
Office of Solid Waste and Emergency Response
Office of Toxic Substances
Office of Pesticides
COMMITTED ACTIVITIES
1. Quarterly conference calls:
10 or less Regional Biologists plus 1-2 ORD Reps, 1-2 Hdqtrs Rep
2. Information exchange in:
Monthly surface water monitoring status report -
Regional monthly reports circulated to regional biologists
NETAC Newsletter . ^
Superfund monthly FORUM report . -
EMSL - Bulletin computer board ,
EMAIL ,
FAX ; . . ' ' _. _./-. .'; '.
181
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EPA BIOLOGICAL ADVISORY COMMITTEE:
ORGANIZATION STRUCTURE
I. BAG Chairperson and Deputy Chairperson
EPA Biological Advisory Committee Steering Committee
A. BAG Chairperson and Deputy Chairperson
B. Subcommittee Chairpersons and Vice-Chairpersons
EPA Biological Advisory Subcommittees
A. Toxicity Assessment Subcommittee
1. Chairperson, Vice-Chairperson, and Steering Committee
B. Toxicity Assessment Subcommittee Steering Committee
C. Ecological Assessment Subcommittee
1. Chairperson, Vice-Chairperson, and Steering Committee
2. Ecological Assessment Subcommittee Steering Committee
182
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COMPOSITION OF EACH FPA BIOLOGICAL ADVISORY COMMITTEE
EPA Biological Advisory Committee Chairperson :<,
The Chairperson of the EPA biological Advisory Committee will be the Chief of the
Bioassessment & Ecotoxicology Branch.
FPA Biological Advisory Committee Deputy Chairperson:
The Deputy Chairperson of the EPA Biological Advisory Committee will be a willing ORD,
Regional or Program Office Representative appointed by the Chairperson of the EPA BAG.
EPA Biological Advisory Committee Steering Committee: ,
The Steering Committee will consist of the present and past BAG Chairpersons and Deputy
Chairperson, Chairpersons and Vice-Chairpersons of the EPA BAG Subcommittees.
COMMITTEE
gTTTON OF F, ACH EPA BIOL
Toxicitv Assessment Subcommittee:
The subcommittee will consist of BAG members willing to serve and a Chairperson nominated
by anyone on the BAG, willing, to serve, and elected by the BAG. The subcommittee will also
include a Vice-Chairperson that is a willing ORD, Regional or Program Office Representative,
nominated and elected by the members of the BAG Subcommittee. Either the Subcommittee
Chairperson or Vice-Chairperson should be from and ORD Laboratory.
Toxicitv Assessment Subcommittee Steering Committee:
The Subcommittee Steering Committee will consist of a minimum of 3 BAG members (in
addition to Subcommittee Chairperson and Vice-Chairperson) willing to serve and confirmed by
Subcommittee Chairperson and Vice-Chairperson. Steering committee should be composed of
.a least 1 member from each of the following organizations: Regional Office, Headquarters
Program Office, and an ORD representative.
Ecological Assessment Subcommittee:
The Subcommittee will consist of BAG members willing to serve and a Chairperson nominated
by anyone on the BAG, willing to serve, and elected by the BAG. The subcommittee will also
include a Vice-Chairperson that is a willing ORD, Regional or Program Office Representative
nominated and elected by the Subcommittee members. Either the Subcommittee Chairperson
or Vice-Chairperson should be from an ORD Laboratory.
183
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Ecological Assessment Subcommittee Steering Committee:
A minimum of 3 BAG members (in addition to the Subcommittee Chairperson and Vice-
Chairperson) willing to serve and confirmed by Subcommittee Chairperson and Vice-
Chairperson. Steering committee should be composed of a least 1 member from each of the
following organizations: Regional Office, Headquarters Program Office and an ORD
Representative.
NOTE: Tentatively the Committee and Subcommittee officers are filled by the following
individuals:
* EPA Biological Advisory Committee (BAG)
Chairperson, Jim Lazorchak, ORD EMSL-Cincinnati
Deputy Chairperson, Vacant
* BAG Steering Committee:
Bill Peltier ,
Teresa Norberg-King
Ron Preston
Don Klemm
Cornie Weber
* Toxlcity Assessment Subcommittee;
Chairperson, Bill Peltier, Region 4 BSD
Vice-Chairperson, Teresa Norberg-King, ORD ERL-Duluth
* Steering Committee:
Joe Cummins, Region 10, BSD
Cornie Weber, ORD EMSL-Cincinnati
Margarete Heber, HDQTS OW
184
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* Toxicitv Assessment Subcommittee Members
Peter Nolan, Region 1
Robert Donaghy, Region 3 .
Chick Steiner, Region 5
Terry Hollister, Robert Vickery, P. Crocker, Region 6
Mike Tucker, Region 7 ; ,
Loys Parrish, Glenn J. Rodriguez, Region 8
Joe Cummins, Region 10
Gary Chapman, ORD ERL-Newport
Doug Middaugh, ORD ERL-Gulf Breeze
Don Klemni, ORD EMSL - Newtown
Phil Lewis, ORD EMSL - Newtown
- Members signed up as of 6/91 BAG Meeting. Others not attending 6/91 meeting can
also join, contact subcommittee chairperson. .
* Ecological Assessment Committee;
Chairperson, Ron Preston, Region 3 BSD
Co-Chairperson, Don Klemm, ORD EMSL-Cincinnati\
* Steering Committee;
Jim Kurtenbach, Region 2, BSD
Chris Faulkner, HDTS Wetlands, Watershed, Oceans
George Gibbons, HDTS, Science and Technology
* Ecological Assessment Subcommittee Members
Peter Nolan, Region 1
Jim Kurtenbach, Region 2
Jim Green, Region 3
Del Hicks, Jerry Stober, Hoke Howard, Region 4
Wayne Davis, Thomas Simon, Region 5
Evan Hornig, Region 6
Gary E. Welker, Region 7
Loys Parrish, Region 8
Peter Husby, Region 9 . ,
Gretchen Hayslip, Gerald Montgomery, Region 10
Teresa Norberg-King, ORD ERL-Duluth
Chris .Faulkner, OWOW AWD
Margarete Heber, OST HESD
Don Klemm, ORD EMSL - Newtown
Brian Hill, ORD EMSL - Newtown .
Prank McCormick, ORD EMSL - Newtown
Phil Lewis, ORD EMSL - Newtown
- Members signed up as of 6/91 BAG Meeting.
Others not attending 6/91 BAG meeting can also join, contact subcommittee Chairperson.
185
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TOXICITY
5NT SUBC<
CEFUNCTIOM
The Subcommittee functions within the EPA EMSL-Cincinnati Biological Advisory
Committee (BAG) that provides guidance in the area of toxicity assessment. The Subcommittee
is directed by a Chairperson with members representing EPA HQ Offices, ORD Laboratories
and Regions who volunteer to serve on the Subcommittee.
The functions of the Subcommittee are as follows:
* Assist in the preparation, and coordination of toxicity test methods prepared for
publication by the EPA for application in freshwater, marine and terrestrial
ecosystems. EPA activities or programs impacted are as follows: NPDES whole
effluent testing, sediment testing, toxicity reduction evaluation, dredge and fill,
ocean disposal, EMAP, CERCLA, RCRA, TSCA and FIFRA.
* Assess existing toxicity test methodologies (test condition, species, endpoints, and
methods of data analyses) that will impact EPA National and Regional
implementation and enforcement of ecological programs.
*' Provide ORD laboratories and HQ programs annually with a technical assistance
needs list to support Regional toxicity assessment activities associated with the
NPDES program, Water Quality Standards, and sediment criteria.
* Serve the Environmental Monitoring Council (EMMC) as a resource for review
of toxicity testing methods proposed by other offices. Provides information on
Subcommittee activities relating to the development and use of new toxicity
testing methods.
* Coordinate with the Ecological Assessment Subcommittee of the BAG on
overlapping activities, such as biomarkers development and statistical analyses.
* Integrate toxicity biomarkers from the agency's strategic planning initiative into
the activities of the Toxicity Assessment Subcommittee.
. * Make available Subcommittee technical expertise to State and EPA Regional
offices and HQ programs for: program, project, or report reviews; judicial,
administrative, or legislative hearings; and adversarial proceedings.
186
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ASSESSMENT SUBCOMMITTEE:
FTINPTTONAT. STATEMENT
The Subcommittee functions, within EPA EMSL-Cincinnati Biological Advisory
The
,
Committee (BAG) that provides guidance in the area of ecological a^essmen^ The
Subcommittee is facilitated by a Chairperson with members representing EPA HQ program
offices, ORD Laboratories and Regions who volunteer to serve on the Subcommittee.
The functions of the Subcommittee are as follows:
* Assist in the preparation, review and coordination of ecological assessment
methods prepared for publications by EPA for application in freshwater, marine
and terrestrial ecosystems. EPA activities or programs impacted are as follows:
water quality monitoring, environmental indicators, biological criteria, non-
regionalization, EMAP, CERCLA, RCRA, TSCA and FIFRA.
* Evaluate existing ecological assessment methodologies (biosurveys, field
procedures, study design and methods of data analyses) that will impact EPA
National and Regional activities utilizing ecological assessments.
* Provide ORD laboratories and HQ programs annually with a technical assistance
needs list to support ecological assessment activities associated with EPA and
State environmental protection programs.
Serve the Environmental Monitoring Management Council (EMMC) as a resource
for review of ecological assessment activities proposed by other offices. Provides
information on Subcommittee activities relating to the development and use of
state-of-the-art ecological assessment methods.
* Coordinate with the Toxicity Testing Subcommittee of the BAG, on overlapping
activities, such as biomarkers development and statistical analyses.
* Integrate ecological indicator goals from the agency's strategic planning initiative
into the activities of the Ecological Assessment Subcommittee.
Make available Subcommittee technical expertise to State and EPA Regional
offices and HQ programs for: program, project of report reviews; judicial,
administrative or legislative hearings; and adversarial proceedings.
187
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STANDARDIZATION OF BIOLOGICAL METHODS
1. INTRODUCTION
1.1 BACKGROUND
Field and laboratory "methods for monitoring the status and trends of the biological
integrity of aquatic communities and the quality of surface waters and effluents have played a
key role in Federal and State water pollution control programs for several decades. The current
Agency biological monitoring methods development and standardization program emerged from
activities of the National Water Quality (Monitoring) Network (NWQN) in Cincinnati in the late
1960's, later renamed successively the Methods Development and Quality Assurance Research
Laboratory (MDQARL), the Environmental Monitoring and Support Laboratory - Cincinnati,
and the Environmental Monitoring Systems Laboratory - Cincinnati. Development, evaluation,
standardization and publication of biological (field) sampling and (laboratory) analysis methods,
first begun by the NWQN in the late 1950's, has preceded uninterrupted to the present.
The need for a formal monitoring methods development and standardization program was
recognized by the federal water pollution control program (Federal Water Quality Agency) in
the late 1960's. As a result, an Agency chemical methods advisory group was created to
recommend and assist NWQN in the standardization and publication of chemical and physical
monitoring methods for water. The first Agency manual of chemical and physical monitoring
methods was published in 1968.
An Agency biological advisory committee was created by MDQARL in 1970, consisting
of representatives from the regions, research laboratories, and headquarters program offices.
The members of the committee were selected to provide a cross-section of technical expertise
in biological monitoring and guidance on state, regional and headquarters program office
requirements for aquatic biology data. At that time, the emphasis in biological monitoring in
the Agency water monitoring program was on the effects of discharges from publicly owned
sewage treatment plants on the structure and function of aquatic communities.
Methods for the collection and analysis of biological samples and interpretation of
biological data were selected from the peer-reviewed literature and techniques then in regular
use by Agency regional and research personnel and state programs. Primary emphasis was
placed on taxonomic composition and standing crop. Data on the identification and enumeration
of aquatic organisms were used to establish the status and trends of biological integrity in terms
of indicator organisms, the proportion of sensitive (clean water) and tolerant( polluted water)
organisms, and species diversity indices. The first Agency biological monitoring methods
manual was published by MDQARL in 1973 (USEPA, 1973). The Biological Advisory
Committee, established in 1970, has continued to function to the present.
188
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In the mid-1970's, primary emphasis on biological monitoring in the Agency and states
began to shift from the biological integrity of ambient waters to the measurement of effluent
toxicity. In response to the new Agency and state programs needs, EMSL, with the assistance
of the Biological Advisory Committee, published the first methods manual for monitoring the
acute toxicity of effluents and surface waters (USEPA, 1978) to aquatic organisms, now in its
fourth edition (USEPA, 1991), and methods manuals for the estimation of the chronic toxicity
of effluents and surface waters to freshwater and marine organisms (USEPA, 1992a, 1992b),
based on methods developed by the Environmental Research Laboratories at Duluth and
Narragansett, respectively.
k
1.2 CURRENT STATUS OF BIOLOGICAL METHODS STANDARDIZATION
During this period of chemical, physical and biological methods development (21965 -to
the present), the Agency was also developing and continually strengthening its quality assurance
program, which rests heavily on the availability of standardized and validated methods. The
development of the Agency's policy for a water quality-based approach to discharge permitting
(USEPA 1984) and the subsequent move to place toxicity limits in discharge permits and to
include effluent toxicity test in the list of "official" EPA (largely chemical and physical)
monitoring methods in Table I, 40 CFR Part 136, have led to questions related to biological
methods standardization, such as, "when (at what point) is a biological method considered to be
standardized or validated?" or, "what process is involved in biological methods standardization
and validation?" The agency currently lacks an official policy on methods standardization.
1.3 EXISTING AGENCY (ORD) GUIDANCE ON METHODS
STANDARDIZATION/VALIDATION
The process of monitoring methods selection,' standardization and validation is essentially
similar for chemical, physical and biological methods. A consensus document or "white paper"
on the subject (USEPA, 1987) was prepared by the staff of the Office of Acid Deposition,
Environmental Monitoring, and Quality Assurance (it has since been renamed the Office of
Monitoring, Modeling and Quality Assurance, or OMMSQA), but the document and its contents
have not yet been "officially" endorsed by OMMSQA or the Agency, or promulgated as Agency
policy. .
Because of the detail and clarity of the 1987 document, it would be senseless to repeat
its contents in full, here. However, it would be advantageous to provide a summary of the
process described in the document, and to indicate how the steps may differ, if at all, for
biological methods. .
189
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The six major steps are described below. In the case of biological methods
development/validation, the Biological Advisory Committee should be consulted during each
step.
1.
2.
tination of method requirements and data quality objective
Determination
- Provided by the program office
Method selection/development
- Potential user, such as program office and/or regions, should be actively
involved. , ,
3. Single-laboratory evaluation
- Includes sensitivity to test method variables (ruggedness) and single
laboratory/single operator precision.
4. Confirmatory testing
- Evaluation by several (minimum of three) independent labs.
5. Interim method description _
- Full method description, information on ruggedness, mandatory and optional test
conditions, guidance on data analysis, single laboratory precision, etc. If
endorsed by the Agency, it would now be considered a "standard" method.
6. Formal collaborative (interlaboratory study)
- Complete, acceptable, data from a minimum of six labs.
190
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METHODS STANDARDIZATION
BIBLIOGRAPHY:
1. AOAC. 1984. Report of the Committee of Collaborative Interlaboratory Studies,
Association of Official Analytical Chemists. J. Assoc. Anal. Chem. 67 (2)
2. ASTM. 1979. Standard practice for conducting an interlaboratory test program to
determine the precision of test methods. Annual Book of Standards, 14.02. Water,
Standard E691-79. American Society for Testing and Materials, Philadelphia,
Pennsylvania.
3. ASTM. 1981. Standard practice of precision and accuracy of methods of Committee D-
22 on Sampling and Analysis of Atmospheres. Annual Book of Standards, 11.03.
Standard E3670-81. American Society for Testing and Materials, Philadelphia,
Pennsylvania. , l
4. ASTM. 1985. -Standard practice of determination of precision and accuracy of methods
of Committee D-19 on Water. Annual Book of Standards, 11.01. Standard D2777-85.
.American Society for Testing and Materials, Philadelphia, Pennsylvania. ',
5. BatteUe Columbus Laboratories. 1982. Development of appropriate statistical techniques
to compare analytical methods across wastewaters. Bishop, T.F., F.E. Brydon, and
B.C. Dutter. BatteUe Columbus Laboratories, Columbus, Ohio. USEPA Contract 68-
03-2624.
6. BatteUe Columbus Laboratories. 1985. Single laboratory validation protocol. BatteUe
Columbus Laboratories, Columbus Ohio. USEPA Contract 68-03-3224, Work
assignment #1. .
7. Glaser, J.A., D.A. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde. 1981. Trace
analyses for wastewater. Environ. Sci. Techn. 15(12): 1426-1435.
8. USEPA. 1983. Guidelines and format for EMSL-Cincinnati methods. J.F. Kopp. U.S.
Environmental Protection Agency, Cincinnati, Ohio. EPA/600/4-83/020.
9. USEPA. 1983. Guidelines for conducting single laboratory evaluations of biological
methods. McKenzie, W., and T. Olsson, m, Bioassay Systems Corporation.
Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency,
Las Vegas, Nevada. EPA/600/4-83/056.
191
-------
10. USEPA. 1984. The development of data quality objectives. Quality Assurance
Management Staff, Office of Research and Development, U.S. Environmental Protection
Agency, Washington, D.C. Unpublished report.
11. USEPA. 1984. Formal collaborative study design for water and wastes. P.W. Britton,
ed. Environmental Monitoring and Support Laboratory, U.S. Environmental Protection
Agency, Cincinnati, Ohio. EPA/600/4-83/020.
12. USEPA. 1987. Guidelines for selection and validation of USEPA'S measurement methods.
Office of Acid Deposition, Environmental Monitoring, and Quality Assurance, Office of
Research and Development, U.S. Environmental Protection Agency, Washington, D.C.
