91 0R9301 4A
vvEFA
United States
Environmental Protection
Agency
Region 10
1200 Sixth Avenue
Seattle WA98101
Alaska
Idaho
Oregon
Washington
Water Division
Surface Water Branch
August 1993
Refinements of
Current PSDDA Bioassays
Final Report Summary
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Detailed data report available
EPA document 910/R4M33-014b
EPA DOCUMENT NUMBER: EPA 910/R-93-014a
SUMMARY REPORT
REFINEMENTS OF CURRENT PSDDA BIOASSAYS
FINAL REPORT
EPA Contract No. 68-C8-0062
Work Assignment No. 3-57
March 19, 1993
Report No. 9210.003/B.016
SAIC Project No. 01-0098-03-1009
Submitted to:
U.S. Environmental Protection Agency
26 West Martin Luther King Drive
Cincinnati. Ohio 45268
Submitted by:
Science Applications International Corporation
Environmental Sciences Division
1 8706 North Creek Parkway, Suite 110
Bothell, WA 98011
RX000003fafa7
An fmptoyee-Owned Company
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PROGRAM OVERVIEW
INTRODUCTION
Sediment larval bioassays are currently used under the Puget Sound Dredged Disposal Analysis
(PSDDA) program to assist in the determination of proposed dredged material suitability for unconfined
disposal at open water sites within Puget Sound. These bioassays are used as a screen for possible
adverse biological effects that occur due to the presence of chemicals of concern within the test
sediment. Included within the suite of tests employed within the PSDDA program are the larval
sediment toxicity tests.
The Pacific oyster Crassostrea gigas, and the Northern Pacific sand dollar Dendraster excentricus
account for most of the larval tests that have been conducted within the PSDDA program, as well as
within other regulatory programs. Larval sediment toxicity tests are not only used in the PSDDA
program, but also within Washington State's sediment quality standards (WAC 173-204) and as a part
of dredged material testing programs throughout the U.S. Within the regulatory programs, both larval
species are presumed to respond similarly to dredged material. There is no data concerning the relative
response of these two organisms to varying conditions.
While larval test protocols have been well documented (ASTM, 1991, PSEP, 1992), these tests are
thought to be sensitive (and may yield false positive effects) 1 to the entrainment of embryos by fine-
grained test materials, and may be sensitive to the presence of ammonia that is frequently found in
organic-rich Puget Sound sediments.
Suspended sediment effects include entrainment of test organisms by material settling in the chamber
during the exposure, or any other potential suspended sediment-induced causes of larval mortality. The
ideal sediment larval bioassay would measure the toxic response of a test organism to anthropogenic
chemicals, and minimize or block effects from sediment conventional and physical influences. Within
the PSDDA program, a reference sediment of similar grain size to the test material is included as a
control for sediment grain size effects on the test organisms. However, these relatively "clean"
reference sediments often have high mortalities exceeding recommended quality control standards for
acceptable test results. This may in part, be due to physical effects such as interference in the test
from larval entrainment by suspended solids, or to physiological stress due to small grain sizes. The
use of varying grain sized sediments in exposures while maintaining the constancy of other variables
could potentially contribute to the understanding of grain size effects.
Issues associated with false positive results from larval sediment bioassays were discussed at the
PSDDA 1990 Annual Review Meeting (PSDDA Third ARM Minutes)2. Based on those discussions, a
commitment was made by the PSDDA regulatory agencies to attempt to refine and resolve the issue
of false positives in larval sediment toxicity tests.. The U.S. Environmental Protection Agency (EPA),
Region 10, issued a Statement of Work to Science Applications International Corporation (SAIC),
entitled Refinements to Current PSDDA Bioassays, dated July 31, 1991 that is intended to meet that
commitment. The data presented in this report are the results of that SOW.
1 A false positive condition occurs when the bioassay results indicate that a toxic response has
occurred, but for reasons unassociated with sediment chemistry. Under these circumstances, the
measured chemicals-of-concern in the sediment do not appear to be sufficiently high to explain the
toxicological response, but the testing results indicate that significant mortality or abnormality have
occurred within the test replicates.
2 ARM Minutes, paragraph 9; Post-ARM Meeting Issue Resolution Summary, bullets on ML/SL
adjustments and Effects of Grain Size, Ammonia, and Sulfides on AET Revisions.
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SAIC was directed by EPA to focus its work on the two larval species that are frequently used in
dredging programs; the Pacific oyster Crassostrea gigas, and the Northern Pacific sand dollar,
Dendraster excentricus. Both of these organisms are found in Puget Sound, and occupy ecologically
important niches. As such, the PSDDA program uses these two organisms as important indicators of
possible deleterious effects due to dredged material disposal.
The objectives of the study were as follows:
1.) to determine the effect of ammonia on larval development. Determination of the LC50 and
EC50 of ammonia to the two larval species will assist the PSDDA agencies in interpreting larval
toxicity.
2.) to compare sensitivities of the sand dollar and oyster in both clean and contaminated
sediments.
3.) to determine if a test protocol could be identified which minimizes the chance of false
positive responses due to suspended sediment in the test chamber.
PROGRAM ORGANIZATION
The work plan was divided into a series of discrete phases that proceed in a linear fashion toward
addressing EPA's objectives. These phases were as follows:
• Phase I. Literature Search
• Phase H. Ammonia Effects on Bivalve and/or Echinoderm Species
• Phase IIIA. Species Sensitivity Comparison to Grain Size Effects
• Phase IIIB. Species Sensitivity Comparison to Contaminated Sediment Effects
This report follows that format in presentation of test results. Each Phase is presented as a discrete
document, with appropriate discussion of importance of the work, methods and materials, results,
discussions, and recommendations based on the findings in the experiments. Each section builds on
the data and information generated in the previous section.
Appendices for each phase are included with that section's report. Level II Quality Assurance Data for
the analytical work conducted in Phase IIIB have been transmitted to EPA as a separate package.
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The objectives of the study were as follows:
1.) to determine the effect of ammonia on larval development. Determination of the LC50 and EC50
of ammonia to the two larval species will assist the PSDDA agencies in interpreting larval toxicity.
2.) to compare sensitivities of the sand dollar and oyster in both clean and contaminated sediments.
3.) to determine if a test protocol could be identified which minimizes the chance of false positive
responses due to suspended sediment in the test chamber.
PROGRAM ORGANIZATION
The work plan was divided into a series of discrete phases that proceed in a linear fashion toward addressing
EPA's objectives. These phases were as follows:
Phase
I.
Literature Search
Phase
II.
Ammonia Effects on Bivalve and/or Echinoderm Species
Phase
IIIA.
Species Sensitivity Comparison to Grain Size Effects
Phase
IIIB.
Species Sensitivity Comparison to Contaminated Sediment Effects
This report follows that format in presentation of test results. Each Phase is presented as a discrete document,
with appropriate discussion of importance of the work, methods and materials, results, discussions, and
recomm'endations based on the findings in the experiments. Each section builds on the data and information
generated in the previous section.
Appendices for each phase are included with that section's report. Level II Quality Assurance Data for the
analytical work conducted in Phase IIIB have been transmitted to EPA as a separate package.
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TABLE OF CONTENTS
PHASE I. LITERATURE SEARCH
OVERVIEW 1-1
ON-LINE LITERATURE SEARCH 1-1
Bibliography 1-1
Ammonia-related Research 1-1
Research Relevant to Sediment Grain Size and to Elutriate Tests I-5
Other Relevant Studies I-8
GRAY LITERATURE SEARCH I-9
TELEPHONE INQUIRIES I-9
PHASE II. AMMONIA EFFECTS ON BIVALVE AND/OR ECHINODERM SPECIES
INTRODUCTION 11-1
METHODS AND MATERIALS II-2
TEST OVERVIEW 11-2
Dendraster excentr/cus 11-2
Crassostrea gigas 11-4
RESULTS 11-6
DISCUSSION 11-15
RECOMMENDATIONS 11-16
REFERENCES II-23
List of Tables
Table 11-1. Results of Ammonia Effects Experiment II-8
Table II-2. Calculation of Regression and Power for the Determination of the Unaerated
Ammonia EC Values 11-17
Table II-3. Calculation of Regression and Power for the Determination of the Aerated
Ammonia EC Values 11-18
Table JI-4. Testing for the Difference between the Aerated and Unaerated Regression
Coefficients 11-19
Table II-5. Summary of No Observed Effect Concentration, and Effective Concentration
values II-20
Table II-6. Calculation of Regression Equation Using Combined Aerated/Unaerated Data
Sets 11-21
Table II-7. Theoretical values for unionized ammonia determined for PSDDA echinoderm
bioassays II-22
List of Figures
Figure 11-1. Oyster Ammonia Vs. Time Aerated Treatments U-9
Figure (1-2. Oyster Ammonia Vs. Time Unaerated Treatments 11-10
Figure II-3. Echinoderm Ammonia Vs. Time Unaerated Treatments 11-11
Figure II-4. Echinoderm Ammonia Vs. Time Aerated Treatment It-12
Figure II-5. Oyster Ammonia Effects Aerated Vs. Unaerated Treatments "-13
Figure II-6. Echinoderm Ammonia Effects Aerated Vs. Unaerated Treatments H-14
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PHASE IIIA. SPECIES SENSITIVITY COMPARISON TO GRAIN SIZE EFFECTS
INTRODUCTION IIIA-1
METHODS AND MATERIALS IIIA-2
TEST OVERVIEW IIIA-2
REFERENCE SEDIMENT COLLECTION AND ANALYSES IIIA-2
Sample Preparation IIIA-3
Source of Broodstock and Spawning Conditions IIIA-4
Experimental Procedure IIIA-4
Data Analysis IIIA-5
RESULTS IIIA-5
DISCUSSION IIIA-11
RECOMMENDATIONS IIIA-14
REFERENCES IIIA-15
List of Tables
Table IIIA-1. Sampling location, conventional and grain size data for reference sediment
samples IIIA-6
Table IIIA-2. Results of Phase IIIA oyster and echinoderm larval tests with varying grain-size
reference sediment IIIA-7
Table IIIA-3. Estimates of Silt and Clay Fractions Present in Bioassay Vessels Based on Grain
Size Results IIIA-12
Table IIIA-4. Comparison of reported grain size distributions vs. mass of material in bioassay
test vessel IIIA-1 3
Table IIIA-5. Predicted Settling Rates of Silt and Clay Particles Sizes in Bioassay Chambers,
based on Stoke's Law IIIA-13
List of Figures
Figure IIIA-1. Oyster Mortality Grain Size and Aeration Effects IIIA-8
Figure IIIA-2. Oyster Abnormality Grain Size and Aeration Effects IIIA-9
Figure IIIA-3. Echinoderm Mortality Grain Size and Aeration Effects IIIA-10
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PHASE IIIB. SPECIES SENSITIVITY COMPARISON TO CONTAMINATED SEDIMENT EFFECTS
INTRODUCTION IIIB-1
METHODS AND MATERIALS IIIB-2
TEST OVERVIEW IIIB-2
SEDIMENT COLLECTION AND ANALYSES IIIB-2
Contaminated Sediment Site Selection IIIB-2
Contaminated Site Sample Collection IIIB-3
Reference Sediment Collection IIIB-3
Construction and Analyses of Contaminated/Reference Site Composites IIIB-4
Analytical Methods IIIB-4
BIOASSAY PROCEDURES IIIB-5
Test Sample Preparation IIIB-5
Source of Broodstock and Spawning Conditions IIIB-6
Experimental Procedure IIIB-6
Data Analysis IIIB-7
RESULTS IIIB-8
SEDIMENT COLLECTION AND ANALYSES IIIB-8
Sediment Conventionals IIIB-8
Sediment Analyses IIIB-9
BIOASSAY RESULTS IIIB-9
Data Acceptability IIIB-9
General Results By Station and Species IIIB-13
Results of PSDDA r-Test Comparisons IIIB-13
Results of Species Responses to Ml Treatments IIIB-13
Results of Differences by Species Between Treatments IIIB-22
Results of Species as Predictors of Apparent Sediment Toxicity IIIB-22
Comparison of Species Reference Toxicant Responses IIIB-22
DISCUSSION IIIB-25
SEDIMENT CHEMISTRY IIIB-25
BIOASSAYS IIIB-26
ANALYTICAL VALUES AS PREDICTORS OF BIOASSAY RESULTS IIIB-27
RECOMMENDATIONS IIIB-27
REFERENCES IIIB-28
List of Tables
Table IIIB-1. Sampling location, conventional and grain size data for IIIB sediment
composites IIIB-8
Table IIIB-2. Concentrations of PSDDA Chemicals of Concern Found in Test Sediments . . IIIB-10
Table IIIB-3. Results of Phase IIIB Larval Exposures IIIB-14
Table IIIB-4. Application of PSDDA bioassay criteria to Oyster as Echinoderm responses to
the (Ml) dilution series and treatments IIIB-20
Table IIIB-5. Application of PSDDA bioassay criteria to Oyster as Echinoderm responses to
the (D1) dilution series and treatments IIIB-21
Table IIIB-6. Two-tailed Mest comparisons between echinoderm vs. oysters responses for
the M1 dilution series by treatment IIIB-22
Table IIIB-7. Phase IIIB. Determination of Tukey's Wholly Significant Differences IIIB-23
Table IIIB-8. Phase IIIB. Determination of Tukey's Wholly Significant Differences IIIB-24
Table IIIB-9. Reference Toxicant LC60 and EC60 Values for Phases IIIA, and IIIB IIIB-25
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List of Figures
Figure 1. Phase 1MB, M1 CRR2 Series and Oyster Mortality . . .
Figure 2. Phase 11 IB, M1 CRR2 Series - Echinoderm Mortality . .
Figure 3. Phase IIIB, M1 CRR2 Series - Echinoderm Abnormality
Figure 4. Phase IIIB, D1/CRR4 Series - Oyster Mortality
Figure 5. Phase IIIB, D1/CRR4 Series - Echinoderm Mortality . .
IIB-15
IIB-16
IIB-1 7
IIB-18
IIB-1 9
CONCLUSIONS
APPENDIX A
Phase II Oyster
Larval Counts
Ammonia Data
Physical Monitoring Data
Reference Toxicant
Phase II Echinoderm
Larval counts
Ammonia Data
Physical Monitoring Data
Reference Toxicant
Phase IIIA
Reference Sediment Conventional Data
Phase IIIA Oyster
Larval Counts
Ammonia Data
Physical Monitoring Data
Reference Toxicant
Phase IIIA Echinoderms
Larval Counts
Ammonia Data
Physical Monitoring Data
Reference Toxicant
Phase IIIB
Sediment Chemistry Values
Phase IIIB Oyster
Larval Counts
Ammonia Data
Physical Monitoring Data
Reference Toxicant
Phase IIIB Echinoderm
Larval Counts
Ammonia Data
Physical Monitoring Data
Reference Toxicant
APPENDIX B
APPENDIX C
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REFINEMENTS TO CURRENT PSDDA BIOASSAYS
FINAL REPORT
PHASE I: LITERATURE REPORT
An Employee-Ownea Compa
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OVERVIEW 1-1
ON-LINE LITERATURE SEARCH 1-1
Bibliography 1-1
Ammonia-related Research 1-1
Research Relevant to Sediment Grain Size and to Elutriate Tests 1-5
Other Relevant Studies 1-8
GRAY LITERATURE SEARCH 1-9
TELEPHONE INQUIRIES 1-9
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PHASE 1 LITERATURE SEARCH
OVERVIEW
As part of ils overall investigations concerning larval elutriate bioassays, the U.S. Environmental Protection
Agency requested that SAIC conduct a review of the available literature prior to conducting the experiments
described in Phases II and III. Specifically, the objective of this first phase was to gather information
concerning recent findings on echinoderm and bivalve larvae comparability and sensitivity to ammonia, grain
size or the existence of sediment in bioassay containers. Information gathered also included research
relevant to sediment larval elutriate test concerns such as the possibility of false positive results due to the
presence of suspended sediment in test chambers.
The literature review consisted of on-line literature searches, a survey of gray literature (unpublished data
and reports), and a telephone survey in order to acquire information on recently published or unpublished
reports and current research. This report documents the results of the surveys, and includes an annotated
bibliography of relevant reports and a telephone inquiry list indicating the laboratories and individuals
contacted and their relative responses. It is not intended to be an exhaustive search or review; but simply a
presentation of information that is relevant to the subsequent experiments.
ON-LINE LITERATURE SEARCH
SAIC conducted an on-line literature search using the University of Washington Fisheries library system, the
National Oceanic and Atmospheric Administration Library system, and the U.S. Environmental Protection
Agency Library system in Seattle, Washington.
Key words used in the search are as follows:
ammonia, bioassay, bivalve, echinoderm, grain size, sediment elutriate test
Numerous references to ammonia toixicity to aquatic organisms were found in the literature. Selected
references were downloaded from each library system and saved onto a personal computer disc. SAIC
project toxicologists reviewed the literature search and obtained articles relevant to the Task Order. An
annotated bibliography of references collected is presented below.
Bibliography
Ammonia-related Research
Ankley GT, Katko A, Arthur JW. 1990. Identification of ammonia as an important sediment-associated
toxicant in the lower Fox River and Green Bay, Wisconsin. U.S. Environmental Protection Agency,
Environmental Research Laboratory-Duluth, Duluth, Minnesota 55804. Environmental Toxicology
and Chemistry, Vol. 9. pp. 313-322.
Toxicity of sediment pore water from 13 sites in the lower Fox River/Green Bay watershed was assessed
using a number of test species. Sediment pore water from the 10 lower Fox River sites exhibited acute
toxicity to fathead minnows (Pimephales promelas) and Ceriodaphnia dubia, and pore water samples from all
13 sites were chronically toxic to C. dubia. Sediment pore water from seven of the sampling sites was toxic
to Selenastrum capricomutum, but none of the samples were toxic to Photobacterium phosphoreum. Toxicity
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characterization, identification and confirmation procedures indicated that a significant amount of the acute
toxicity of the pore water to fathead minnows and C. dubia was due to ammonia. The identification of
ammonia, a naturally occurring compound in sediments, as a potentially important sediment-associated
toxicant has implications for sediment toxicity assessment and control, not only in the Fox River and Green
Bay, but in other freshwater and marine systems as well.
Cardwell RD, Olsen S, Carr MI, Sanborn EW. 1979. Causes of oyster larvae mortality in south Puget
Sound. Washington State Dept. of Fisheries, Olympia. NOAA Technical Memorandum ERL
MESA-39, April 1979. 79 p.
Water samples were collected from the southern Puget Sound (SPS) basin in September 1977 and
characterized for acute toxicity to Pacific oyster larvae (Crassostrea gigas), chemical composition, and
biological composition. Certain receiving waters containing the dinoflagellates Ceratium fusus and
Gymnodinium spiendens were also tested specially to determine if they were toxic to oyster larvae, as was a
laboratory culture of C. fusus. Toxicity tests of two sewage treatment plant effluents, ammonium chloride,
and salinity were also conducted. The causes of oyster larvae mortality seemed clear from the laboratory
and special receiving water bioassays of the dinoflagellates. Several multi-parameter statistical tests
attempted to ferret and rank 16 biologic and chemical parameters in terms of their association with receiving
water toxicity. Sewage plant effluents had such low toxicity that they could impart only localized toxicity in
situ. The recurring receiving water toxicity problem in SPS is believed to affect, at a minimum, other species
of bivalve molluscs. Evidence is presented suggesting the susceptibility of adult Pacific oyster, Olympia oyster
(Ostrea lurida), and Manila littleneck clam (Venerupis japonica). (NOAA)
Fitt WK, Haymans DE, Coon SL. 1989. Production and role of ammonia, an inducer of settlement of
veiiger larvae of oysters. Dep. Zool., Univ. Georgia, Athens, GA. J. Shellfish Res.; vol. 8, no. 2, p.
456
Laboratory experiments with ammonium chloride have shown ammonia to be an inducer of settlement
behavior of veligers of oysters in the genus Crassostrea. In spite of the fact that most animals and bacteria
produce ammonia as a by-product of protein catabolism, natural levels of dissolved ammonia in seawater are
typically low. This is confirmed in Georgia salt marshes, but increasing concentrations of
ammonia/ammonium occur in proximity to the substrate. High concentrations have been documented from
oyster beds in salt marshes. Eyed veligers exposed to oyster-conditioned seawater responded only to
seawater containing > 100 mu M ammonia/ammonium, suggesting that ammonia is a natural cue. As with
other invertebrate larvae, veligers of oysters can be induced to settle by an adult-produced cue. A live and
productive oyster bed, with its associated bacteria and assemblage of other invertebrates, has the potential of
providing both settlement cues and appropriate substrate for veiiger larvae.
Kingzett BC, Bourne N, Leask K. 1990. Induction of metamorphosis of the Japanese scallop Patinopecten
yessoensis Jay. Dep. Fish. Oceans, Biol. Sci. Branch, Pacific Biol. Stn., Nanaimo, B.C. V9R 5K6,
Canada. J. Shellfish Res.; vol. 9, no. 1, pp. 119-125.
Hatchery reared larvae of the Japanese scallop, Patinopecten yessoensis, were treated with different levels of
neurotransmitters including, norepinephrine, epinephrine, L-DOPA, serotonin to test the ability of these
compounds to increase percent metamorphosis in the absence of a suitable substrate. Thermal shock and
the addition of ammonia were also tested for their effect on mature larvae. Norepinephrine, epinephrine and
L-DOPA produced significant increases in percent metamorphosis. Results with ammonia were variable and
significant increases in percent metamorphosis depended on concentration and exposure time. No consistent
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significant increase in percent metamorphosis was observed when mature larvae were treated with serotonin
or subjected to cold temperature shock.
Kobayashi N. 1980. Comparative sensitivity of various developmental stages of sea urchins to some
chemicals. Biological Laboratory, Doshisha University; Kyoto, 602, Japan. Marine Biology 58, pp.
163-171.
The sensitivity to some chemical agents was examined comparatively at sperm, fertilization, cleavage, blastula,
gastrula, pluteus and metamorphosis stages of a sand dollar from Japanese waters (Peronella japonica) and a
sea urchin from the Pacific coast of Australia (Heliocidaris erythrogramma). These agents included Cu
sulphate, ABS and NH3 chloride. Responses observed included departures from control rates of fertilization
and developmental reduction at the attainment of first cleavage, gastrula, pluteus or metamorphosis stages.
Using minimum effective concentrations of the 3 chemicals at various developmental stages of P. japonica, it
was found that sensitivity to chemicals varies from fertilization to metamorphosis. It seems that sperm
activity is the most sensitive, and that fertilization and gastrulation are more sensitive than first cleavage,
blastulation and pluteus formation. H. erythrogramma seems to show nearly the same responses to Cu, but is
more sensitive at metamorphosis.
Pierson KB, Ross BD, Melby CL, Brewer SD, Nakatani RE. 1983. Biological testing of solid phase and
suspended phase dredged material from Commencement Bay, Tacoma, Washington. Washington
Univ., Seattle (USA). Fisheries Research Inst., 71 pp.
Sediments from nine sites in Blair and Sitcum Water-ways, Commencement Bay, were tested for potential
acute chemical toxicity using chinook salmon (Oncorliynchus tshawytscha) smolts, Pacific oyster (Crassostrea
gigas) larvae, and phoxocephalid amphipods. Survival of salmon smolts was not affected by 96 hr exposure to
elutriates of up to one part per thousand by volume from 5 sites. Oyster larvae developed abnormal shells
following 48 hr exposure using undiluted water drained from defrosted sediment from 4 sites, but were not
affected by 1:5 dilutions; of artificially prepared elutriates. 240 hr exposure to sediments from each of the
nine sites neither decreased survival of amphipods nor altered the time spent in the sediment or the
amphipod's ability to rebury in sand. Ammonia-nitrogen concentrations in artificially prepared 1:5 elutriates
at ambient pHs would be potentially toxic to salmonids and other fishes; therefore dredging methods that
dilute the elutriate are recommended. An elutriate dilution of 1:1000 was shown to be safe; elutriate
concentrations greater than 1:1000 could be toxic to salmonids and other fishes. Amphipod bioassays should
not be used to assess potential chemical toxicity of dredged sediments until further research clarifies
confounding factors such as anoxia and starvation.
PRC Environmental Management Inc., 1992. Results of a spiked ammonia sediment porewater study. Navy
CLEAN Contract No. N62474-88-D-5086.
Ammonia spiked sediment bioassays were conducted with the polychaete Nephtys caecoides; and porewater
eliutriate bioassays with the mysid Hoiniesimysis costata, and the amphipod Ampelisca abdita. Results of the
study indicate that sediment porewater ammonia levels have potential to cause toxicity to infaunal and
cpibenthic species. Porewater ammonia levels were highly toxic to mysids (less than 24-48 hours) and
amphipods at 96 hours. The 96-hour LC50as total ammonia was similar for both species, but the LCsoat 48
hours was significantly different between species. In spiked sediments under flow-through conditions, initial
sediment ammonia levels were rapidly decreased and no significant toxicity was found in infaunal polychaete
worms. In the future, it is suggested that polychaetes be tested under static renewal or static conditions to
assess the toxic potential of sediment absorbed ammonia.