Unpublished report. 57pp.
13. USEPA. 1983. s Validation of testing/measurement methods. L.R. Williams.
Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency,
Las Vegas, Nevada. EPA/600/X-83/060.
192
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STEPS IN BIOLOGICAL METHODS DEVELOPMENT.
STANDARDISATION. AND VALIDATION
1. Determination of Methods Requirements and DQO's
2. Candidate Method Selection/Development
3. Method Evaluation
- Single Laboratory
- Precision, Bias, Ruggedness
4. Confirmatory Testing
- Evaluation by 3 Labs
\
5. Interim Method Description
- Now a Standard Method
" =' " . " <
6. Formal Collaborative Study
- Data from 6 Labs
193
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EMMC Methods Format
William Telliard, U.S. EPA Office of Science and Technology
I. Scope and Application - Tabular format whenever possible
A. Analyte list
B. CAS numbers .
C. Matrices /=
D. Method sensitivity (expressed as mass and as concentration with a specific
sample size)
E. Data quality objectives
n. Summary of Method "
. \ .
DDL. Definitions
IV. Interferences
V. Safety
A. Above and beyond good laboratory practices
B. Disclaimer Statement (look at ASTM disclaimer),
C. Special precautions
D. Specific toxicity of target analytes or reagents
E. Not appropriate for general safety statements
VI. Equipment and Supplies
VH. Reagents and Standards
i
Vm. Sample Collection, Preservation and Storage
A. Provides information on sample collection, preservation, shipment and
storage conditions. -
B. If holding times are exceeded, data may have changed and should be
flagged for data user's attention.
IX. Quality Control
A. This section provides a summary of the QC requirements of the method.
213
-------
X. Calibration and Standardization
A. Should include calibration steps that are not followed daily; daily
calibration steps will be included in the procedure section.
XI. Procedure
XII. Data Analysis and Calculations
Xm. Method Performance
A. A precision/bias statement should be incorporated in the section, including
detection limits, and source/limitations of data.
XIV. Pollution Prevention
A. Cite good laboratory practices for pollution prevention.
XV. Waste Management
A. Cite how waste and samples are properly disposed.
XVI. References
A. Source documents ,
B. Publications.
XVn. Tables, Diagrams, Flowcharts, and Validation Data
A. Location of these items will be left to the judgement of the individual
work group.
(Finalized as a result of January 24, 1992, balloting by Members of EMMC Methods Integration
Panel and Work Group Tri-chairs.)
214
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Freshwater Sediment Toxictiy Assays:
Necessary and Desirable Attributes
G. Allen Burton, Jr., Wright State University, Department of Biological Sciences - Dayton, OH
I. What's Available?
H. Assay Strengths/Weaknesses
ffl. Assay Requirements
A. Necessary attributes (from a scientific perspective)
B. Desirable attributes (from a regulatory/program/project perspective)
. . - ' , ; >
IV. Examples of Assay Evaluations
V. Preliminary Recommendations
227
-------
-------
Toxicity Testing
usefulness improving rapidly
- method refinement
- short-term chronic assays
- toxicant interactions simplified
- reduced uncertainty
229
-------
Selected Freshwater Sediment
Organism Group
Amphibians
Fish
Zooplankton
Toxicity Tests
Test Species
Response Measures
Embryo-larval survival, Terata Xenopus laevis
Embryo-larval survival, Length
Weight, Terata
Survival, Reproduction
Benthic Invertebrates Survival, Size, Reproduction,
Molting, Emergence, Avoidance
Microbial
Phytoplankton
Macrophytes
Benthic Indigenous
Communities
Luminescence (Microtox)
Cell number, 14C uptake
Frond number, chlorophyll,
biomass, root and shoot length,
peroxidase
Structure indices, functional
indices, chlorophyll, respiration,
enzyme activities
Pimephales promdas
Oncornynchus mykiss
Oryzins latipes
Daphnia magna
Ceriodaphnia dubia
Brachionus sp.
Colpidium campylum
Panagrdlus redivivus
Caenorhabditis elegans
Tubifex tubifex
Stylodrihts heringianus
Pristina Iddyi
Lumbriculus variegatus
Hyaldla azteca
Diporeia sp.
Gamrnarus pulex
Gammarus fasciatus
Corbicula fluminea
Anodonta imbedllis
Chironomus tentans
Chironomus riparius
Hexagenia limbata
Hexagenia bilieata
Photobacterium phosphorewn
Selenastrum capricormttum
Lemna sp.
Hydrilla verticillata
Bacteria
Protozoan
Periphyton
Phytoplankton
Macroinvertebrate
230
-------
Optimal Tpxicity Assay Issues
Validation Resources ,
Relevance Organism availability
Sensitivity/discriminatory Laboratory resources
Exposure design Expertise
Response dynamics Expense, time
Standardization
Methods
QA/QC criteria
Adequate database
231
-------
Selection of Optimal Assessment Endpoints
Project objectives
Site characteristics .
Available methods
*
Key components represented
232
-------
Assay Sensitivity: effect vs. control or reference
Realistic protection of study ecosystem . .
(i.e., nationwide, ecoregion, site-specific)
Relevance
Validity
Significance
233
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Organism type
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Sample manipulation
235
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236
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DISCRIMINATION
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237
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Necessary Attributes
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Validity (field verified, few false +/-)
238
-------
Desirable Attributes
/
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Standardized
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Uniqueness (non-redundant)
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Significance (ecosystem, commercial, societal)
. 239
-------
What Makes An Assay Relevant?
Consider the:
Test ecosystems' characteristics
Sample manipulation artifacts
Organisms' route of exposure(s)
Organisms' ecological niche
Measured response sensitivity
. (e.g., mortality vs. reproduction)
Organism stress
. 240
-------
For example:
Trout * Carp
Hyalella * Aquatic worms (e.g., Tubifex)
Elutriate * Solid phase
Pore - Solid phase ?
Benthos - Nonbenthos ?
Lab response i- In situ response ?
241
-------
Total Quality Assurance
Study design
Sample collection/manipulation
Exposure design
Assay performance criteria
242
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Principal Components Factor Analysis:
ARCS Assay Comparison
Factor 1 Factor 2 Factor 3 Factor 4
P. .promelas (wt.) D. magna (survival) Hexagenia (survival) Hexagenia (molting)
Ii azteca (survival) C dubia (survival) C dubia (young)
D. magna (young)
P. promelas fterata)
Variance explained
40% 23% 13% n%
250
-------
Summary Ranking
(Ingersoll et al., 1992)
Protectiveness Similarity Sum
;" -
H. azteca 14-D 1 3 4
H. azteca 28-D 2 5 7
C. riparius 14-D 3 1 4
Microtox 4 2.6
D. magna 5 49
C. tentans 10-D 6 6 12
251
-------
Sediment Toxicity Sensitivity Comparisons
Twelve studies by Ankley, Burton, Cairns, Giesy, Hoke, Ross,-et al.
Comparisons of 3 to 20 assays/study
Most sensitive assays
Group A: Hyalella azteca 7-14 d survival
Chironomus riparius 7 d survival
Daphrtla magna 2-7 d survival
Group B: Ceriodaphriia dubia 7 d reproduction
Pimephales promelas 7 d larval growth
Chironomus tentans 10 d growth
A = Most sensitive in at least 2 studies
B = Second and third in sensitivity in at least 3 studies
252
-------
Test Battery Recommendations
Burton et al. Giesv-Hoke flC ITC
Daphnia magna (48 h)
/ (7d)
-'- .'(or)
Ceriodapnnia dubia (7 d)
Hyalella azteca (7-14d)
(28 d) (or), ' *
Chironomus riparius (10 d) (or)
(or)
Chironomus tentans (10 d) . , *
(or) (or)
Hexaeenia limbata (10 d) *
Microtox
J \
Selenastrum capricornutum
Algal fractionation bioassay
Ames
253
-------
General Purpose Short List
I. a. Microtox (screen in tandem)
b. Ceriodaphnia dubia or Daphnia magna (3 brood)
c. Hyalella azteca, Chironomus tentans or C. riparus.
or Hexagenia limbata (7-14 day)
n. a. Pimephales promelas (early life stage)
b. Selenastnim capricornutum
ffl. Other assays in tandem with above
254
-------
Extreme
Slight
I
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Need to Integrate Assessment Tools
Burton & Scott 1992)
255
-------
Conclusions
Consider all assessment issues '
Test multiple, relevant species
Test multiple trophic levels
Validate laboratory responses
Use proven methods
Expect site variance
256
-------
Midge Whole Sediment Bioassays
John P. Giesy andJody A. Kubitz, Michigan State University - East Lansing, MI
. midge Chironomus tentans has been effectively used as a bioassay organism
to predict the toxicity of sediments to benthic organisms. This organism is easy to culture and
maintain and standardized protocols exist to produce a continuous supply of known-aged
individuals. The organisms are hardy and easy to manipulate in bioassays. Growth of ^
tentans is a sensitive measure of response, which gives good discrimination power among
^edim^nts Studies have been conducted to calibrate the response of the survival and growth
studies to community structure, and some information on the relative sensitivity of the midges
to chemicals is available.
There are several issues that need to be addressed if C, tentans assays are to be adopted
for routine use. Theses include: more studies of the relative sensitivity of the midges to
chemicals; more comparisons between laboratory and field studies; investigations of the genetic
drift of laboratory cultures; and comparisons of partial life cycle tests with whole life tests for
toxicants of several modes of action.
257
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Desirable and Necessary Attributes for Freshwater Sediment Toxicity Tests:
Hyaletla azieca , ,
Chris Ingersoll, U.S. Fish and Wildlife Service, NFCR - Columbia, MO
*
I. Objectives
A. Life history of HyjteHa azteca
B. Culture and test methods
C. Research needs for standard development .
H. HvaleUa azteca Life History
A. Species: HyaMla azteca (Saussure; talitrid amphipod)
B. Habitat: lakes, ponds, streams
C. Distribution:, North America and Caribbean
D. Salinity: Euryhaline; fresh .water up to about 22 g/L, culture 10 to 15 g/L
E. Life stages: Immature (1st 5 instars), juvenile (6th & 7th instar), adult (8th instar
and older; about 35 d at 20 degrees C) ,
F. Growth: Indeterminant; male larger than female; male enlarged gnathopods
G. Feeding: Omnivore; bacteria and algae < 65 um
H. Behavior: Epibenthic, burrow in sediment w/o vegetation
in. HvaleUa azteca Culture Methods
A - Flow: Static renewal, or flow through
B. Temperature: 20 to 25 degrees C
C. Light: 16:8 photoperiod; 50 to 100 ft. candles
D. Chamber: 1 L to 100 L
E. Age of animals: Known age vs. mixed age
F. Water quality: Soft water (ERL-Duluth) vs. hardjvater (ERL-Corvalis) strain
G. Aeration: Moderate
H. Feeding: Maple leaves. Tetramin. Rabbit Chowj diatoms
I. Substrate: Maple leaves. Mitex screen, cotton gauze, 3-M base web plastic
269
-------
IV. Hvalelia azteca Test-Methods
A. Flow: Static, renewal, flow through
B. Temperature: 2Q to 25 degrees C
C. Light: 16:8 photoperiod; 25 to-50 ft. candles
D. Chamber: 25 mL, 1 L. up to 100 L
E. Sediment ration: 1:1 to .1:4 ratio sediment: water
F. Age of animals: Known age (0 to 7 d, 7 to 14 d) vs. mixed age (size about 7 to
14 d)
G. No. animals: 5 to 20/beaker; 4 to 5 replicates/treatment
H. Duration: 7 d, 10 d. 14 d, 28 d
I. Endpoints: Survival, length, weight, sexual maturation (males), young production
J. Water Quality: Soft water (ERL-Duluth) vs. hard water (ERL-Corvallis) strain
K. Aeration: None or moderate
L. Feeding: None, Rabbit Chow, YCT, maple leaves, Tetramin
M. Acceptability: survival (80%). length, weight
N. Particle size: low sensitivity (with sufficient food)?
O. NH3 and H2S: low to moderate sensitivity?
P. Sediment contact: Mayflies = Midges > Amphipods > Daphnids
Q. Sensitivity: Daphnids > Amphipods = Mayflies > Midges
R. Reliability: Amphipods = Daphnids > Midges > Mayflies
V. Research Needs for Standardization of Hvalelia azteca
A. Culture and testing: know-age and' feeding
B. Culture and testing: reconstituted water
C. Reconstituted sediment
D. Abiotic factors
E. Reference toxicants
F. Species and strain sensitivity
G. Inter-laboratory comparisons
H. Life history and chronic indicators of toxicity
I. Spiking methods and positive controls
J. Dilution studies and mixtures
K. Laboratory to in situ comparisons
270
-------
OBJECTIVES
Life history of Hvalella azteca
Culture and test Methods
Research Needs for Standard Development
271.
-------
HYALELLA AZTECA LIFE HISTORY
Species:
Habitat:
Distribution:
Salinity:
Hvalella azteca (Saussure; talitrid amphipod)
lakes, ponds, streams
North America and Caribbean
Euryhaline; fresh water up to about 22 g/L
Culture 10 to 15 g/L
272
-------
HYALELLA AZTECA LIFE HISTORY (cont.)
Life stages:
Growth:
Feeding:
Behavior:
Immature (1st 5 instars) " '
Juvenile (6th and 7th instar)
Adult (8th instar and older; about 35 d at 20ฐC)
Indeterminant; male larger than female; male enlarged gnathopods
Omnivore; bacteria and algae < 65 urn
Epibenthic; burrow in sediment w/o vegetation
273
-------
HYALELLA AZTECA CULTURE METHODS
Flow:
Temperature:
Light:
Chamber:
Static, Renewal, or flow through
20to25ฐC
/
16:8 photpperiod; 50 to 100 ft. candles
1 L to 100 L
274
-------
HYALELLA AZTECA CULTURE METHODS (cont.)
Age of Animals: Known age vs. mixed age
Water Quality:
Aeration:
Feeding:
Substrate:
Soft water (ERL-Duluth) vs. hard water (ERL-
Corvallis) strain
Moderate
Maole leaves. Tetramin, Rabbit Chow, diatoms
Maple leaves. Nitex screen, cotton
gauze, 3-M base web plastic
275
-------
HYALELLA AZTECA TEST METHODS
Flow:
Temperature:
Light:
Chamber:
Sediment ratio:
Age of animals:
No. animals:
Static. Renewal, flow through
20to25ฐC
16:8 photoperiod; 25 to 50 ft. candles
25 ml, 1_L, up to 100 L
1:1 to 1:4 ratio sediment: water
Known age (0 to 7 d, 7 to 14 d)
vs. mixed age (size about 7 to 14 d)
5 to ZQ/beaker; 4 to 5 replicates/treatment
2-76
-------
HYALELLA AZTECA TEST METHODS (cont.)
Duration:
Aeration:
Feeding:
7d. 10d.14d, 28 d
Endpoints: SyiylvaJ, length, weight, sexual maturation (males), young production
Water Quality: Soft water (ERL-Duluth) vs. hard water (ERL-Corvallis) strain
None or moderate
None, Rabbit Chow, YCT, maple leaves, Tetramin
Acceptability: Survival (80%). length, weight
277
-------
HYALELLA AZTECA TEST METHODS (cont.)
Particle size: Low sensitivity (with sufficient food)?
NH3 and H2S: Low to moderate sensitivity?
Sediment contact: Mayflies = Midges > Amphipods > Daphnids
Sensitivity: Daphnids > Amphipods = Mayflies > Midges
Reliability: Amphipods = Daphnids > Midges > Mayflies
278
-------
RESEARCH NEEDS FOR STANDARDIZATION OF HYALELLA AZTECA
CULTURE AND TESTING: KNOWN-AGE AND FEEDING
CULTURE AND TESTING: RECONSTITUTED WATER
/ ., i ,
RECONSTITUTED SEDIMENT
ABIOTIC FACTORS
REFERENCE TOXICANTS '
SPECIES AND STRAIN SENSITIVITY
279
-------
RESEARCH NEEDS FOR STANDARDIZATION OF HYALELLA AZTECA (cont).
INTER-LABORATORY COMPARISONS
LIFE HISTORY AND CHRONIC INDICATORS OF TOXICITY
SPIKING METHODS AND POSITIVE CONTROLS
DILUTION STUDIES AND MIXTURES
LABORATORY TO IN SITU COMPARISONS
280
-------
Discussion of Desirable and Necessary Attributes for Marine and Estuarine
Sediment Toxicity Tests, and the Use of Ampelisca abdita. Rhepoxvnius
abronius. Leptocheirus plumulosus. and Eohaustorius estuarius in Marine and
Estuarine Sediments
Richard C. Swam, U.S. EPA Environmental Research Laboratory - Pacific Division
I. Description of Acute Amphipod Sediment Toxicity Test
n. Research and Regulatory Applications
m. Limitations and Advantages
, " ' " ' ' ' ' ' ' ; ' v - '
IV. Necessary and Desirable Attributes of Acute Sediment Toxicity Tests
A. Species selection
1. Relative sensitivity (field and lexicological data)
a. Other species
b. Size
c. Sex
2. Ecological importance/relevance
3. Economic importance ,
4. Habitat -,.'.'.'
5. Substrate relation
a. Pelagic
b. Epibenthic
c. Infaunal - tube dwelling
d. Infaunal - free burrowing
6. Availability
, a. Field collection ,
b. Culture method
7. Laboratory compatibility .
8. Zoogeography
9. Compatibility with bioaccumulation/chronic tests
281,
-------
B. Method development and standardization
. 1. Written standard method
2. Sediment toxicity database
a. Field sediment
b. Spiked sediment
3. Control responses
a. QA/QC - collection/culture sediment
b. QA/QC - reference toxicant
c. Experimental - negative
d. Field - reference sediment
4. Statistical power
5. Tolerance limits of species/method
a. Sediment grain size
b. Temperature
c. Salinity
d. Sediment organic enrichment
e. Ammonia
f. Seasonality
6. Sediment collection/processing/storage
7. Field validation
8. Interlaboratory comparison
V. Amphipod Species
A. Rhepoxynius abrom'us
B. Ampelisca abdita
C. Eohaustorius estuarius
D. Leptocheirus plumulosus
VI. Chronic Test Methods
. Other Species
282
-------
TABLE IPrecision of the benthic bioassay in relation
to sample size and replication.*
Number of Amphipods per Replicate
10 20
No. of
Replicates
2
4
6
8
10
12
14
16
18
20
6
6.80
2.66
1,94
1.60
1.38
1.25
1.14
1.05
0.98
0.93
6/c
71.6
28.0
20.4
16.8
14.5
13.2
12.0
11.0
10.3
9.8
No. of
Replicates
2
3
4
5
6
7
8
9
10
(
a
8.55
4.44
3.35
2.80
2.45
2.20
2.02
1.89
1.76
d/c
45.0
23.4
17.6
14.7
12.9
11.6
10.6
10.0
9.3
a6 is the difference between two survival means for which the bio-
assay is 75% certain of detecting statistical significance (P < 0.05)
[76]. 6/c expresses the precision estimate as a percent of the normal
control survival (c = 19.0 for n = 20; c = 9.5 forซ = 10).
283
-------
FIELD VALIDATION
PARAMETER
BENTHIC BIOMASS
BENTHIC DENSITY
SPECIES RICHNESS
AMPHIPOD DENSITY
RHEPOXYNIUS DENSITY
EH
BOD
OIL/GREASE
CADMIUM
CHROMIUM
DEHP
DDE
Toxic
SITES
9.4
450
21
0,8
0
-54
13
15
29
670
16
5
NON -Toxic
SITES
50.3
2000
70
33
23
+223
4
4
11
370
4
3
284
-------
1. Written Protocol
Biology of Test Species.
. all1 species selection factors
Limitations of Method
environmental factors (grain size, salinity, etc.)
variability/statistical power , .
field relevance
Logistics >
exposure chamber ,
duration , ' . -
sequence of events
quarantine
285
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290
-------
Other Species
Grandidierella japonica
T.epidactvlus dytiscus
CoroDhium spp
Neanth.es ar.enaceodentata
Meiofauna , ; ,
other Tests
Microtox .. - , ,
- Bivalve Larval Survival/Growth/Development
Echinoderm Larval Survival/Growth/Development
Benthic Recolonization
291
-------
Issues and ResearchNeeds
Written Protocol - ASTM/EPA format
Culture Protocol: Leptocheirus/Ampelisca
Sensitivity of Cultured vs Field-collected Amphipods
* Shipping/Handling/Acclimation
Nutrition
Comparative Toxicology
* Reference Toxicant Control
* Reference Sediment QA/QC
Statistical Power: Compare variability among species
Tolerance Limits
* Ammonia
* Grain.Size, except Rhepoxynius
Organic Enrichment
Salinity, except Rhepoxynius
Seasonality
Light: Intensity/photoperiod
* Field Validation '
* Interlaboratory Comparison
* Sediment Collection, Processing, Storage
292
-------
Chronic Test Methods
Test Species
Leptocheiriis plumulosus. Ampelisca ' abdita
Response Criteria
Mortality, Growth, Reproduction, Population
Dynamics
Key Issues .
Nutrition
' Narrow Tolerance Limits .
'n Chronic Control QA/QC " . , "
Relative Sensitivity of Acute and Chronic Tests
293
-------
-------
Bioaccumulation of Sediment-Associated Contaminants:
Significance, Current Status, and Future
Peter Landrum, NOAA Great Lakes Environmental Research Laboratory - Ann Arbor, MI
I. Significance of Bioaccumulation
A. Role in toxicity assessment
1. Aquatic species
2. Human health .
n. Picture of the Problem
A. Complexity of the exposure,environment
B. Current mechanistic model
El. Criteria for Bioaccumulation Organisms
A. Examples of organisms
* - . '
IV. Factors Affecting Bioaccumulation
-'.".,
A. External factors ;
B. Physiological factors
1. Behavior
a. Importance of feeding
b. Feeding mechanism
c. Feeding selectivity
V. Status ,
A. Field data
VI. Future - Kinetics
A. Models -
B. Field validation
295
-------
-------
BIOACCUMULATION
The accumulation of contaminants from all sources,
food and water.
BIOACCUMULATION FACTOR
The ratio of the steady state concentration in the
organism resulting from multiple source accumulation
to one of the source concentrations
297
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Accumulation Factors
For PCB Congeners
52 101 151 118 153 138 128 180 195 194
lUPACNo.
N. incisa
Glycera
M. mercenana
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Bioaccumulation of Sediment-Associated Contaminants:
Present Status, Laboratory Methods, and Related Research Needs
Henry Lee II, U.S. EPA Environmental Research Laboratory - Pacific Division
I. Important of Bioaccumulation of Sediment-Associated Compounds
n. Methods to Measure/Predict Bioaccumulation of Sediment-Associated Compounds
A. Criteria to choose among the methods
HI. Equilibrium Participating Bioaccumulation Model
A. Use as screening tool
B. Limitations/uncertainties with equilibrium predictions
C. Lab/kinetic alternatives to partitioning paradigm
IV. 28-Day Bedded Sediment Bioaccumulation Test
A. Status of "standard 28-day" sediment bioaccumulation test
B. Test duration ,
. " 1. Why need a 28-day test vs. 10-day test
2. Adequacy of 28-day tests for slowly accumulated compounds
C. Organism selection
1. Selection criteria .'.,",
2. Why need sediment ingesting organism
3. Recommended bioaccumulation species
D. Laboratory methods
1." No feeding of test organisms
E. Experimental design
1. Number of replicates and statistical power
2. Pseudoreplication >
F. How proposed methods differ from those in the "green book"
V. Toxicokinetic Bioaccumulation Models/Tests
A. Equilibrium vs. non-equilibrium exposures
'. B. Modifications of laboratory procedures .
315
-------
VI. Evaluation of Bioaccumulation Results
A. . Criteria .for "reference" sites
VH. Research Needs
A. Related to 28-day bioaccumulation test
B. Field validation
C. Round robin
D. Lipid methods
E. Research needs related to other methods of predicting bioaccumulation
F. Resuspended sediment tests
316
-------
317
-------
PREDICTING BIO ACCUMULATION OF SEDIMENT -
ASSOCIATED POLLUTANTS BY INFAUNAL ORGANISMS
Field Approach:
measure tissue residues in species collected
at surrogate site
Bioaccumulation Test:
measure tissue residues in species exposed to
sediment collected at a surrogate site
Bioaccumulation Factors (BAF):
ratio of concentrations in tissue to sediment .
Thermodynamic Partitioning Model:
based on chemical partitioning normalizing
for lipids and TOC but only for neutral
organics
1st Order Kinetic Model:
tissue residues modeled as balance between
rate of uptake and rate of loss
Toxicokinetic Model:
tissue residues modeled as function of
bioenergetics of the organism
' 318
-------
Figure 2.1: Summary of questionnaire choices. Numbers above decision boxes refer to
questionnaire choices.
I'.'''1" ' '"
Predict or model tissue residues
1 Use EqPB Model 1
Section 4 1
Compound is
polar organic?
12
I UseBAFmc
1 Section 3
' """"^
Use a standard model to
residues.
s metal or Yes
__^__ Go to Next Page for Field or Lab
questions (15) ' ' , '
Yes . " '
predict
Model used as quick screening tool
for bioaccumulation?
Yes "' ' . 5'
xiel, 1
Model more man one u]
(as opposed to modeling
from bedded sediment?
6
No
stake route
uptake only
1 Collect field s:
regression moc
implcs to derive a 1
lei. Section 11. 1
. .
Iljsc bioenerge
toxicokinetic u
No 7
Will sediment pollute cone, change?
8
No
Predict time to specific tissue
residue value (time to steady-state
or elimination time)?
Kre site-speeificYAAFs
/
11
No
Are specific k2 and ks Yes
values available?
'Yes
9
No
Will organism grow substantially
during test or modeling?
10
Yes Changes in test
are likely due t
characteristics.
Yes 1 Use Ist-order
1 Section 6
No
Is compound metal or polar
organic?
,'..
14
Is measured AF available for
compound and sediment of concern?
1 Use Ist-order kinetic 1 '
model 1
Section 6 |
ics-based ' 1
nodel. Section 5. 1
'Yes '-..-.'
organism behavior
o changes in sediment
No .
kinetic model 1
Yes
No Specific ks and k2 values are both
available
Yes .
[Use EqPB Model 1
Section 4 |
Accurate est
(box 4) or n
No
imate not possible. 1
screening prediction 1
lake site-specific 1
t fRnr 1 SI I
319
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320
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. Table 4.1 Continued ,
COMPOUND
HCBP
HCBP
HCBP
HCBP
HCBP
, HCBP
HCBP
HCBP
HCBP
HCBP
HCBP
HCBP
HCBP
HCBP
HCBP
'HCBP
HCBP
HCBP
HCBP
HCBP
'' , HCBP
HCBP
HCBP
HCBP '
HCBP
PESTICIDES
a-chlor
a-chlor
, ; g-chlor
g-chlor
DDD
DDD
DDD
DDD
DDD
DDD
DDD
DDD
DDD
DDD
DDE
DDE
DDE
DDE
DDE
1 DDE
- - DDE '
DDE
DDE
ORGANISM
Yoldia limarula
Yoldia limatuia
Yoldia limatuia
Yoldia limatuia
Nephrys incisa
Nephtys incisa
Nephrys incisa
Nephrys incisa
Nephrys incisa
Nephrys incisa
Gtycera sp.
Mercenaria mercenaria
Nereis virens
Nereis virens
Nereis virens
Nereis virens
Macoma nasuta
Macoma nasuta
Macoma nasuta
Macoma nasuta
Macoma nasuta
Macoma nasuta
Diporeia sp.
Oligochaetes
Palaemonetes pugio
Bivalve Mean
Polychaete Mean
' OveraU Mean
Yoldia limatuia
Nephrys incisa
Yoldia litnatula
Nephtys incisa
OveraU Mean
Macoma nasuta
Macoma nasuta
Macoma nasuta
Macoma nasuta ' .
Macoma nasuta
Macoma nasuta
Yoldia limatuia ^"
Yoldia limatuia
Nephrys incisa
Nephtys incisa
Bivalve Mean
Polychaete Mean
Overall Mean
Macoma nasuta
Macoma nasuta
Macoma nasuta
Macoma nasuta
Macoma nasuta
Macoma nasuta
Oligochaetes
Oligochaetes
Oligochaetes
Bivalve Mean
Overall Mean
'
AF
1.7
0.9
S.26
5.1'
0.4-
0.2
16.6'
7.1'
7.1'
3.81
3.9
1.5
4.8.
1.4 ',
4.8
1.4
4.8
0.5
1.6
2.7
, 4.0
1.8
0.7s
1.2"
2.1
4.4
4.7; '
4.4
4.0
4.2
4.5
5.9
4.7
1.0
0.4
0.7
0.7
0.5
0.5
4.0 ,
4.2
4.2
4.8
1.5
4.5
,2.1
2.8
0.7
1.3
1-1
0.7.
1.1
0.9'ฐ
1.0'ฐ
1.3
1.3
1.2
TOC% .
1.9
4.0
1.9
4.0
1.1 '
5.7-
1.1
0.8 ,
1.3
2.5
5.7
0.7
" 3.6
5.7
0.9 .
0.8
3.7
4.0
5-l
7.4
0.9
0.8
3.7
4.0
5.1
7.4
3.6
4.6
6.0
321
LIPID%
7.6
4.6
4.6
4.6
1.3
6.6
1.6
5.5
5.5
5,5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
6.6
6.6
6.6
' .i
REFERENCE ,
McElroy and Means, 1988 ' :
McElroy and Means,' 1988 . .
Lake et al.. 1990
Lakeetal.. 1990
McElroy and Means. 19S8 '
McElroy and Means. 1988
Lake et al.. 1990
Lake et al., 1990
Lakeetal.. 1990 .
Lake et al.. 1990
Lake et al:. 1990
Lake et al., 1990
' Brannonet al., (manuscript) '
Brannonetal., (manuscript)
Brannon et al.. (manuscript)
Pruell et al.. (manuscript)
Pruell et al., (manuscript)
Pruell et al., (manuscript)
Boese et al.. (manuscript)
Boese et al., (manuscript)
Boese et al., (manuscript)
Pruell et al., (manuscript)
Landrum (pers. comzn.) .
. Olivier, 1984
Pruell et al., (manuscript)- <
Lake et al.. 1987 ' '. ,'
. Lakeetal.. 1987 '
Lakeetal.. 1987
Lake et al., 1987
Ferraro et al., 1990
Ferraroetal., 1990
Ferraroetal.. 1990
Ferraroetal., 1990
Ferraro et al.. 1990
Ferraro et al.. 1990 ,
Lake et al.. 1987
Lake et al., 1987 ' .
Lakeetal., 1987
Lakeetal., 1987
Ferraro et al., 1990
Ferraro et al., 1990
Ferraro et al., 1990
Ferraroetal.. 1990
Ferraro et al., 1990 '
Ferraro et al.. 1990
Olivier. 1984
Olivier, 1984
Olivier, 1984
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Idealized Uptake. - Elimination Curve
' Cf(f).= Cs * Ks/|<2 ( l-e~k2' ')
Uptake Phase 'Elimination Phase
.-Cs = k 2* CI ;
Sleody-slale \ Fosf component
Linear uptake phase
Cs ป k.
CI
Slow component
I
(Contaminated Sediment) \ (Clean Sediment)
Ci = tissueconcentration (ug/g)
Ks = sediment uptake rote coetd'cienl (
Cs = sediment concentration (ug/g)
k2 = elimination constant (I/time)
f = time
edimcn> .
* ''mฐ
Figure, 2. Idealized uptake elimination curve.
BIOENERGET1C-BASED TOXICOKINET1C MODEL
MAJOR UPTAKE ROUTES FOR DEPOSIT-FEEDERS
Sorplion/Dermol Uptoke
Tissue Residue Ow + fw 4- Si . * Ss + SprpJiori Eliminalion
/ Flux
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\ ( Pbllulanl
; ' V Cone.
.V ( ExIrocJion \
/ "\ CffJciency ./
/
Flux Sgdtment = /fWgtqhl. Activiiy. TQCJ
Ss Susp-ended Solids Up-lako Roule
Qw = Ovgrlyin-q Wgter Up-ltrtte Rrrule
Si = lh'g*S'Jซ''d Se-dim-e-rH Upซcrtce R:ovte
(w =
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EVALUATION OF DREDGE MATERIAL BEDDED -
SEDIMENT BIOACCUMULATION TEST
OBJECTIVE: Measure tissue residues in infaunal
organisms resulting from exposure to dredge
material
PREVIOUS APPROACH: 10-day bedded-sediment
bioaccumulation test to estimate
"bioaccumulation potential"
"i
PRESENT APPROACH: 28-day bedded-sediment
bioaccumulation test to estimate "steady-state"
tissue residues (10 day - test if only metals
present). Used in Tier HI
STATUS: EPA Guidance Document produced and
referenced in Implementation Manual. Guidance
document in ASTM review
326
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ERLN-N111
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UssyEPA
GUIDANCE MANUAL:
BEDDED SEDIMENT
BIQACCUMIILAHOJNL TESTS
U:S. EnvironmentarPr otec tioh "Agency
ERL-N
Pacific Ecosystems Branch
Bioaccumulatlon Team
Newport, OR/
328
-------
Sediment Bioaccumulation Test
Key Procedures
1. 28-DAY EXPOSURE DURATION.
' * '
2. SEDIMENT-INGESTING ORGANISM
REQUIRED.
3. NO SUPPLEMENTAL FOOD USED.
4. S P E C I E S EXPOS ED
INDEPENDENTLY.
5. 80% OF STEADY-STATE TISSUE
RESIDUES RECOMMENDED
ACCURACY.
6. L Q.N.-G -TERM T E S T S OR
TOXICOKINETIC APPROACHES USED
FOR > 80% ACCURACY OR SLOWLY
ACCUMULATED COMPOUNDS.
329
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Appendix Figure 1: Acceptable and Unacceptable Reference
Sediments. (A) Acceptable reference sediment with test confidence
intervals not bracketing the tissue criterion. (B) Acceptable
with test confidence intervals bracketing the tissue criterion.
(C) Unacceptable because reference confidence intervals overlap
the confidence intervals of a mean test residue exceeding the
criterion. CD) Unacceptable because reference confidence
intervals exceed the tissue criterion (test confidence intervals
ฑnBnarerฑarrr~~Th'e points "represenfth'e tissue residues "that "would
result in organisms exposed to a particular sediment
concentration. A BAF of 2 was used for illustrative purposes
only.
339
-------
vs..