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Sullivan BK, Ritacco PJ. 1985. Ammonia toxicity to larval copepods in eutrophic marine ecosystems: A
comparison of results from bioassays and enclosed experimental ecosystems. Mar. Ecosystems
Res. Lab., Grad. Sch. Oceanogr., Univ. Rhode Island, Narragansetl, RI 02882. Aquat. toxicol.; vol.
7, no. 3, pp. 205-217.
In an experiment designed to simulate eutrophication of a shallow coastal ecosystem, nutrients were added to
experimental ecosystems (MERL mesocosms) in six different treatment levels. The authors observed large
reductions in the numbers of normally dominant copepods of the species Acartia tonsa and A. hudsonica
associated with high concentrations of unionized ammonia (NH sub(3)) in the two most nutrient enriched
treatments. Comparison of 48 h LC sub(50) values of 10-15 mu M multiplied by I super(-l) NH sub(3)
obtained from laboratory bioassays with concentrations of NH sub(3) associated with increased mortality in
the MERL tanks indicated that bioassay data correctly predicted trends of high and low mortality as well as
fluctuations in the numbers of copepods in MERL tanks. Actual mortality rates of the mesocosm copepods
was sometimes higher than predicted, however.
Sumathi VP, Chetty AN. 1990. Ambient ammonia clearance by bivalve mollusc Lamellidens corrianus
(Lea). Dep. Zool., Sri Venkateswara Univ., Tirupati 517502, India. Environ. Ecol.; vol. 8, no. 4, pp.
1333-1334.
Freshwater mussels Lamellidens corrianus were used in ambient ammonia clearance for 5 days. The mussels
cleared the ammonia efficiently during the 5 day period. The ammonia uptake may possibly be one of the
indices of its biopurification potentials.
Viana ML de. 1987. Efecto de compuestos nitrogenados en el crecimiento de Schizopera elatensis
(Copepoda: Harpaticoidea) (Effect of nitrogenated compounds on the growth of Schizopera
, elantensis (Copepoda: Harpacticoidea)). Univ. Nac. Salta, Buenos Aires 177, Salta, Argentina. An.
Mus. Hist. Nat. Valparaiso.; vol. 18, pp. 21-27. Language - Spanish
•
The inhibitory effect of ammonia, nitrites and nitrates at different concentrations on the growth of Schizopera
elatensis was analyzed. Compounds were added in two ways: at the beginning of the experiment and daily
during the experiment. In the former, growth was inhibited; when the compounds were added daily, there
was no growth inhibition. Ammonia inhibited growth at all concentrations. Nitrites affected mainly the
nauplii stage. Ammonia and nitrates affected all development stages. The results are compared with other
reports on crustaceans, fishes and larvae.
Walch M. Dagasan L, Coon SL, Weiner RM, Bonar DB, Colwell RR., 1988. Mechanisms of microbial
induction of oyster larval settlement behavior and metamorphosis. Cent. Mar. Biotechnol., Univ.
Maryland, Baltimore, MD. First International Symposium on Marine Molecular Biology, October 9-
11, 1988, Baltimore Maryland.; vp
A close relationship exists between specific bacterial films on inert substrata and settlement of competent
larvae of the oysters Crassostrea gigas and C. virginica. Products of one particular bacterium, Alteromonas
colwelliana, are especially active in inducing spat set. Identification of the soluble metabolites of A.
colwelliana that induce search behavior has revealed at least two different kinds of active compounds. One is
ammonia, a product of amino acid degradation. The other is a group of closely related products of the
enzyme tyrosinase, including L-dihydroxyphenylalanine (L-DOPA) and one or more trihydroxyphenylalanines.
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Wang X-Y, Zhang D-H, Ji D-R, Zhang S-Q. 1985. The toxic effect of ammonia on larvae and juveniles of
oyster (Cmssostrea gigas). Shandong Mar. Cultivation Inst., Qingdao, People's Rep. China. Trans.
Oceanol. Limnol./Haiyang Huzhao Tongbao.; no. 4, pp. 66-71. Language - Chinese
Role of ammonia in toxicity tests used in evaluation of dredged material. 1992. U.S. Environmental
Protection Agency, Task 3 of Work Assignment #13.
A 96 hour range find and two 96 hour definitive assays were completed as water-only assays to determine the
level of toxicity of unionized ammonia to Ampelisca abdita. The geometric mean for the two 96 hour
definiitive LC50 is 2.27 mg/1 unionized ammonia. A 10 day solid phase assay with Ampelisca abdita shows a
correlated dose response with toxicity of unionized ammonia. The LCsoof unionized ammonia for overlying
water is 1.29 mg/1. Ammonia porewater values indicate that the amphipods were exposed to ammonia
through both the porewater and overlying water. However, the lower pH of the sediment porewater (7.2-7.5)
as compared to the overlying water (8.06-8.09) had a strong influence on the concentration of unionized
ammonia. The LC50 calculated with the porewater unionized ammonia values was 0.21 mg/1.
Ambient aquatic life water quality criteria for ammonia. U.S. Environmental Protection Agency, Office of
Research and Development, Environmental Research Laboratory, Duluth Minnesota.
This document presents a comprehensive report on the effects of ammonia on freshwater and marine
organisms. Within this document, the following data pertaining to marine bivalves and echinoderms is
presented: The LC50 for the Eastern oyster, Crassostrea virginica is 24-37 mg/L NH3 at 46-62 mm in length,
8.3-13 mg/L at 13-17 mm. The mean acute value is reported at 18.3 mg/L NH3. The LCsofor the Quahog
clam, Mercenaria mercenaria is 3.2-5.0 mg/L NH3 at 28-33 mm in length, 4.6-7.2 mg/L at 4.7-5.2 mm in
length. The mean acute value is reported as 5.01 mg/L NH3. For the mussel, Mytilus edulis, exposure of
0.097 mg/L NH3 for < 1 hour caused 50% reduction in ciliary beating rate. 0.11 mg/L exposure for <1 hour
caused 90% reduction. 0.11-0.12 exposure caused complete inhibition of cilia.
Research Relevant to Sediment Grain Size and to Elutriate Tests
Daniels SA, Munawar M, Mayfield CI. 1989. An improved elutriation technique for the bioassessment of
sediment contaminants. In Environment bioassay techniques and their application., pp. 619-631;
Hydrobiologia., vol. 188-189. ed. by Munawar M. Dixon G., Mayfield CI, Reynoldson T, Sadar MH.
Res. and Appl. Branch, Natl. Water Res. Inst., CCIW, P.O. Box 5050, 867 Lakeshore Rd.,
Burlington, Ont. L7R 4A6, Canada.
An improved method is proposed for the preparation of sediment elutriates which permits relatively realistic
determination of bioavailable contaminants. It suggests the use of rotary tumbling in a cycle of 3-4 rpm to
achieve sediment-water mixing. Experiments were undertaken to evaluate the mixing efficiency of the rotary
tumbler as compared to that of the compressed air, wrist-action shaker, and reciprocal shaker methods.
Sediment to water ratios of 0:1, 1:20, 1:10, and 1:4 were tested over 0.5, 1.0, 24, and 48-h elution periods.
Elutriate evaluations were based on chemical, physico-chemical and gravimetric determinations; and also on
super(14)C-phytoplankton bioassays using Chlorclla \11lgaris (Beyerinck). Results indicated that rotary
tumbling produced the most consistent bioassay-supportable data. It was also the most efficient procedure
when used for 1 h with 1:4 sediment-water mixtures.
1-5
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Davis HC. 1960. Effects of turbidity-producing substances in sea water on eggs and larvae of the clam
(Venus (Mercenaria) mercenana). U.S. Fish and Wildlife Service, Milford, Conn. Biological Bulletin;
vol. 118, pp. 48-54.
The effects of several concentrations of silt, clay (kaolin), Fuller's earth, and chalk on development of the
eggs of the hard clam (Venus (Mercenaria) mercenaria) and the effect on the survival and growth of their
larvae are reported. Although some clam eggs developed normally in concentrations of 4.0 g/1 of clay, chalk
or finely ground Fuller's earth, the percentage decreased as the concentration of these suspended materials
increased. In silt concentrations of 0.75 g/1 or lower, the percentage of clam eggs developing normally was
not significantly different from that in control cultures, but decreased progressively in successively higher
concentrations. Results appear to indicate that larger particles (coarse silt 62-31 microns) have the greatest
effect on clam egg development and larval growth.
Davis HC, Hidu H. 1969. Effects of turbidity-producing substances in sea water on eggs and larvae of
three genera of bivalve mollusks. Bureau of Commercial Fisheries, Milford, Conn. Biological Lab.
The Veliger; vol. 11, no. 4, pp. 316-323.
As little as 0.188 g/1 of silt caused a significant decrease in the percentage of american oyster (Crassostrea
virginica) eggs developing normally, as did 3 g/1 of kaolin (silt, clay) or 4 g/1 of Fuller's earth (dusting
powder). The percentages of oyster eggs developing normally was not affected by concentrations of silicon
dioxide of 4 g/1, regardless of particle size. Clam eggs were affected only at 4 g/1 of the smallest particles
(less than 5 microns). These smallest particles of silicon dioxide had, however, the greatest effect on survival
and growth of clam and oyster larvae. Larger particles (5-25 microns and 25-50 microns) had little effect on
survival of either species or on growth of clam larvae. Growth of oyster larvae decreased progressively as the
size of silicon dioxide particles was decreased. Bivalve larvae grew faster in low concentrations of turbidity-
producing substances than in clear seawater, possibly because the suspended particles chelate or adsorb
toxins present in larval cultures. (Legore-Washington)
Kloechner K. Rosenthal H, Willfuehr J. 1985. Invertebrate bioassays with North Sea water samples. 1.
Structural effects on embryos and larvae of serpulids, oysters, and sea urchins. Biol. Anst.
Helgoland (Zent.), Notkest. 31, D-2000 Hamburg 52, FRG. Helgol. Meeresunters.; vol. 39, no. 1, pp.
1-19.
Structural effects of bottom and surface water samples from two dumping grounds in the inner German
Bight on the development of three meroplanktonic organisms (Pomatoceros triqueter: Polychaeta,
Psammechinus miliaris: Echinodermata, and Crassostrea gigas: MoIIusca) were investigated, the
titaniumdioxide dumping site was sampled immediately after dumping (within the visible waste trail 1 km
behind the vessel), and 10 h after dumping. Samples were taken in the sewage sludge deposition area in the
intervals between the usual dumping activities, regardless of the exact dumping schedule. The preserved
bioassay test organisms were inspected microscopically to count percentages of "normal" larval hatch in test
water samples, reference water samples, and laboratory aged control water samples (5 to 10 replicates). The
relative water quality of various dumping sites was expressed in terms of "net risk" -values (Woelke, 1972)
compared to hatching rates observed in the controls.
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Meador JP, Ross BD, Dinnel PA, Picquelle SJ. 1990. An analysis of the relationship between a sand-dollar
embryo elutriate assay and sediment contaminants from stations in an urban embayment of Puget
Sound, Washington. NMFS, Environ. Conserv. Div., 2725 Montlake Blvd. E., Seattle, WA 98112.
Mar. Environ. Res.; vol. 30, no. 4, pp. 251-272.
A sand-dollai embryo test was used to assess the toxicity of contaminants in sediment elutriate samples from
Puget Sound, Washington. A synoptic chemical data set of priority pollutants was reduced and subjected to
combinatorial clustering which grouped stations by the amount of chemicals present. Clustering was done
for metals and organic compounds together and separately. Analysis of variance revealed that the embryo
test was able to predict the group of stations considered least contaminated by organic chemicals but not for
metals, although copper and lead could not be excluded due to confounding effects. The results generally
support the additivity hypothesis of toxicity in that as total contamination increased toxicity increased. Due
to a possible change in redox conditions or the release of bio-organically bound metals, it was concluded that
the elutriate test may not be appropriate for assessment of metal contaminants associated with sediment.
Ramsdell KA, Strand JA, Cullinan VI. 1989. Amphipod bioassay of selected sediments from Sequim Bay,
Washington. Northeastern Illinois Univ., Chicago, IL 60625. Marine Technology Soc., Washington,
DC., Institute of Electrical and Electronics Engineers, New York, NY. Oceans '89: The Global
Ocean. Volume 2: Ocean Pollution, pp. 443-448; Oceans '89.
Amphipod (FUiepoxynius abronius) bioassays performed in surveys of Sequim Bay suggested possible
sediment toxicity at three sites. These findings were not supported by other biological analyses and tests
(dominant infauna, oyster larvae test) nor by the finding of relatively low levels of priority pollutants. A re-
examination of the sites demonstrated that the Sequim Bay sediments were clearly nontoxic. Mean
survivorship ranged from 89 to 100%. It was hypothesized that earlier indications of toxicity may have been
due to a relatively high percentage of fines (greater than or equal to 80%) and/or a relatively low interstitial
salinity (24 ppt) encountered at one or more of the 1983-1984 sites. The continued use of Sequim Bay as
both a reference bay and a source on control sediment in future marine research is recommended.
Dinnel, Paul A. 1990. Annotated bibliography of bioassays related to sediment toxicity testing in
Washington State. Fisheries Research Institute, University of Washington, School of Fisheries. US
Army Corps of Engineers, Seattle District, Seattle, WA: Contract No. E318900PD
Jones, R. Anne. 1978. Evaluation of the elutriate test as a method of predicting contaminant release
during open-water disposal of dredged sediments and environmental impact of open-water dredged
material disposal. Vol I: discussion: final report. US Army Corps of Engineers; US Army
Engineer Waterways Experiment Station; University of Texas at Dallas. US Army Engineer
Waterways Experiment Station.
Lee, Ci. Fred. 1978. Evaluation of the elutriate test as a method of predicting contaminant release during
open-water disposal of dredged sediments and environmental impact of open-water dredged
material disposal. Vol II: data report: final report. US Army Corps of Engineers; US Army
Engineer Waterways Experiment Station; University of Texas at Dallas. US Army Engineers
Waterways Experiment Station.
Factors influencing the development of Pacific oyster larvae in 48-hour bioassays of spent sulfite liquor.
National Council for Stream Improvement, Inc.; EPA Dallas, TX: DOC NCASI 115
1-7
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Development of a modified elutriate test for estimating the quality of effluent from confined dredged
material disposal areas. US Army Engineer Waterways Experiment Station; EPA Boston, MA:
DOC 01A0004899
Hiological assessment of the soluble fraction of the standard elutriate test final report. 1977. US Army
Corps of Engineers, Waterways Experiment Station, Environmental Effects Laboratory;
Other Relevant Studies
Okubo K, Okubo T. 1962. Study on the bioassay method for the evaluation of water pollution-II. Use of
the fertilized eggs of sea urchins and bivalves. Bulletin of the Tokai Regional Fisheries Research
Laboratory, No. 32, pp. 131-140. English Summary.
A method of bioassay has been developed by using artificially fertilized eggs of sea urchins and bivalves as
test organisms. The procedures of bioassay with fertilized eggs of the test organisms are described and the
results obtained for various pollutants are compared. The method proposed in this report is advantageous
because the effects of pollutants on the development of eggs were easily recognizable in disturbed
metamorphosis. Four species tested, two species each for sea urchins and bivalves, were similar in sensitivity
to pollutants. The sensitivity of the present test organisms to pollutants was much higher than that shown by
other test organisms. The morphologically ineffective concentration of some pollutants for the embryonic
development of sea urchins and bivalves may be directly equal to the safe level of the pollutant concentration
for littoral fishes. (Katz-Washington)
Phelps HL, Warner KA. 1990. Estuarine sediment bioassay with oyster pediveliger larvae (Crassostrea
gigas). Biol. Dep., Univ. District Columbia, Washington, DC 20008. Bull. Environ. Contam. toxicol.;
, vol. 44, no. 2, pp. 197-204.
There are several standard bioassays for toxicants in water but few for sediment, the pediveliger larva of the
Pacific oyster, Crassostrea gigas, was explored as a sediment bioassay organism because of nearly year round
commercial availability, and common use in previous work with culture and toxicity testing. One comparison
bioassay was also made with the native Chesapeake Bay species, Crassostrea virginica, which may become
more readily available in the future.
Considerations in selecting bioassay organisms for determining the potential environmental impact of
dredged material. 1981. US Army Engineers Waterways Experiment Station.
Development and validation of a field bioassay method with the Pacific oyster, Crassostrea gigas, embryo: a
thesis submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy. EPA
HQ, Wash, DC: BKS OH91.57.B5W6
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GRAY LITERATURE SEARCH
Gray literature is defined as federal, state, local or private-entity sponsored research that is not generally
distributed to public library systems. Inquiries were made by phone and mail to the following institutions
that are known to sponsor or be repositories of such work, using the key search words identified above:
ammonia, bioassay, bivalve, echinoderm, grain size, sediment elutriate test
• Woods Hole Marine Biological Library
Susan Bertraux
Woods Hole Oceaoographic Institute
Woods Hole, MA 02543
508-543-1400 ext.2269
Informed that use of library requires fee and search must be done in person. Was
suggested that local SAIC office near Woods Hole be contacted to do search.
• Environment Canada
Telephone inquiries have not been acknowledged.
• Environmental Protection Agency
Literature search performed at the Seattle EPA Library using the EPA's Online Library
System (OLS), National Catalog
No information was found in this effort.
TELEPHONE INQUIRIES
Telephone inquiries were made to both public and private laboratories in order to gather information
concerning recent findings on echinoderm or bivalve larval comparability or sensitivity to ammonia, grain
size, or the existence of sediment in bioassay containers. Questions asked of each individual contacted are as
follows:
1. Has your organization been involved in any research associated with the following topics?
a. The effects of ammonia on either bivalve or echinoderm larval species (especially
development).
b. The effects of grain size distributions or the amount of suspended sediment in sediment
elutriate tests using either bivalve or echinoderm larval species.
c. Research comparing the relative sensitivities of bivalve vs. echinoderm larval species.
d. Any other research relevant of the issue of sediment larval elutriate tests (i.e., the possibility
of false positive results due to the presence of suspended sediment in the test chamber).
1-9
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2. If yes are copies of those reports available?
3. If those reports represent private clients, to whom would EPA need to address a request for
information release?
4. Are you aware of any other person or institute conducting relevant or related research to whom we
could address an inquiry?
For each inquiry, a telephone log containing the above questions was completed. A list of the organizations
and individuals contacted is provided below. A brief synopsis of conversations with each individual is also
included.
Batelle Pacific Northwest Laboratories, Sequim, WA.
Dr. Jack Word - 206-683-4151
prefers the Green Book methods - found that sediment in the chambers hinders an accurate
count of the organisms for survival and abnormality assessments - performed a "quick and
dirty" evaluation using oyster larvae - found low survival when using the PSEP method and a
much higher survival using the Green Book method for the same sediment tested - with
respect to sensitivity, they found that there was a greater sensitivity (higher mortality and
abnormality) in the controls, during certain periods of the year, if water was collected near
shore - this occurred even if water was filtered - suspected toxins from dinoflagellates - if
water was collected off shore in the straights (where diatoms were prevalent instead of the
dinoflagellates) during these periods, there was no toxic response in the controls
California Marine Pollution Lab, Granite Canyon, Monterey, CA.
Mr. Brian Anderson - 408-624-0947
generally have used sediment toxicity tests to indicate contamination - have made crude
ammonia measurements using HACH kit - found that in some of the controls which
exhibited a toxic response, there were high levels of ammonia - this data was submitted to
the San Francisco Regional Water Resources Control Board (project manager Karen
Tabersky)have done sediment elutriate tests using oyster larvae and a small number of pore
water extraction tests using echinoderms
EBASCO Environment, Rellevue, WA.
Frank S. Dillon - 206-451-4500
has studied freshwater molluscs conducting filtering assays using environmental samples -
have found a correlation between filtering rates and presence of ammonia - found
detrimental effects from low levels of ammonia over long periods - have used pH levels as
an indicator of the amount of the non-ionized ammonia fraction - has not done work on
sediments (suspended particles or grain size) - generally uses centrifuge extraction to obtain
the interstitial water. Mr. Dillon provided copies of the following articles:
Zischke JA, Arthur JW. 1987. Effects of elevated ammonia levels on the fingernail clam,
Musculium transversum, in outdoor experimental streams. Arch, of Environ. Contain.
1-10
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Toxicol., 16:225-231.
Arthur JW, West CW, Allen KN, Hedtke SF. 1987. Seasonal toxicity of ammonia to five
fish and nine invertebrate species. Bull. Environ. Contam. toxicol., 38:324-331.
Environmental Protection Agency (EPA)
Athens, Georgia, EPA Region 4; Bill Peltier - 404-546-2296, main office number - 404-546-2294
mainly deals with bioassays strictly as a regulatory measure - does not really work with
research aspects
Environmental Research Lab, Duluth, MN: Gerald T. Ankley - 218-720-5603
did not have information regarding these issues
Gulf Breeze Laboratory, FL: Barbara Albrecht - 904-934-9351
currently not doing any work with echinoderm or bivalve larvae; has been aware of effects
on urchin larvae in the past, although is not aware of specific reports from their laboratory
documenting this concern
Manchester laboratory, WA: Joe Cummins - 206-895-4347 and Margaret Stinson - 206-871-8821
did not have information regarding these issues
Newport, OR: Gary A. Chapman - 503-867-4041 and Rick Swartz - 503-867-4031
did not have information regarding these issues - not involved at this time with any work
pertaining to echinoderm and bivalve larvae, or elutriate tests
EVS Consultants, Ltd., North Vancouver, B.C.
Dr. Peter M. Chapman - 604-986-4331
involved in research concerning some of the topics although the information was confidential
- expects it to be released in June 1993
Fisheries Research Institute, University of Washington, Seattle, WA.
Dr. Paul Dinnel - 206-543-7345
currently involved in revising/writing ASTM bioassay protocols - has been more involved in
effluent studies and water column assays at this time - is not aware of current research
being performed regarding the issues of concern
Gulf Coast Research Lab, Ocean Springs, MS.
Dr. Tom Lytle - 601-875-2244
is expecting to begin studies in association with the Gulf Breeze Labs including field
validation and sediment tests to improve bioassay techniques, and revising and developing
laboratory and field sediment bioassays - some of the work will focus on bio-availability of
particular compounds and sediment structure as it affects bioavailability - will also be
examining sediment in association with the organisms's life stages
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Dr. William Walker - 601-872-4261
working with water column chemicals and effects on early life stages of fish, as well as shell
deposition and its effects on oysters - is not aware of anyone currently conducting research
on the topics of concern
Jo Ellen Hose, Shell Beach, CA - 805-773-6715
has researched the development of echinoderm and bivalve larvae, but has not examined
larval sensitivity to ammonia or grain size
MEC Analytical Systems, Inc., San Rafael, CA.
Mark J. Burke - 415-435-1847
currently involved in TIE form using echinoderms - sanitation effluent studies - is predicting
that ammonia is a problem
National Council for Air and Stream Improvement (NCASI), Shannon Point Marine Center, Anacortes,
WA.
Mr. Tim Hall - 206-293-4748
generally have been working with effluent studies and not sediments - has done some work
with methodology concerning sperm and egg ratios, fertilization, and stocking density - also
found that if they used on-line filter systems in which there is dead water for a period of
time, their echinoderms did not do well - suspected toxicity was due to hydrogen sulfide -
was not a problem when used flow through systems
National Oceanographic and Atmospheric Administration (NOAA), Seattle, WA.
Dr. Ed Casillas - 206-553-7740
currently not doing research with larvae - have done some studies with the reproductive
stages - juvenile to adult - have monitored ammonia and sulfides
Mr. Ed Long - 206-526-6338
involved in studies using the fertilization success of sea urchins as a toxicity test - extremely
sensitive - studies using undiluted pore water - results of analysis of ammonia in the pore
water indicated that there was a poor correlation (R2 = .100) with toxicity (using undiluted
pore water) and sea urchin fertilization - unpublished information at this time - will be
released in spring of 1993 - this work is with Dr. R. Scott Carr, U.S. Fish and Wildlife,
Corpus Christi, Texas
does not feel ammonia is the problem - feels it is more of a regional or site specific issue
other work has/will included studies in San Francisco Bay using the Green Book methods
(reference provided below), and other urchin fertilization tests in Tampa Bay and Galveston
(EPA, Region 6) - mentioned work by Jo Ellen Hose with echinoderm and bivalve larvae
looking at subcellular abnormality endpoints for bioassays
Long ER, Markel R. 1992. An evaluation of the extent and magnitude of biological effects
associated with chemical contaminants in San Francisco Bay, California. NOAA Technical
Memorandum NOS ORCA 64. Seattle, WA.
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South California Coastal Water Research Project, Long Beach, CA.