"Guidance Manual" and "Synthesis of Methods"
GUIDANCE/SYNTHESIS DOCUMENTS
DREDGE MANUAL
EqP BIOACCUMULATION MODEL SCREEN
FOR NEUTRAL ORGANICS
"THEORETICAL BIOACCUMULATION
POTENTIAL"
GOAL OF TEST = BIOACCUMULATION
POTENTIAL"
10 DAYS FOR METALS
28 DAYS FOR ORGANICS
N = 5
. EqP SCREEN FOR NEUTRAL ORGANICS
BAF'S SCREEN FOR METALS/POLAR
EQUIVALENT TO USING EqP
BIOACCUMULATION MODEL WITH AF = 4
GOAL OF TEST = TISSUE RESIDUES
> = 80% OF STEADY STATE RESIDUES
28 DAYS FOR ALL COMPOUNDS AS
"STANDARD" SINGLE POINT ESTIMATE
REPLICATION (n~ =8) BASED ON:
TYPE I ERROR = TYPE II ERROR
DETECT 2-FOLD DIFFERENCE
INCLUDES FILTER-FEEDING ORGANISMS ONLY SEDIMENT-INGESTING ORGANISMS
TO CONTROL SAMPLES ANALYZED IF
"DISCREPANCIES"
ALWAYS PURGE FOR 24 HOURS '
CONDUCT TIER IV KINETIC OR FIELD
EVALUATION ONLY IF FAIL TIER III
KINETIC TEST OR FIELD EVALUATION IF
FAIL TIER IE
KINETIC APPROACH WITH kl AND k2
ESTIMATED FROM UPTAKE CURVE ONLY
NO QUANTITATIVE CRITERIA FOR
CONTROL SEDIMENTS
NO QUANTITATIVE CRITERIA FOR
REFERENCE SITES
TO CONTROL SAMPLES ALWAYS
ANALYZED
PURGE FOR 24 HOURS EXCEPT FOR
TROPHIC TRANSPORT STUDIES AND
RAPIDLY METABOLIZED COMPOUNDS
KINETIC, LONG-TERM EXPOSURES, OR
FIELD STUDY DEPEND ON GOALS,
COMPOUNDS, RESOURCES, AND
ACCURACY
(FOCUS ON EFFECTS OF TISSUE RESIDUES
RATHER MORE ACCURATELY ESTIMATING
TISSUE RESIDUES)
KINETIC APPROACH WITH kS (=kl) AND k2
ESTIMATED INDEPENDENTLY
GUIDELINES FOR ACCEPTABLE SEDIMENT
POLLUTANT CONC. IN CONTROLS
STATISTICAL CRITERIA BASED ON
OVERLAP WITH TISSUE RESIDUE
"CRITERIA"
340
-------
-------
342
-------
BEDDED SEDIMENT BIOACCUMULATION TEST
RESEARCH NEEDS
I. INTERLABORATORY ROUND ROBIN
H. FIELD VALIDATION
IE. ADEQUACY OF 28 DAY TESTS
IV. REFINEMENT OF CULTURING AND EXPOSURE METHODS FOR
STANDARDIZED SPECIES
1. BIOLOGICAL: TEMPERATURE, SALINITY, GRAIN SIZE,
TOC
2. EXPOSURE: SEDIMENT/ORGANISM MASS, GUT PURGING
V. EFFECTS OF SEDIMENT COLLECTION, STORAGE, AND SPIKING ON
BIOAVAILABILITY
VI. "STANDARDIZATION" OF LIPID METHOD
VH. TESTING/GUIDANCE FOR NEED FOR 2 BIOACCUMULATION TEST SPECIES
'
VIE. DEVELOPMENT OF REFERENCE BIOACCUMULATION SEDIMENT
K. REFINEMENT OF STATISTICAL DESIGNS
/. ' ' - .
X. DEVELOPMENT /VALID ATIONOF KINETIC ALTERNATIVES TO 28-DAY
TEST
XI. TEST SPECIES FOR SUBTROPICAL SUBARCTIC, AND OLIGOHALINE
HABITATS , .
343
-------
BIOAVMLABELITY OF SEDIMENT CONTAMINANTS
RESEARCH NEEDS
I. QUANTIFY UNCERTAINTY/ASSUMPTIONS OF EQUILIBRIUM
PARTITIONING BIO ACCUMULATION MODEL
H. DEVELOP SCREENING MODEL/APPROACH FOR METALS
m. DEVELOP METHODS TO QUANTIFY EXPOSURE, INCLUDING
INGESTED DOSE, OF BENTHIC SPECIES
IV. DEVELOP/VALIDATE TOXICOKINETIC APPROACHES FOR BENTHIC
SPECIES
V. COUPLE TOXICOKINETIC AND TOXICODYNAMIC APPROACHES TO
PREDICT TISSUE RESIDUE EFFECTS ON BENTHOS
VI. PREDICT TISSUE RESIDUE EFFECTS FROM "WEIGHT OF EVIDENCE"
OR USE AS INDEPENDENT VARIABLE IN AET'S
344
-------
(log) RISK
-------
-------
Discussion of the Use of Lumbriculus variegatus
in Freshwater Sediments
Gary Ankley, U.S. EPA Environmental Research Laboratory - Duluth, MN
I. Desirable Attributes in Selection of Species for Sediment Bioaccumulation Testing
A. Readily available
B. Known exposure history
C. Adequate tissue mass for trace analyses
D. Easily handled
; E. Amenable to long-term exposures -,.-" .
F. Reflect concentrations of contaminants in field organisms (i.e., exposure is
realistic)
G. Tolerant of a wide range of physico-chemical conditions in sediments (e.g.,
particle size) ,
n. Freshwater Species Uses for Sediment Bioaccumulation Testing
A. Chironomids
B. Amphipods "., .''.
C. Mayflies
D. Clams
E. Fishes
F. Oligochaetes" . .
ffl. Oligochaetes as Bioaccumulation Test Species
A. Certain species easily cultured and, therefore, readily available with known
exposure history
B. Provide adequate tissue mass for trace residue analysis
C. Can be used in long-term exposures
D. Easily handled and tolerant of a wide range of physico-chemical conditions
E. Realistic exposure to sediment-associated contaminants
TV. Attributes of Lumbriculus variegatus
A. Relatively large (-5-10 mg/organism) V
B. Easily .handled ',. ,
C. Tolerant of wide range of physico-chemical conditions (e.g., DO, particle size)
D. Tolerant of many contaminants-
E. Can be used in long-term tests
F. Standard culture and test methods have been developed /
G. Some field validation. '>..-.
347
-------
V. Lumbriculus variegatus in Aquatic Toxicological Studies
A. Single chemical toxicity testing (Bailey and Liu, 1980; Ewell et al., 1986;
Nebekeretal., 1989)
B. Sediment toxicity testing (Ankley et al., 1991a; 1991b; Gall et al., 1991; Carlson
et al., 1991; Phipps et al., 1992; West et al., 1993; Dermott and Munawar, 1993)
C. Metal bioaccumulation studies (Ankley et al., 199Ib; 1993; Carlson et al., 1991)
D. Nonionic organic bioaccumulation studies (Ankley et al., 1992; Call et al., 1991;
Nebeker et al., 1989; Schuytema et al., 1990)
348
-------
,, Identification of Long Term Needs for Assessing Sediments
Norm Rubinstein, U.S. EPA Environmental Research Laboratory - Narragansett, RI
I. Major Goals
A. Identify "problem" sediments
- f
B. Assess potential impact of contaminated sediments on aquatic habitats,
wildlife, and human health
C. Remediate contaminated sediment sites in a cost-effective and
environmentally consistent manner :.'.-'
n. Scientific Questions and Research Needs
A. What are the most technically valid and cost-effective approaches for
deriving sediment quality criteria?
B. How can we best identify and quantify contaminated sediment exposure
regimes?
C. What are the key physico-chemical factors controlling the biological
availability of sediment-^associated contaminants?
D. What is the ecological and human health significance of sediment mediated
tissue residues in aquatic food chains?
E. What kinds of assessment methods are needed to best identify ecologically
relevant endpoints and how can these techniques fit within a tiered testing '
strategy for eco-risk assessment?
F. Which specific fractions or individual constituents of sediment-associated
pollutants are of significant lexicological concern?
G. What are the best approaches for identifying and cleaning up contaminated
sediment sites?
H. To what extent is natural recovery sufficient to remediate contaminated
sites?
349
-------
Research Areas
A. Sediment quality criteria
B. Contaminated sediment assessment methods
* '
C. Remediation technology
D. Monitoring
IV. Sediment Quality Criteria (SQC)
A. Field validation of EqP approach
B. SQC development for ionizable organic and metallics (e.g., AVS
approach)
C. Development of SQC tissue residue approach to address wildlife and
human health concerns
V. Exposure Assessment
A. Exposure assessment modeling for aquatic disposal of dredged materials
B. Wasteload allocation models to evaluate contaminated sediments and
source control options
C. . Chemical analytical methods development
VI. Effects Assessment
A. Development and validation of acute and chronic testing protocols for
contaminated sediments in freshwater and marine systems
B. Development and validation of contaminated sediment bioaccumulation test
methods
C. Development of tissue residue thresholds .
D. Development of trophic transfer models for sediment mediated tissue
residues
350
-------
VH. Remediation Methods
A. Development and validate methods for in situ containment and treatment
of contaminated sediments
i.' ' * . '' .
B. Develop methods to examine rates of natural recovery for benthic
communities . ,
Vin. Ecological Risk Assessment
A. Develop methods to integrate stress response relationships for ecologically
relevant endpoints
351
-------
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Break-Out Workgroup for Freshwater Sediment Issues:
Overview of the Day
Gary Ankley, U.S. EPA Environmental Research Laboratory - Duluth, MN
I. General Discussion Format
A. Brief presentation of survey results on culturing and testing
1. Hyalella azteca
2. Chironomus tentans
3. Lumbriculus variegatus
B. Discussion of major culturing and testing issues
1. List of proposed issues (by organism)
2. Resolution (if possible) of issues
ri. Selection of Test Species
A. Current and historical technical acceptance of test organisms
B. Logistical concerns (e.g., organism availability)
C. Availability of some test methods . "
HI. Survey Techniques
A. Questionnaires on culturing/testing distributed to workshop participants and others
testing target species
B. Focused on IL azteca. C.. tentans/riparius. L^ variegatus. but other species
identified as well
C. Results summarized by issue
IV. General Culturing Issues
A. Substrate
B. Genetic drift/stream differences '
C. Density
D. Known age systems
E. Water , ,
F. Nuisance organisms
G. Flow-through vs. static - . .
H. Light/photoperiod
I. Feeds/feeding
J. Temperature
K. QA/QC (e.g., reproduction, reference toxicants)
357
-------
General Testing Issues
A. Test lengths/endpoints
B. Organism age
C. Water renewal (volumes, frequency,'method)
D. Physical test system (sediment volume, etc.)
E. Test condition and design (chambers, lighting, etc.)
F. Interpretation of sediment variables (e.g., organic carbon particle size) on test
results
G. Feeds/feeding regimes
H. QA/QC (for acceptable test)
NOTE; For discussion purposes, the proposed issues initially were ranked based upon:
Immediacy of issue ,
Generality across tests
358
-------
SURVEY RESPONDENTS
Institution
Columbia FWS
Athens FWS
Duluth EPA
University of WI-
Superior
NOAA
(Ann Arbor)
Wright State
ABC Laboratories
Environment
Canada
EVS Consultants
Region 8 EPA
Old Dominion
Cincinnati EPA
Region 1 EPA
University of
Mississippi
Michigan State
University
Maryland DE
Canada Oceans &
Fishes
Miami University
Washington State
DE
H. azteca
X
X
X
x
X
X
X
X
X
X
x
X
X
X
X
X
X
X
C. tentans/riparius
X
X
X
x
. '
X
X
x
X
>
x
x
x
x
L. variegatus
X
x
x
x
X
1
-
' ' 1
.
359
-------
-------
Development of a Standard Protocol for Testing Hvalella azteca
Teresa Norberg-King, U.S. EPA Environmental Research Laboratory - Duluth, MN
I. Summary of Culture Methods Used as Reported in Questionnaires
ri. Summary of Test Methods Used as Reported hi Questionnaires
HI. Proposed Key Issues for Discussion
361
-------
-------
RANKED ISSUES FOR CULTURING H. AZTECA
Known Age Systems
Feeds/Feeding Regimes
Water (Reconstituted vs. Surface Waters)
Flow-through vs. Static
QA/QC (e.g., Reproduction, Reference
Toxicants)
Genetic Drift/Strain Differences
Substrate
Density
Nuisance Organisms
' Light/Photoperiod
Temperature
Other
363
-------
RANKED ISSUES FOR TESTING H. AZTECA
Test Lengths/Endpoints
[
Organism Age
Water Renewal
(Volumes, Frequency, Method)
Interpreting Effects of Sediment
Variables on Test Results
(e.g., organic carbon, particle size)
Feeding in Tests
QA/QC (Criteria for Acceptable Test)
Test System
(e.g., Chamber Size,
Construction, Replicates,
Temperature, Light/Photoperiod,
et cetera) ,
Water and Sediment-Quality Monitoring
Sediment Volumes (Physical test system)
Other _
364
-------
Frequency of Culture Re-starts
1 month 1 '' ,
2 month 6
3 month 1
4 month 1
continuous 1
quarterly 2
2x/year
variable
365
-------
Variety of Foods Tried (listed singly)
yeast
Cerophyllฎ
wheat grass
Chlorella
diatom (Spirulina)
alfalfa grass
Tetraminฎ
Nutrafinฎ
YCT
paper towels
S. capricornutum
Ankistrodesmus
maple leaves
paper towels innoculated with Tetramin
rabbit food
brine shrimp
astroturf
aquatic plants
sediment
366
-------
Feeding Frequency in Cultures
Flow-through cultures
Static cultures
3x/wk 1
1x/d 1
1x/wk 1
Ix/mpnth 1
3x/wk 3
2x/wk 8
1x/wk 2
1x/day 1
2x/day 1
as nee 1
367
-------
Waters Used for Culturinq
tap 7
well 4
surface 3
reconstituted 3
mix of sources 1
368
-------
Foods fed Cultures of:
Laboratory
Food(sl> Using
How Long
ABC Lab.
Dept Fish/Oceans
Environ. Canada
EPA-Duluth
EPA Region 1
EPA Region 8
EPA Newtown
EVS Consultants
MD Dept. Env.
Miami Univ.
Mich. State
NFCRC-Athens
NFCRC-CoIumbia
Old Dominion
State of Wash.
Univ. of Miss.
Univ. of WI-Sup.
Wright State
Yeast, Cerophyllฎ, Chlorella,
wheat grass, diatom, alfalfa
Tetraminฎ flakes
Nutrafinฎ flakes
YCT & diatoms
rabbit pellets
paper towels &
flake fish food
Cerophyllฎ &
S. capricornutum
YCT and S. capricornutum
Tetraminฎ & leaves
digested paper towels '
inoculated with Tetramin
Tetraminฎ
leaves
leaves & Tetraminฎ
leaves & rabbit chow
rabbit food & leaves .
leaves & rabbit chow
YCT & Ankistrodesmus
rabbit pellets, Tetraminฎ
5-6 y
~1 y
~1 y
~1 y
~1 y
3 mo
2y
~3 y
2y
1.5 y
5y
5y
2y
5y
3y
6 mo
4 mo
369
-------
Reconstituted Water
7 labs have used it
4 with good success
370
-------
Characteristics of Culture Water (n = 18)
very soft 1
soft 5
moderately hard 7
hard 4
very hard 1
371
-------
Types of Water Renewal in Cultures
flow-through 3
static, static renewal 14
372
-------
Culture Records Desirable/Maintained
Parental survival 56%
Age brood animals started 50%
Routine chemistries 94%
Quality of food 69%
Freq. of culture initiations 100%
373
-------
Reference Toxicants Used (n = 14)
cadmium 6
copper 3
KCI 5
NaCI 1
zinc 1
chromium 1
374
-------
Types of Substrates Currently in Use
plastic mesh 2
gauze 4
nitex 1
sediment/towels 1
towels 2
sand/towels/nitex 1
plastic/leaves 1
leaves 4
mesh/towel 1
none 1
375
-------
Substrates Used
Laboratory Choice Others Tried
ABC Lab. nylon mesh maple leaves
Dept Fish/Oceans sterile gauze aquatic plants, none,
nitex, leaves, astroturf
Environ. Canada gauze . sediment
EPA-Duluth gauze leaves, sediment
EPA Region 1 plastic mesh pad, leaves leaves only
EPA Region 8 sediment
EPA Newtown kraft paper towel leaves
EVS Consult, silica sand, leaves, nitex cones gauze
MD Dept. Env. leaves
Miami Univ. paper towels (unbleached) -
Mich. State gauze, unsterilized towel strips
NFCRC-Athens leaves
NFCRC-Columb leaves & 3M plastic web -,
Old Dominion leaves
State of Wash. leaves
Univ. of Miss. leaves
Univ. of WI-Sup. gauze -
Wright State leaves, paper towels
polyethylene mesh
376
-------
Culture Temperature (ฐCV
15 ' . '.'' ..;;".' '',' ..;;.
20 3
23 8
25 4
19-23 1
377
-------
Culture Chambers Used
Aquaria Sizes 1 L - 39 L
Water Volume 0.75 L - 38 L
378
-------
Test Lengths
96 h 3
7d 4
10 d 8
10-14 d 1
14 d 4
20 d 1
28 d 4
379
-------
Test Age
known age 7
sieve for size/age 8
mixed age 2
unknown 1
380
-------
Water Renewal and Frequency
Static
no water replacement 10
top off 2
Renewal
4-6 h 1
24 h 2
72 h 1
1.5ad.dn/d 1
4addn/d 2
2x/wk 1
not specified 1
381
-------
FEEDING IN SEDIMENT TESTS
7x/wk 5
3x/wk 5
2x/wk 2
1x/wk 1
every 48 h 1
initiation only 1
none 1
382
-------
Test Acceptability Criteria
Survival
60% 1
i " '
70% 2
80% 13
90% 1
383
-------
What is Reasonable to Evaluate
Test Acceptability
Survival
yes 18
no 0
Minimum growth
yes 3
no 8
Reference Toxicity Test
yes 7
no 7
may be 1
384
-------
Test Temperatures (ฐC) (n = 17)
20 6
25 5
23 4
20 1
20-25 1
385
-------
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Development of a Standard Protocol for Chironomus tentans
Robert Hoke, AScI - Duluth, MN
I. Summary of Culture Methods Used as Reported in Questionnaires
n. Summary of Test Methods Used as Reported in Questionnaires
ffl. Proposed Key Issues for Discussion
387
-------
-------
SURVEY RESULTS
Ckironomus tentans (riparius) Culture Conditions
No. of Responses - 8 (2)
CULTURE TYPE
TEMPERATURE
LIGHT QUAL./INT.