Steven M. Bay - 310-435-7071
has done some work with echinoderm larvae and sperm in interstitial water has a report in
press which includes ammonia studies with echinoderm larvae - is sending this information
as well as a report completed for NOAA comparing 5 bioassays in San Francisco:
Long ER, Buchman MF, Bay SM, Breteler RJ, Carr RS, Chapman PM, Hose JE, Lissner
AL, Scott J, Wolfe A. 1990. Comparative evaluation of five toxicity tests with sediments
from San Francisco Bay and Tomales Bay, California. Environmental Toxicology and
Chemistry, 9:1193-1214
Bay S, Burgess R, Nacci D. in press. Environmental toxicology and risk assessment (1st
symposium). Status and applications of echinoid (phylum Echinodermata) toxicity test
methods.
Mr. Bruce Thompson - 310-435-7071
is involved in some work with echinoids and ophiuroids and their sensitivity to contaminants
in sediments; current research includes examining sensitivity of urchins to interstitial sulfides
- doing ammonia work with urchin sperm in straight water tests - is not aware of research
occurring with bivalves or sand dollars in southern California related to sediment bioassays
Toxscan, Inc., Watsonville, CA.
Dr. Phil Carpenter - 408-724-4522
have performed some studies concerning grain size effects, in particular with Ampelisca
U.S. Fish and Wildlife, Corpus Christi, TX.
Dr. Scott Carr - 512-888-3366
have performed work with echinoderms and their sensitivities to ammonia and grain-size
mentioned sperm cell test - prefer use of pore-water rather than elutriate
WES, Vicksburg, MS.
Mr. Tom Dillon - (>01-636-3111
not currently involved with any work concerning echinoderm and bivalve larvae - have done
work in fresh water with elutriate testing - trying to determine physical and chemical factors
in mortality
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Telephone Inquiry List
Batelle Pacific Northwest Laboratories, Sequim, Wa.
Dr. Jack Word - 206-683-4151
California Marine Pollution Lab, Granite Canyon, Monterey, CA.
Mr. Brian Anderson - 408-624-0947
EBASCO Environmental, Bellevue, WA.
Frank S. Dillon - 206-451-4500
Environmental Protection Agency (EPA)
Athens, Georgia, EPA Region 4: Bill Peltier - 404-546-2296, main office number -
404-546-2294
Environmental Research Lab, Duluth, MN: Gerald T. Ankley - 218-720-5603
Gulf Breeze Laboratory, Florida: Barbara Albrecht - 904-934-9351
Manchester Laboratory, WA: Joe Cummins - 206-895-4347
Margaret Stinson - 206-871-8821
Newport, OR: Gary A. Chapman - 503-867-4041
Rick Swartz - 503-867-4031
EVS Consultants, Ltd., North Vancouver, B.C.
Dr. Peter M. Chapman - 604-986-4331
Fisheries Research Institute, University of Washington, Seattle, WA.
Dr. Paul Dinnel - 206-543-7345
Gulf Coast Research Lab, Ocean Springs, MS.
Dr. Tom Lytle - 601-875-2244
Dr. William Walker - 601-872-4261
Jo Ellen Hose, Shell Beach, CA - 805-773-6715
MEC Analytical Systems, Inc., San Rafael, CA.
Mark J. Burke - 415-435-1847
National Council for Air and Stream Improvement (NCASI), Shannon Point Marine Center, Anacortes,
WA.
Mr. Tim Hall - 206-293-4748
National Oceanographic and Atmospheric Administration (NOAA), Seattle, WA.
Dr. Ed Casillas - 206-553-7740
Mr. Ed Long - 206-526-6338
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South California Coastal Water Research Project, Long Beach, CA.
Steven M. Bay - 310-435-7071
Mr. Bruce Thompson - 310-435-7071
Toxscan, Inc., Watsonville, CA.
Dr. Phil Carpenter -408-724-4522
U.S. Fish and Wildlife, Corpus Christi, TX
Dr. Scott Carr - 512-888-3366
WES, Vicksburg, MS.
Mr. Tom Dillon - 601-636-3111
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REFINEMENTS TO CURRENT PSDDA BIOASSAYS
FINAL REPORT
PHASE II: AMMONIA EFFECTS ON BIVALVE AND/OR ECHINODERM SPECIES
An Employee-Owned Company
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INTRODUCTION 11-1
METHODS AND MATERIALS 11-2
TEST OVERVIEW 11-2
Dendraster excentricus 11-2
Crassostrea gigas 11-4
RESULTS 11-6
DISCUSSION 11-15
RECOMMENDATIONS 11-16
REFERENCES II-23
List of Tables
Table 11-1. Results of Ammonia Effects Experiment N-8
Table II-2. Calculation of Regression and Power for the Determination of the Unaerated
Ammonia EC Values 11-17
Table II-3. Calculation of Regression and Power for the Determination of the Aerated
Ammonia EC Values 11-18
Table II-4. Testing for the Difference between the Aerated and Unaerated Regression
Coefficients 11-19
Table II-5. Summary of No Observed Effect Concentration, and Effective Concentration
values H-20
Table II-6. Calculation of Regression Equation Using Combined Aerated/Unaerated Data
Sets 11-21
Table II-7. Theoretical values for unionized ammonia determined for PSDDA echinoderm
bioassays II-22
List of Figures
Figure 11-1. Oyster Ammonia Vs. Time Aerated Treatments II-9
Figure II-2. Oyster Ammonia Vs. Time Unaerated Treatments 11-10
Figure II-3. Echinoderm Ammonia Vs. Time Unaerated Treatments 11-11
Figure II-4. Echinoderm Ammonia Vs. Time Aerated Treatment 11-12
Figure II-5. Oyster Ammonia Effects Aerated Vs. Unaerated Treatments 11-13
Figure II-6. Echinoderm Ammonia Effects Aerated Vs. Unaerated Treatments 11-14
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PHASE II AMMONIA TOXICITY TO LARVAL SPECIES
INTRODUCTION
Ammonia has long been recognized as a toxicant to aquatic organisms. Numerous experimental studies
and reviews concerning ammonia toxicity exist in the literature (see EPA, 1985). The Environmental
Protection Agency utilized many of these published results to establish a water quality criterion for
ammonia (EPA, 1985). The criterion document, and published values for ammonia toxicity to a wide
range of organisms, are useful for interpreting water-column toxicity effects in regulatory effluent
bioassays.
By contrast, the role of ammonia in sediment toxicity bioassays has only recently begun to be
investigated. In a literature survey, only the paper by Ankley et al. (1990) discussed ammonia as an
important sediment-associated toxicant in freshwater sediments.
Within the context of regulatory dredged sediment testing, understanding the conditions under which
sediment-associated ammonia can cause significant bioassay responses, is of particular importance.
Biological testing of dredged sediments for disposal suitability decisions is based on the assumption
that positive (mortality) responses are correlated with the presence of contaminants of concern in the
test sediments. Positive responses in the bioassays trigger a regulatory decision that the material is
unsuitable for either open ocean or deep water disposal. Guidelines for making a suitability decision for
open water disposal are defined under the Puget Sound Dredged Disposal Analysis program (PSDDA)
based on statistically significant percentage differences as compared to reference sediment responses.
Guidelines defined in EPA's manual for Evaluation of Dredged Materia/ Proposed for Ocean Disposal
(1991) utilize statistical difference, relative to the control or reference sediment.
Ammonia is not considered to be a chemical of concern. Failure of a bioassay due to the presence of
ammonia, is not viewed by the resource agencies as sufficient reason for rejecting dredged sediment
for open water disposal. However, in the absence of information on the conditions under which
ammonia can be identified as causal agent, the regulatory agencies will reject for open-water disposal
tested material that has exceeded the bioassay interpretive guidelines for suitability.
The U.S. Army Corps of Engineers Dredged Material Management Office, Seattle District conducted
statistical analyses on echinoderm larval data submitted by bioassay laboratories under the PSDDA
program. In that study, the test sediments were found not to have elevated levels of the 58 PSDDA
chemicals-of-concern, but yet demonstrated significant biological responses, exceeding the PSDDA
guidelines. Their study found statistical correlations (a = 0.05, r2 = 0.91) between levels of
echinoderm mortality, and reported levels of aqueous total ammonia (USACE, 1991). Furthermore,
those analyses indicated that when test vessels were aerated, the apparent toxic effects of ammonia
were ameliorated. Those relationships have subsequently been used in the PSDDA program in
interpreting anomalous false positive bioassay data. In addition, all larval bioassays are currently
conducted using aeration in test vessels.
The resource agencies implementing the PSDDA' program identified the need to have corroborative,
experimentally-derived, ammonia toxicity data to support their decision making process. Furthermore,
data are required for the two larval tests organisms principally used in dredged sediment
characterization programs, the Pacific oyster Crassostrea gigas, and the sand dollar Dendraster
excentricus. While other molluscs and echinoderms are used (eg., mussels or sea urchins), oysters and
sand dollars to date have made up the bulk of sediment testing programs.
'Those agencies include the Environmental Protection Agency, the Corps, and the Washington
Departments of Ecology, and Natural Resources.
Phase II: Ammonia Effects
11-1
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The objectives of this study were as follows:
To establish the No Observed Effect Concentration (NOEC) and both the Lethal
Concentration (LC) and Effective Concentration (EC) to 20%, 30%, and 50% of oyster
and sand dollar larvae.
To determine if simple aeration of the experiment vessel during the test could affect
the test results.
METHODS AND MATERIALS
TEST OVERVIEW
Unless otherwise specified, ammonia will be taken to mean the total measured complex of NH3 and
NH4. Maintaining that convention simplifies comparisons to the selected range of nominal spiked
concentrations. However, it must be emphasized that the unionized form (NH.,) is comprises most of
the toxicity, and that the amount of unionized ammonia present in the system is dependent upon the
temperature, pH, and salinity of the test system. Unionized values will be given for the NOEC, LC, and
EC discussions.
While it was the original intent of this study to conduct the oyster and sand dollar exposures
concomitantly, gravid broodstock oysters were unavailable, or larval survival was poor during the late
winter/early spring, when the program was initiated. The initial experiment that provided the sand dollar
data was set up with both species, but oyster larval survival did not meet the PSEP-prescribed survival
guideline of 50%.
The sarcd dollar larval exposures were conducted at the laboratory of Parametrix, Inc., at its facilities
in Bellevue, WA in January, 1992. The oyster exposures were performed at SAIC's Environmental
Testing Center in Narragansett, Rhode Island in June, 1992. The basic protocols followed at both
facilities were those described in the Puget Sound Estuary Protocols fPSEP, J991) for oyster and sand
dollar larvae. Procedures followed during the testing at each facility, are described below.
Dendraster excentricus
Sample Preparation
The test dilution series was set at a range of nominal 0.0, 0.28, 0.63, 1.25, 2.5, 5.0, and 10 mg/L
as NH3-N. This dilution series was based on previous in-house work that had found an EC50 of
between nominal 0.275 and 0.625 mg/L NH3-N. Ammonium chloride was obtained from the Sigma
Chemical Corporation (ACS Reagent Grade). A 10,000 ppm NH,-N stock solution was prepared by
dissolving the ammonium chloride in distilled water, using a magnetic stir bar to ensure complete
dissolution. The stock solution was prepared fresh the day prior to the exposures.
Seawater was collected from deep, upwelled water approximately 800 meters offshore of Duwamish
Head in Seattle, Washington. Water was collected via a submersible pump from a depth of 15 meters,
and transported back to the laboratory in well-seasoned polyethylene containers. At the lab the
seawater was filtered to 0.22 ij, passed through an ultra-violet sterilizer, and the salinity adjusted to
28 ppt using deionized water. Water for testing was used within 8 hours of collection. Quarterly
priority pollutant scans conducted at the lab have shown no chemicals of concern detected in the
seawater.
Borosilicate glass jars were used for the exposure containers, with 900 mL of the filtered-adjusted
seawater. In these exposures, the ammonium dilutions were prepared by dispensing a pre-determined
Phase II: Ammonia Effects
II-2
-------
volume of the stock solution into each replicate container. To approximate conditions that would occur
under PSDDA testing, the test solutions were prepared four hours prior to inoculation of the embryos
into the test vessels. For each test concentration, six replicates were prepared: five of those were for
organism exposures, and the sixth was used to withdraw aliquots for ammonia measurements at time
points 0 (test solution preparation), 4 hours (point of organism inoculation), 24 hours, and 48 hours
(test termination).
Source of Broodstock and Spawning Conditions
Adult sand dollars were collected by divers off of West Beach on Whidbey Island. Approximately 200
adults were collected, and transported in water to the laboratory. At the lab, the organisms were
placed into a temperature controlled growth chamber held in a 15° C growth chamber in fresh
seawater with vigorous aeration. The organisms were collected 48 hours prior to testing.
Spawning of the sand dollars was conducted by injection of between 0.5 and 1.0 milliliter (mL) of a
0.5 molar solution of potassium chloride prepared in de-ionized water. Spawning occurred in the
growth chamber at 1 5° C. Adults were inverted over cups with fresh seawater to collect the gametes.
Eggs were selected by examination of the each individual females discharge under the microscope.
Only those eggs that had dense, non-vacuolate cytoplasm, and were fully-rounded, were selected for
use in the experiments. Sperm from five males were selected by examination under the microscope
for vigorously swimming cells. Counts were made of both the egg and sperm prior to fertilization.
Fertilization was controlled by dispensing a volume of sperm solution to deliver approximately 100
sperm cells per egg. Post-fertilization, the developing embryos were washed twice by allowing the
eggs to settle to the bottom, decanting the overlying water, and replacing it with fresh seawater.
Thereafter, until inoculation, the embryos were kept in solution by frequent agitation with a perforated
plunger.
Counts of the embryos were made, and a determination made as to the volume of embryo stock
solution to be dispensed to deliver a final density of between 20 and 30 organisms per mL. Volumetric
pipettes were gravimetrically calibrated to deliver the determined volume of embryos. Spawning
occurred three hours prior to, and fertilization two hours prior to test inoculation. Embryos had
developed to approximately the 1 6 cell stage at inoculation.
Experimental Procedure
For each test concentration, five test replicates were run without aeration, and five replicates with
gentle aeration. Aeration was accomplished by dispensing the air through a glass pipette set at a rate
of less than 100 bubbles per minute. As noted above, samples for ammonia measurement were taken
for each concentration from a separate sixth replicate, in aerated and unaerated treatments.
Post inoculation, physical monitoring measurements (pH, salinity, dissolved oxygen, and temperature)
were taken for each test series. Those measurements were taken again at 24 and 48 hours.
Inoculation of embryos was performed using a volumetric pipette, calibrated to deliver between 20 -
30 embryos/mL. To ensure a homogeneous distribution of embryos in the stock solution, a perforated
plunger was use to mix the solution. Post-inoculation, two 10 mL aliquots were withdrawn from each
of five seawater controls, and counted to determine the actual number of larvae dispensed into
replicates. All subsequent mortality determinations were made by comparison to the initial seawater
control counts.
To ensure consistency with previous echinoderm tests, a reference toxicant was run in a gradient series
using cadmium chloride. The series included 0, 1.5, 3, 6, and 12 mg/L as cadmium.
Phase II: Ammonia Effects
II-3
-------
The end of the test is taken as the point at which greater than 90% of the organisms in the seawater
control reach the pluteus larval stage, as defined by the PSDDA program. For each test replicate, two
10 mL aliquots were withdrawn, fixed with 5 % buffered formalin, and scored microscopically as normal
(pluteus larvael or abnormal. It should be noted here that abnormality can include larval forms that
may be embrvolooicallv correct (eg., prism stage larvae). Failure to achieve the same developmental
state as the controls is scored as abnormal - the test is interpreted as having a deleterious effect on
larval development.
Ammonia Measurements
The method for ammonia analysis are those found in the Standard Methods for the Examination of
Water and Wastewater, (1989). Ammonia, as NH3-N, was measured using an Orion Model 95-12 ion
specific electrode coupled with an Orion model 720A meter. Ammonium chloride standards "certified
traceable to National Bureau of Standards material" were purchased from the manufacturer. All
dilutions of standards were performed with volumetric glassware, using deionized water. A calibration
slope of the probe response is made using dilutions of the calibration standard, and compared to
established Control Chart values. The 720A meter, once calibrated, provides a direct read-out of NH^-N
values.
Crassostrea oioas
Sample Preparation
The test dilution series was set at a range of nominal 0.0, 1, 5, 10, 20, and 40 mg/L as NH3-N. This
dilution series was based upon previous published data by Cardwell, et al (1979) who reported an EC50
of 15.0, and an LC50 of 21.7 m/L as NH4CI. Ammonium chloride for these experiments was obtained
from Aldrich Chemical Company (Reagent Grade). In contrast to the echinoderm experiments, the
concentration exposures were made in batch (6 L), as opposed to each individual replicate. A stock
solution of NH4CI was made up in seawater immediately prior to making the appropriate dilutions. This
method proved to be more accurate in approximating the nominal values, than did the previous method.
Seawater was obtained from the University of Rhode Island's Graduate School of Oceanography
seawater intake from Narragansett Bay. This intake is proximal to the EPA's Environmental Research
Lab at Narragansett seawater intake (within approximately 300 ft.), and has been used successfully
as control water for numerous saltwater criterion work for bivalve larvae. Seawater was filtered to
0.45 ju , and transported to the laboratory in polyethylene carboys. Water for the experiments was
used within eight hours of collection.
Glass Mason jars were used for the exposure containers, with 800 mL of the filtered-adjusted
seawater. To standardize the initial ammonia for both aerated and unaerated treatments, the test
concentrations were first made up in batch, and then dispensed into the replicate vessels. To
approximate conditions that would occur under PSDDA testing, the test solutions were prepared four
hours prior to inoculation of the embryos into the test vessels. For each test concentration, six
replicates were prepared: five of those were for organism exposures, and the sixth was used to
withdraw aliquots for ammonia measurements at time points 0 (test solution preparation), 4 hours
(point of organism inoculation), 24 hours, and 48 hours (test termination).
Source of Broodstock and Spawning Conditions
Gravid shellfish were obtained from two sources: Coast Oyster Company of Quilcene, WA, and from
Hog Island Oyster Company in Bodega Bay, California. This was done to ensure maximum opportunity
for acquiring gravid animals. Animals were shipped dry on overnight freight in blue-ice cooled
containers. Upon arrival to the lab, the blue ice was removed, and the shellfish allowed to come to
room temperature prior to being placed back in the water. During that approximately one-hour period,
Phase II: Ammonia Effects
II-4
-------
shells were cleaned of fouling organisms (barnacles, mussels) and detritus to the best extent possible.
After coming to room temperature, the animals were placed in a 20° C re-circulating saltwater bath,
and allowed to siphon for one hour before thermal stimulation. While both sets of shellfish spawned,
only the Coast Oyster parental stock was used for the ammonia exposures.
Thermal stimulation to induce spawning was accomplished by quickly raising the temperature of the
water bath to approximately 30°. The oysters were closely observed, and when an individual
commenced spawning, it was transferred to a shallow tray to complete discharge of gametes.
Eggs were collected by first pouring the egg suspension gently through a 64 /v Nitex screen to remove
excess gonadal or fecal material. Prior to fertilization, the eggs were examined microscopically to
ensure that they had the normal "tear-drop" shape, and that there were no other unusual features
which would preclude the use of the eggs (e.g., large vacuoles within the eggs). Sperm was also
examined microscopically to ensure that the cells were actively swimming. Sperm density was
determined and a volume of solution that yielded approximately 100 sperm cells/egg was used for
fertilization.
As was done for the echinoderms, spawning took place 3 hours before inoculation, with fertilization
occurring just two hours prior to test exposures.
Experimental Procedure
The experimental procedure was identical to that described for the echinoderms. For each test
concentration, five test replicates were run without aeration, and five replicates with gentle aeration
accomplished by dispensing the air through a pipette set at a rate of less than 100 bubbles per minute.
Samples for ammonia measurement were taken for each concentration from a separate sixth replicate,
in aerated and unaerated treatments.
Post inoculation, physical monitoring measurements (pH, salinity, dissolved oxygen, and temperature)
were taken for each test series. Those measurements were taken again at 24 and 48 hours. The pH
of each nominal concentration was measured at the time of each ammonia sampling effort ( 0, 4, 24,
and 48 hours).
A reference toxicant of cadmium chloride was run using a concentration series of 0, 7.5, 1.5, 3, and
6 mg/L as cadmium.
The end of the test is taken as the point at which > 90% of the organisms in the seawater control
reach the prodissoconch I, or "D-shaped", larval stage, as defined by the PSDDA program. For each
test replicate, two 10 ml. aliquots were withdrawn, fixed with 5% buffered formalin, and scored
microscopically as normal (prodissoconch) or abnormal. As noted previously, abnormal larvae could
include developmentallv correct stages; failure to achieve prodissoconch within the same time frame
as the controls was interpreted as abnormal.
Ammonia Measurements
Ammonia measurements were measured using an Orion Model 95-12 probes, coupled to an Orion
model 250A meter. Ammonium chloride standards "certified traceable to National Bureau of Standards
material" were purchased from the manufacturer. All dilutions of standards were performed with
volumetric glassware, using diluent adjusted to the salinity of test samples with reagent sodium
chloride. Ammonia was measured using the standard addition method; the probe response to each
unknown sample is measured before and after addition of a known volume of standard. Because the
slope of the probe response is an important term in the calculation of results for the standard addition
method, it was checked with duplicate observations of standard before, and duplicate observations
after each set of unknown samples was read, or every two hours, whichever came first.
Phase it: Ammonia Effects
11-5
-------
Data Analyses
Calculations for determining the unionized ammonia component in the samples were based on the
methods of Whitfield (1974). A simple spreadsheet program for determination of unionized ammonia
in seawater was written by Dr. Glen Thursby of SAIC, and utilized in this report. By Dr. Thursby's
permission, the spreadsheet is provided to EPA with this report, and may be used by EPA at its
discretion.
Larval response data were first tabulated in a spreadsheet, percentage mortality and abnormality
determined by replicate, and the mean of all replicates for an exposure treatment was reported in the
data summary. Calculations are those recommended by ASTM (1991) for larval bioassays:
% Mortality = [ 1 -(number of normal plus abnormal larvae/mean initial seawater control countsll * 100
% Abnormality = (number of abnormal larvae/number of normal plus abnormal larvae) *100
This method of data expression was selected over the PSDDA combined mortality (mortality plus
abnormality) endpoint. This was specifically done to distinguish be able to distinguish the lethal
concentration (LC) from the effective concentration (EC). Data were then normalized to the seawater
control mortality and abnormality responses by simple subtraction of the respective seawater values
from the associated exposure concentration.
There were insufficient intermediate data points to calculate the regression line for determination of
oyster LC and EC responses As such, a geometric mean for the median response was calculated (Zar,
1989). To calculate the equation of the line for the echinoderm EC responses, least squares regression
was used (Zar 1984). Calculation of the regression equation was done using Microsoft's Excel 4.0,
with confirmation using equations defined in Zar (1984). All other statistics were calculated by
formulae entered into the spreadsheet.
Determination of the probability of encountering a Type II (beta) error in the regression was performed
using the Fisher z transformation of the critical value of r, following the methods of Zar (1984).
To determine if significant differences occurred between the aerated and unaerated treatment, the
method described by Zar for the testing of two sample regression coefficients was followed.
Determination of the NOEC was by visual examination of the data - statistical analyses were not
necessary.
For calculation of LC, EC, and NOEC concentrations, the total ammonia, and un-ionized ammonia,
values recorded at the initiation of the experiments (T0 ) were used. EC and LC 20 and 30 values were
determined as they correspond to the PSDDA regulatory guidelines for suitability determinations in
dredged sediment testing.
RESULTS
Results of the ammonia effects experiment appear in Table 11-1. Larval data tables, the physical
monitoring data, and ammonia measurements may be found in the Phase II appendix tables (Appendix
A).
Data overall exceeded PSEP quality assurance requirements for larval bioassays. For the oysters,
control mortality was less than 20% for the unaerated treatment, and 24% for the aerated treatments.
Abnormality was 1.9% and 0.6%, respectively. Mortality for the unaerated echinoderm control was
Phase H: Ammonia Effects
II-6
-------
5.6%, and 2.4% for the aerated control. Abnormality in the unaerated control was 11.3%, which
exceeded the PSEP-specified limit by 1.3%. The effect on overall data quality was interpreted to be
negligible. Abnormality for the aerated control was 2.1 %. The physical monitoring data also show that
for both species tests, all parameters were well within established guidelines.
Table 11-1 presents the results of Phase II, with data normalized to final seawater mortality and
abnormality values. It should be re-emphasized that % mortality is the percent larvae not recovered,
relative to the number of larvae in the seawater control. Abnormality is the number of abnormal larvae,
divided by the total number of larvae recovered in that replicate.
For the oyster ammonia solutions, the actual measured values were close to the nominal values. Table
11-1, and Figure 11-1 and II-2 show that the levels of ammonia remained constant throughout the course
of the experiment, in both un-aerated and aerated treatments. In contrast with the oyster results, the
ammonia levels in both the echinoderm aerated and unaerated treatments dropped over time (Figures
11-3, 11 - 4); by as much as 1.6 mg/L in the nominal 10 mg/L vessel.
Larval oysters were not affected by ammonia concentrations up to 4.68 mg/L (0.13 mg/L un-ionized
NH,), but showed high mortality and abnormality responses to concentrations at 9.79 mg/L, and above.
It is interesting to note that above 9.79, the response was asymptotic Figure II-5; there was no further
increase in mortality or abnormality at higher concentrations. All of the surviving larvae in
concentrations > 9.79 mg/L were truly abnormal; i.e., were non-identifiable cellular masses that did
not resemble any developmental stage. No differences were observed in the unaerated and aerated
treatments.
In contrast to the oysters, echinoderm larval mortality was negligible at all concentrations tested (Figure
II-6). However, larval development was impacted in concentrations as low as 1.82 mg/L (0.01 mg/L
unionized NH3). As with the oyster larvae, the response appears to become asymptotic at 4.26 mg/L.
During larval scoring, most of the abnormal larvae scored appeared to be embryologically distinct forms.
The preponderance of abnormal larvae scored at 1.82 mg/L were still at the pre-pluteus prism form (see
Dinnel and Stober, 1985). Larvae exposed to 4.26 mg/L were still at the gastrulation phase.
In these exposures, the NOEC for oyster larvae can be set at 4.68 mg/L total ammonia, corresponding
to an unionized value of 0.08 mg/L. Calculation of lethal or effective concentration values is dependent
upon observing intermediate values on a dose/response continuum. Unfortunately, in the case of
oysters, the response was "all or nothing" for both mortality and abnormality. As such, the best
estimate for both LC50 and EC50 is the geometric mean of the two ammonia values associated with
the low and high responses, which was calculated as 6.83 mg/L, which would correspond to an
unionized value of 0.13 mg/L.
For echinoderm larvae, there was no test concentration that produced lethality during the 48 hour
exposure. For the abnormality response, the NOEC was established as 1.24 mg/L (0.014 mg/L
unionized ammonia). Calculation of the regression equation for the two treatments yielded different
equations of the line, but virtual similarity in expression of EC50 values (Tables ll-2,3). When tested,
the two regression equations were found to be statistically different at an alpha level of 0.05 (Table
II-4).
Testing for a Type II (beta) error indicated that the probability of a beta error is less than 0.01 % for
both aerated and unaerated treatments (see Tables 11-2 and 11-3).
Phase II: Ammonia Effects
11-7
-------
Table 11-1 Results of Ammonia Effects Experiment
Crassostrea gigas
UnAerated
Measured Tota
NH3 (mg/L)
Aerated
Measured Total NH3 (mg/L)
Nominal NH3
Time
Time
Concentration (mg/l)
% Mortality
% Abnormality
To
T4
T24
T48
% Mortality
% Abnormality
To
T4
T24
T 48
0
000
0 00
000
N D
N D
ND
000
0 00
0 00
ND
N D
ND
1
1 70
020
089
1 06
111
1 02
000
030
0 89
1 04
099
1 07
5
O.OO
1 00
468
502
4 92
4 88
860
1 70
4 68
508
488
458
10
69 30
94 50
9 79
10 31
10 02
994
74 30
96 90
9 79
10 20
977
10 61
20
77 60
98 10
19 06
1952
18 26
1920
75 10
99 40
1906
18 94
17 26
1890
40
7970
98 10
37.81
40 65
3921
41 00
7530
99 40
37 81
41 83
37 35
41 40
Dendraster excentricus
Un-Aerated
Measured Total
NH3 (mg/L)
Aerated
Measured Total NH3(mg/L)
Nominal NH3
Time
Time
Concentration (mg/L)
% Mortality
% Abnormality
To
T4
T24
T48
% Mortality
% Abnormality
To
T4
T24
T48
000
000
000
000
000
0 02
002
000
000
000
002
0 03
0.28
000
350
0 21
021
024
020
5 10
050
0 27
022
0 20
0 20
0 63
200
1 00
063
045
0.51
0 48
1 50
1 60
052
0 46
043
034
1 25
630
1 60
1 14
1 01
1 01
094
000
1 00
1 24
0 92
084
076
250
640
46 30
1 82
1 92
1 92
1 82
390
31 80
232
1 93
1 66
1 52
500
460
88 50
4 26
407
329
3 15
230
97 60
386
3 79
366
3 07
1000
260
88 40
7 64
7 84
800
605
320
97 70
7 32
8 12
711
608
Phase II Ammonia Effects
II-8
-------
FIGURE II-1 OYSTER AMMONIA & TIME
AERATED TREATMENTS
a
E=S
~fcB-
P 20 6
0
~r~
12
24 36
TIME FROM INITIATION (hrs)
-v
48
NOMINAL 10mg/L --ST- NOMINAL 20 mg/L -ffl~ NOMINAL40m
-------
5
s»
o
3
3
Q
3
5'
5!
5"
n
0
FIGURE II - 2 OYSTER AMMONIA VS. TIME
UNAERATED TREATMENTS
50
I
£40
30
g20
Q
g10
a
izd
0
' " "1 '
12
i
~T-
24
TIME FROM INITIATION (hrs)
X- NOMINAL 1 rne/L •- NOMINAL 5 mg/L ¦- NOMINAL 10 mgO. ^ NOMINAL 20 mg/L -®- NOMINAL 40 mg/L
-------
$
85
«6
i.
I
8
i
E
<6
z
o
E3-
--ffi-
FIGURE Ii-3 ECHINODERM AMMONIA & TIME
IJNAERATED TREATMENTS
.. • - -¦ : H . - - -- -
8
Q
a,
D *
to
<
0
i
f
X
0
12
24
TIME FROM INITIATION (hrs)
36
48
NOMINAL 0 28 mg/L -X~ NOMINAL 0.63 mg/L NOMINAL I 25 mg/L
-U~ NOMINAL 2.5 mg/L -•*- NOMINAL 5 mg/L -ffl- NOMINAL JO mg/L
-------
FIGURE II - 4 ECHINODERM AMMONIA VS.
UNAERATED TREATMENTS
Lb
m-
Y
¦-a—
ill} •
12 24 36
TIME FROM INITIATION (hrs)
48
-*r- NOMINAL 0.28 mg/L ~X- NOMINAL 0.63 mg/L NOMINAL 125 mg/L
a NOMINAL 2 5 mg/L -*B*- NOMINAL 5 mg/L -Bi~ NOMINAL 10 mg/L
-------
FIGURE II-5 OYSTER AMMONIA EFFECTS
DERATED VS UN AERATED TREATMENTS
0.89
4 68
9.79
19.06
0
MEASURES TOTAL AMMONIA
-------
5
to
Crt
(V
I
0
3
5
1
-------
DISCUSSION
The results of the ammonia stability over time between the two species appear on the surface to be
dichotomous. Ammonia levels in the oyster exposures remained stable, wttile the higher nominal
concentrations in the echinoderm dropped over time. Re-examination of the laboratory analysis records
for the echinoderm exposures revealed that the standards measured were off by 10 - 20% of the
expected value (eg., 10 mg/L standard measured as 9 mg/L), and the T48 matrix spike run with the
batch recovered only 80% of the spike. The measurements made for the oyster tests did not show
a similar variance. While this is a modest degree of imprecision that is within laboratory OA
requirements, a 20% difference could account for the apparent drop. Therefore, it is concluded that
ammonia concentrations remained stable over the course of the 48 hour exposure in both unaerated
and aerated treatments.
Based on the limited data, it appears that for oysters, the mortality response is not different from the
abnormality response. The appearance of misshapen larval forms indicates that the oyster development
is severely impacted by the levels of aqueous ammonia. By contrast, the sand dollar larvae were not
acutely affected by ammonia, but the chronic effect was readily apparent. The main effect observed
during these experiments was an inhibition of normal larval development. It should also be pointed out
that the sand dollar larvae were more sensitive to the effects of ammonia, than were oyster larvae.
The effects of ammonia toxicity on sand dollars are in general agreement with those found by
Kobayashi (1977) for the Japanese sand dollar, Peronella japonica, and the sea urchin
Stronglyocentrotus droebachiensis (Kobayashi, 1981). For both of those echinoderm species, ammonia
as low as 2 mg/L caused a retardation of the developmental processes, and at 5 mg/L, the larvae
developed to the blastula stage where development was arrested.
That there were statistical differences noted between the echinoderm aerated and unaerated treatments
is consistent with the Corps' findings (1991). However, the findings of this study are not in complete
agreement with the Corps'. In addition to different regression equations (y = 1 5.09 + 35.46x vs. this
study), the Corps' data suggest 40% mortality at 0.6 mg/L total-ammonia. Under salinity, temperature,
and pH conditions prescribed by PSEP, the level of unionized ammonia would be 0.01, a value that this
study suggests should have no effect. However, it must be emphasized that the data used by the
Corps was taken in whole sediment exposures, as opposed to the spiked seawater exposures in these
experiments.
These findings are only useful if a threshold level for evaluating potential ammonia false positive
responses can be set. In order to strengthen the data set for setting a critical ammonia value, we
elected to combine the aerated and unaerated data sets. This decision was based on evaluating the
expected EC values, and determining that for values of EC50 and greater, the level of unionized
ammonia under standard conditions was virtually the same. The summation of both species NOEC and
EC values is given in Table 11-5, the combined regression is given in Table 11-6.
There was sufficient statistical power within the experimental design to set with confidence the levels
of unionized ammonia associated with 20 and 30% effects. What limits the application of these
conclusions in the regulatory environment would be the lack of power associated with only one
ammonia reading associated with five replicate larval readings per test sediment. With these few data,
the difference in effects would need to be approximately 50% in order to detect a significant difference
at an o = 0.05. As such, the EC50 values for echinoderm, set at 0.03 mg/L unionized ammonia, would
be the proposed ammonia criterion value.
However, the percent variance allowed for in the specific ion probe must be accounted for in setting
the criterion. In this study, the testing laboratories allow for a variance of ± 15 in the calibration
slope. Thus, two values may be off by as much as 30%, and still reported as an allowable detect by
the analytical operator.
Phase II: Ammonia Effects
11-15
-------
Based on the above argument, it is proposed that an echinoderm effect threshold be set at 0.04 mo/L
as unionized ammonia. This value is derived by taking the minimum detectable difference of 50%
(0.03 mg/L unionized), and allowing for a 30% variation on the analytical measurements. It is of
interest to note that EPA's Ammonia Criterion Document sets as its 4-day criterion concentration for
pH = 8.0, 15°C, in freshwater at 0.036 mg/L.
It is further proposed that the NOEC unionized ammonia levels be employed as a warning level.
Unionized ammonia measurements of 0.014 mg/L (NOEC) would be used as to indicate that additional
ammonia monitoring during the test would be required.
Insufficient dose/response data was generated for the oyster larvae to develop an oyster ammonia
threshold value. However, a value for echinoderms was proposed that corresponded to the EC50. As
such, an interim value equal to the ovster larval LC/EC50 of 0.13 mo/L unionized ammonia is proposed,
until such time as additional work is conducted to more rigorously define the number.
Table II-7 presents a compilation of theoretical values for unionized ammonia that were determined over
the range of parameters allowed for a PSDDA echinoderm bioassay. Total ammonia values are
presented in the left-hand column, while unionized ammonia values for the allowable temperature
(1 5°C ± 1°) and pH (7.5 - 8.5) at a constant salinity of 28 ppt. Values shaded represent those
concentrations of unionized ammonia exceeding the proposed criterion. Mortality results falling within
those shaded areas, could be considered for false positive effects due to the levels of unionized
ammonia.
RECOMMENDATIONS
An ammonia testing criterion of 0.04 mg/L unionized ammonia is proposed for the echinoderm
test. Data may be qualified as a possible false positive response if un-ionized ammonia values
in echinoderm tests are greater than or equal to 0.04 mg/L.
The above criterion value relates specifically to echinoderm abnormality, not mortality. For an
acute criterion to be set for echinoderm larval mortality, additional work is necessary.
A warning level of 0.014 mg/L unionized ammonia is recommended. The warning level would
be used as to indicate that additional ammonia monitoring during the test would be required.
An interim oyster-specific criterion is proposed as 0.13 mg/L unionized ammonia. Some caution
is recommended in using this number for interpretation, as it is an estimate. Additional work
is recommended to better define that number.
Aeration appears to have an effect on ammonia toxicity on echinoderm. PSDDA should
continue to use aeration in the larval bioassays.
All laboratories performing PSDDA bioassays should be required to express all ammonia values
as the un-ionized form. The SAIC spreadsheet format could be made standard for data
submittal.
Phase //: Ammonia Effects
111 6
-------
Table II - 2. Calculation of Regression and Power (or the Determination of the Unaerated Ammonia EC Values for Echinoderms
DATA
Measured
Proportional
Adjusted
Total Ammonia
Abnormality
Abnormality
1.14
0.13
0.00
1 14
009
-0 04
1 14
0 10
-0 02
1 14
013
000
1 14
oto
-0 03
1 14
010
-0 02
1 14
0 17
004
1 14
0 16
003
1 14
0 15
002
1 14
0 15
0 02
1 82
0 71
058
1 82
0 65
053
1 82
057
044
1 82
0 65
053
1 82
066
0.53
1 82
0 67
054
1 82
0 76
0 63
1 82
072
0.59
1.82
028
015
1 82
029
0 16
426
1 00
0.87
426
1 00
087
426
099
086
426
1 00
087
426
1 00
087
426
1 00
087
426
1 00
087
426
1 00
087
426
099
086
426
1 00
087
Regression Statistics
Multiple R 0 90
R Square 0.82
Adjusted R Square 0.81
Standard Error 0 16
Observations 30 00
Analysis of Variance
df
Sum of Squares Mean Square
Significance F
Regression
Residual
Total
1.00
28 00
29 00
332
0 74
406
332
003
125.32
0.00
Coeffict
Standard Error
t Statistic
P- value
Lower 95%
ier 95%
Intercept
x1
-0 15
0.25
006
002
-2 49
11 19
002
0.00
-0 28
0.20
-0 03
029
Equation of the line =
y = mx ~ b =
0.25x -0.1S
EC20 =
EC30 =
EC50 =
(0.2+0.15^0.25 =
(0 3+0 15)/0.2S =
(0 5+0.15)/0.25 =
1 40
1 80
260
Calculation of f) Error using Fisher's z Transformation (Zar, 1984)
zp(2) = (z-z,alpha)"sqrt(n-3)
n = 30 00 degrees of freedom = 29
r = 0 9 a = 0.05
z = 0 5*(LN((1+r)/(1-r))
1 49
r 0 05(2),39 = 0 308
z 0 05 = 0.3183
Zp(2)= (1 49-0 3183)*(sqrt(30-3)) = 6.088331793685
P= P(Z>6.088) <0 0001
Therefore, the power of the test = 1 - p = 0 9999
-------
Table II - 3. Calculation of Regression and Power (or the Determination of the Aerated Ammonia EC Values for Echinoderms
DATA
Measured
Total Ammonia
Proportional
Abnormality
Adjusted
Abnormality
1 24
004
0.01
1 24
004
001
1 24
003
-0 00
1 24
002
-0 02
1 24
0 02
-0 02
1 24
003
-0 00
1 24
003
-001
1 24
003
-0 00
1 24
004
0 01
1 24
004
001
232
042
0 39
2 32
0.29
026
2 32
031
028
2 32
041
0 38
2 32
029
026
2 32
0.29
026
2 32
035
0.32
2 32
034
031
2 32
030
0.27
232
039
036
386
1 00
0 97
386
1 00
096
3 86
1 00
096
386
1 00
097
386
1 00
096
386
1.00
097
386
1 00
097
386
099
096
386
1 00
097
386
1 00
096
Regression Statistics
Multiple R 0 99
R Square 0.98
Adjusted R Square 0.98
Standard Error 0.05
Observations 30
Analysis of Vanance
df
Sum of Squares Mean Square
Significance F
Regression
Residual
Total
1.00
28
29
4 82
0 08
490
482
0 00
1726 78
000
Coeffici
Standard Error
t Statistic P-value Lower 95% Upper 95%
Intercept
x1
-0 50
0 37
0 02
001
-20 56
41 55
000
000
-0 55
035
-0 45
0.39
Equation of the line =
EC20 =
EC30 =
EC50 =
y = mx + b =
(0.2+0.5)/0 37 =
(0 3+0 5)/0 37 =
(0.5+0 5)/0 37 =
0.37x -0.5
1 89
2.16
270
Calculation of p Error using Fisher's z Transformation (Zar, 1984)
Zp(2) = (z-z,alpha)*sqrt(n-3)
n = 30
r = 0.99
z= 0 5*(LN((1 +r)/(1-r))
276
degrees of freedom = 29
a = 0.05
r0.05(2),39 =
z 0 05 =
031
032
Z[3(2) =
(2 76-0 3183)"(sqrt(30-3))
12 69
P(Z>12 69) <0 0001
Therefore, the power of the lest =
l -p =
0 9999
-------
Table II - 4 Testing for the Difference between the Aerated and Unaerated Regression Coefficients
Null Hypothesis: Unaerated Regression Coefficient = Aerated Regression Coefficient
Unaerated Aerated
n=
30
n=
30
Mean Sum X =
2.41
Mean Sum X =
2.47
Mean Sum Y =
0.45
Mean Sum Y =
0.43
Sum Squares X =
227.596
Sum Squares X =
218.196
Sum Square Y =
10.0056
Sum Square Y =
10.30806
Sum X*Y =
85.6848
Sum X*Y =
86.9808
b =
-0.15
b =
-0.5
m =
0.25
m =
0.37
residual SS =
0.74
residual SS =
0.08
residual df =
28
residual df =
28
1 Pooled Residual Mean Square
= (residual SS Unaerated + residual SS Aerated)/(residual df of Unaerated + residual df Aerated)
(0.74 + 0.08)/(28 + 28) = 0.014643
2. Standard error of the difference between regression coefficients
= square root [(pooled residual mean square/sum x-squared unaerated) + (pooled residual mean square/sum x-squared aerated)]
sqrt((0.014643/227.6)+(0.014643/218.2)) = 0.011465
3. Calculation of t statistic
= (slope ot unaerated - slope of aerated)/combmed standard error
-0.15-0.5/0.014643 = 33.99601
4. Critical t for two -tailed test at alpha 0.05 2.005
Calcualted t > Critical t
Therefor, reject the null hypothesis
Phase 11: Ammonia Effects
II - 19
-------
Table 11-5. Summary of No Observed Effect Concentration, and Effective Concentration values.
SPECIES
MORTALITY
ABNORMALITY
NOEC
NOEC
EC20
EC30
EC50
Total
Union.
Total
Union.
Total
Union
Total
Union
Total
Union
OYSTER
4.68
0.08
4.68
0.08
N.D.
N.D.
N.D.
N.D.
6.83
0.13
SAND DOLLAR
N.D.
N.D.
1.24
0.014
1.63
.019
1 .97
.022
2.63
.03
Phase II: Ammonia Effects
11-20
-------
Table II - 6. Calculation of Regression Equation Using Combined Echinoderm Aerated/Unaerated Data Sets.
Measured
Total Ammonia
Adjusted
Abnormality
1.14
0.00
1 14
-0 04
1 14
-0 02
1 14
000
1 14
-0 03
1 14
-0 02
1 14
004
1 14
0 03
1 14
0 02
1 14
0 02
1 24
0 01
1 24
001
1 24
-0 00
1 24
-0 02
1 24
-0 02
1 24
-0 00
1 24
-0 01
1 24
-0 00
1.24
0 01
1 24
0 01
1 82
058
1 82
0 53
1 82
044
1 82
053
1 82
0 53
1 82
0 54
1 82
0 63
1 82
059
1 82
015
1 82
0 16
2 32
0 39
2 32
026
2 32
0 28
2 32
038
232
026
232
026
2.32
0.32
2 32
0.31
Regression Statistics
Multiple R 0 93
R Square 0 87
Adjusted R Square
Standard Error
Observations
087
0.14
60
Analysis of Variance
df
Sum of Squares
Mean Square
F
Significance F
Regression
Residual
Total
1.00
58
59
779
1 17
896
779
0 02
385 82
0.00
Coefflc/
Standard Error
t Statistic
P-value
Lower 95%
Upper 95%
Intercept
x1
o o
g 8
0 04
002
-701
20
0.00
000
-0 37
0.27
-0 21
033
Equation of the line =
y = mx*b-
0.30x -0.29
EC20 = (0.2+0 29)/0.3 = 1 63
EC30 = (0 3+0 29)/0 3 = 1 97
EC50 = (0 5+0.29)/0.3 = 2_63
Measured
Total Ammonia
Adjusted
Abnormality
386
0.97
386
096
386
096
386
097
386
096
386
0 97
386
0 97
3 86
096
386
0 97
386
097
Measured
Adjusted
Total Ammonia
Abnormality
4.26
087
426
087
426
086
4 26
087
426
087
426
087
426
087
4 26
087
426
086
426
087
-------
Table 11-7. Theoretical values for unionized ammonia determined for PSDDA echinoderm bioassays.
Measured Total Ammonia
ppm
Unionized Ammonia at
14 degrees C
Unionized Ammonia at
15 degrees C
Unionized Ammonia
16 degrees C
pH = 7.5
pH = 8.0
pH = 8.5
1
0.01
0.02
0.06
2
0.01
0.04
0.13
3
0 02
0.08
0.19
4
0.03
6.08
0.25
5
0 03
0.10
0.32
6
0 04
0.13
0.38
7
O.OS
0.15
044
8
0 05
0.1?
0.51
9
0.06
0.1$
6.$?
10
6.07
0.21
06 3
11
007
0.23
089
12
0.08
0.25
0 76
13
0.09
0.27
0.62 *
14
0.09
0 29
0.88
15
0.10
0.31
0.95 ~
16
0.11
0.33
W
17
0.11
0.35
1.07 :
18
0.12
0.38
t.14
19
0.13
0.40 .
1.20 .
20
0.13
0.42
1 26
pH = 7.5 pH = 8.0 pH = 8.5 pH = 7.5 pH = 8.0 pH = 8.5
001 0.02
0.01 . 0.04
0.02 5 007
0 03 0.09
0\ * v 1 ^
• . M
m •.»
foe 0.18
.\SVAftt-.Viw.SW.,.W.,MV.,.V>,*V.V.WA
0.20
. 0.0? 0J22
" 0.08- 0.23
* 0.09 6.27
•\ 0.00
; o,
-------
REFERENCES
APHA 1989. Standard Methods for the Examination of Water and Wastewater. American Public Health
Association. 1015 Fifteenth Street NW. Washington, D.C. 20005
Ankley, G.T., A. Katko, and J.W. Arthur. 1990. Identification of Ammonia as an Important Sediment-
Associated Toxicant in the Lower Fox River and Green Bay Wisconsin. Environmental
Toxicology and Chemistry 9:313 - 322.
American Society For Testing Materials. 1991, Standard Guide for Conducting Static Acute Toxicity
Tests Starting with Embryos of Four Species of Saltwater Bivalve Molluscs. Method E 724-89.
1991 Annual Book of ASTM Standards Volume 11.04. ASTM 1916 Race Street, Philadelphia,
PA 19103-1187.
Cardwell.R.D., C.E. Woelke, M.I. Carr, and E. W. Sanborn. 1979. Toxic Substance and Water Quality
Effects on Larval Marine Organisms. State of Washington Department of Fisheries, Technical
Report No. 45.
Dinnel, P.A., and Q.J. Stober, 1985. Methodology and Analysis of Sea Urchin Embryo Bioassays.
Circular No.85-3. Fisheries Research Institute, University of Washington. Seattle, WA.
Kobayashi, N. 1977. Preliminary experiments with sea urchin pluteus and metamorphosis in marine
pollution bioassays. Publ. Set Marine Biol. Lab 24:9-21
Kobayashi, N. 1981. Comparative toxicity of various chemicals, oil extracts, and oil dispersant
extracts to Canadian and Japanese sea urchin eggs. Publ. Seto Marine Biol. Lab 26: 123 -
133.
PSEP, 1991. Recommended Guidelines for Conducting Laboratory Bioassays on Puget Sound
Sediments. U.S. EPA, Region 10, Office of Puget Sound. Seattle, WA.
USACE, Seattle District. 1991. Memorandum for Record. Decision on the Suitability of Dredged
Material Tested under PSDDA Guidelines for Bellingham Maintenance Dredging in Whatcom
Creek Waterway, Squalicum Creek Waterway, and l&J Street Waterway to be Disposed of at
the Bellingham Bay Nondispersive Open Water Disposal Site and Rosario Straits Dispersive Site.
CENPS-OP-DMMO. 3 June 1991.
U.S. Environmental Protection Agency, 1991. Evaluation of Dredged Material Proposed for Ocean
Disposal, Testing Manual. United States Environmental Protection Agency and the U.S. Army
Corps of Engineers. EPA - 503 /8-91 / 001.