PHOTOPERIOD
CULTURE CHAMBER SIZE
CHAMBER WATER VOLUME
CHAMBER WATER RENEWAL RATE
NO. OF CHAMBERS
NO OF ORGANISMS/CHAMBER
CHAMBER RESTART INTERVAL
AGE OF RESTART ORGANISMS
ORGANISM REMOVAL
FEEDING REGIME
CHAMBER CLEANING
AERATION
CULTURE WATER
FT-2.SWR-8
10-25ฐ c
ambient lab fluor./50-120 ft-c
16L/8D
1-40L
1 - 30 L
once/day - evaporative loss only
4-40
,50-800
2X weekly - every 6 months '
egg cases - <24 h old larvae
daily - as needed
bleached-l/unbleached-7/sand-2
(paper towels)
none - weekly
yes-10 " . / ; .
soft/moderately hard/very hard,
lake-l/tap-3/well-5/recon-l
389
-------
SURVEY RESULTS
Chironomus tentans (riparius) Test Conditions
No. of Responses - 8
TEST TYPE . S(6); R(3)-(4/d, 2X or 3x/wk)
TEST DURATION
TEMPERATURE
LIGHT QUAL./INT.
PHOTOPERIOD
TEST CHAMBER SIZE
CHAMBER SEDIMENT VOLUME
CHAMBER OVERLYING WATER
VOLUME
NO. OF CHAMBERS (REPS/SAMPLE)
NO. OF ORGANISMS/CHAMBER (REP)
AGE OF TEST ORGANISMS
SIZE OF TEST ORGANISMS
FEEDING REGIME
TEST CHAMBER CLEANING
AERATION
TEST WATER
TEST ACCEPTABHITY CRITERION
MINIMUM SURVIVAL - 7
MINIMUM LENGTH - I/WEIGHT - 7
20(2), 22(2), 23(2), 25(2), ซC
ambient lab fliior./25-120 ft-c
16L/8D
50 ml(2), 250(2), 300, 1000(2), 2000
10-200 ml
40-1800 ml (1:4, S:W, 6 of 8)
2-15; 3-4 (6), 15 (2)
15-80
0-16d; 10-14d (7)
daily (5), 2X-3X weekly (3)
none
yes(6), no(2)~ERLD,UM
soft/moderately hard/very hard,
lake-l/tap-3/well-5/iecon-l
>40% saturation (2), temp. (1)
70% (4), 75-80% (1), 80% (2)
390
-------
Chironomus tentans (riparius) CULTURE CONDITION ISSUES
4- SUBSTRATE
4 CULTURE WATER .
* FOOD/FEEDING REGIME
4- QUALITY ASSURANCE/QUALITY CONTROL
391
-------
Chironomus tentans (riparius) TEST CONDITION ISSUES
4 ORGANISM AGE
4 TEST DESIGN (TYPE, RENEWAL, FEEDING)
+ EFFECTS OF ABIOTIC FACTORS
4* TEST LENGTH AND ENDPOINTS
4 QUALITY ASSURANCE/QUALITY CONTROL
392
-------
ism** fnr Lumhriculus varie^atus
Peter Landrum, NOAA Great Lakes Environmental Research Lab - ^n^or- MI
Gary Ankley, U.S. EPA Environmental Research Laboratory - Duluth, MN
I. Culturing
A.. Substrate
B. Density
C: Water
D. Feeding
E. Temperature
F. Light
G. QA/QC
H. Testing
A. Age
B. Loading rates
C. Test lengths
1. Example
D. Water renewal
- E. Sediment volume
' , F. ' Interpreting effects of sediment variables
HI. To Feed or Not To Feed?
IV. Sediment Avoidance - Effect on Exposure
V. Gut Purging for Bioaccumulation
393
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Development of a Standard Acute Amphipod Protocol
Richard C. Swartz, U.S. EPA Environmental Research Laboratory -Pacific Division
I. Generic .Protocol Design
n. Generic Technical Issues
HI. Species Specific Mo
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M. Redmond
LIFE CYCLE AT 25ฐC
METHODS
Newly-released juveniles isolated from
brooding females in seawater, then held 8-
10 days
10 juveniles/replicate
25ฐC, SOppt, 16 hrs light
Fed the flagelfate Pseudoisochrysis
paradoxa
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food)
3 replicates sampled/week for 7 weeks
DRAFT
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M. Redmond
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EOHAUSTORIUS
415
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J. Larriberson
EOHAUSTORIUS ESTUARIUS (HAUSTORIDAE)
Geographic range:
Habitat:
Nutrition: .
Life cycle:
Source of amphipods:
Life stage tested:
Central B.C. to central California
(other haustorids along Atlantic,
Pacific and Gulf coasts)
Free-burrowing sand dweller,
upper intertidal to shallow subtidal
Probable deposit feeder
Probably annual, not cultured
Field collected, sandy sediment
(shovel, sieve, bucket)
Large immature to mature,"' both sexes
416
-------
3. Larribefson
ACUTE SEDIMENT TOXICITY TEST CONDITIONS FOR EOHAUSTORIUS ESTUARIUS
Temperature:
Salinity:
Photoperiod:
Light quality:
Light intensity:
Test chamber sediment depth:
Test chamber water volume:
Number of organisms per
test chamber:
15ฐC (5 to 25ฐC)
28 ppt (2-35 ppt)
Continuous light
Fluorescent lights
Normal room lights
(subdued light for water only tests)
2 cm
Fill to 950 ml
20
417
-------
J. Larrfoerson
ACUTE SEDIMENT TOXICITY TEST CONDITIONS FOR EOHAUSTORIUS ESTUARIUS
(continued)
Feeding regime:
Aeration:
Test endpoints:
Control sediment:
Grain size:
Reference toxicant test:
No food added during acute test
Air bubbled through a 1-ml diposable
glass pipette
Emergence, mortality, reburial
Collection site sediment, 0.5-mm sieved
92% survival in 80% silt-clay
97% survival in sandy sediments
cadmium chloride, 4-day water only
Mean LC50 = 13.05 (4.38-21.72) ** .
Mean EC50 = 7.07 (1.81-12.33) **
** Numbers in- parentheses are reference toxicant warning limits
(95% confidence limits ='mean +/.- 2 standard deviations)
418
-------
J. Laraberson
EOHAUSTORIUS ESTUARIUS. ACUTE SEDIMENT TOXICITY TEST
ADVANTAGES , *
Ease of handling and collection from field
Salinity tolerance over a broad range
Grain size tolerance
Year round availability, can be shipped
'LIMITATIONS ' , ,'
Annual life cycle
Cannot culture ;
- Cannot use for chronic tests
Variable response to reference toxicant (cadmium) _
RESEARCH NEEDED
Factors affecting sensitivity to reference toxicant
Interlaboratbry comparison of test method
Tolerance limits - salinity, temperature, grain size
Field validation ,
419
-------
Ji- *i
2. Fluoranthene Tolerance: Eohaustorius estuarius was siigr
" more sensitive to fluoranthene than Hyalella azteca at 2 ppt
salinity, and slightly less sensitive than Rhepoxynius
abronius at 28 ppt. There was.no significant interaction
between salinity and fluoranthene tolerance for E. estuarius.
arson
Eohaustorius-Salinity-Fluoranthene
10'
Spec
Hya
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-------
B. McGee
SUMMARY OF 10 D SEDIMENT TOXICITY TEST CONDITIONS
USING Leptocheirus plumulosus
1. Temperature
2. Salinity
3. Photoperiod
4. Volume of test chamber
5. Water:sediment ratio
6. Size/age of test organism
7. No. of organisms/chamber
8. Overlying water
9. Negative control
10. Positive control
11. Source
20; 25 ฐC
Dependent on objectives of
the study
16:8 light:dark
1 L
ซ 4:1 (v:v)
,3 - 5 mm
20
Synthetic sea salts;
natural seawater
Fine sediment (>85% silt
clay); salinity ?
96h aqueous CdCl2
@ 20 ฐC and 20 %
. Field collected; cultured
421
-------
B. McGee
Research topics
Effects of salinity on test results
- Is acclimation necessary?
- Will the acclimation salinity influence
test sensitivity?
Sensitivity to common sediment contaminants
- Expand the chemical database
- Field validation
- Interspecies comparisons
Sensitivity differences among sources of amphipods
- Laboratory reared versus field collected
422
-------
B. McGee
Effect of acclimation salinity on survival of
juvenile Leptocheirus in 10 d exposures
100
Acclimation salinity
7ppt i20ppt
Test salinity (ppt)
423
-------
B.
Laboratory Reared Versus Field Collected
Amphipods in Sediment Toxicity Tests .
ADVANTAGES
Laboratory reared
Year round availability
Geographic availability
Reared under known,
controlled conditions
Field collected
Subsample of a "natural"
population
Easy to obtain large numbers
with minimal effort and cost
DISADVANTAGES
$ Genetic effects
(e.g., inbreeding, selection)
$ Influence of culture
condition dn test sensitivity
Cost (time & money)
Limited availability (seasonal,
geographic)
$ Seasonal &/or geographic
variation in sensitivity
$ Potential research topics
424
-------
R. Alden
Lepidactylus dytiscus Distribution and Ecology:
Lepidactylus dytiscus is broadly distributed
throughout the upper Chesapeake Bay and its
tributaries to Florida. It is an intertidal species
found 1-2 meters above the low tide mark in moist
coarse sand in summer to approximately 1-3
meters below low tide in winter months. L.
dytiscus burrows freely in coarse sand throughout
the year generally burrowing to 4 cm in summer
and 6-7 cm in winter. Densities can be 1200-1500
individuals/M2. Average densities are
approximately 150-200/M2 where L. dytiscus
occurs.
Bousfield (1970) reports feeding in L. dytiscus
is by suspension, although it may supplementarily
deposit feed. Lab held animals are fed small
amounts of Anemia salina and algae.
Reproduction generally occurs spring through
fall with large females overwintering. Bimodal
reproduction appears to be the rule with early
spring and late summer recruitment appearing to
be greatest.
425
-------
R. Alden
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426
-------
CULTURE CONDITIONS FOR LEPIDACTYLUS DYTISCUS AT AMRL
,R. Alden.
CULTURE CONDITION
CONDITION USED BY LABORATORY
1. Test type
2. Temperature
3. Light quality
4. Light intensity
5. Photoperiod
6. Culture chamber size
7. Culture water volume
8. Restart duration
9. Renewal of culture water
10. Removal of offspring
(frequency)
11. Age of restart organisms
12. No. of organisms/culture
chamber
13. No. of culture tanks
14. Feeding regime
15. Substrate used
16. Chamber cleaning
17. Aeration
18. Culture water
Static non-renewal
20 + 2ฐC
ambient laboratory
ambient laboratory
16:8 L:D
20 gallons
18 gallons
I vr
50% weeklv
2/vr
Various
1200
arterriia (twice/week)
native coarse sand
occasional stirring and siphoning
moderate w/filtration
DI w/ artificial sea salts
'427
-------
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REFERENCE TOXICANT DATA:
R. Alden
r ADMTIJM CHLORIDE TEST:
At 28 ppt the measured LC50 was 6.33 mg/L Cd (95% CI
11.22-4.35) with a control survival of 97% (EPA Probit
Analysis, Version 1.4).
At 20 ppt the measured LC50 was 6.13 mg/L Cd (95% CI
7.25 and 5.3), with a control survival of 97% (EPA Probit
Analysis, Version 1.4).
Results of reference toxicant tests conducted at intervals
throughout the year showed sensitivities in the range of 6.3
to 2.5 mg/L Cd, with a trend toward increasing sensitivity
in the early fall population.
CADMIUM CHLORIDE REFERENCE TOXICANT TEST
(96-Hour, Water Only)
LCSO's as mg/L Cadmium
12
10
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Dec. '89 Dec. '89 May '90 Sep. '90 Oct. '90
Date of Exposure
429
20 ppt D 28 ppt
-------
R. Alden
TOXICANT DATA:
NE TEST:
An average survival of 94% was found in both the
sediment and the acetone controls.
%
Calculations using the Moving Average Angle Method
indicated a nominal LC50 of 1.44 ing/kg (95% confidence
intervals 1.33 and 1.55 ing/kg) and a measured LC50
value of 0.793 mg/kg (95% confidence intervals 0.698 and
0.887 mg/kg) fluoranthene.
COMPARISON OF AMPHIPOD SENSITIVITIES
(10-Day Fluoranthene Exposure, 28ppt)
mg/kg
14
R. abronius* El E. estuarius* L. dytiscus
*From DeWitt et ai., 1989
430
-------
R. Alden
IALINITY TEST:
Statistical analysis using anova and regressions showed no
significant difference in survival of amphipods tested in
salinities from 5 to 40 ppt.
Average survival in the controls was 93.3%.
Test survival averaged over all test groups was 93.3% over
the 14 day test period, with no less than 90% survival in
any group.
SALINITY TOLERANCE
(10-Day Exposure)
Percent Survival
120
100
80
60
40
20
0
ii
WA
>//////
10 20 30
Salinity (ppt)
431
40
D Control salinity
NOTE: 95% Confidence Limits Shown
-------
R. Alden
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438
-------
Development of a Standard Chronic Amphipod Protocol
John Scott, SAIC - Narragansett, RI
with contributions from:
Ted DeWitt, AScI - Newport, OR
Michelle Redmond, AScI - Newport, OR
Chris Schlekat, Maryland Department of the Environment r Baltimore, MD
I. Introduction (Scott)
A. Historieal background
B. Applications of chronic procedures
C. Minimum requirements of the methods ,
Chronic Procedures Using Leptocheirus plumulosus (Dewitt)
1 A. Preliminary Leptocheirus plumulosus chronic method
1. Species selection
a. Culturability
b. Contaminant sensitivity
c. Handling ,
2. Culture methods
' a. Physical requirements !
- i. Tubs
';... , ii. Salinity
iii. Temperature
iv. Light
v. Sediment
3. Test animals ,
a. Life stage
b. Number per replicate
c. Feeding
d. , Handling and recovery
e. Responses
i. Mortality *
-.ii.. Growth
iii. Reproduction
iv. Others
, f. Tolerance limits
i. Grain size
ii. Salinity
iii. Organic enrichment
iv. Seasonality
g. Special requirements
i. Quarantine laws and practices
439
-------
4. Exposure logistics
a. Chamber characteristics
b. Exposure duration s
c. Sediment depth and volume
d. Water change
e. Aeration
f. Temperature
g. Salinity
h. Dissolved oxygen
i. Light
i. Photoperipd
ii. Intensity and quality
j. Other environmental variables
5. Experimental design
a. Treatments
b. Replication
c. Statistics
i. Analytical methods
ii. Variability and power
d. Controls
i. QA/QA
- Performance (health)
- Reference toxicant
ii. Experimental
- Carrier (spiked sediment)
- Site (dilution series)
- Environmental (grain size, TOC, salinity, temperature)
e. QA/QC
i. Environmental conditions
ii. Response criteria (mean, variability)
iii. Controls . . '.
B. Research findings
1. Sensitivity to sediment variables
a. Mortality and growth
2. Sensitivity to phenanthrene-spiked sediment
a. Relative sensitivity of mortality, growth, and fertility
3. Sensitivity to contaminated sediment dilution series
a. Relative sensitivity of mortality, growth, and fertility
4. Nutrition affects growth
C. Comparison of EPA and Maryland Department of Environment methods
440
-------
D. Research needs
i . , i
/ "
1. Influence of nutrition on lexicological sensitivity
2. Influence of other variables on toxicological sensitivity
a. Temperature
b. Salinity
c. Sediment grain size
d. Other variables
3. QA/QC issues
a. Development of reference toxicant method for growth and fertility
b. Environmental conditions during exposure
\ - ,
4. Relative sensitivity of cultured and field-collected animals
5. Simplification of culture and feeding methods
6. Relative sensitivity to other species (acute and chronic)
7. Experimental design optimization
8. Development of toxicological database (chemical diversity)
9. Inter-laboratory comparison
10. Field validation
n. Chronic Procedures Using Leptocheirus plumulosus (Schlekat)
A. Characteristics of the test organism
B. Selection of the test endpoints ._.-.''
C. Elements of the procedure '
D. Representative results
E. Research needs
HI. Chronic Procedures Using Ampelisca abdita (Redmond and Scott)
A. . Characteristics of the test organism
B. Selection of the test endpoints
C. Elements of the procedure
D. Representative results
E. Research needs
441
-------
IV. Discussion (Workgroup)
A. Input from workgroup these procedures
B. Experience with other species
V. Summary and Recommendations (Scott)
442
-------
J. Scott
CHRONIC TEST METHODS
LEPTOCHEERUS
STATUS:
ISSUES:
GOAL:
PRELIMINARY METHOD
28-DAY SURVIVAL, GROWTH
REPRO
MULTIPLE LABORATORY USE
MINOR DIFFERENCES
CULTURE/FIELD COLLECTED
NUTRITION-DIET QUANT/QUAL
INTER-INTRA LAB VARIABILITY
FIELD VALIDATION
ROLE OF SEDIMENT NUTRITION QUAL
REF TOX FOR CHRONIC ENDPOINTS
DRAFT STANDARD 9/93
GUIDE?