U.S. Environmental Protection Agency, 1985. Ambient Aquatic Life Water Quality Criteria for
Ammonia. NTIS PB85-22711 4.
Whitfield, M. 1974. The hydrolysis of ammonium ions in sea water -- a theoretical study. J. Mar. Biol.
Assoc. U.K. 54:565 - 580.
Zar, J.H. 1984. Biostatistical Analysis, Second Edition. Prentice-Hall, Inc., Englewood Cliffs, N.J.
07632. xv + 718 pp.
Phase II: Ammonia Effects
li-23
-------
REFINEMENTS TO CURRENT PSDDA BIOASSAYS
FINAL REPORT
PHASE IIIA: SPECIES SENSITIVITY COMPARISON TO GRAIN SIZE EFFECTS
An Employee-Owned Company
-------
INTRODUCTION
ill A-1
METHODS AND MATERIALS IIIA-2
TEST OVERVIEW IIIA-2
REFERENCE SEDIMENT COLLECTION AND ANALYSES IIIA-2
Sample Preparation IIIA-3
Source of Broodstock and Spawning Conditions IIIA-4
Experimental Procedure IIIA-4
Data Analysis IIIA-5
RESULTS IIIA-5
DISCUSSION IIIA-11
RECOMMENDATIONS IIIA-14
REFERENCES IIIA-15
TABLES
Table IIIA-1. Sampling location, conventional and grain size data for reference sediment
samples IIIA-6
Table IIIA-2. Results of Phase HI A oyster and echinoderm larval tests with varying grain-size
reference sediment IllA-7
Table IJIA-3. Estimates of Silt and Clay Fractions Present in Bioassay Vessels Based on Grain
Size Results IIIA-1 2
Table JIIA-4. Comparison of reported grain size distributions vs. mass of material in bioassay
test vessel IIIA-13
Table IIIA-5. Predicted Settling Rates of Silt and Clay Particles Sizes in Bioassay Chambers,
based on Stoke s Law IIIA-13
FIGURES
Figure IIIA-1. Oyster Mortality Grain Size and Aeration Effects IIIA-8
Figure IIIA-2. Oyster Abnormality Grain Size and Aeration Effects IIIA-9
Figure IIIA-3. Echinoderm Mortality Grain Size and Aeration Effects IIIA-10
-------
PHASE IIIA: SPECIES SENSITIVITY COMPARISON TO CLEAN REFERENCE SEDIMENTS
INTRODUCTION
Sediment larval bioassays are currently used under regulatory dredged sediment testing programs to
help determine the suitability of proposed dredged material for unconfined open water disposal. These
elutriate bioassays are used as a screen for possible adverse biological effects that occur due to the
presence of chemicals of concern in the dredged test sediment.
One problem with the larval sediment bioassays is that the physical presence of fine-grained material
in the test vessels may entrain embryos, which could result in a false positive response.1 Within the
context of the current PSDDA regulatory environment, exceedences of the regulatory guidelines in
bioassays are interpreted as being a toxic response to the presence of detected, or non-detected,
chemicals of concern. Disposal at the open-water sites is not allowed when such exceedences occur.
Within the PSDDA program, a reference sediment of similar grain size to the test material is included
to account for potential sediment grain size effects on the test organisms. However, these relatively
"clean" reference sediments often have high mortalities exceeding recommended quality standards for
acceptable test results. This may, in part, be due to physical effects such as interference in the test
from larval entrainment by suspended solids, or to physiological stress due to small grain sizes.
The PSDDA agencies identified the need for sponsoring research in defining the conditions under which
grain-size effects could cause larval mortality or abnormality using regulatory dredged sediment testing
protocols. The study was constructed so as to determine if criteria can be identified that indicate when
the chance of false positive responses due to suspended sediment in the test chamber could occur.
This would necessarily include the two classes of organisms most frequently used in dredged sediment
elutriate testing; bivalves and echinoderms.
One further variable that the agencies identified was to compare the effects of the different exposure
protocols that have been used for dredged sediment testing. There are two principal means of elutriate
preparation: 1) the Puget Sound Estuary Program (PSEP, 1991) procedure used under the PSDDA
program, and 2) the "Green Book" method (EPA, 1991) for evaluating dredged material for open ocean
disposal. Within the PSDDA program, three variations have been employed on the PSEP protocol.
These are:
• Allow suspended materials 4 hours to settle prior to testing, with no aeration during
testing (standard PSEP)
• Allow suspended materials 4 hours to settle, using aeration during testing
• Allow suspended materials 24 hours to settle, with no aeration during testing.
1 A false positive condition occurs when the bioassay results indicate that a toxic response has
occurred, but for reasons un-associated with sediment chemistry. Under those circumstances, the
measured chemicals-of-concern do not appear to be sufficiently high to explain the toxicological
response, but the testing results indicate that significant mortality or abnormality has occurred
within the test replicates.
Phase IIIA: Clean Sediment Effects
IIIA-1
-------
The intent was to compare the methods to determine if there were any differences in organism
responses, and if not, to select a method for regulatory testing that would retain environmental
sensitivity, but potentially would minimize the possibility of false positives (eg., aeration to maintain
high levels of dissolved oxygen, longer settling times - 24 hours - to minimize fine sediment
entrainment of larvae).
The objectives of this study were as follows:
• Compare the sensitivity of oyster and sand dollar embryo exposures to clean reference
sediment of varying grain sizes, and test procedures.
• Within a single species exposure, compare the responses to varying grain sizes and test
procedures.
• Determine the conditions under which either the bivalve or echinoderm larval bioassay
methods would be susceptible to false positive results due to the presence of
suspended sediment in the test chamber.
METHODS AND MATERIALS
TEST OVERVIEW
The Pacific oyster, Crassostrea gigas, and the eastern Pacific sand dollar, Dendraster excentricus, were
selected for study. While sand dollars are the most frequently used species for testing under this
program, bivalve larvae have been used in other Puget Sound programs, as well as in testing conducted
under the EPA/USACE Ocean Disposal program.
A target range of grain-sizes was pre-selected in consultation with the PSDDA agencies. The four grain-
size distributions were chosen to reflect the range of materials most frequently seen within Puget
Sound dredged sediments. The targeted test range was less than 30% fines, 45 - 60% fines, 65 -
75% fines, and greater than 85% fines.
Larvae were exposed for 48 hours to the test sediments/test conditions, and the responses compared
to that observed in a seawater control.
All testing was conducted at SAIC's Environmental Testing Center, in Narragansett, Rhode Island.
REFERENCE SEDIMENT COLLECTION AND ANALYSES
Reference sediment samples were collected in Carr Inlet using 3 reference stations that have been
previously used under the PSDDA testing program.2 Reference sediments within Carr Inlet have been
documented to be relatively free of the PSDDA chemicals of concern and represent a wide range of
grain sizes (PTI, 1991). The three reference stations, identified as CRR 2, CRR 4, and CRR 6, have
2These site designations are independent from those identified in PTI (1991), and bear no
relationship to stations identified by PTI as CARR 2, 4, or 6.
Phase III A: Clean Sediment Effects
IIIA-2
-------
been shown to possess 45, 65, and 85% fines, respectively. During the collection trip, an additional
station possessing less than 30% fines was located, and assigned the station name CRR A.
Samples were collected using the Washington State Department of Natural Resources' 0.1m2 stainless
steel van Veen grab, aboard the research vessel Brendan D II. Station locations were determined using
both the Global Positioning System (GPS) and Loran-C. When a sample was collected, the station's
position and water depth along with the time of sample collection were recorded in a field log.
All sample collection, handling, processing, and chain of custody procedures were conducted in
accordance with PSEP (1986, 1989). Prior to deployment, the van Veen was thoroughly washed with
Alconox soap, followed by a sequence of nitric acid (10%), deionized water, and methanol rinses.
Between stations, the sampler was washed with Alconox and rinsed with seawater.
Once a grab sample was taken, the overlying water was siphoned off from one side of the sampler.
Surface sediments were inspected for disturbance, and depending on their acceptability, sampled or
rejected. As 8 liters of material were required for analytical and biological testing, multiple grabs were
necessary at each station to achieve the required volume. To avoid possible adverse effects associated
with high sediment sulfate levels, the sampling was restricted to the oxic layers (i.e., less than or equal
to 2 cm). This was especially important at CRR 2, where the anoxic layers smelled strongly of
hydrogen sulfide. Samples for total sulfide were collected from one, randomly selected grab and
preserved in zinc acetate to minimize oxidation. Samples were homogenized on-board with stainless-
steel, decontaminated utensils. After compositing, sediments were transferred to appropriate glass
containers, labelled, and taped shut. Proper chain-of-custody procedures were followed in all transfers.
To ensure that the grain-size distribution required from each station was met, care was taken in the
field to verify the percent fines using a wet sieving technique. This involved measuring 100 mL of the
sediment into a graduated cylinder. The 100 mL of sediment was then carefully transferred into a 62
fj screen, and thoroughly washed to push the fine materials through the screen. After washing, the
retained material was transferred back into the graduated cylinder, and the volume recorded. The
percent fines was inferred by subtracting the retained volume of sand from the original 100 mL.
For this study, only PSDDA sediment conventional analyses were required. Conventional analyses (i.e.,
total solids, total organic carbon, total volatile solids, ammonia, and total sulfide) were conducted by
Analytical Resources Incorporated. Grain size analyses were performed by Soil Technology. Analyses
for PSDDA conventional parameters were performed in accordance with those methods employed
during Phase 1MB.
Sample Preparation
Each of the four test sediments were prepared according to the following procedures:
• PSDDA 4-Hour Settlement. Twenty grams of test sediment per liter were measured into pre-
labelled glass Mason jars. Filtered seawater was added, and the contents vigorously
shaken for approximately 10 seconds. The test vessels were then placed in the
temperature-controlled water bath and allowed to settle for 4-hours prior to inoculation
of test embryos. One set of five replicates prepared this way was aerated; a second
set of five replicates was run without aeration.
Phase ll/A: Clean Sediment Effects
IIIA-3
-------
PSDDA 24-Hour Settlement. These treatments were prepared exactly as above, with the
exception of an allowance for 24 hours of settling time. These replicates were
prepared a day ahead of time, so that the end of the settling time would coincide with
the inoculation. All replicates prepared this way were not aerated during the test.
Green Book. The method for preparing an elutriate as defined by the EPA involved making a
1 part sediment to four parts seawater mixture, that is vigorously stirred for 30
minutes. While the Green Book recommends use of a magnetic stirrer, the volume of
material prepared in batch (8 L) prevented effective use of a stir bar. In these
experiments, vigorous aeration was used. Test treatments were thoroughly mixed
manually at the initiation of the mixing, and at 10 minutes intervals thereafter. The
resultant slurry was allowed to settle for 30 minutes, and the liquid portion was
carefully poured off so as to not disturb the settled material. The retained liquid was
then dispensed into the test vessels.
For each sediment, a total of 10 replicate samples were prepared. All 10 replicates
were inoculated and held under the same conditions during testing. As defined by the
Green Book protocol, aeration was not used unless dissolved oxygen levels fell to less
than 40% saturation. At the end of the exposure, aliquots for larval counts were taken
from one set of five replicates by carefully decanting off the overlying water into a
second container, taking care not to include any of the bottom material (in accordance
with the "PSDDA counts" method). In the second set of five replicates, the entire
vessel contents were first mixed, and then aliquots were withdrawn for larval counts
(in accordance with the Green Book method). This latter procedure was an effort to
determine if larvae could be identified that had been entrained in the sediment during
exposure.
Source of Broodstock and Spawning Conditions
Gravid sand dollar and Pacific oyster broodstock were obtained from the same sources as described
for Phase II. All organisms were transported to the SAIC Testing Center in Narragansett on overnight
freight, and were used on the day of arrival. Prior to spawning, the organisms were acclimated to test
temperature.
Spawning procedures were identical to those described in Phase II.
Experimental Procedure
For each organism, and test procedure, an individual set of five seawater control replicates was set up
to duplicate the sediment test procedure. For example, for the four-hour settling-aerated treatments,
there were five aerated seawater controls that were placed under the same conditions as the test
replicates prior to inoculation. The controls for the 24-hour settling test were set up at the same time
as the test treatments, and allowed to "settle" for 24 hours. Green Book controls were mixed and
handled identical to the test treatments.
For each organism and test procedure, six replicates were prepared. Where required, aeration was
accomplished by dispensing the air through a glass pipette set at a rate of less than 100 bubbles per
minute. The sixth replicate was used for taking physical monitoring and ammonia measurements. Post
inoculation, physical monitoring measurements (pH, salinity, dissolved oxygen, and temperature) were
Phase MA: Clean Sediment Effects
IIIA-4
-------
taken for each test series. Those measurements were taken again at 24 and 48 hours. Ammonia was
taken at the time of inoculation, and at test termination.
Inoculation of embryos occurred using a volumetric pipette, calibrated to deliver between 20 and 30
embryos per mL. To ensure homogenous distribution of embryos in the stock solution, a perforated
plunger was use to mix the solution. Post-inoculation, two 10 mL aliquot were withdrawn from each
of five seawater controls, and counted to determine the actual number of larvae dispensed into
replicates. All subsequent mortality determinations were made by comparison to the initial seawater
control counts. Separate control counts were made for the "Green Book" procedures, due to the
difference in test volume, as described above.
For both organisms, a single set of reference toxicant was run in a gradient series using cadmium
chloride. The series was identical to that identified in Phase II. To confirm cadmium levels, a sample
of the highest concentration was analyzed.
The end of the test is taken as the point at which > 90% of the organisms in the seawater control
reach the prodissoconch (oyster), or pluteus larval (sand dollars) stage, as defined by the PSDDA
program. Beginning at T = 45 hours, laboratory staff withdrew 10 mL aliquots from each treatment
controls, and counted the number of normal and abnormal larvae. The procedure was repeated hourly
until the 90% criterion was achieved, at which time the test was terminated.
For each test replicate, two 10 mL aliquot were withdrawn, fixed with 5% buffered formalin, and
scored microscopically as normal (pluteus larvae) or abnormal. As a quality assurance procedure, 20%
of all larval counts were re-scored by a second counter. In the event of a discrepancy between the
counters, a third count was made, and procedures reviewed prior to proceeding further with counts.
Data Analysis
Larval response data were first tabulated in a spreadsheet, and then percentage mortality and
abnormality determined by replicate, and then the mean of all replicates for an exposure treatment
reported in the data summary. All data in the spreadsheets were checked and confirmed against the
original data sheets, prior to proceeding with analyses.
For each treatment, the seawater control final counts were taken, and then compared to the initial
inoculum counts for determination of percent control survival. Mortality in these exposures is
expressed as the PSDDA combined mortality/abnormality endpoint. All sediment treatment
comparisons are made against the number of surviving, normal larvae in the controls.
RESULTS
Reference sampling locations, depth of sampling, and conventional analyses results for the four Carr
Inlet stations are found in Table IIIA-1. The analytical laboratory report may be found in Appendix B.
The field efforts were successful in collecting material within the desired grain size ranges. In this
effort, the wet-sieving method was an effective predictor of the laboratory determined percent fines.
In these four cases, the field-predictive method was generally within 10% of the actual value. While
bulk ammonia in CRR2 was relatively high, the aqueous unionized ammonia level measured in the
experimental beakers didn't exceed 0.01 mg/L.
Phase ///A: Clean Sediment Effects
IIIA-5
-------
Table IIIA-1. Sampling location, conventional and grain size data for reference sediment samples.
Location
Station
GPS
Loran TD
Depth
(m)
% Field
Retained
Sand*
%
Lab
Fines
Total
Solids
%
Tvs
(mg/kg)
TOC
(%>
Ammonia
(mg/kg)
Sulfide
(mg/kg)
CRR 2
47° 19.94'N
122° 40.74'W
27951 .1
42214.0
23
60
28
64.74
12,700
0.4
74.85
0.13
CRR 4
47° 19.99'N
122° 40.48'W
27953.4
42214.9
14
40
51
62.59
13,700
0.4
3.72
0.1 1
CRR 6
47° 21.98'N
122° 38.82'W
27960.6
42223.1
18
10
87
32.49
21,800
1.2
9.87
0.15
CRR A
47° 20.18'N
122° 40.88'W
27956.4
4221 4.0
13
80
6
78.32
10,100
0.6
2.59
0.1
* = Percent material retained on the 62p screen in field wet-sieving of test material.
Data, overall, were judged to be within acceptable PSDDA quality assurance parameters. In the oyster
exposures, control mortality was less than 30%, with less than 10% abnormality. One exception was
the control for the four-hour, aerated group, which had a mortality of 34.3%; this value was still within
PSEP guidelines. Salinity was between 28 - 30 ppt, pH and temperature at acceptable levels. The
levels of unionized ammonia were less than 0.04 mg/L, accept for in the Green Book preparations,
where significantly higher levels were encountered. Additional problems occurred in the dissolved
oxygen (DO) levels in the Green Book elutriate preparations. On Day 2 (24 hours) after inoculation,
it was found that DO levels had fallen below 40% saturation in all of the Green Book elutriate vessels,
and in some cases to less than 1 mg/L.
Echinoderm mortality for the seawater controls ranged between 8 - 13.5%, with abnormality exceeding
the PSEP 10% maximum in one set (13.5%). Salinity was between 28 - 30 ppt, with pH range of 7.8
- 8.3. Temperature mean for all replicates was 16° C, which is higher than the PSEP-required limit of
15° C. However, the overall effect on data quality is thought to be negligible. Unionized ammonia
values were less than 0.04 mg/L, except for the Green Book elutriates. DO levels were within
acceptable limits, except for the Green Book treatments, where the DO again fell to less than 40%
saturation.
Intra-sample variability for some treatments within these experiments is somewhat problematic. Within
both oyster and sand dollar treatments, standard deviations in some replicates exceeded 33%, with
corresponding Coefficients of Variance greater than 20%. There are no specific PSDDA, PSEP, or
ASTM guidance on maximum allowable intra-replicate variability. Exercising best professional
judgement, these data cannot provide definitive guidance on the relationship between grain size and
mortality. However, they are judged to be of sufficient quality to indicate overall trends in that
relationship.
Table IIIA-2 presents a summation of the experimental treatments. Larval data tables, physical
monitoring data, and ammonia measurements may be found in Appendix B.
Phase III A: Clean Sediment Effects
IIIA-6
-------
Table IIIA-2. Results of Phase IIIA oyster and echinoderm larval tests with varying grain-size reference sediment.
Phase IIIA Oyster Mortality and Abnormality
PSDDA 4 Hour Setting
(Aerated)
Mortality Abnormality
% %
PSDDA 4 Hour Settling
(Urwerated)
Mortality Abnormality
% %
PSDDA 24 Hour Sorting
(Unaerated)
Mortality Abnormality
' % %
Elutriate Green Book
protocol1
Mortality Abnormafity
- %
Sutrfet* Qreen Book
w/PSBDA Count2
Mortality Abnortnafit
% y %
Carr 6
W% fine*)
87.5
70.2
47.0
8.7
39.5
16.0
99.9
99.8
98.6
20.2
Carr 4
17.1
1.4
20.5
0.7
-1.9
0.3
83.0
80.1
92.4
5.5
Cart 2
(28% fine*)
11.0
2.1
9.8
1.0
-10.1
0.5
87.7
59.8
94.5
11.1
I Carr A .
| («% fines)
14.1
2.7
14.9
1.6
-2.8
0.4
99.9
99.4
93.6
10.5
Phase lltA Sand Dollar Mortality and Abnormality
PSDDA 4 Hour SetlKng
(Aeratod)
PSDDA 4 Hour Setting
(Unaeratedt
PSDDA 24 Hour Settling
(Unaerated)
Elutriate Green Book
protocol1
Efutrmte Green Book
wIPSODA Cotnti
Mortality
*
Abnormality
Mortality
Abnormality
y;':^::.v .
Mortality
Abnormality
%
Mortality
%
Abnormality
Mortality
%
Abnorma&t
y%
Carr 6
(87% fiDMl
16.8
8.7
45.5
23.6
-23.8
8.0
100.0
100.0
100.0
100.0
Cart 4
{51% fine*)
-2.3
8.3
47.2
25.2
-2.1
7.8
100.0
100.0
94.6
8.6
Carr 2
(29% fine*)
13.3
17.8
31.6
13.7
-10.4
7.1
100.0
100.0
92.6
9.5
Carr A
-------
Figure IIIA-1. Oyster Mortality
Grain Size and Aeration Effects
100
90
80
70
60
50
40
30
20
10
0
n»
CRR6 (87%) CRR4 (51%) CRR2 (28%) CRRA (6%)
%
Carr Inlet Reference Station (% fines)
Four Hour, Aerated
Four Hour,Unaerated
24 Hour, Unaerated
-------
Figure IIIA-2. Oyster Abnormality
Grain Size and Aeration Effects
100
5
0)
(6
(6
S
C/5
!
§
I
r>
E>
>
I
CO
cd
§
o
c
a
<
T3
a>
c
«¦*
¦O
£
o
J
£
CRR6 (87%)
CRR4 (51%) CRR2 (28%)
Carr Inlet Reference Station (% fines)
CRRA (6%)
Four Hour, Aerated
Four Hour,Unaerated
24 Hour, Unaerated
-------
Figure IIIA-3. Echinoderm Mortality
Grain Size and Aeration Effects
$
(o
£
n>
9.
3"
(b
5
I
o
3
cd
t:
o
TJ
o
100
90
80
70
60
50
40
30
20
10
0
CRR6 (87%)
CRR4 (51%) CRR2 (28%)
Carr Inlet Reference Station (% fines)
CRRA (6%)
Four Hour, Aerated
Four Hour.Unaerated
24 Hour, Unaerated
-------
Mortality in all Green Book elutriate treatments for both species was virtually 100%. The principal
reason for the high mortality appears to be low levels of dissolved oxygen in all the Green Book
elutriate procedures. There is likely a compounding factor due to grain size and entrainment, but low
DO is believed to be the principal cause.
As a result of the loss of all the Green Book larval data, the remaining comparisons and discussions will
focus on the three PSDDA-treatments only.
Figure IIIA-1 presents a bar chart summation of the oyster larval mortality data by stations. In all
treatments, the CRR 6 reference sediment showed significantly greater responses, relative to the other
three Carr stations. CCR4, CRR2, and CRRA were not significantly different from each other, within
the respective treatments. Figure IIIA-2 shows the effect of grain size and treatment on oyster
abnormality. As with mortality, the greatest effect was observed in the CRR6, 4-hour aerated
treatment. Percent abnormality for CRR6 was low for the other two treatments, but higher than that
observed for the other three stations.
The response of the sand dollar larvae to the exposures and treatments can be seen in Figure IIIA-3.
Of immediate note is the higher apparent mortality in all four reference sediments of the unaerated
treatments. These high responses represent both low numbers of live larvae counted at the end of the
exposure, and a relatively high abnormality response. Those larvae reported as abnormal were
developmentally retarded, suggesting that some factor within those treatments was inhibiting cellular
development. For the aerated 4-hour settlement the mortality/abnormality was low (< 20%). In all four
24-hour treatments, there was virtually no mortality or abnormality.
DISCUSSION
The results for the oyster tests suggest that at higher percent fine concentrations, oyster mortality and
abnormality are effected. At 87% fines, there were large effects observed at all three treatments. The
high response observed in the aerated treatment may be due to the finer material being forced to
remain in suspension longer, entraining or otherwise affecting the developing oyster larvae. These data
suggest that for grain-sizes of fines percentages s 51%, oyster larvae are not affected by the finer
material under any treatment. PTI (1991) attempted to define a relationship between larval mortality
and abnormality for oyster with Carr Inlet samples. They found a relatively weak correlation (r =
0.557), but those experiments had high larval mortality (>50%) for all stations.
The results for the echinoderm larval tests are somewhat more problematic. The data suggest that for
the four-hour settling, aerated treatments, and the 24 hour unaerated treatments, sand dollar larval
responses do not exceed PSDDA reference sediment performance criteria. The unexpected result in
the four-hour settling test was the high mortality (as non-recovered larvae), and higher retarded
develoment, encountered for all the reference stations. This was especially surprising for CRR A, which
was largely sand and shells. There is no indication that the response was related to any ammonia or
other test chemical parameters. Numerous PSDDA regulatory bioassays have been conducted using
unaerated controls with West Beach sand, and had virtual 100% survival. At the very best, the most
that can be concluded from those data is that the mortality effect is not related to grain-size effects.
That finding would be consistent with the work conducted by PTI (1991) for correlating Carr Inlet
reference samples with grain size effects.
Phase III A: Clean Sediment Effects
IIIA-11
-------
To date, there has been little work addressing the effects of fine grained material on larval survival in
sediment elutriate bioassays. The effects of particulate matter on bivalve larval development were
addressed by Davis (1960) and by Davis and Hindu (1969). In those papers, the authors described
inhibition of larval development to the prodissoconch stage (hinged or "D"-shaped) by silt/clay
concentrations in as low as 3-4 gms/L.
While direct extrapolation of those data to elutriate testing is not possible (owing to major differences
in exposure procedure) the ability to express inhibitory effects in gms/L of silts and clays has some
utility. In order to compare these results to the findings of Davis, the grams of silt and clay present
in the test vessels for each test sediment was calculated based on the percent solids, and percent
fractional components, according to the following formula:
Test Sediment (gms) x Percent Solids x Percent Silt and/or Clay
These calculations are presented in Table IIIA-3.
Table IIIA-3. Estimates of Silt and Clay Fractions Present in Bioassay Vessels Based on Grain
Size Results.
Reference
Test Sediment
Total Silt
Total Clay
Clay Fractions by Particle Size
Station
(gms)
(gms)
(gms)
<3.9
<1.9
<0.9
CRR 6
20
4.1 6
1.56
0.39
0.31
0.84
CRR 4
20
5.63
0.88
0.10
0.10
0.68
CRR 2
20
2.83
1.16
0.34
0.00
0.82
CRR A
20
0.47
0.94
0.16
0.00
0.78
Carr 4 has a total fines concentration of 6.51 grams/L, while Carr 6 has a fines concentration of 5.7
g/L.. Based on the hypothesis that total higher fines loads correspond to higher mortality, Carr 4
should have had a higher percent mortality than Carr 6. When compared to the data, Carr 6 had
mortality in the oysters, but was not significantly different from Carr 4 in the echinoderm exposures.
When compared to the values published by Davis, both Carr 4 and Carr 6 should have had no surviving
larvae to the prodissoconch stage.
As an example of how this procedure could be applied for data interpretation, the grain size data from
the Reference Area Performance Standards (PTI, 1991) were re-expressed as grams of material per liter
in Table IIIA-4. In reporting the percent fines for stations SM30 (96%) and HM05 (58%), there is a
mathematical difference of 38%. However, when the percent solids are factored into the calculation,
the total amount of silt and clay in an elutriate test vessel is only 0.26 gms different. When HM05 is
compared to SM34 (74% fines), the calculated grams of material is actually higher for HM05 (6.5 vs.
5.55 gms) for HM05.
Phase III A: Clean Sediment Effects
IIIA-12
-------
Table IIIA-4. Comparison of reported grain size distributions vs. mass of material in bioassay test
vessel. Data taken from PTI (1991).
Reported Grain Size Distribution
Actual Material In Test Replicate
Station
To«t
Sediment
Solida
Fines
sat
Clay
Silt
Clay
Total
(gms)
(%)
(%l
(%)
(%)
(gms)
(gms)
(Total)
SM30
20
35
96
63
34
4.41
2.38
6.79
SM34
20
37
74
48
27
3.55
2
5.55
HM05
20
56
58
40
18
4.48
2.02
6.5
CR23
20
58
49
43
6
4.99
0.7
5.68
SM33
20
60
29
19
10
2.28
1.2
3.48
CR21
20
71
14
9
5
1.28
0.71
1.99
SM31
20
89
13
8
5
1.42
0.89
2.31
It is suggested that the more important component in this evaluation is the clay content. Table IIIA-4
presents the predicted settling rates of silt and clay particles in the bioassay chambers, based on the
physical principal of settling, known as Stoke's Law. The assumptions made to generate these settling
rates (eg., spherical particles, seawater having a viscosity of 1) are under ideal circumstances, and are
probably not equivalent to those observed in bioassay chambers. However, the table is useful in
demonstrating what materials should theoretically remain in suspension after 4, 24, and 48 hours of
settling. Actual rates are probably slower.
Table IIIA-5 indicates that the only particles that are of concern after four hours of settling would be
the clay fraction of particle size < 1.9 */, and after 24 hours the :£ 0.9 /; fraction. Based on the data
in Table IIIA-3, Carr 6 would have the greatest amount of material in suspension at 4 hours, but that
all stations would have an equivalent amount of material in suspension after 24 hours settling. It
should be noted that changes to the viscosity of the seawater with the addition of the sediment,
coupled with non-ideal behavior of sediment particles in test suspension, would likely result in the
settling rates being much slower than shown here. Aeration of the test vessels would likely create
small currents in the test chambers that also would effect the rate of settling.
Table IIIA-5. Predicted Settling Rates of Silt and Clay Particles Sizes in Bioassay Chambers, based on
Stoke's Law.
Particle
Settling
Height of
Settled Distance of Particles
Size
Rate
Vessel
4 hours
24 hours
48 hours
(cm/s)
(cm)
(cm)
(cm)
(cm)
62
0.1400
15
2016.00
12096.00
24192.00
31
0.0500
15
720.00
4320.00
8640.00
3.90
0.0023
15
32.61
195.69
391.37
1.90
0.0005
15
7.36
44.15
88.30
0.90
0.0001
15
1.65
9.31
19.81
Phase MA: Clean Sediment Effects
IIIA-13
-------
We caution that at this level, the relationship between clay particles and oyster mortality is still not
clearly defined. More data is necessary to evaluate the strength of the relationship, and to define the
level at which relative amounts of silts or clays in bioassay vessels becomes a problem. We do
suggest, however, that the traditional means of examining the data in terms of "percent fines" is
probably invalid, and that to do comparative analyses between stations, the data must be expressed
as grams of fines/Liter.
RECOMMENDATIONS
Caution should be exercised in utilizing Crassostrea gigas larvae in sediments known
to have a high proportion of clays and silts.
Larvae of Dendraster excentricus, when tested under current PSDDA protocols, do not
show an adverse response to increasing silt and clay fractions. Under conditions of
expected high silts/clay, the sand dollar test should be utilized.
For the purposes of selecting suitable reference material for larval bioassay
comparisons, the percent grain-size data should be converted to grams/Liter of silts and
clays.
Aeration is recommended for use in the Green Book larval bioassay.
Phase III A: Clean Sediment Effects
111 A-14
-------
REFERENCES
Davis, H., 1960. Effects of Turbidity-Producing Materials in Sea Water on Eggs and Larvae of the
Clam Venus (Mercenaria) mercenaria. Biol. Bull. 118: 48 - 54.
Davis, H., and H. Hindu. 1969. Effects of Turbidity-Producing Substances in Sea Water on Eggs and
Larvae of Three Genera of Bivalve Mollusks. The Veliger 11 (4): 316 - 323.
PSEP. 1986. Puget Sound Estuary Program. Recommended Protocols for Measuring Selected Environ-
mental Variables in Puget Sound. Final Report. Prepared for the U.S. Environmental Protection
Agency Region X, Office of Puget Sound, and the U.S. Army Corps of Engineers. Tetra Tech
Inc., Bellevue, Washington.
PSEP. 1989. Puget Sound Estuary Program. Recommended Guidelines for Measuring Metals/Organic
Compounds in Puget Sound Sediment and Tissue Samples. Prepared for the U.S.
Environmental Protection Agency Region X, Office of Puget Sound, and the U.S. Army Corps
of Engineers. PTI Environmental Services, Inc., Bellevue, Washington.
PSEP. 1991. Puget Sound Estuary Program. Recommended Guidelines for Conducting Laboratory
Bioassays on Puget Sound Sediments. U.S. EPA, Region 10, Office of Puget Sound, Seattle,
WA.
PTI Environmental Services (1991). Reference Area Performance Standards for Puget Sound. U.S.
EPA, Region 10, Office of Coastal Waters, Seattle, WA
U.S. EPA 1991. Evaluation of Dredged Material Proposed for Ocean Disposal, Testing Manual. United
States Environmental Protection Agency and the U.S. Army Corps of Engineers. EPA - 503 /8-
91 / 001.
Phase III A: Clean Sediment Effects
1(1 A-15
-------
REFINEMENTS TO CURRENT PSDDA BIOASSAYS
FINAL REPORT
PHASE IIIB: SPECIES SENSITIVITY COMPARISON TO
CONTAMINATED SEDIMENT EFFECTS
Art Employee-
-------
INTRODUCTION IIIB-1
METHODS AND MATERIALS IIIB-2
TEST OVERVIEW IIIB-2
SEDIMENT COLLECTION AND ANALYSES IIIB-2
Contaminated Sediment Site Selection IIIB-2
Contaminated Site Sample Collection IIIB-3
Reference Sediment Collection IIIB-3
Construction and Analyses of Contaminated/Reference Site Composites IIIB-4
Analytical Methods IIIB-4
BIOASSAY PROCEDURES IIIB-5
Test Sample Preparation IIIB-5
Source of Broodstock and Spawning Conditions IIIB-6
Experimental Procedure IIIB-6
Data Analysis IIIB-7
RESULTS IIIB-8
SEDIMENT COLLECTION AND ANALYSES IIIB-8
Sediment Conventionals IIIB-8
Sediment Analyses IIIB-9
BIOASSAY RESULTS IIIB-9
Data Acceptability IIIB-9
General Results By Station and Species IIIB-13
Results of PSDDA r-Test Comparisons IIIB-13
Results of Species Responses to Ml Treatments IIIB-13
Results of Differences by Species Between Treatments IllB-22
Results of Species as Predictors of Apparent Sediment Toxicity III B-22
Comparison of Species Reference Toxicant Responses IIIB-22
DISCUSSION IIIB-25
SEDIMENT CHEMISTRY IIIB-25
BIOASSAYS IIIB-26
ANALYTICAL VALUES AS PREDICTORS OF BIOASSAY RESULTS IIIB-27
RECOMMENDATIONS IIIB-27
REFERENCES IIIB-28
-------
List of Tables
Table 11 IB-1. Sampling location, conventional and grain size data for 1MB sediment
composites IIIB-8
Table IIIB-2. Concentrations of PSDDA Chemicals of Concern Found in Test Sediments . . IIIB-10
Table IIIB-3. Results of Phase IIIB Larval Exposures IIIB-14
Table IIIB-4. Application of PSDDA bioassay criteria to Oyster as Echinoderm responses to
the
-------
PHASE IIIB: SPECIES SENSITIVITY COMPARISON TO CONTAMINATED SEDIMENTS
INTRODUCTION
Sediment larval bioassays are currently used under regulatory dredged sediment testing programs to
help determine the suitability of proposed dredged material for unconfined open water disposal. These
elutriate bioassays are used as a screen for possible adverse biological effects that occur due to the
presence of chemicals of concern in the dredged test sediment.
Both the Puget Sound Dredged Disposal Analysis (PSDDA) program, and the Ocean Dumping Testing
Manual (EPA/USACE, 1991) allow a project proponent to select the test organism for the larval test.
Most frequently, the Pacific oyster Crassostrea gigas, and the North American sand dollar Dendraster
excentricus, are used in the programs. The regulatory agencies with jurisdiction in dredged testing
programs currently regard all larval species as being equivalent in their response to test materials.
However, to date, there have been no definitive data comparing the responses of sand dollars and
oysters to the same sediments.
The Environmental Protection Agency identified the need to characterize and compare the responses
of these two species to both clean and contaminated sediments. Phase III A of this document compares
the response of the two species to clean reference sediments, the experiments of Phase IIIB compares
the responses to sediments of known contamination.
The PSDDA agencies also wanted to compare the same test protocols described for Phase IIIA, with
the contaminated sediments in these exposures. The intent was to compare the methods to determine
if there were any differences in organism responses, and if not, to select a method for regulatory
testing that would retain environmental sensitivity, but potentially would minimize the possibility of
false positives (eg., aeration to maintain high levels of dissolved oxygen, longer settling times - 24
hours - to minimize fine sediment entrainment of larvae). Finally, the issue of whether the protocols
developed for elutriate testing under the PSEP program were as effective as the procedures for elutriate
testing in the Green Book in indicating sediment toxicity.
The objectives of this study are as follows:
• To determine if oysters and sand dollars have equivalent responses to the same level
of contaminants in a sediment.
• To determine if oysters and sand dollars responses to test sediments are equivalent in
predicting sediment toxicity under the PSDDA program.
• To determine how the various test protocols compare in terms of predicting sediment
toxicity to both organisms.
Phase IIIB: Contaminated Sediments Effects
-------
METHODS AND MATERIALS
TEST OVERVIEW
The preliminary experimental design was to evaluate six different contaminated test sediments over
the five test procedures. In order to try and minimize confounding influences of different grain sizes,
sediment total organic carbon, sulfide, or other sediment conventional parameters, it was decided to
collect just 2 contaminated sediments with known chemical distributions and positive bioassay
responses. Those sediments would then be "diluted" by 50% and 75% with the clean Carr Inlet
reference sediments identified in Phase IIIA. If similar conventional parameters could be matched, the
only effect that would be measured would be varying contaminant load. This approach is similar to
that employed by Pastorok and Becker (1989) who had attempted to examine both larval species
responses in the same study. In that study, however, oyster larval survival was poor, preventing an
effective comparison of the two species.
The Pacific oyster, Crassostrea gigas, and the eastern Pacific sand dollar, Dendraster excentricus, were
selected for study. These two species are the most frequently tested under the elutriate sediment
testing programs. Larvae were exposed for 48 hours to the test sediments/test conditions, and the
responses compared to that in a seawater control, and a reference sediment.
All testing was conducted at SAIC's Environmental Testing Center, in Narragansett, Rhode Island.
SEDIMENT COLLECTION AND ANALYSES
Contaminated Sediment Site Selection
Prior to selecting the appropriate stations for field collections, a review was conducted of sediment
data sets from both EPA's and the Corps' data bases. The objective was to locate stations with
sufficiently high levels of contaminants, and a past history of having a positive, partial larval bioassay
response The review included stations in Commencement Bay, Elliott Bay, Eagle Harbor, and
Bellingham Bay. Upon evaluation of the data, two stations were identified from a data set associated
with a former shipyards on the West Waterway and Elliott Bay. Location and justification for utilization
of these two sites is as follows:
Site D1: This site is located at the western edge of the shipyard property line, in proximity to
a pole-creosote facility, approximately 450 ft. offshore. Previous data indicated that
the site had high levels of low and high molecular weight polyaromatic hydrocarbons
(LPAH, HPAH), but no exceedances of the PSDDA SL for metals. The grain size was
reported as 68% fines, and previous bioassays on sediment collected from D1 indicated
an echinoderm response of 27.7% combined effects mortality, and Rhepoxynius
abronius mortality of 42.5%.
1 The stations used by Pastorok and Becker were not used in this study due to the high level of
mortality observed in those sediments. In the present study, stations were sought for which partial
larval mortality responses were observed.
Phase MB: Contaminated Sediments Effects IIIB-2
-------
Site Ml: Site M1 is located on the West Waterway, approximately 525 ft. south of the north
end of Pier 21, and 75 ft. eastward of the Pier. Data for M1 showed a mixture of
contaminants, with high concentrations of copper, lead, and zinc. For copper and lead,
the MLs were exceeded by approximately 2 and 3 times, respectively. Total LPAH, but
not HPAH, exceeded the ML. Previous echinoderm larval bioassay data showed a
response of 25% combined effects.
Contaminated Site Sample Collection
Samples were collected using a stainless steel 0.1 m2 Young van Veen grab, aboard the research vessel
KITTIWAKE. Station location was determined using both a Global Positioning System, and a Loran C
system. Location, depth, and time of sample were recorded for each grab. PSEP protocols were
followed for sampling procedures and sample acceptability. The van Veen sampler was thoroughly
washed with Alconox detergent and seawater before the start of sampling, and between each sampling
station. To obtain 20 L of sediment required to formulate the dilution series, it was necessary to take
multiple grabs. All of the sediment from each acceptable grab was retained, except the sediment
touching the sides of the sampler. Samples were collected with decontaminated stainless steel spoons
and 1 9 L stainless pans.
For chemical analysis of the whole parent sediment, volatile organic and total sulfides samples were
taken from a randomly selected grab from each station. Samples were placed into appropriate glass
containers according to PSEP procedures. For total sulfide analyses, the samples were fixed in the field
with zinc acetate. Samples were stored in coolers on ice at 4° C, and taken back to the SAIC
laboratory to composite and prepare the dilution series.
Reference Sediment Collection
Reference sediments necessary to complete the sediment dilution series were collected in Carr Inlet.
Carr Inlet has been recognized as a reference sediment site (PTI, 1991), and has been previously used
under the PSDDA testing program. Two stations, CRR2 and CRR4 were identified to represent 45%
and 65% fines for sediment grain size, respectively. As with Phase IIIA, CRR2 and CRR4 are site
designations independent from those identified in PTI 1991. Samples were collected using a stainless
steel 0.1 m2 Young van Veen grab, aboard the University of Washington's 17 foot Boston whaler. Two
field events were necessary to collect sufficient sediment to make the composites. Of note is that on
the first collection trip the Loran C receiver, a Micrologic ML-3000, was faulty making positioning of
the vessel at the proper Loran coordinates questionable. For the second field event, a Northstar 800
Loran C navigation system was acquired for positioning, which proved much more reliable. At each
station, the Boston whaler was positioned according to the proper Loran C time delays (TDs), and for
each replicate grab, the TDs never exceeded 0.1 from the original TDs.
To ensure that the grain size distribution required for each station was met, the wet sieving technique
used and described in Phase IIIA was also used. All sampling procedures and judgement of sample
acceptability followed the PSEP protocols, unless stated otherwise. To avoid possible adverse effects
associated with high sediment sulfide levels, only the top 2 cm were collected from each sediment
grab. Volatile organics and total sulfides samples were taken from a randomly selected grab from each
station. A total of 20 L of sediment was collected at each station in the two trips, stored in clearly
labeled polyethylene bags, and brought back to the SAIC laboratory to be homogenized. Samples were
transported in coolers with ice to keep sediments at 4 degrees C.
Phase MB: Contaminated Sediments Effects tlIB-3
-------
Construction and Analyses of Contaminated/Reference Site Composites
The dilution series was prepared as 50%, and 25% dilutions of the parent contaminated sediment.
Dilutions were constructed on a volume to volume basis, with a final volume of 10 L per test sediment.
An industrial mixer was used to thoroughly mix each sample, before creating the dilutions. The
stainless steel bowl and mixer were decontaminated between each sample. For each 100%
contaminated sediment, the sample was thoroughly homogenized, and then sub-sampled for PSDDA
sediment conventional chemistry and chemicals of concern analyses. The remaining sediment was then
put into 2 L glass jars, purged with nitrogen gas, and refrigerated at 4° C until bioassay testing. To
create the dilutions, samples were measured volumetrically with 4 L decontaminated glass beakers and
thoroughly mixed with the industrial mixer. After thorough mixing of the dilution sample, samples for
PSDDA sediment conventionals and chemicals of concern were collected. The remaining sediment was
then put into 2 L glass jars, purged with nitrogen gas, then refrigerated until bioassay testing.
Analytical Methods
All of the chemical analytical procedures used in this program were performed in accordance with the
most current PSEP (1986, 1989) and PSDDA (1989) documentation with modifications specified in
the annual reviews (PSDDA, 1990 and 1991). Sediments were analyzed for the PSDDA chemicals of
concern and conventional parameters specified in the PSDDA Management Plan Report (MPR)
Unconfined, Open-water Disposal of Dredged Material, Phase II fNorth and South Puget Sound),
September 1 989. Appropriate PSDDA/PSEP QA/QC analyses (i.e., matrix spike/matrix spike duplicate)
were performed for both Batch B442 and B656 samples. A QA2 data package was also prepared by
the laboratory and was submitted with the final data reports under separate cover.
Sediment samples for contaminant chemistry were submitted to Analytical Resources, Inc., in two
batches. The first batch, B442, consisted of Samples M1, D1, M1C2-50/50, and D1C4-50/50 for
contaminant chemistry analysis. Two Carr Inlet samples (Carr2 and Carr4) were also submitted in this
batch for conventional chemistry testing. The second batch, B656, was composed of Samples D1C4-
25/75 and M1C2-25/75, and samples were analyzed for the PSDDA chemicals of concern. Raw
analytical results are presented by batch as are the QA1 review forms (see appendices).
Batch B656 sediments were submitted to the analytical laboratory beyond holding times for some
parameters (see QA1 review) following an unplanned change in the work plan. For this reason,
samples were not analyzed for volatile organic compounds.
Specific analytical methodologies and problems encountered during analyses are described in the
following paragraphs.
Organics
Batch B442 samples were analyzed for volatile organics (VOAs) using Method 8240 (purge and trap).
VOAs were analyzed using approximately 5.0 grams of sample (wet weight); no problems were noted
during analysis.
Semivolatile organic compounds were prepared using extraction Method 3550 and analyzed by Method
8270 (GC/MS). Both PSEP-recommended modifications to the extraction method and the use of
calibration standards of lower dilution concentrations were implemented to lower detection limits. In
Batch B442, some surrogate recoveries were reported out of the recommended control limits. The
laboratory postulated that twice the amount of surrogate/spike compound mixture was added at the
time of extraction, however, the analytical results as reported were not affected. In addition, all
samples required analysis at diluted concentrations because analytes were detected above the linear
Phase IIIB: Contaminated Sediments Effects 111B-4
-------
calibration range. Sample D1C4-25/75, in batch B656, required re-analysis at a 1 to 10 dilution due
to several analyte levels above the linear calibration range.
Pesticides and PCBs were extracted using Method 3550 and analyzed by Method 8080 (GC/ECD). The
presence of pesticides and PCBs were verified using dual column confirmation. For both batches,
matrix interferences caused the laboratory to raise detection limits for pesticides and PCBs.
Metals
Sediment samples were analyzed for the PSDDA metals of concern (except mercury) using the total
acid digestion (TAD) method described in the PSEP (1989) documentation. Sediments were digested
in a Teflon bomb using nitric, hydrochloric, and hydrofluoric acids. ARI utilized a pressure-controlled
microwave heating technique to complete the digestion process. The digestion products were analyzed
for arsenic, antimony, cadmium and silver using graphite furnace atomic absorption (GFAA). Copper,
lead, nickel, and zinc were analyzed by inductively coupled plasma emission spectroscopy (ICP).
Mercury was digested using nitric and sulfuric acids and oxidized using potassium permanganate and
potassium persulfate. The digestate for mercury was analyzed using Cold Vapor Atomic Absorption
ICVAA).
No problems during the metals analyses were reported by the laboratory.
Conventionals
The PSDDA conventional parameters include grain size distribution, total solids, total organic carbon,
total sulfides, ammonia, and total volatile solids. Methods for conventional parameters followed those
provided in PSEP (1986) and PSDDA (1989) with modifications specified in the annual reviews
(PSDDA, 1990 and 1991). Ammonia measurements were determined using Plumb (1981). Particle
grain size distribution for each sample was determined in accordance with ASTM D 422 (modified).
Wet sieve analysis was used for the sieve sizes U.S. No. 4, 10, 18, 35, 60, 120, and 230. (Hydrogen
peroxide was not used in preparations for grain size analysis which may break down organic aggregates
causing an overestimation of the percent fines found in undisturbed sediment.) Hydrometer analysis
was used for particle sizes finer than the 230 mesh, and water content was determined using ASTM
D 2216. Sediment classification designation was made in accordance with U.S. Soil Classification
System, ASTM D 2487.
No analytical problems were noted by the laboratory for all conventionals analyses.
BIOASSAY PROCEDURES
Test Sample Preparation
Each of the six test sediments were prepared according to the following procedures:
• PSDDA 4-hour Settlement, Aerated. Twenty grams per liter of test sediment were measured
into pre-labeled glass Mason jars. Filtered seawater was added, and the contents
vigorously shaken for approximately 10 seconds. The test vessels were then placed
in the temperature-controlled water bath, and allowed to settle for 4 hours prior to
inoculation of test embryos. This treatment was aerated throughout the entire 48 hour
exposure.
Phase MB: Contaminated Sediments Effects HIB-5
-------
PSDDA 4-hour Settlement, Unaerated. These treatments were prepared identical to that
described above, but were not aerated during exposures.
• PSDDA 24-Hour Settlement. These treatments were prepared exactly as above, with the
exception of an allowance of 24 hours of settling time. These replicates were prepared
a day ahead of time, so that the end of the settling time would coincide with the
inoculation. All replicates prepared this way were not aerated during the test.
• Green Book. The method for preparing an elutriate involves making a 1 part sediment to four
parts seawater mixture, that is vigorously stirred for 30 minutes. While the Green Book
recommends use of a magnetic stirrer, the volume of material prepared in batch (8 L)
prevented effective use of a stir bar. In these experiments, vigorous aeration was
used, with a complete manual mixing at the start, and every 10 minutes. The resultant
slurry was allowed to settle for 30 minutes, and the liquid portion was carefully poured
off so as to not disturb the settled material. The retained liquid was then dispensed
into the test vessels.
For each sediment, a total of 10 replicate samples were prepared. All 10 replicates
were inoculated and held under the same conditions during testing. Based on the
experiences of Phase IIIA, where the dissolved oxygen levels fell below acceptable
levels, a decision was made to begin aerating these sediments from the point of
inoculation. At the end of the exposure, aliquots for larval counts were taken from one
set of five replicates by carefully decanting off the overlying water into a second
container, taking care not to include any of the bottom material ("PSDDA counts"). In
the second set of five replicates, the entire vessel contents were first mixed, and then
aliquots were withdrawn for larval counts.