FALLASTM
ATVrPRT.TSr.A ,
STATUS: PREUMINARY/SUBLETHAL METHOD
20-DAY - GROWTH
LIFE CYCLE 30-40 DAY-DEVELOPING
CULTURE METHODS - DEVELOPING
ISSUES: TEST DESIGN FOR REPRO ENDPTS
OTHER SAME AS LX,
GOAL: DRAFT STANDARD SUBLETHAL -
GUIDE? CHRONIC?
9/93 -> FALL ASTM
443
-------
J. Scott
TOXICITY TEST INTERPRETIVE GUIDANCE
STATISTICAL SIGNIFICANCE
DETECTABLE SIGNIFICANCE
BIOLOGICAL SIGNIFICANCE
444
-------
i Scott
INTERPRETATIVE GUIDANCE
DETECTABLE SIGNIFICANCE
Statistical significance incorporates within
test variability among replicates.
but what about test performance
variability???
445
-------
J. Scott
INTERPRETATIVE GUIDANCE
DETECTABLE SIGNIFICANCE
CALCULATION OF THE LEAST
SIGNIFICANT DIFFERENCE (LSD)
o Conduct one-way Mest
o Assume unequal variances
o Generate a t value.
o Conduct ANOVA to generate MSE
LSD = ^.0.05 SqRt ((1/NC + 1/N,) * MSE)
446
-------
J. Scott
Frequency
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40.1-45
447
-------
cc
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range: 2.3 - 32.4%
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the control 95% of the time.
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449
-------
J. Scott
INTERPRETATIVE GUIDANCE
BIOLOGICAL SIGNIFICANCE
ESTABLISH BASIC POPULATION PARAMETERS
o Age-specific survival and fertility
o Model-based population estimation
o Leslie Projection Matrix
TEST DESIGN
o Test duration of 70 days
o Regular non-destructive sampling
o Examine mortality effects at 10-day intervals
450
-------
J. Scott
FINITE GROWTH RATE
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J. Scott
AMPfflPOD CHRONIC TESTS
WHY DO CHRONIC TESTS?
More sensitive than acute
More relevant ecological processes
Understand population biology
Determine sensitive life stages
EPA/COE guidance
Sediment Management Strategy
452
-------
J. Scott.
AMPHIPOD CHRONIC TESTS
RESEARCH GOAL
o Understand population biology
o Optimize design for:
information content
cost efficiency
453
-------
J. Scott
AMPfflPOD CHRONIC TESTS
EARLY DESIGNS WITH AMPELISCA
o Population sampling approach:
initiate with ovigerous females
two generations
o Suspended sediments
o What we learned:
amphipods will reproduce in the laboratory
r ' '
growth is a sensitive endpoint
developed crude population models
o Limitations:
high variability
no age standardization
not amenable to model-based approaches
454
-------
J. Scott
MINIMUM REQUIREMENTS
FOR TEST PROTOCOL
Responsive to chemical contaminants
Relatively sensitive
Intra- and interlaboratory variability low
Organism available
Well documented
455
-------
-------
T. "DeWitt
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460
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T. DeWitt
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461
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462
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T. DeWitt
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Newborn
SubAdult SubAdult
SubAdult
SubAdult
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No No No Yes Yes Yes Yes
No No
No Yes Yes' Yes
Food Added
Yes
463
-------
-------
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SUMMARY OF TEST CONDITIONS FOR PARTIAL LIFE-CYCLE TEST
CONDUCTED WITH Leptocheirus plumulosus
1) ORIGIN OF TEST ANIMALS:
2) NO. AMPHIPODS/CHAMBER:
3) VOLUME OF TEST CHAMBER:
4 ^WATER: SEDIMENT RATIO:
5) PHOTOPERIOD:
6)fWATER SOURCE:
7)"*TEST WATER TEMPERATURE:
8)tTEST WATER SALINITY:
9) TEST DURATION:
10)fTEST ENDPOINTS:
Obtained by isolating gravid 9s
in control sediment for < 1
week. Offspring collected on
250 Jim sieve are utilized in
test. Size has ranged from 1.2
to 1.5 mm.
20
1 L
4:1 (v:v)
16:8 (light:dark)
Artificial seawater diluted with
distilled and spring water. :
20 ฑ 2ฐC
Ambient interstitial salinity
of test sediment
28 d; Static-renewal 2 X week
Survival, length, measures of
reproduction
11) POSITIVE CONTROL:
12)fFEEDIN6 REGIME:
i3)tcoNTROL SEDIMENT:
Aqueous cadmium chloride @ 20ฐC
and 20 %o
6-12 mg TetraMin
Conditioning 3 X week
Tetra
Amphipod collection site
sediment (Corsica River, Queen
Anne's County, MD;
93% silt-clay)
Different from EPA-Newport
466
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471
-------
-------
APPENDIX A:
WORKSHOP AGENDA
473
-------
-------
Tiered Testing
Issues for Freshwater and Marine Sediments
Wednesday, September 16, 1992
8:30 Introduction and Description of EPA Sediment Strategy
Elizabeth Southerland, Workshop Moderator, U.S. EPA Office of Science and
Technology
9:00 Discussion of EPA Program Office Sediment Evaluation Needs
Tom Armitage, U.S. EPA Office of Science and Technology
9:30 Discussion of EPA Regional Sediment Evaluation Needs
William Peltier, Environmental Services Division, U.S. EPA Region 4
10:00 Break
i -
10:15 Tiered Sediment Testing Conceptual Overview
Elizabeth Southerland, U.S. EPA Office of Science and Technology
' i
Tom Armitage, U.S. EPA Office of Science and Technology
10:45 , Summary of ASTM Activities on Freshwater and Marine Sediment Test
Methods
Chris Ingersoll, U.S. Fish and Wildlife Service National Fisheries Contaminant
Research Center
11:15 Discussion of Approaches for Test Standardization: Historical Perspective and
Present Guidance
Jim Lazorchak, U.S. EPA Environmental Monitoring and Systems Laboratory,
Cincinnati
Bill Telliard, U.S. EPA Office of Science and Technology
475
-------
12:15 Lunch
1:15 Discussion of Desirable and Necessary Attributes for Freshwater Sediment
Toxicity Tests, and the Use of Hvalella azteca and Chironomns teritans in
Freshwater Sediments
Allen Burton, Wright State University
John Giesy, Michigan State University, Department of Fisheries
Chris Ingersoll, U.S. Fish & Wildlife Service National Fisheries Contaminant
Research Center
2:00 Discussion of Desirable and Necessary Attributes for Marine and Estuarine
Sediment Toxicity Tests, and the Use of Ampelisca abdita. Rhepoxvnius
abronius. Leptocheirus plumulosus. Eohaustorius estuarius in Marine and
Estuarine Sediments
Rick Swartz, U.S. EPA Environmental Research Laboratory, Pacific Division
3:15 Break
3:30 Discussion of Desirable and Necessary Attributes for Freshwater, Marine,
and Estuarine Bioaccumulation Test, and the Use of Neries and Macoma in
Marine and Estuarine Sediments .
Peter Landrum, NOAA Great Lakes Environmental Research Laboratory
Henry Lee, U.S. EPA Environmental Research Laboratory, Pacific Division
4:45 Discussion of the Use of Lumbriculus variegatus hi Freshwater Sediments
Gary Ankley, U.S. EPA Environmental Research Laboratory, Duluth
5:00 Identification of Long Term Needs for Assessing Sediments
Gary Ankley, U.S. EPA Environmental Research Laboratory, Duluth
Norm Rubinstein, U.S. EPA Environmental Research Laboratory, Narragansett
5:30 Adjourn
476
-------
Break-Out Workgroup for Freshwater Sediment Issues
Thursday, September 17
8:00 am Introduction and Overview of Day
Moderator/Discussion Leader: Gary Ankley
General description of survey/summary of methods
Number of labs responding, who responded on which organisms, et
cetera.
Discussion of issues for each method
8:30 am Development of a Standard Protocol for Testing Hvalella azteca
Discussion Leader: Teresa Norberg-King
, Rapporteur: Chris Ingersoll
Summary of culture methods used as reported in questionnaires
(-10 mm)
Summary of test methods used as reported in questionnaires
(~ 10 min)
Proposed key issues for discussion
10:15 am Break
10:30 am Continuation of Discussion of Development of a Standard Protocol for Testing
Hvalella azteca
477
-------
11:30 am Development of a Standard Protocol for Chironomus tentans
Discussion Leader: Bob Hoke
Rapporteur: Jody Kubitz
Summary of culture methods used as reported in questionnaires
(-lOmin)
Summary of test methods used as reported in questionnaires
(~10min)
* Proposed key issues for discussion
12:15 pm Lunch
1:30 pm Continuation of Chironomus tentans
2:30 pm Development of a Standard Protocol for Lumbriculus variegatus
t
Discussion Leader: Peter Lahdrum
Rapporteur: Allen Burton
Summary of culture methods used as reported La questionnaires
(~10min)
Summary of test methods used as reported in questionnaires
(~10min)
Proposed key issues for discussion
3:30 pm Break
3:45 pm Continuation of Lumbriculus variegatus
5:00 pm General Adjournment
5:30 pm Meeting of Discussion Leaders, Moderators, and Rapporteurs to Write
Conclusions
478
-------
Break-Out Workgroup for Marine and Estuarine Sediment Issues
Thursday, September 17
8:00 am Overview of Day
Discussion of Issues for Each Method
Norm Rubinstein, U.S. EPA Environmental Research Laboratory, Narragansett
8:30 am Development of a Standard Acute Amphipod Protocol
Summary of methods used
Applications of the test
Minimum requirements for the method
Technical issues
Rick Swartz, U.S. EPA Environmental Research Laboratory, 'Pacific Division
10:15 am Break .
10:30 am Development of a Standard Chronic Amphipod Protocol
Summary of methods used
Applications of the test
Minimum requirements for the method
John Scott, SAIC
Ted DeWitJ, AScI
12:15 pm Lunch
1:30 pm Continuation of Chronic Protocol Discussion
479
-------
2:30 pm Development of a Standard Bioaccumulation Protocol
Summary of methods used
Applications of the test
Minimum requirements for the method
Technical issues
Henry Lee, U.S. Environmental Research Laboratory, Pacific Division
3:30 pm Break
3:45 pm Continuation of Bioaccumulation Protocol Discussion
5:00 pm Development of Other Test Methods and Standard Protocols
5:30 pm Adjourn
480
-------
Tiered Testing
Issues for Freshwater and Marine Sediments
Friday, September 18
9:00 am Overview of Day
" >
Elizabeth Southerland, Workshop Moderator, U.S. EPA Office of Science and
Technology
9:15 am Report of Issues Covered in Marine Methods Session and Discussion of
Conclusions/Next Steps
; '
Rick Swartz, U.S. EPA Environmental Research Laboratory, Pacific Division
10:15 am Report of Issues Covered in Freshwater Methods Session and Discussion of
Conclusions/Next Steps
Gary Ankley, U.S. EPA Environmental Research Laboratory, Duluth
[
11:15 am Workshop Summary and Wrap-up
Elizabeth Southerland, Workshop Moderator, U.S. EPA Office of Science and
Technology
11:45 am Adjourn
481
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-------
APPENDIX B:
SEDIMENT TOXICITY TESTS
UNDER DEVELOPMENT BY
ENVIRONMENT CANADA
483
-------
-------
DEVELOPMENT OF A 10-DAY MARINE/ESTUARINE AMPHIPOD ASSAY
FOR SEDIMENT TOXICITY IN SUPPORT OF THE CANADIAN OCEAN
PUMPING PROGRAM (CEPA, PART VI).
D.J. McLeay, S.C. Yee, K.G. Doe and L.M. Porebski, McLeay Associates Limited, West
Vancouver, British Columbia, Environment Canada, and Aquatic Toxicity Laboratory, North
Vancouver, British Columbia, and Environment Canada, Laboratory Division, Dartmouth, Nova
Scotia, and Environment Canada, Office of Waste Management, Environment Canada, Ottawa,
Ontario.
ABSTRACT
Beginning in 1988, Environment Canada commenced the development of a test method
for measuring the acute toxicity of sediment samples, using a number of marine or estuarine
sediment-burrowing amphipods common to Canada's coastal waters. The evolution of this
toxicity test included five series of inter-laboratory assessments using various candidate test
organisms, sediment samples, and a reference toxicant. These studies are reviewed briefly.
Additionally, the test method and its applications are summarized. ,
INTRODUCTION
The Ocean Dumping Control Act has been consolidated into Part VI of the Canadian
Environmental Protection Act (CEPA). The Act requires valid ocean dumping permits before
dumping of any substance at sea is allowed. Biological testing and assessment can be an integral
component of this permit process (Sergy, 1988; Anthony, 1991; Porebski, 1991). In order for
Environment Canada to perform its regulatory responsibilities associated with this Act, biological
screening tests using marine or estuarine organisms may be required to determine if material is
suitable for unconfmed open-water disposal, and to perform environmental-effects monitoring
at dump sites. Interim contaminant testing guidelines for ocean disposal, associated with
Environment Canada's Ocean Dumping Control Program, specify several toxicity tests for use
in screening materials. The 10-day assay for sediment toxicity, using one or more species of
estuarine or marine amphipods common to Canada's coastal waters, is included on the lists of
biological screening tests (Environment Canada, 1990a, 1990b).
In consideration of the above, Environment Canada's Atlantic and Pacific & Yukon
regional laboratories commenced inter-laboratory studies in late 1988, using a number of marine
or estuarine sediment-burrowing amphipods and various samples of control, reference or test
(contaminated) sediments. The objectives of this testing program were twofold: (1) to study
several candidate species of amphipods, selecting those suitable for use in acute lethality tests
with samples of sediment or other test material; and (2) to evaluate conditions, procedures and
biological endpoints appropriate for use in a standard biological test method to be developed to
meet Environment Canada's testing requirements in this respect. The past status of this and
other biological test methods under development on behalf of Environment Canada has been
reported (McLeay etal., 1991a).. ,
485 --.''
-------
Following is a brief summary of the inter-laboratory studies with marine or estuarine
amphipods, performed to date by Environment Canada's Pacific & Yukon and Atlantic regional
laboratories.1 Also presented is a list of those species of infaunal amphipods presently
recommended for use in Environment Canada's draft biological test method "Acute Test for
Sediment Toxicity Using Marine or Estuarine Amphipods" (Environment Canada, 1991).
Tentative checklists of recommended conditions and procedures for holding and acclimating
amphipods, and for testing them in 10-day static assays, are provided. Finally, some of the
applications of this biological test method are indicated.
LABORATORY EVALUATION AND DEVELOPMENT OF TEST METHOD
In 1988, Environment Canada's Atlantic and Pacific-& Yukon regional laboratories
undertook a preliminary (Phase-I) evaluation of the 10-day sediment assay, using only
Rhepoxynius abronius (McLeay et al., 1989). The effects of holding amphipods in control
sediment for periods of up to 81 days, on their 10-day survival and subsequent reburial rates
under test conditions (control sediment only), were investigated. Their acute tolerance (96-h
LC50/EC50) to the reference toxicant cadmium chloride (seawater-only exposures) was
monitored during the prolonged holding period. Although 109-day survival and reburial rates
were acceptable (> 90%) in all trials, the reference toxicant tests indicated a declined tolerance
of these organisms with extended > 13 days) periods of holding in the laboratory.
The second (Phase-IE) inter-laboratory study (McLeay et al., 199Ib) measured and
compared the 10-day survival, emergence and reburial rates for a population of Rhepoxynius
abronius exposed to control,2 reference,3 or test* sediment. At each laboratory, survival and
reburial rates in control sediment were high (>90%). No consistent differences in these
endpoints were caused by exposure to reference or test sediments, indicating that neither were
highly toxic. However, increased rates of emergence from the test sediment were observed.
'Basic test conditions and procedures used for the 10-day static assays were according to Swartz et al. (1985)
and ASTM (1990).
2Control sediment is clean sediment taken from the site where the test organisms were collected, and intended
for use in the 10-day test*with amphipods. This sediment must contain 'no test material. It is used to determine the
absence of measurable toxicity due to basic test conditions (e.g., temperature, health or handling of test organisms).
3Reference sediment is a field-collected sample of sediment, taken from a site thought to be relatively free of
contaminants (i.e., "clean" sediment), and intended for use in the 10-day test with amphipods. It is often collected
from a site within the general vicinity of a test sediment, and is frequently selected for biological testing because
of its geochemical similarity (e.g., particle size, compactness, total organic content) to the test sediment(s).
^Test sediment is a field-collected sample of solid-phase sediment, taken from a site thought to be contaminated
with one or more chemicals, and intended for use in the 10-day test with amphipods. In this study, the sample of
test sediment was collected from the vicinity of a B.C. coastal pulp mill discharging bleached kraft mill effluent.
486
-------
A Phase-m study was performed by each laboratory using both Rhepoxynius abronius
and Corophium volutator as test organisms (McLeay et al., 1991b). For each species, ten-day
survival and subsequent reburial rates were determined for three clean5 (control or reference)
sediments and three test sediments. The clean sediments varied appreciably in grain size, with
silt/clay contents of 1%, 82% or 99%. Tests with R, abronius showed highest survival rates
(96% and 100%) for control sediment, and lowest survival rates (43% and 78% for the reference
sediment containing 99% fines (silt and clay). R, abronius survival rates for the three test
sediments ranged from 72 to 93%. Unlike these findings, survival rates for Corophium
volutator were high (93 % and 97%) in the extremely fine-grained reference sediment. For both
species, reburial and/or survival data6 showed no consistent response for any of the test
sediments examined, although emergence rates indicated an avoidance response to one of the test
sediments.