Source of Broodstock and Spawning Conditions
Gravid sand dollar and Pacific oyster broodstock were obtained from the same sources as described
for Phase II. All organisms were transported to the SAIC Testing Center in Narragansett by overnight
freight, and were used on the day of arrival. Prior to spawning, the organisms were acclimated to test
temperature.
Spawning procedures were identical to those described in Phase II.
Experimental Procedure
For each organism, and test procedure, an individual set of five seawater control replicates were set
up to duplicate the conditions of the procedure. For example, for the 4-hour settling/aerated
treatments, there were five aerated seawater controls that had sat under the same conditions as the
test replicates prior to inoculation. The controls for the 24-hour settling were set up at the same time
as those treatments, and allowed to "settle" for 24 hours. Green Book controls were mixed and
handled identically to the test treatments.
For each organism and test procedure, six replicates were prepared. Where required, aeration was
accomplished by dispensing the air through a glass pipette set at a rate of less than 100 bubbles per
minute. The sixth replicate was used for taking physical monitoring and ammonia measurements. Post
inoculation, physical monitoring measurements (pH, salinity, dissolved oxygen, and temperature! were
Phase MB: Contaminated Sediments Effects |||B-6
-------
taken for each test series. Those measurements were taken again at 24 and 48 hours. Ammonia was
taken at the time of inoculation, and at test termination.
Inoculation of embryos occurred using a volumetric pipette, calibrated to deliver between 20 - 30
embryos/mL. To ensure homogenous distribution of embryos in the stock solution, a perforated plunger
was used to mix the solution. Post-inoculation, two 10 mL aliquots were withdrawn from one set of
five seawater controls, and counted to determine the actual number of larvae dispensed into replicates.
All subsequent mortality determinations were made by comparison to the initial seawater control
counts. To ensure that all the various treatment controls were identical, an additional two 10 mL
aliquots were taken from two replicates of each seawater control. These counts were made and
compared to the other 10 initial counts.
To compare responses of the two organisms to a metal and organic reference toxicant, two sets of
reference toxicants were run in a gradient series using cadmium chloride and phenol. The cadmium
series was identical to that identified in Phase II. For the oysters, a phenol series of 0, 15.6, 31.25,
62.5, and 125 mg/L was run, and for the echinoderms 31.25, 62.5, 125, and 250 mg/L.
The end of the test is taken as the point at which > 90% of the organisms in the seawater control
reach the prodissoconch (oyster), or pluteus larval (sand dollars) stage, as defined by the PSDDA
program. For each test replicate, three 10 mL aliquots were withdrawn, fixed with 5% buffered
formalin, and two were scored microscopically as normal (pluteus larvae) or abnormal. As a quality
assurance procedure, 20% of all larval counts were re-scored by a second counter. In the event of a
discrepancy between the counters, a third count was made, and procedures reviewed prior to
proceeding further with counts. If further resolution was needed, the third aliquot was scored for that
replicate.
Data Analysis
Larval response data were first tabulated in a spreadsheet, percent mortality and abnormality
determined by replicate, and then the mean of all replicates for an exposure treatment reported in the
data summary. All data in the spreadsheets were checked and confirmed against the original data
sheets, prior to proceeding with analyses.
For each treatment, the seawater control final counts were taken, and then compared to the initial
inoculum counts for determination of percent control survival. Mortality in these exposures is
expressed as the PSDDA combined mortality/abnormality endpoint. All sediment treatment
comparisons are made against the number of surviving, normal larvae in the controls.
Each of the test sediments and treatments were analyzed to determine how the results would compare
to a PSDDA open-water disposal determination. The mortality data were compared to the control, and
the appropriate reference sediment data from IIIA, using the arcsin/square root transformation of
proportional data, and conducting one-way r-test analyses. The 20% and 30% guideline values were
then applied, to determine "suitability" under PSDDA.
To evaluate differences between the two species by station and treatment, multiple two-way r-tests
were run on the transformed mortality data using Excel 5.0. An alpha level of 0.05 was used for all
comparisons.
To compare differences between test treatments, an Analysis of Variance (AN0VA) was conducted,
followed by using Tukey's Wholly Significant Differences test on the transformed mortality data. The
Statgraphics statistical package was used to run the ANOVA and derive the pooled standard error, and
Phase IIIB: Contaminated Sediments Effects |||B-7
-------
the differences determined by following the procedures of Zar (1984) to determine the g statistic and
critical q values.
Determination of reference toxicant LC50 and EC50 values was done using EPA's probit program on
proportional mortality and abnormality data.
RESULTS
SEDIMENT COLLECTION AND ANALYSES
Sediment Conventional
Station coordinates, conventional, and grain size analyses are presented in Table IIIB-1. The blended
sediments for the D1 series achieved a fair agreement on grain size distribution (58 - 68% fines), and
percent total solids (approximately 52 to 65%). However, the analysis of CRR2 showed that there was
a much lower level of percent fines than was anticipated (14% vs. the 31% measured during Phase
111 A). As a result, the Ml dilution series ranged between 14 and 43% fines, and total solids ranging
from 43 to 76%.
Table IIIB-1. Sampling location, conventional and grain size data for 1MB sediment composites.
Location
Station
GPS
(North by
West)
Loran TD
Water
Depth
(m)
% Fines
Total
Solids
Tvs
(mg/kg)
TOC
(%)
Ammonia
(mg/kg)
Sulfide
(mg/kg)
Ml
47 °35.1 1 N
122°21.67
W
27989.3
42296.1
16
43
42.94
6.06
2.90
17.60
238.00
M1C 25/50
...
...
--
25
64.77
1.36
1.00
9.72
275.00
M1C2
25/75
...
---
--
13
73.48
2.50
0.80
18.60
73.00
CRR2
NONE
27954.1
42214.4
21-23
14
74.55
6.74
0.40
5.41
<1.66
D1
47°35.1 5N
1 22 °22.02
W
27991 .1
42295.3
14
58
51.85
2.54
1.80
1 1.00
297.00
D1C4
50/50
...
...
--
63
55.63
3.25
1.30
13.10
99.60
D1C4
25/75
...
...
--
66
62.12
1 1 ,50
0.80
33.12
175.00
CRR4
NONE
27953.1
42215.4
13
68
64.65
12.70
0.50
1 1.9
<1.81
Phase MB: Contaminated Sediments Effects lllB-8
-------
Sediment Analyses
The results of the screening for PSDDA chemicals of concern are presented in Table IIIB-2. PSDDA
screening levels, maximum levels, and the 1988 oyster Apparent Effect Threshold (AET) values (PTI,
1988) are also shown on Table IIIB-2. The original laboratory data sheets, chain-of-custody records,
and QA1 memoranda are included in the appendix of this section. QA2 data have been provided to
EPA under separate cover.
In general, the mixing of contaminated sediments with the clean reference sediment achieved the
desired effect of diluting the chemicals of concern. Table IIIB-2 shows that for metals, and most of
the LPAH and HPAH, the chemicals of concern were decreased proportionally. Some exceptions were
noted in the D1 series for napthelene and acenapthene at the 50% dilution. For napthelene, the
measured value doubled, while the acenapthene level remained diluted. Review of the laboratory
procedures indicated that the measured values were valid data points. One possible explanation is that
while every effort was made to achieve thorough mixing of the test sediment, "pockets" of undiluted
sediment remained, which may have had higher levels of these compounds.
All test sediments exceeded at least the PSDDA SL for several analytes, and Station M1 and all of the
D1 series dilutions exceeded the ML and AET for some of analytes. As expected, M1 had a high level
of copper,lead, mercury, and zinc, and exceeded the SL for most of the low polyaromatic hydrocarbons
(LPAH) and high PAH. The 50% and 25% dilution samples for M1 had no ML or AET exceedences,
but several SL exceedences for metals, and the LPAH and HPAH fractions. Station D1 had lower levels
of metal contaminants that were reduced to at or below the SL in the 50 and 25% dilution series. By
contrast, the D1 series had extremely high levels of LPAH and HPAH at all dilutions. The total LPAH
level for D1 exceeded the AET value by 3 times. Oyster AET exceedences were measured at all D1
series dilutions.
Both Ml and D1 showed SL exceedences for phenols, miscellaneous extractabfes, and Total PCB. D1
and D1C4 50/50 had dibenzofuran levels exceeding the ML and AET. Neither series showed any
detected chlorinated hydrocarbons, phthalates, volatile organics, or pesticides.
Based on the chemical data alone, all of the D1 dilution series, and Station Ml, would not be suitable
for open water disposal under the PSDDA program. M1C2 50/50 and Ml C2 25/75 had numerous SL,
but no ML exceedances, and would be eligible for biological testing under the PSDDA program. All test
stations exceeded the Washington State Sediment Quality Standards and the Impact Zones Maximum
Chemical Criteria for total LPAH and HPAH (WAC 173-204-320 and 420).
BIOASSAY RESULTS
Data Acceptability
Oyster larval survival to the prodissoconch stage was >95% for all seawater controls, with
abnormality less than 4%. Unionized ammonia levels were less than 0.04 mg/L for all controls and
most treatments at inoculation and conclusion. Noted exceptions were many of the Green Book
preparations which had exceeded the guideline value at the end of the test.
Echinoderm larval survival to the pluteus larval stage in the seawater controls was >70%, with less
than 10% abnormality, with the exception of the Green Book controls, where the abnormality was
recorded as 12.1%. The effect on data quality by a 2% exceedance is thought to be negligible.
Unionized ammonia levels were all less than 0.04 mg/L at the test initiation, but values for two of the
Green Book M1 treatments exceeded that value at test termination.
Phase It IB: Contaminated Sediments Effects MlB-9
-------
Table 1KB 2. Concentrations of PSDDA Chemicals of Concern Found in Test Sediments.
PSDDA 1988 OYSTER
PSDDA PARAMETER SL ML AET
CONVENTIONALS
Total Solids (%l
Total Volatile Solids (%)
Total Organic Carbon (%)
Total Sulfides (mg/kg)
Ammonia (mg/kg)
Grain Size (Percent Fines)
METALS Ippm. dry weight)
Antimony
Arsenic
Cadmium
Copper
Lead
Mercury
Nickel
Silver
Zinc
ORGANICS (ppb. dry weightl
LPAH
Napthelene
Acenapthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
2-Methylnaphthalene
Total LPAH
20
57
0.96
81
66
0.21
140
1 .2
1 60
210
64
63
64
320
130
67
610
200
700
9.6
810
660
2.1
6 1
1 600
2100
640
630
640
3200
1 300
670
6100
700
9.6
390
660
0.59
>0.56
1600
2100
>560
500
540
1500
960
670
5200
Boxes around the vdlues mdicjles SL exeedeoces
Shading of the values indicates ML exceedences
STATION
Ml M1C2 M1C2 D1 D1C4 D1C4
50/50 25/75 50/50 25/75
42.94
6.06
2.90
238.00
17.60
43,00
64.77
1.36
1.00
275.00
9 72
25.00
73.48
2.50
0.80
73.00
1 8.60
1 3.00
51 .85
2.54
1.80
297.00
1 1 .00
58.00
55.63
3.25
1.30
99,60
13.10
63.00
62.12
1 1 .50
0.80
1 75.00
33.1 2
66.00
60.10
77.40
0.40
355.00
141.00
0.18
29.00
0.42
366 00
1 9.90
28.80
0.21
171.00
69.30
0.09
22.80
0.25
178.00
7.30
20.70
1.43
174.00
139.00
0.43
36.00
0.72
290.00
3.40
9.78
0.72
80.50
~66.00 I
0.21
35.00
0.34
126.00
1.15
7.36
0.50
50.30
42.10
0.08
39.40
0.23
86.00
76.00
21.00
170.00
170.00
1300.00
350.00
46.00
2133.00
23.00
15.00
59.00
79.00
500.00
1 10.00
24.00
81 0.00
1700.00
3400-00
260.00
120.00
1909.00
1 $00,00
1600.00
1400.00
SftOOOO
SgfixJ.oO
3300.00
1300.00
530.00
490.00
15790.00
U&10.00
840.00
88.00
710,00
1500.00
1000.00
290.00
5258.00
-------
Table IIIB 2. Concentrations of PSDDA Chemicals of Concern Found in Test Sediment
PSDDA PARAMETER
PSDDA 1988 OYSTER
SL ML AET
6300 2500
7300 3300
4500 1600
6700 2800
8000 3600
6800 1600
5200 690
1200 230
5400 720
51000 17000
350 50
>170
260 120
64 64
230 230
160
>73
1400
>470
1900
>420
1200 420
72 63
1200 670
50 29
690 >140
HPAH
Fluoranthene 630
Pyrene 430
Benzolajanthracene 450
Chrysene 670
Benzofluoranthenes 800
Benzo(a)pyrene 680
lndeno(1,2,3-c,d)pyrene 69
Dibenzo(a,h|anthracene 120
Benzo(g,h,i)perylene 540
Total HPAH 1800
CHLORINATED HYDROCARBONS
1.2-Dichlorobenzene 19
1.3-Dichlorobenzene 170
1.4-Dichlorobenzene 26
1,2.4-Trichlorobenzene 13
Hexachlorobenzene (HCB) 23
PHTHALATES
Dimethlyt phthalate 1 60
Diethyl phthalate 97
Din-butyl phthalate 1400
Butyt Benzyl phthalate 470
Bis(2-ethylhexyl)phthalate 3100
Di-n-octyl phthalate 6200
PHENOLS
Phenol 120
2 Methylphenol 20
4 Methylphenol 120
2,4-Dimethylphenol 29
Pentachlorophenol 100
Boxes around the values indicates SL exeedences.
Shading of the values indicates ML exceedences
STATION
M1C2
50/50
M1C2
25/75
D1
D1C4
D1C4
50/50
25/75
9300.00
6300.00
$300,06
AtmiM
2700.00
2100.00
4800.00
3300.00
7700.00
4100.00
3300 00
1900.00
1500.00
880.00
820.00
280.00
1300.00
490.00
41320.00
30350.00
2500.00
1800.00
1200.00
1500.00
2200.00
1 100.00
1000.00
320.00
620.00
12240.00
1 100.00
1200.00
470.00
570.00
1000.00
470.00
280.00
75.00
150.00
5315.00
6TOCM5C
20000.00
3700.00
2000:00
2500.00
92700,00
1.3U
1.3U
1.3U
1.3U
14U
NO DATA
NO DATA
NO DATA
NO DATA
13U
2.4U
2.4U
2.4U
2.4U
23U
1,4U
1.4U
1 4U
1,4U
17U
NO DATA
NO DATA
NO DATA
NO DATA
15U
14U
14U
21.00
59.00
410.00
14U
13U
13U
10M
54.00
190.00
13U
23U
23U
23U
23U
400.00
23U
17U
17U
17U
17U
160M
17U
130.00
160.00
180.00
97.00
42.00
13J
23U
7.9M
30.00
23.00
23U
28.00
16M
13U
23U
18M
230.00
98.00
110U
84U
15U
15U
15U
15U
75.00
15U
10M
15U
1 1M
15U
75U
-------
Table 1MB 2 Concentrations of PSDDA Chemicals of Concern Found in Test Sediments.
STATION
PSDDA
1988 OYSTER
Ml
M1C2
M1C2
D1
D1C4
D1C4
PSDDA PARAMETER
SL
ML
AET
50/50
25/75
50/50
25/75
MISCELLANEOUS EXTRACTABLES
Benzyl Alcohol
25
73
73
72U
13M
1 3U
23U
17U
15U
Benzoic acid
400
690
650
72U
61J
1 30U
220U
170U
1 50U
Dibenzofuran
54
540
540
240.00
I 110.00
47.00 j;
1200 OQ
530.00
Hexachloroethane
1400
14000
72U
14U
13U
23U
17U
1 5U
Hexachlorobutadiene
29
290
270
72U
14U
1 3U
23U
17U
15U
N-Nitrosodiphenylami ne
28
220
130
72U
14U
1 3U
23U
17U
1 5U
VOLATILE ORGANICS
Tnchloroethene
160
1 600
1 ,7U
1.3U
NO DATA
2.4U
1,4U
NO DATA
T etrachloroethene
1 4
210
140
1,7U
1 3U
NO DATA
2.4U
1 4U
NO DATA
Ethylbenzene
10
50
37
1.7U
1 3U
NO DATA
2.4U
1,4U
NO DATA
Tolal Xylene
12
1 50
120
3.4U
2.5U
NO DATA
4.7U
2.1 M
NO DATA
PESTICIDES and PCBs
Tolal DDT
4,4*-DDD
4,4'-DDE
4,4'DDT
Aldnn
Chlordane
Dieldrin
Heptachlor
Lindane
PCB 1016/1242
PCB 1248
PCB 1254
PCB1260
Total PCBs
6.9
10
10
10
10
10
29U
11U
2.4U
13U
1 ,2U
5.0U
1,2U
1,2U
72U
72U
270.00
280U
9.90
6.3U
12U
2.5U
1 2U
2 9U
1 .2U
1 2U
38U
1 20 U
130 00
1 50 U
4-.7U
2.1 U
5 OU
0.8U
0.8U
1 .6U
0.8U
0.8U
40 U
SOU
71.00
68C
13U
6.2U
2.4U
3.2U
1.2U
6.4U
2.5U
1.8U
96U
20OU
180.00
450U
130
2500
1100
270.00
190.00
139.00
180.00
11.00
3U
2.4U
1 ,4U
1 2U
2.4U
1 ,2U
1 2U
43U
85U
71.00
150U
71 .00
1 ,6U
1.6U
1,6U
0.8U
0.8U
1.6U
0.8U
0.8U
48U
25.00
28.00
65U
53.00
Data Qualifiers
U
J
M
C
Indicates compound was analysed for, but not detected at the given detection limit
Indicates an estimated value when result is less than specified detection limit.
Indicates an estimated value of analyte found and cofirmed by analyst, but with low spectral matach parameters
This flag is used when the analyte is detected in both the sample and blank. Indicates possible/probable blank contamination
Boxes around the values indicates SL exeedences.
Shading of the values indicates ML exceed en ces
-------
General Results By Station and Species
In general, the echinoderms showed a greater magnitude of response in all aerated treatments, than
did the oysters for the M1 series, but neither species showed a great deal of response to any of the
D1 series (Table IIIB-3). For the unaerated treatments, there was virtual similarity in the percent
mortality for both species. For the M1 series, both oysters and echinoderms showed an decreased
response with decreased percent of the contaminated material for all treatments, except the 24 hour
settlement. In the latter treatments, the dose/response was flat.
Virtually all of the oyster response to exposure was in mortality (Figure IIIB-1); the percent abnormality
was less than 10% for ail sediments and treatments. By contrast, the echinoderms showed a much
higher level of response to the M1 series (Figure IIIB-2), with abnormality comprising a large part of the
combined endpoint (Figure IIIB-3).
Oyster larval mortality responses to the D1 series are shown in Figure IIIB-4. A significantly elevated
response was observed only at the whole-DI sediment for the Green Book/PSDDA. The standard
Green Book method for the D1 series did not exhibit a dose/response relationship. Echinoderm larvae
showed very little mortality or abnormality response to exposure to D1, with the exception of the 4-
hour unaerated treatment (Figure IIIB-5). While overall the mortality was generally low, the pattern of
highest echinoderm response in the 4-hour unaerated was observed in the D1 treatments. The
abnormality response to D1 for the PSDDA elutriate preparations was low; < 15%. Both Green Book
preparations showed abnormalities ranging from 15 - 35%.
Results of PSDDA f-Test Comparisons
The results of PSDDA r-test and trigger levels comparisons show that both species' response to the
test sediment/treatments show good correspondence when compared to PSDDA guideline values.
Table IIIB-4 shows that for the M1 series, both species were in agreement on eight of nine treatments
that exceeded the 30% over the reference sediment guideline. Some differences were noted in
comparisons with the 20% guideline value. The oysters exceeded the 20% trigger value for the 24-
hour unaerated treatment of M1C2 50/50, whereas the echinoderm mortality was only 4.5%.
Conversely, the echinoderms exceeded the 20% mark for the Green Book/PSDDA treatment of M1C2
25/75, but the oyster mortality was only 8.5%.
For the D1 series, both species were in agreement on identifying the two 30% trigger exceedances,
and one 20% exceedance (Table IIIB-5). The oysters exceeded the 20% triggers for two of the D1C4
series, but there were no exceedences for the echinoderms.
Results of Species Responses to M1 Treatments
Relatively little response that occurred with exposure to D1, thus between species comparisons were
limited to the M1 treatments. The results of f-test comparisons between species for mortality
responses to the M1 dilution series is given in Table IIIB-6. These comparisons reinforce the earlier
observation that in the aerated treatments, echinoderms were more sensitive to the exposures than
were the oysters. In the 4-hour settling/unaerated treatments, there were no significant differences
between the two species. For the 24-hour settling/unaerated treatments, the oysters exhibited
significantly greater mortality response.
Phase MB: Contaminated Sediments Effects IM B-13
-------
Table IIIB-3. Results of Phase IIIB Larval Exposures.
PHASE I1IB OYSTER MORTALITY AND ABNORMALITY
STATION
PSDDA 4-HOUR SETTLING
PSDDA 4-HOUR SETTLING
PSDDA 24-HOUR SETTLING
GREEN BOOK
GREEN BOOK WITH
(AERATED)
(UNAERATED)
(UNAERATED)
PSDDA COUNTS
Mortality Abnormality
Mortality
Abnormality
Mortality
Abnormality
Mortality
Abnormality
Mortality
Abnormality
Ml
61.03
3.70
41 47
7 03
15 51
2 65
59 88
5 98
45 34
4 84
M1C2-51)%
44 71
1.66
35 81
3 70
25 04
3.05
32 55
6 49
55 13
6.35
N1IC2-25%
35.88
0.57
18.29
1 54
18.26
4 31
18.96
1 53
8 50
0 50
C2
11 00
2 10
9 80
1 00
0.20
0 50
**
* *
**
**
D1
24.63
I 50
29 30
5 06
15 88
3 14
11 87
1 69
43 54
I 70
DIC4 - 50/50
17.43
0 94
12 88
4 91
14 27
5.22
7.07
1 57
34 70
0 88
D1C4-25/75
21 32
0.63
13 41
3 10
15 88
4 25
4.58
2 36
13 46
1 61
C4
17.10
1 40
20.50
0 70
0.10
0.30
*»
**
»*
**
PHASE IIIB ECHINODERM MORTALITY AND ABNORMALITY
STATION
PSDDA 4-HOUR SETTLING
PSDDA 4-HOUR SETTLING
PSDDA 24-HOUR SETTLING
GREEN BOOK
GREEN BOOK WITH
(AERATED)
(UNAERATED)
(UNAERATED)
PSDDA COUNTS
Mortalitv Abnormality
Mortality
Abnormal ily
Mortality
Abnormality
Mortality
Abnormality
Mortality
Abnormality
Ml
99 02
98 80
46 89
2191
12 20
9 15
98 39
98 63
99 63
98 27
M1 C2-50%
84 06
79 21
28 71
11 86
4 46
8.7.3
67.02
75.55
56.07
49.63
MIC2-25%
49.87
45 75
19 08
8 83
I 65
7 96
15 11
36 88
34.98
31.23
C2
13.30
17 80
31 60
13.70
7.10
-10 40
**
**
*~
**
D1
17.48
13 57
39.53
7 81
11.77
10 46
1083
25 38
25 16
14 51
DIC4 - 50/50
19 33
13 05
24 96
7 02
8.62
7 48
11.22
31 55
62.10
15.04
D1C4 -25/75
9 87
10 81
21.83
7 70
3.24
8 31
15.99
29.22
67.21
22.03
C4 *
-2 30
8 30
47.2
25.2
-2.1
7 80
*~
**
**
**
* Values for C2 and C4 included from 111A summary table
** Green Book treatment data from for C2 and C4 not included due to lugh mortality and abnormalit) in Phase III A
Phase IIIB Contaminated Sediments Effects
NOTE: Bioassay results for reference sediments C2 and C4 were included from
Phase IIIA testing. Sediments C2 and C4 were not run synoptically
with the IIIB treatments due to logistical constraints.
-------
Phase NIB, Figure 1
M1/CRR2 Series and Oyster Mortality
03
O
i-
C/5
>.
o
c
tu
o
u
u
CL
'/?W\
' tw.
mz\
100
90
80
70
60
50
40
30
20
10
0
Ml
M lC2-50%
M1C2
-25%
4 Hour Aerated
4 I lour Unaerated
24 Hour Unaerated
Green Book
Green Book/PSDDA
-------
t:
o
u
-o
o
UJ
a
v
o
—
u
a.
Phase NIB, Figure 2
M1/CRR2 Series - Echinoderm Mortality
MIC2-50%
MlC2-25%
4 Hour Unaerated
24 Hour Unaerated
Green Book
Green Book/PSDDA
4 Hour Aerated
-------
Phase HIB, Figure 3
• ^
c3
O
C
_o
<
e
u
o
u
u
Ou
Ml
M1 C2-50%
MlC2-25%
4 Hour Aerated
4 Hour Unaerated
24 Hour Unaerated
Green Book
Green Book/PSDDA
MI/CRR2 Series and licliinodenn Abnormality
-------
Phase IIIB, Figure 4
DI/CRR4 Series - Oyster Mortality
DIC4 - 50/50
DIC4 -25/75
Sediment Series
4 Hour Aerated
4 Hour Unaerated
24 Hour Unaerated
£4 Green Book
Green Book/PSDDA
Experimental Treatments
-------
Phase IIIB, Figure 5
D1\CRR4 Series - Echinoderm Mortality
4 Hour Aerated
4 Hour Unaerated
D1C4 - 50/50
24 Hour Unaerated
Green Book
DIC4 -25/75
Green Book/PSDDA
-------
Table IIIB-4. Application of PSDDA bioassay criteria to Oyster as Ecbinoderm responses to the (Ml) dilution
series and treatments.
PERCENT MORTALITY
STATION
MEAN
STANDARD
DEVIATION
COMPUTED t
CRITICAL t
STATISTICAL
SIGNIFICANCE
20% OVER
CONTROL
.10% OVER
REF
OYSTERS
SW
0
0
CARR2
0
0
OMHUA
*1.5
10,4
13.95
1.77
•
•
QM54UA
3*.*
8 0
15.06
1.78
n
•
OM24UA
18 3
8 8
7 83
1 .77
•
0M14A
61,1
2
26,49
1,77
~
0
~
0M54A
44,7
88
17,00
1,77
~
OM24A
35.9
8.8
14.15
1.77
•
l-J
•
OM124UA
15.5
18 3
2.18
1 77
•
OM624UA
2&,1
13 0
7-45
1 r77
~
0
OM224UA
18 4
10 3
6 40
I 80
•
GM1G
00.0
3 5
53.17
1.77
•
OMSG
32.6
73
18.93
1.77
•
•
OM2G
17 9
15 0
3 60
1.78
•
0M1GP
45,3
88
17.97
T ,77
~
-
OM5GP
55,2
12,6
14 09
1.77
•
¦->
•
QM2GP
84
8 4
2.63
1.77
•
ECHINODERMS
SW .0 0
CARR2 154 98
£M14UA
46.7
7 8
5,35
1.77
•
•
FM54UA
28.6
22.1
99
1 77
EM24UA
19 1
12.6
;ig
1 77
EM14A
99,0
1.8
15.32
1.77
•
•
EM64A
840
10.2
a 01
1.77
• - ¦
•
EM24A
499
15.1
4 44
1.77
•
•
EM124UA 12 2 8 9 44 1 77
EM524UA 4 5 7 9 2 12 1 77
EM224UA 1 7 5 4 3 56 1.77
EM1G
90,5
17
14,73
1,77
~
~
EM5G
67,0
1*5
6.31
1.77
• ¦
•
FM2G
15.1
18 6
40
1 77
EMlGf*
99 e
.7
17 IB
t 77
•
•
pyibgp
58 1
12,2
a.©9
1.77
•
o •
EM2GP
35,0
1*0
2.90
1.77
•
O = oyster M = Ml 1 = 100% 4UA = 4 hour unaerated 24UA = 24 hour unaerated
E = echinoderm D = D1 5 = 50% 4A = 4 hour aerated G = Green Book
2 = 25% GP = Green Book/PSDDA
Phase IIIB: Contaminated Sediments Effects lllB-20
-------
Table IIIB-5. Application of PSDDA bioassay criteria to Oyster as Echinoderm responses to the (D1)
dilution series and treatments.
PERCENT MORTALITY
STATION
MEAN
STANDARD
DEVIATION
COMPUTED t CRITICAL t STATISTICAL
SIGNIFICANCE
20% OVER
CONTROL
30% OVER
REF.
OYSTEFS
sw
0
0
CARR4
2 2
4 9
0014UA
29,3
6.8
e,63 1 ,yy *
OD64UA
12.9
7.9
3.22 1.77 •
OD24UA
13 6
9 4
3 06 1.77 •
0014A
24,6
12.6
4.13 1,77 •
¦ ¦ o ¦¦
OD64A
1 7.6
9 2
3.82 1.77 •
0024A
21,6
7.0
6 67 1,77 ~
o
OD124UA
16 0
10.2
3 36 1 77 •
OD624UA
14 3
9 1
3 29 1.77 •
(J0224UA
16 8
9 4
J.66 1 77 •
OD1G
1 1 8
7 0
3 60 17 7 •
ODbG
? 2
1 1 T
1.13 1 77
OD2G
4 6
6 0
1.30 1 77
001GP
43.4
20.3
6 67 1.77 ~
o
• ¦
OOBGP
34.7
12.6
7 18 1.77 ~
0
OD2GP
13.6
16 6
197 1 77 •
ECHINOOERMS
SW
n
0
CARR4
12.6
22.8
CD14UA
19.6
12.6
3 67 1.77 ~
o
FD64UA
26.0
16.2
1.60 177
tD24UA
?1 6
16 0
164 1.77
f DMA
1 7 6
13 6
66 1 89
( D64A
19 3
1 6 7
73 1 77
f 024 A
9 9
10 7
12 1 77
mi 24UA
1 1 8
12 0
23 177
BD624UA
8 6
9 6
16 T 77
FD224UA
I 2
4 2
1 20 1 77
FD1G
10 7
9 7
17 1.77
FD6G
113
1.3.2
10 1 77
F02G
16.1
111
91 1.77
fcDIGP
26.2
12 2
1.76 177
f06GP
62,1
14.2
6 44 1.77 ~
o
•
E02GP
67.1
13,8
6.88 1.77 •
o
•
O =t oytier M * M1
' - echinoderm
0 * 01
1 =. 100% 4UA
6 *
¦ 4 hour ururarftted 24UA ¦ 24 hour unMrafM
60% 4A * 4 hour Mfatvd G « Gr««n Book
2-26% QP a Groan Book/PSDDA
Phase IIIB: Contaminated Sediments Effects iuB-21
-------
Table IIIB-6. Two-tailed f-test comparisons between echinoderm vs. oysters responses for the M1
dilution series by treatment. Positive values indicate greater echinoderm response; negative values
greater oyster response. Shaded areas indicate statistical significance at a - 0.05.
4-HOUR UA
4-HOUR A
24-HOUR UA
GREEN BOOK
GREEN BOOK/PSDOA
M1
1 .33
16.91
0.24
18.33
24.52
Calculated t
2.10
2.10
2.1 1
2.10
2.10
Critical t
M1C2 50/50
1.06
7.20
-5.1 5
6.66
0.18
2.11
2.10
2.10
2.10
2.10
M1C2 25/75
0.34
2.48
-5.84
0.72
5.08
2.10
2.10
2.12
2.11
2.10
Results of Differences by Species Between Treatments
The results of the Tukey's Wholly Significant Differences test on arcsin/square root transformed data
for oysters and echinoderms are presented in Tables IIIB-7 and IIIB-8. In general for both species,
mortality was greatest in the 4-Hour Aerated and Green Book, and least for the 24-Hour treatments.
The 4-Hour Unaerated treatments for both species had significantly less mortality than the 4-Hour
Aerated treatments.
Results of Species as Predictors of Apparent Sediment Toxicity
In general, the PSDDA SL/ML's and the 1988 Oyster AET values were adequate predictors of sediment
toxicity to both species for the M1 dilution series. This was true for all treatments, with the exception
of those vessels where the sediment was allowed to sit for 24 hours prior to testing.
In contrast, the responses for D1 did not match the expectations of either the ML or AETs. For both
species, the mortality and abnormality responses for D1 were relatively negligible.
Comparison of Species Reference Toxicant Responses
The data collected for the two species for the two reference toxicants is consistent with the
observation that oyster response to the toxicant over the range tested was principally mortality, while
sand dollars exhibited abnormal development. For the oysters, low numbers of total larvae were
recovered at increasing concentrations. Abnormality did occur with increasing frequency, but in a
decreasing number of larvae. In contrast, the total number of echinoderm larvae recovered for both
reference toxicants were at or near the number of embryos inoculated into the test vessels. However,
with increasing dosage, there were increasing numbers of abnormal larvae.
Phase ll/B: Contaminated Sediments Effects 111B-22
-------
Table 1118 - 7 Results of Tukey's Multiple Comparison Test for Oyster Mortality by Test Protocol
Shaded areas indicate statistically significant differences between treatments.
Oyster Mortality for Ml by Treatment
Calculation of Tukey Q statistic Pooled
Level Average Stnd. Error
Calculated q value
OM14A 0M1G 0M1GP
OM14UA
OM14A
0.898
0.049 j
OM1G
0.885
0.049
0.271
0M1GP
0.738
0.049
3.27
3.00
OM14UA
0.698
0.049
4.08
3.32
0M124UA
0.306
0.049
12.0$
11 >82
k = 4
D.F. = 45
8 00
Critical q 0.05, 4,45 = 3.81
Oyster Mortality for M1C2 50/50 by Treatment
Calculation of Tukey Q statistic Pooled
Level Average Stnd. Error
Calculated q value
0M5GP 0M54A 0M54UA 0M5G
OM5GP
0.838
0.035 I
OM54A
0.731
0.035
3.06|
OM54UA
0.624
0.035
641
3.061
0M5G
0.605
0.035
3.60
OM524UA
0.513
0.035
9.29
1WMM
D.F. = 44
Critical q 0.05, 4,45 = 3.81
2.63
Oyster Mortality for M1C2 25/75 by Treatment
Calculation of Tukey Q statistic Pooled
Level Average Stnd. Error
Calculated q value
OM24A OM24UA 10M224UA
0M2G
0M24A
0.639
0.052 1
OM24UA
0.431
0.052
4.00 ¦
0M224UA
0.425
0.058
4.12
0.12
OM2G
0.390
0.055
4.7$
0.79
OM2GP
0.236
0.052
7.7$
3.75
0.60
2.80
D.F. = 42
Critical q 0.05, 4,45 = 3.81
0 = Oyster 1 = 100% M1
M = M1 Sediment Series 5 = 50% M1
2 = 25% M1
4A = 4 Hour Aerated
4 UA = 4 Hour Unaerated
24 UA = 24 Hour Unaerated
G = Green Book Procedure
GP = Green Book with PSDDA counts
Phase l/IB: Contaminated Sediment Effects
-------
Table IIIB-8. Results of Tukey's Multiple Comparison Test for Echinoderm Mortality by Test Proto
Shaded areas indicate statistically significant differences between treatments.
Echinoderm Mortality for M1 by Treatment
Calculation of Tukey Q statistic
Pooled
D.F. = 45
Calculated q value
Level
Average
Stnd. Error
EM1GP
EM1GP
1.538
0.036
EM14A
1.520
0.036
0.51
EM 1 G
1.476
0.036
1.75
EM14U
0.754
0.036
22.08
EM124UA
0.313
0.036
34.81
EM14U
21*58
34.00
\2M
Critical q 0.05, 4,45
Echinoderm Mortality for M1C2 50/50 by Treatment
Calculation of Tukey Q statistic Pooled
Level Average Stnd. Error
EM54A
Calculated q value
EM5G EM5GP
EM54UA
EM54A
1.192
0.062 |
EM5G
0.966
0.062
3-65 J|
EM5GP
0.849
0.062
$.53
1.89
EM54UA
0.512
0.062
10.97
7.32
EM524UA
0.148
0.062
16.84
13,19
D.F. = 45
Critical q 0.05, 4,45 = 3.81
5.87
Echinoderm Mortality for M1C2 25/75 by Treatment
Calculation of Tukey Q statistic Pooled
Level Average Stnd. Error
k = 4
Calculated q value
EM24A EM2GP EM24UA EM2G
EM24A
0.782
0.065 |
EM2GP
0.628
0.065
2.37 |
EM24UA
0.403
0.065
5.83
3.46
EM2G
0.300
0.065
7A2
5.05
EM224UA
0.042
0.065
11.38
wz
D.F. = 45
1.581
5.5S 3 .$7
Critical q 0.05, 4,45 = 3.81
E = Echinoderm 1 = 100% M1
D = D1 Series Sediments 5 = 50% M1
2 = 25% M1
4A = 4 Hour Aerated
4 UA = 4 Hour Unaerated
24 UA = 24 Hour Unaerated
G = Green Book Procedure
GP = Green Book with PSDDA counts
Phase MB: Contaminated Sediment Effects
-------
The oyster LCS0 and the echirioderm EC60 are presented in Table IIIB-9. Species were comparable in
their responses to phenol, but the ECB0 response to cadmium was higher for the echinoderms.
Table IIIB-9. Reference Toxicant LC60 and ECS0 Values for Phases IIIA, and IIIB
PHASE EC/LCS0 (mq/L)
OYSTERS
IIIA - CdCI2 1.65
IIIB - CdCI2 1.24
IIIB - Phenol 112.83
ECHINODERMS
IIIA - CdCI? 3.90
IIIB - CdCI? 3.54
IIIB - Phenol 96.52
DISCUSSION
SEDIMENT CHEMISTRY
With the exceptions noted above, the "dilution" procedure was successful in producing a range of
contaminants for testing. The levels of chemical values observed in these test sediments are such that
only two of the six sediments would have ever been considered for biological testing under the PSDDA
program: the 50% and 25% dilutions of the M1 test sediment. The other four sediments had ML
exceedences for several analytes. As noted above, all of the test sediments exceed the state Sediment
Quality Standards. Thus, these six test sediments represented a good range contaminants by which
to compare regulatory standards to larval bioassay results. It is interesting to note that if those two
sediments were being evaluated for disposal under the PSDDA program, both larval bioassays exceeded
the 30% trigger value and would be designated unsuitable for open water disposal.
To determine why there was a major difference between the reported grain size values for CRR4
between Phase IIIA and IIIB, the field collection notes were reviewed. During collection of Phase IIIA
sediments, the field crew navigated by use of the GPS, and reported both GPS and Loran TD values.
During Phase IIIB, the field crew only had Loran C, and went to the coordinates provided by the Corps.
During IIIB sediment collection, the crew reported problems with the Loran unit, which likely resulted
in their being on a different site relative to the IIIA - CRR4 site. Field wet sieving indicated that the
material was 35% fines; the discrepancy between field and actual is considerable. While the grain
sizes of the M1 dilution series did range between the two "parent" sediments (see Table IIIB-2), it is
not likely to impact the interpretive results associated with the between species and between test
effects. The silt/clay content of sample M1 were below those levels shown to cause an effect to
oyster larvae in Phase IIIA. However, the differences associated with the changes in conventional
Phase IIIB: Contaminated Sediments Effects IIIB-25
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parameters (grain size, sulfides, total organic carbon) across the M1 series does limit discussions
regarding sediment chemistry effects.
BIOASSAYS
The data produced during these exposures suggest that oyster mortality and echinoderm abnormality
respond similarly to toxicant exposure, over the 48 hour exposures. When compared to PSDDA
guideline values for bioassays, both species showed similar exceedences. However, there also appears
to be varying degrees of response to the different treatments and sediments, with the abnormality
response showing greater sensitivity.
The reference toxicant data further buttress the observed relationship between larval oyster mortality
and sand dollar abnormality. In the presence of both the metal and organic toxicant, oyster larvae
showed positive dose response, but fewer larvae were recovered with higher concentration. The
echinoderms also showed positive dose response to increasing toxicant concentration, but the total
number of larvae recovered remained stable while the frequency of abnormality increased.
These data, along with the following arguments, suggest that Dendraster excentricus is a more suited
organism for use in larval elutriate testing. In these experiments, the sand dollar showed greater
sensitivity over the range of contaminants in the M1 dilution series, and thus was a better predictor
of sediment contamination. Data from Phase IIIB show that sand dollar larvae, under current PSDDA
testing procedures, do not appear to be sensitive to increasing silt/clay components in bioassay
chambers. D. excentricus is native to, and is widely distributed throughout Puget Sound, as well as the
North-Eastern Pacific coast. Gravid adult sand dollars can be obtained year-round, and the method of
spawning (potassium chloride injection) is quicker and simpler than for oysters (thermal shock). Finally,
the sand dollar has a better track record in meeting minimum quality assurance guidelines for control
survival (Tim Thompson, personal observation).
An unexpected result was the occurrence of the elevated mortality when the M1 series samples were
aerated. The 4-hour settlement with aeration is the current standard practice for all PSDDA larval
bioassays. Exactly why that phenomenon occurred is not clear, but it does suggest that some caution
should be exercised when recommending aeration during regulatory testing.
An additional unexpected result was that the standard PSDDA elutriate test produced results for both
species that were similar to the Green Book elutriate method. Given that the latter method has a bulk
sediment loading factor in the test that is roughly 10 times the PSDDA method, one would predict
greater mortality and/or abnormality. While a conclusion could be inferred that the two methods are
equivalent in predicting sediment contamination, it must be cautioned that this is a limited data set.
Further careful documentation would be required before a definitive statement comparing the two
methods could be made.
The comparison of the two methods for larval recovery from the Green Book elutriate at test
termination with the Green Book elutriate were confounding. No conclusions could be drawn from
these data as to which method showed greater larval recovery.
Based on the results of these tests, a rough order of protocol sensitivity for both species could be set
as follows:
4-hour Aerated = Green Book > 4-Hour Unaerated > 24-Hour Unaerated.
Phase IIIB: Contaminated Sediments Effects 111B-26
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ANALYTICAL VALUES AS PREDICTORS OF BIOASSAY RESULTS
A discussion concerning the levels of chemicals observed and the percent response in the larval species
should be restricted to the current accepted PSEP/PSDDA testing scheme (4-hour settlement and
aeration). Under that restriction, the responses to the M1 sediment series were as one would predict;
generally decreasing response with decreasing contaminant load. In contrast, the lack of response to
the D1 series is contrary to the expectations of the ML and 1988 Oyster AET values. With LPAH and
HPAH exceeding by several times the ML/AET's, considerable toxicity is predicted in these sediments.
M1 sediments, overall, contained more and higher levels of metals, and fewer and lower concentrations
of organic compounds relative to the sediments collected from D1. The high levels of copper alone are
probably sufficient to drive all of the toxicity observed in these sediments. Although several organic
compounds (especially LPAHs and HPAHs) were measured above the PSDDA SL in M1 samples, only
two organic compounds, phenanthrene and 2,4-dimethylphenol, were detected above the PSDDA ML.
In contrast, D1 samples were predominantly contaminated with organic compounds. For example, in
Sample D1, most LPAHs and HPAHs and dibenzofuran were detected above the PSDDA ML values.
A total of 5 PSDDA metals were detected above the PSDDA SL in Sample D1 compared to 6 PSDDA
metals detected above the SL in Sample M1, one of which (copper) was detected above the ML.
These data suggest a possible link between high larval mortality and high metals concentrations. This
correlation requires additional study and, at this time, is viewed as an observation only.
RECOMMENDATIONS
•Crassostrea gigas and Dendraster excentricus larval responses to dredged sediment can be considered
equivalent predictors of contamination under the PSDDA program.
•Dendraster excentricus is recommended as the primary test organism for sediment characterization.
•The continued use of the combined mortality/abnormality endpoint is recommended for all bivalve and
echinoderm elutriate larval testing.
•The Four-hour settlement and aeration treatment is a good predictor of sediment contamination, and
is recommended for continuous use under the regulatory program.
•The Twenty-four settling time was the least accurate in predicting sediment contamination, and is
therefore not recommended for use in the regulatory program.
Phase MB: Contaminated Sediments Effects tUB-27
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REFERENCES
Pastorok, R.A., and D.S. Becker. 1989. Comparison of Bioassays for Assessing Sediment Toxicity in
Puget Sound. U.S. Environmental Protection Agency, Region 10. Office of Puget Sound.
Seattle, WA
Plumb, R.H. 1981. Procedures for handling and chemical analysis of sediment and water samples.
Technical Report EPA/CE-81-1. U.S. Army Corps of Engineers, Vicksburg, MS.
PSEP, 1991. Puget Sound Estuary Program. Recommended Guidelines for Conducting Laboratory
Bioassays on Puget Sound Sediments. U.S. EPA, Region 10, Office of Puget Sound, Seattle,
WA.
PSEP. 1989b. Puget Sound Estuary Program. Recommended protocols for measuring metals in Puget
Sound sediment and tissue samples. Prepared for the U.S. Environmental Protection Agency
Region X, Office of Puget Sound, and the U.S. Army Corps of Engineers. PTI Environmental
Services, Inc., Bellevue, Washington.
PSEP. 1986. Puget Sound Estuary Program. Recommended protocols for measuring selected environ-
mental variables in Puget Sound. Final Report. Prepared for the U.S. Environmental Protection
Agency Region X, Office of Puget Sound, and the U.S. Army Corps of Engineers. Tetra Tech
Inc., Bellevue, Washington.
PTI Environmental Services 1991. Reference Area Performance Standards for Puget Sound. U.S. EPA,
Region 10, Office of Coastal Waters, Seattle, WA
PTI Environmental Services, 1988. 1988 Update and Evaluation of Puget Sound AET. Vol I and II.
U.S. EPA, Region 10, Office of Puget Sound, Seattle, WA.
U.S. EPA 1991. Evaluation of Dredged Material Proposed for Ocean Disposal, Testing Manual. United
States Environmental Protection Agency and the U.S. Army Corps of Engineers. EPA - 503 /8-
91 / 001.
WAC 173-204. Sediment Management Standards. Washington State Department of Ecology. April,
1990.
Zar, J.H. 1984. Biostatistical Analysis, Second Edition. Prentice-Hall, Inc., Englewood Cliffs, N.J.
07632. xv + 718 pp.
Phase IIIB: Contaminated Sediments Effects HIB-28
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REFINEMENTS TO CURRENT PSDDA BIOASSAYS
FINAL REPORT
CONCLUSIONS
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CONCLUSIONS
This program was designed to try and answer the objectives posed in the Program Overview at the
beginning of this document. Those objectives, and the conclusions/recommendations suggested by
the data are re-summarized below.
The first objective was to identify the effects of ammonia on larval development. This study
provided dose/response data for ammonium chloride to both oyster and sand dollar larvae. Those
data were the basis for the following conclusions:
• An ammonia testing criterion of 0.04 mg/L unionized ammonia is proposed for the
echinoderm test. Data may be qualified as a possible false positive response if un-ionized
ammonia values in echinoderm tests are greater than or equal to 0.04 mg/L.The criterion
value relates specifically to echinoderm abnormality, not mortality. For an acute criterion to
be set for echinoderm larval mortality, additional work is necessary.
• An interim oyster-specific criterion is proposed as 0.13 mg/L unionized ammonia. Some
caution is recommended in using this number for interpretation, as it is an estimate.
Additional work is recommended to better define that number.
• Aeration appears to have an effect on ammonia toxicity on echinoderm. PSDDA should
continue to use aeration in the larval bioassays.
• All laboratories performing PSDDA bioassays should be required to express all ammonia
values as the un-ionized form. The SAIC spreadsheet format could be made standard for
data submittal.
The second objective was to compare echinoderm and oyster responses to clean and contaminated
sediments. In Phase IIIA, the response of both organisms to both a range of grain sizes, and
experimental treatments, were tested. While variability in the larva! data prevent definitive
conclusions from being drawn, the following trends were strongly suggested by the data:
• Crassostrea gigas larvae appear to be sensitive to sediments having a high proportion of
clays and silts.
• Dendraster excentricus, when tested under current PSDDA protocols, do not show an
adverse response to increasing silt and clay fractions. Under conditions of expected high
silts/clay, the sand dollar test is recommended.
• The current convention of comparing sediments on the basis of percent sands, silts and
clays, may not be a useful parameter in attempting to correlate biological observations. For
the purposes of selecting suitable reference material for larval bioassay comparisons, the
percent grain-size data should be converted to grams/Liter of silts and clays.
Phase IIIA set as objectives the comparison of the two species as predictors of sediment toxicity,
and to determine which of the experimental procedures was most accurate in predicting presence
of contaminants in marine sediments. To that end, this report finds the following:
• Crassostrea gigas and Dendraster excentricus larval responses to dredged sediment can be
considered equivalent predictors of contamination under the PSDDA program. During
IV-1
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experimental exposures to contaminated, the oyster larval response was high mortality, but
low abnormality. Conversely, the echinoderms had high abnormality, but low mortality.
• Dendraster excentricus is recommended as the primary test organism for sediment
characterization based upon general availability, consistent control performance, and ease of
handling.
• These results support the continued use of the combined mortality/abnormality endpoint
bivalve and echinoderm elutriate larval testing.
• These data suggest that an order of protocol sensitivity to contaminants in sediments was
as follows:! 4-hour Aerated = Green Book > 4-Hour Unaerated > 24-Hour Unaerated.
The Four-hour settlement and aeration treatment is a good predictor of sediment
contamination, and is recommended for continuous use under the regulatory program. »The
Twenty-four settling time was the least accurate in predicting sediment contamination, and
is therefore not recommended for use in the regulatory program.
IV-2
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