A fourth inter-laboratory assessment,7 using six species of marine or estuarine mfaunal
amphipods8 common to Canada's coastal waters, was undertaken during late 1990 and early
1991 (Paine and McPherson, 1991a). The objectives of this (Phase-IV) study were to determine
and compare the relative sensitivity of each of the six test species to four test sediments, two
reference sediments (fine-grained and coarse-grained), and control sediments (one for each
species) For the four test sediments examined, each of the six species of amphipods used in
these assays distinguished the same two sediments as clearly toxic, and the remaining two as
marginally or not toxic. Percentage survival at 10 days was the most useful biological endpoint;
little if any additional information was obtained using the other two endpoints (% emergence,
% of survivors that did not rebury in control sediment within 1 h following test completion).
R washinetonianus and K abronius were most sensitive to the test sediments; C volutator and
E estuarius were least sensitive. Two of the six species studied (R washingtomanus and A,
vh-giniana) showed unacceptably low (< 90%) 10-day survival rates in control sediment;
Depending on species, grain-size effects were minimal or not evident. This study also
demonstrated that, if care is taken, amphipods can be shipped across the country without
influencing the test results. . .. ... '
Additional studies were performed by Environment Canada's Atlantic regional laboratory
in early 1991 to assess the laboratory hardiness and worth of Amphiporeia virginiana as a test
organism (Doe, 1991). Two populations of field-collected specimens were tested for 10-day
survival rates in control sediment only. Animals were acclimated and tested at temperatures of
10 or 15 C (Series 1); and at 5, 10 or 15 C (Series 2). Results from these studies showed_that
acceptable (>90%) 10-day survival rates could be obtained at 5 or 10 C? but not at 15 C.
Seasonally-cold (2 to 4 C) seawater temperatures at collection sites likely contributed to these
findings. ,
*Clean sediment is sediment (e.g., control or reference sediment) that does not contain concentrations of
contaminants which cause discernible distress to the test organisms or reduce their survival in 10-day assays.
'Since a volutator do not rebury readily in control sediment within 1 hour, this biological endpoint cannot be
determined for this species. ~ .
Participating laboratories included Environment Canada's Atlantic and Pacific & Yukon regional laboratories,
and EVS Consultants Ltd.
Test organisms were Rhepoxvnius abronius. Fo^EbaM xiximeus, Eohaustorius estuarius, Eohaustorius
washingtonianus. Corophium volutator. and Ampfiiporeia virginiana.
487
-------
A fifth inter-laboratory9 appraisal of the 10-day test for sediment toxicity, using multiple
species of marine or estuarine amphipods, was conducted during June 1991 (Paine and
McPherson 199Ib). For this study, seven species of infaunal amphipods10 were collected and
examined for their laboratory adaptability and sensitivity to each of the three test sediments.
Biological endpoints (% survival and % emergence at 10 days; % reburial of survivors in
control sediment at test end) were determined for each species in these sediments as well as in
a fine-grained (79% silt-clay) reference sediment and respective control sediments. Assays with
Amphiporeia virginiana were performed at both 10 and 15 C; all other species were tested at
15 C only. Unlike the previous (Phase-IV) inter-laboratory study, none of the three test
sediments used in this study were highly toxic to any of the species of amphipods examined.
Once again, ten-day survival rates in control sediment were unacceptably low (,90%) for
Amphiporeia virginiana (both temperatures) and Eohaustorius washingtonianus. but acceptable
for all other species studied including Leptocheirus pinguis. As in the Phase-IV study, %
survival at 10 days was the most useful biological endpoint, with little if any additional
information provided by the secondary endpoints (% emergence, % of survivors not reburying
in control sediment at test end).
RECOMMENDED TEST SPECIES
Recent attempts have been made to identify suitable Canadian collection sites," and to
collect the following species of amphipods from Canadian coastal waters, in order to evaluate
their worth as candidate test organisms in 10-day sediment assays:
Pacific Coast Atlantic Coast
Monoculodes spinipes Rhepoxynius hudsoni
Grandifoxus grandis Phoxocephalus holbolli
Ampelisca abdita .
Ampelisca vadorum
Amphiporeia lawrenciana
Pontoporeia femorata
The absence or relative scarcity of these species at the Canadian collection sites investigated,
prevented their inclusion in the present test program.
'Environment Canada's Atlantic and pacific & Yukon regional laboratories.
10Test organisms included Rhepoxvnius abronius. Foxiphalus xiximeus. Eohaustorius estuarius. Eohaustorius
washingtonianus. Corophium volutator. Amphiporeia virginiana. and Leptocheirus pinguis.
"Specimen collections held by the National Museum of Natural Sciences (Ottawa, Ontario) were examined by
Dr. E.L. Bousfield, together with historical records available to him (e.g., Bousfield, 1990a, b; Bousfield, 1991;
Bousfield and Laubitz, 1972). Based on these and other distributional and life-history information, initial
recommendations were made for prospective test specimens.
J
488
-------
Based on the results of the laboratory studies mentioned previously (see section
"Laboratory Evaluation and Development of Test Method"), as well as those for other 10-day
assays performed with the candidate species of irifaunal amphipods under consideration by
.Environment Canada, the following species of marine or estuarine amphipods are presently
recommended for use in 10-day static sediment assays (Environment Canada, 1991):
Recommended Pacific Species
Rhepoxynius abronius
Foxiphalus xiximeus
Eohaustorius estuarius ,
Recommended Atlantic Species
Corophium volutator
Leptocheirus pinguis
Additional studies, with Ajnphiporda virgihiana and Eohaustorius washingtonianus are
required to demonstrate that satisfactory survival rates in control sediment can be achieved for
these species, before they can be recommended by Environment Canada as standard test
organisms. ,
RECOMMENDED CONDITIONS AND PROCEDURES
FOR ACCLIMATING AND TESTING AMPHIPODS
\ _ . ',
Environment Canada's draft biological test method (Environment Canada, 1991) provides
details regarding conditions and procedures for holding and acclimating amphipods to be used
in 10-day assays, as well as those necessary to perform the test in a standardized manner. The
test methods of Swartz et al. (1985) and ASTM (1990) formed the basis for Environment
Canada's (1991) acute test for sediment toxicity using marine or estuarine amphipods common
to Canadian coastal waters. Acclimation and test procedures recommended in Environment
Canada (1991) are reproduced here (see Tables 1 and 2). Since this biological test method is
not yet finalized and approved by Environment Canada, these procedures and conditions are
subject to change.
489
-------
Table 1 Checklist of Recommended Conditions and Procedures for Holding and
Acclimating Amphipods12
Source of amphipods:
Life stage:
Sorting:
Holding sediment:
Holding seawater:
Acclimation conditions:
Lighting:
Feeding:
Duration of acclimation:
Health criteria:
Collected subtidally or intertidally
from clean sediment
juveniles or young adults, 3-10 mm
length (depending on species)
Sieve through 1.0-mm screen to
confirm species and select
appropriate size; use seawater within
2 C and 2 ppt salinity of the^
seawater in transport container
Control sediment, 2-4 cm in depth,
previously sieved through 0.5-mm
mesh
Reconstituted or clean natural
seawater
Salinity of seawater same as that for
overlying seawater in test;,
temperature normally 15 + 2 C;
dissolved oxygen > 90% of air
.saturation; temperature, salinity, and
dissolved oxygen measured and
recorded daily
Constant overhead illumination, >
100 lux at surface of sediment in
holding/acclimation container(s)
None
3 to 10 days
Select amphipods able to bury
quickly in control sediment; remove
inactive amphipods that have
emerged from sediment or do not
bury; discard population if ^5 %
dead or emerged and inactive during
48-h period preceding test
12From Environment Canada's (1991) draft biological test method - subject to change.
"490
-------
APPENDIX C:
FRESHWATER SURVEYS
491
-------
-------
Culturing Questionnaire
Species: , :
Laboratory: ' :
Contact:
1. Is the culture intermittent or continuous? If continuous, how long have animals been in
culture at your facility?
2. What was original source and approximate date you started the culture?
3. Have the animals been taxonomically identified? If so, when and by whom?
4. what records on culture animals are maintained?
circle one Frequency
parental survival ves no
age of brood animals yes t no .
temperature ves no
dissolved oxygen ...... yes no . :
pH yes no
quality/age of foods yes no , .
frequency of new culture chambers yes no : =
5. What is source of the water used for culturing? List all used.
6. What are the characteristics of each water?
water hardness alkalinity BS " . - conductivity
7. Have you used any reconstituted waters for culturing? If so, are they successful? If no,
what problems did you experience?
8 What foods have you tried to culture the animals? What is your choice of food for
routine culture? '
9. How long has this regime been used?
10. Do you culture under controlled temperature (ฑ2ฐC) and lighting? If so, specify
conditions.
11. What substrates have you used for culturing? Please list types/quantities evaluated and
current choice.
493
-------
12. Do you conduct any reference toxicant tests with this organism?
A. If so, with what toxicant? What duration and was test conducted with or without
sediment? If with sediment, specify source/type.
B. Have results been reproducible? Have any control charts been established?
13. Do you feel reference toxicant tests are relevant for monitoring the
adequacy/performance of the culture organisms?
14. Provide any additional information or comments that are pertinent to this species below:
494
-------
Testing Questionnaire .
1 -, > '
Species: . !
Laboratory: ^_ - '; ' ';
Contact: ;
1. Describe test (toxicity, bioaccumulation).
2 Are the test organisms cultured at your laboratory? If yes, provide description in
' Attachment 1. If no, please provide details of how test animals are obtained.
3. What is the "routine" test performed with this organisms at your laboratory? Select the
appropriate time frame.
. 4-d 7-d ' 10-d 14-d 21-d
other, specify -
4 Do you use known age or size or unknown age or size of organisms to initiate tests?
What is known age or size? Specify age or size. How do you obtain these known
age/size organisms? .
5. Is it important that animals are a minimum age (or size) for sediment:water exposures
to ensure recovery (of organisms or sediments)?
6. Have you specified a certain test design of sediment:water? If so, what and why? -
7. What is the renewal frequency of the overlying water for the duration of the test? What
procedure is used to renew the water?
8. Do you feed during the test? Is it the same rate and frequency as in the culture? If not
specify what and why. :
9. What statistical analyses are performed on the data? Cite specific procedures and types
of statistical analysis.
10. What are the test endpoints? How do you express the effect?
11 Have you conducted any reference toxicant tests in your laboratory sedimentrwater
exposures? If yes, please identify chemicals and explain general trend of test results.
Manuscripts can be attached.
" V
12. What water quality characteristics of the overlying water are measured during the test?
How often are these parameters measured?
495
-------
13. Provide any additional information or comments that are pertinent to this species below:
PLEASE PROVIDE DATA IN SPACE BELOW
CULTURE CONDITIONS FOR HYALELLA AZTECA
CULTURE CONDITION
1. Culture type (static or renewal)
(specify rate)
2. Temperature:
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Culture chamber size:
7. Culture water volume:
8. Frequency of starting new culture
9. Renewal of culture water:
10. Removal of offspring (frequency):
11. Age of restart organisms:
12. No. organisms/culture chamber:
13. No. of culture tanks:
14. Feeding regime:
15. Substrate used:
16. Chamber cleaning:
17. Aeration:
18. Culture water:
CONDITION USED BY LABORATORY
496
-------
PLEASE PROVIDE DATA IN SPACE BELOW
CULTURE CONDITIONS FOR CHIRONOMUS TENTANS
CONDITION CONDITION USED BY LABORATORY
1. Culture type (static or renewal) .'".'
(specify rate) '_ . -
2. Temperature: , '. .
3. Light quality:
4. Light intensity: : -^
5. Photoperiod: / -
6. Culture chamber size:
7. Culture water volume:
8. Frequency of starting new culture ;
9. Renewal of culture water: /
10. Removal of offspring (frequency): . -.. ,
11. Age of restart organisms: ; ;.
12. No. organisms/culture chamber: :
13. No. of culture tanks: . --r^
14. Feeding regime: ;
15. Substrate used: :
16. Chamber cleaning: -, -
17. Aeration: -:; :
18. Culture water: = ~^-
497
-------
PLEASE PROVIDE DATA IN SPACE BELOW
CULTURE CONDITIONS FOR LUMBRICULUS VARIEGATUS
CULTURE CONDITION
1. Culture type (static or renewal)
(specify rate)
2. Temperature:
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Culture chamber size:
7. Culture water volume:
8. Frequency of starting new culture
9. Renewal of culture water:
10. Removal of offspring (frequency):
11. Age of restart organisms:
12. No. organisms/culture chamber:
13. No. of culture tanks:
14. Feeding regime:
15. Substrate used:
16. Chamber cleaning:
17. Aeration:
18. Culture water:
CONDITION USED BY LABORATORY
498
-------
PLEASE PROVIDE INFORMATION IN SPACES BELOW IF APPROPRIATE
CONDITIONS FOR HYALELLA AZTECA SEDIMENT TOXICITY .TESTS
TEST CONDITION ' CONDITION USED BY LABORATORY
1. Test type (static or renewal)
(specify rate) ;
2. Test duration: - ;
3. Temperature:
4. Light quality: -
5. Light intensity: . ,. -
6. Photoperiod: : : -^
7. Test chamber size: . :
8. Test sediment volume: . ; :
9. Overlying water volume: chamber: . : -
10. Age of test organisms: . .
11. Size of test organisms: .* _ = = :
12. No. organisms/test chamber: . ;
13. No. replicate test chambers/sediment: _
14. No. organisms/sediment: ;
15. Feeding regime: . ; -. ::
16. Test chamber cleaning: . . .
17. Aeration: . ;
18. Overlying water quality characteristics: _; ;
19. Test acceptability criterion:,
(1) minimum control survival ^
(2) length or weight minimum criteria: . -,
20. Eridpoint(s) .
NOTE: If an item listed does not seem pertinent, please explain why.
499
-------
PLEASE PROVIDE INFORMATION IN SPACES BELOW IF APPROPRIATE
CONDITIONS FOR CHIRONOMUS TENTANS SEDIMENT TOXICITY TESTS
TEST CONDITION CONDITION USED BY LABORATORY
1. Test type (static or renewal)
(specify rate)
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test sediment volume:
9. Overlying water volume: chamber:
10. Age of test organisms:
11. Size of test organisms:
12. No. organisms/test chamber:
13. No. replicate test chambers/sediment:
14. No. organisms/sediment:
15. Feeding regime: '
16. Test chamber cleaning:
17. Aeration:
18. Overlying water quality characteristics:
19. Test acceptability criterion:
(1) minimum control survival
(2) length or weight minimum criteria:
20. Endpoint(s)
NOTE: If an item listed does'not seem pertinent, please explain why.
500
-------
PLEASE PROVIDE INFORMATION IN SPACES BELOW IF APPROPRIATE
CONDITIONS FOR LUMBRICULUS VARIEGATUS SEDIMENT TOXIC1TY TESTS
TEST CONDITION CONDITION USED BY LABORATORY
1. Test type (static or renewal)
(specify rate) , ; _
2. Test duration:
3. Temperature: : ;
4. Light quality: , : ;
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test sediment volume:
9. Overlying water volume: chamber: _ :
10. Age of test organisms: ' -
11. Size of test organisms: '
12. No. organisms/test chamber: ; :
13. No. replicate test chambers/sediment:, _ :
14. No. organisms/sediment: ; : -.
15. Feeding regime:
16. Test chamber cleaning: ; :
17., Aeration: : : :
18. Overlying water quality characteristics: :
19. Test acceptability criterion: _ :
(1) minimum control survival :_ ;
(2) length or weight minimum criteria: , =:. . -
20. Endpoint(s) *
NOTE: If,an item listed does not seem pertinent, please explain why.
501
-------
General Culturing Issues
Substrate
Density
Water
Flow-through vs. Static
Feeds/Feeding
Genetic drift/stream differences
Known age systems
Nuisance organisms
. Light/photpperiod
Temperature
QA/QC (e.g., reproduction, reference toxicants)
General Testing Issues
Test lengths/endpoints
Organism age
Water Renewal (volumes, frequency, method of renewal)
Physical Test System (sediment volume, etc.)
Test condition and design (chambers, lighting, etc.)
Interpretation of sediment variables (e.g., organic carbon, particle size) on test results
Feeds/feeding regimes
QA/QC for acceptable tests
502
-------
Table 1. Survey respondents for sediment test organisms.
Laboratory '
Department of Fisheries & Oceans
Environment Canada
EPA-Duluth
EPA Region 1
EPA Region 8
EPA Newtown
EVS Consultants
Maryland Department of Environment
Miami University
Michigan State
NFCRC-Athens ,
NFCRC-Columbia
NO A A- Ann Arbor
Old Dominion
State of Washington
University, of Mississippi
University of Wisconsin-Superior
Wright State
H. azteca
X
X
X
X
X
X
X
X
' ' x
X
X
X
X
X
X
X
X
C. tentans/
C. riparius L. variegatus*
' '
X
X X
X
X
X~
X X
X
x x
X X
x
X X
X
a Two responses were on two different species.
Note: EPA region 6, Dallas, Texas, and the FDA-Center for Veterinary Medicine,
Maryland, responded to survey but did not have organisms in culture.
503
-------
-------
APPENDIX P:
BIBLIOGRAPHIES
FOR
FRESHWATER TEST SPECIES
505
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Group: C:\REF\CfflRSCHl.GRP
Temporary group for searching
Sorted by: Authors, Year, Title
Using Format LMNOL OCEANOGR
Current Search: term=chir
Last Search run on 9 Sep 1992, at 11:18
Last modified on 9 Sep 1992, at'12:53
Listed with Format LIMNOL OCEANOGR
Listing Created 9 Sep 1992, at 12:55
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GRANEY R. L., T. J. KE3LTY, AND J. P. GIESY. 1986. Free.amino acid pools of five species .of freshwater
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estuarine polychaete Nereis diversicolor using inherited metal tolerance. Mar. Pollut Bull. 20: 235-
238. . . .
GUNN A. M., D. T. E. HUNT, AND D. A. WINNARD. 1989. The effect of heavy metal speciation in sediment
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HORNIG C. E. 1980. Use of aquatic oligochaete, Lumbriculus variegatus, for effluent biompnitoring, p. 1-9. In
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531
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KAISER M., U. IRMER, AND K. WEILER. 1989. Monitoring of water quality: seasonal variations of heavy metals
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KLERKS P. L., AND P. R. BARTHOLOMEW. 1991. Cadmium accumulation and detoxification in a Cd-resistant
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KNEZOVICH J. P., F. L. HARRISON, AND R. G. WILHELM. 1987. The bioavailability of sediment-sorbed organic
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KOERTING-WALKER C., AND J. D. BUCK. 1989. The effect of bacteria and bioturbation by Clymenella torquata
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KREZOSKI J. R., AND J. A. ROBBINS. 1985. Vertical distribution of feeding and particle-selective transport of
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MATISOFF G., J. B. FISHER, AND S. MATIS. 1985. Effects of benthic macroinvertebrates on the exchange of
solutes between sediments and freshwater. Hydrobiologia 122: 19-33.
MCELROY A. E. 1990. Polycyclic aromatic hydrocarbon metabolism in the polycbaete Nereis virens. AquaL
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MCMURTRY M. J. 1984a. Avoidance of sublethal doses of cooper and zinc by tubificid oligocbaetes. J. Great Lakes
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155- ' - '. *> *
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535
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APPENDIX E:
WORKSHOP PARTICIPANTS
537
-------
-------
Name: Ray W. Alden
Address: Old Dominion University
Applied Marine Research Lab.
1034 W.45th Street
Norfolk, VA 23529-0456
Phone: 804-683-4195
Fax: 804-683-5293
Name: Gary Ankley
Address: ERL-US EPA
6201 Congdon Blvd.
Duluth, MN 55804
Phone: 218-720-5603
Fax: 218-720-5539
Name: Tom Armitage
Address: US EPA/OST
401 M Street, SW
Washington, D.C. 20460
Phone: 202-260-5388
Fax: 202-260-9830
Name: Ray Arnold
Address: Exxon Biomedical Sciences
Mettlers Road
East Millstone, NJ 08875-2350
Phone: 908-873-6305
Fax: 908-873-6009
Name: Tom Bailey
Address: US EPA
401 M Street, SW
Washington, D.C. 20460
Phone: 703-305-6666
Fax: 703-305-6309
Name:
Address:
Phone:
Fax:
Bev Baker
US EPA
401 M Street, SW
Washington, D.C.
202-260-7037
202-260-9830
20460
Name: Celeste Philbrick Ban-
Address: US EPA Region I Laboratory
'. 60 Westview St.
Lexington, MA 02173
Phone: 617-860-4612
Fax: 617-860-4397
Name:
Address:
Phone:
Fax:
Walter Berry
Sciences Applications International Cor
c/o US EPA ,
27 Tarzwell Dr.
Narragansett, RI 02882
401-782-3101
401-782-3030
Name: G. Allen Burton
Address: Biological Sciences Dept.
Wright State University
Dayton, OH 45435
Phone: 513-873-2201
Fax: 513-873-3301
Name:
Address:
Phone:
Fax:
Carrie Buswell
Viar&Co
Environmental Services Division
300 N. Lee Street, Suite 200
Alexandria, VA 22314-2695
539
-------
Name: Peter Chapman
Address: E.V.S. Consultants
195 Pemberton Avenue
North Vancouver, EC
CANADA V7P2R4
Phone: 604-986-4331
Fax: 604-662-8548
Name: Thomas Chase
Address: EPA/OWOW
401 M Street, SW
Washington, D.C. 20460
Phone: 202-260-1909
Fax: 202-260-6294
Name:
Address:
Phone:
Fax:
Ted Coopwood
EPA/OWEC
401 M Street, SW
Washington, D.C. 20460
202-260-8327
Name: . Pat Cotter
Address: US EPA Region K
75 Hawthorne Street
San Francisco, CA 94105
Phone: 415-744-1163
Fax: 415-744-1072
Name: Philip Crocker
Address: US EPA-Region VI
1445 Ross Avenue, 12th fir., Suite 1200
Dallas, TX 75202-2733
Phone: 214-655-7145
Fax: 214-655-7145
Name: Joe Cummins
Address: EPA Region X
Manchester Laboratory
, 7411 Beach Drive East
Port Orchard, WA 98366
Phone: 206-871-8708
Fax: 206-895-4357
Name: Kristin Day Name:
Address: National Water Research Institute Address:
Rivers Research Branch
867 Lakeshore Rd., PO Box 5050
Burlington, Ontario, Canada L7R4A6 Phone:
Phone: 416-336-4659 Fax:
Fax: 416-336-4972
Ted Dewitt
AScI
2111 S. Marine Science Drive
Newport, OR 97365
503-867-5030
503-867-4049
Name: Robert Donaghy
Address: ESD/Region m-Wheelirig
303 Methodist Bldg.
Wheeling, WV 26003
Phone: 304-234-0241
Fax: 304-234-0260
Name: Steve Ells
Address: US EPA/Technical Oversight Section
401 M Street, SW
Washington, D.C. 20460
Phone: 202-260-9803
Fax: 202-260-3106
540
-------
Name: Dorothy England
Address: ABC Laboratories, Inc.
7200 East ABC Lane
Columbia, MO 65205
Phone: 314-474-8579
Fax: 314-443-9089
Name: Gary Evereklian
Address: Microbics Co.
11908 CanfieldRd,
Potomac, MP 20854
Phone: 301-762-3793
Fax: 301-762-2534
Name: Jim Ferretti
Address: ESD/AMS-Region E
2890 Woodbridge Ave.
Edison, NJ 08837
Phone: 908-321-6728'
Fax: 908-321-6616
Name:
Address:
Phone:
Fax:
Catherine Fox
US EPA
401 M Street, SW
Washington, D.C.
202-260-1327
202-260-9830
20460
Name: Howard Fribush
Address: US EPA/Analytical Operations Branch
401 M Street, SW
Washington, D.C. 20460
Phone: 202-260-2239
Fax: 202-260-0524
Name: John Giesy
Address: Fisheries and Wildlife
Michigan State University
East Lansing, MI 48824-1222
Phone: 517-353-2000
Fax: 517-336-1699
Name: David. Gorodetzky
Address: Abt Associates Inc.
Hampden Square-Suite 600
4800 Montgomery Lane
Bethesda, MD 20814
Phone: 301-913-0500
Fax: 301-652-3618
Name: Carol Haley
Address: FDA/CVM
7500 Standish Place
Rockvffle, MD 20855
Phone: 301-295-8695
Fax: 301-295-8297
Name: Dave Hansen
Address: ERL-ORD
27 Tarzwell Drive
Narrangansett, RI 02882-1154
Phone: 401-782-3027
Fax: 401-782-3030
Name: MikeHarrass
Address: AMOCO Corporation
200 E. Randolph Drive
Chicago, IL 60601-7125
Phone: . 312-856-5116
Fax: 312-856-7584
541
-------
Name:
Address:
Phone:
Fax:
Robert Hoke
SAIC
411 Hackensack Ave.
Hackensack, NJ 07061
Name: Katherine Hollar
Address: . US EPA-Region VI
1445 Ross Ave., 12th Fir., Suite 1200
Dallas, TX 75202-2733
Phone: 214-655-6680
Fax: 214-655-6490
Name: Chris Ingersoll
Address: US Fish & Wildlife Service
4200 New Haven Rd.
Columbia, MO 65201
Phone: 314-875-5399
Fax: 314-876-1896
Name: Peter A. Jones
Address: Bureau of Water Quality Mgt.
50 Wolf Rd.
Albany, NY 12233 .
Phone: . 518-457-3651
Fax: 518-485-7786 '
Name: Darryl Keith
Address: US FJPA/ERL-Narragansett
27 TarzweU Rd.
Narragansett, RI 02882-1154
Phone: 401-782-3135
Fax: 401-782-3030
Name: Mike Kravitz
Address: US EPA/OST
401 M Street, SW
Washington, D.C. 20460
Phone: 202-260-8085
Fax: 202-260-9830
Name:
Address:
Phone:
Fax:
Jody Kubitz
Michigan State University
Institute of Water Research
334 Natural Resources Bldg.
East Lansing, ME 48824-1222
517-353-9174
513-336-1699
Name: Janet Lamberson
Address: ERL-Pacific
Hatfield Marine Science Center
2111 S.E. Marine Science Drive
Newport, OR 97365-5260
Phone: 503-867-4043
Fax: 503-867-4049
Name: Peter Landrum Name:
Address: Great Lakes Environmental Research Lab Address:
2205 Commonwealth Blvd.
Ann Arbor, MI 48105
Phone: 313-668-2276 Phone-
Fax: 313-668-2055 Fax-
Jim Lazorchak
EMSL/Newtown Facility
3411 Church St.
Cincinnati, OH 45244
513-533-8114
513-533-8181
542
-------
Name: Alex Lechich
Address: US EPA - Region H
26 Federal Plaza '
New York, NY 10278
Phone: '212-264-1302
Fax: 212-264-4690
Name: Henry Lee
Address: ERL-Pacific
Hatfield Marine Science Center
2111 S.E. Marine Science Drive
Newport, OR 97365-5260
Phone: 503-867-4042
Fax: 503-867-4049
Name: Charlie MacPherson ,
Address: Tetra Tech Inc.
10306 Eaton Place, Suite 340
Fairfax, VA 22030
Phone: 703-385-6000
Fax: 703-385-6007
Name:
Address:
Phone:
Fax:
John Malek
US EPA-Region X
1200 Sixth Ave.
Seattle, WA 98101
206-442-1286
Name: Victor McFarland
Address: US Army Corps of Engineers
Waterways Experiment, Station
, 3909 Halls Ferry Road
Vicksburg, MS 39180
Phone: 601-637-3721
Fax: 601-634-3120
Name:
Address:
Phone:
Fax:
BethMcGee
US EPA
401 M Street, SW
Washington, D.C. 20460
Name:
Address:
Phone:
Fax:
Suzy McKinney
US EPA-Region VI
1445 Ross Ave., 12th fl., Suite 1200
Dallas, TX -75202-2733
Name:
Address:
Phone:
Fax:
Brian Melzian
US EPA-Region DC
c/o ERL-Narragansett
27 Tarzwell Drive
Narragansett, RI 02882
Name: Ossi Meyn
Address: US EPA/OPPTS
401 M Street, SW
Washington, D.C. 20460
Phone: 202-260-1264
Fax: 202-260-1283
Name: William Nelson .
Address: US EPA/ERL-Narragansett
27 Tarzwell Drive
Narragansett, RI 02882-1154
Phone: 401-782-3053
Fax: 401-782-3030
543
-------
Name: Teresa Norberg-King
Address: ORD-ERL-Duluth
6201 Congdon Blvd.
DuIuth.MN 55804
Phone: ' 218-720-5529
Fax: 218-720-5539
Name: Warren Norwood
Address: Dept of Fisheries and Oceans
867 Lakeshore Rd., PO Box 5050
Burlington, Ontario, Canada L7R4A6
Phone: 416-336-4694
Fax: 416-336-6437
Name: Joe Olha
Address: US Army Coips of Engineers
26 Federal. Plaza
New York, NY 10278
Phone: 212-264-5621
Fax: 212-264-4260
Name: Jim Oris
Address: Dept. of Zoology
Miami University
Oxford, OH 45056
Phone: 513-529-3194
Fax: 513-529-6900
Name:
Address:
Phone:
Fax:
Bill Peltier
ESD
College Station Road
Athens, GA 30613
706-546-2296
Name: Harriet L. Phelps
Address: University of the District of Columbia
Biology Department
4200 Connecticut Ave., NW
Washington, D.C. 20008 :
Phone: 202-282-7364
Fax: 301-345-6017
Name: Michele Redmond
Address: AScI Corporation
2111 S.E. Marine Science Dr.
Newport, OR 97365-5260
Phone: 503-867-5031
Fax: 503-867-4049
Name:
Address:
Phone:
Fax:
Mary Reiley
US EPA
401 M Street, SW
Washington, D.C. 20460
202-260-9456
Name: John H. Rodgers, Jr. Name:
Address: Biological Field Station, Dept. of Biology Address:
University of Mississippi
Oxford, MS 38677
Phone: 601-232-5479 Phone-
Fax: 601-232-5144 Fax-
Lisa Rosman
US Army Corps of Engineers
26 Federal Plaza
New York, NY 10278
212-264-5621
212-264-4260
544
-------
Name: Norm Rubinstein
Address: ERL-US EPA
27 Tarzwell Drive
Narragansett, RI 02882-1154
Phone: 401-782-3002
Fax: 401-782-3030
Name: Keith Sappington
Address: Abt Associates Inc.
Hamden Square-Suite 600
4800 Montgomery Lane
Bethesda, MD 20814
Phone: 301-913-0500
Fax: 301-652-3618
Name:
Address:
Phone:
Fax:
Chris Schlekat
SAIC
165 Dean Knauss Dr.
Narragansett, RI 02882
(401)782-1900
Name:
Address:
Phone:
Fax:
Richard Schwer
Dupont Company
P.O. Box 6090
Newark, DE 19714-6090
Name: John Scott Name:
Address: Science Applications International Corp. Address:
165 Dean Knauss Drive
Narragansett, RI 02882
Phone: ,401-782-1900 Phone:
Fax: 401-782-2330 Fax:
Richard Scroggin
Asticou Centre
241 Cite Des Jeunes Blvd.
Hull, Quebec, Canada K1AQH3
819-953-5907
819-997-8427 ...
Name:
Address:
Phone:
Fax:
Carol Ann Siciliano
US EPA, Office of General Counsel
401 M Street, SW
Washington, D.C. 20460
202-260-8653
Name:
Address:
Phone:
Fax:
Cindy Simbanin
VIAR & Co.
Environmental Services Division
300 N. Lee Street, Suite 200
Alexandria, VA 22314-2695
Name: Jerry Smrchek
Address: US EPA/OPPT
401 M Street, SW
Washington, D.C, 20460
Phone: 202-260-1268
Fax: 202-260-1283
Name: Betsy Southerland
Address: US EPA ,
401 M Street, SW
.Washington, D.C. 20460
Phone: 202-260-3966
Fax: 202-260-9830
545
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Name: Mark D. Sprenger
Address: US EPA/ERT
2890 Woodbridge Ave., Bldg. 18
Edison, NJ 08837-3679
Phone: 908-906-6826
Fax: 908-321-6724
Name: Alexis Steen
Address: American Petroleum Institute
1220 L St., NW .
Washington, D.C. 20005
Phone: 202-682-8339
Fax: 202-682-8270
Name:
Address:
Phone:
Fax:
William Stelz
EPA/ORD
401 M Street, SW
Washington, DC 20640
202-260-5798
Name: Eric A. Stem
Address: US EPA-Region n
2-WMD, 26 Federal Plaza
New York, NY 10278
Phone: 212-264-5283
Fax: 212-264-4690
Name: Margaret Stinson
Address: Washington Dept. of Ecology
7411 Beach Dr. East
Port Orchard, WA 98366
Phone: 206-871-8708
Fax: 206-895-4357
Name: Elaine Suriano
Address: US EPA
401 M Street, SW
Washington, D.C. 20460
Phone: 202-260-7739
Fax: 202-260-3106
Name: Irene Suzukida Name:
Address: US EPA Address:
Office of Wastewater Enforcement and
Compliance
401 M Street, SW Phone:
Washington, D.C. 20460 Fax:
Phone: 202-260-9536
Fax: 202-260-1460
Rick Swartz
ERL-US EPA
2111 S.E. Marine Science Drive
Newport, OR 97365-5260
503-867-4031
Name:
Address:
Phone:
Fax:
Bill Telliard
US EPA/OST
401 M Street, SW
Washington, D.C. 20460
202-260-5131
Name: Sharon Thorns
Address: Tetra Tech Inc.
10306 Eaton Place, Suite 340
Fairfax, VA 22030
Phone: 703-385-6000
Fax: 703-385-6007
546
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Name; Dave A. Tomey
Address: USEPA-Regiori I
JFK Federal Bldg.
Boston, MA 02203
Phone: 617-565-4425
Fax: 617-565-4940
Name:
Address:
Phone:
Fax:
Brian Walls .
Army Corps of Engineers
Baltimore District
P.O. Box 1715
Baltimore, MD 21203
Name: Bill Wilber
Address: USGS/413 National Center
12201 Sunrise Valley Drive
Reston, VA 22092
Phone: 703-648-6878
Fax: 703-648-5295
Name: Joseph G. Winfield
Address: Old Dominion University
Applied Marine Research J-ab.
1034 W. 45th Street
Norfolk, VA 23529-0456
Phone: 804-683-4195
Fax:: 804-683-5293
Name: Parley Winger Name:
Address:: US FWS/School of Forest Resources Address:
University of Georgia
Athens, GA 30602 Phone:
Phone: 404-546-2146 Fax:
Fax: 404-546-2186 "
BobWischer
13454 Cotley Lane
Richmond, VA 23233
804-780-5202
804-649-9661
Name: Douglas A. Wolfe
Address: NOAA/ORCA
6001 Executive Blvd.
Rockville,MD 20852
Phone: 301-443-8933
Fax: 301-231-5764
Name: Robert Wood
Address: US EPA
Office of Wastewater Enforcement
and Compliance
, 401 M Street, SW
Washington, D.C. 20460
Phone: 202-260-9536
Fax: 202-260-1460
Name: Thomas D. Wright
Address: US Army Corps of Engineers
Waterways Experiment Station
3909 Halls Ferry Road
Vicksburg, MS 39180
Phone: 601-634-3708
Fax: . 601-634-3120
Name:
Address:
Phone:
Fax:
Maurice Zeeman
USEPA/OPPT
401 M Street, NW
Washington, D.C. 20460
202-260-1237
547
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