EPA 910/9-90-009
Puget Sound Estuary Program
EFFECTS OF SEDIMENT
HOLDING TIME ON
SEDIMENT TOXICITY
June 1990
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PTI Environmental Services
15375 SE 30th Place
Suite 250
Bellevue, Washington 98007
EFFECTS OF SEDIMENT HOLDING TIME
ON SEDIMENT TOXICITY
By
D. Scott Becker and Thomas C. Ginn
Prepared for
U.S. Environmental Protection Agency
Region 10, Office of Puget Sound
1200 Sixth Avenue
Seattle, Washington 98101
EPA Contract 68-D8-0085
PTI Contract C744-11
June 1990
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CONTENTS
Page
LIST OF FIGURES iii
LIST OF TABLES iv
LIST OF ACRONYMS v
EXECUTIVE SUMMARY 1
INTRODUCTION 3
BACKGROUND 3
STUDY OBJECTIVE 3
METHODS 5
FIELD COLLECTION 5
LABORATORY ANALYSIS 5
Sample Homogenization and Storage 5
Chemical Analyses 8
Bioassay Analyses 8
DATA ANALYSIS 12
RESULTS AND DISCUSSION 14
CHEMICAL ANALYSES 14
SEDIMENT BIOASSAYS 14
Amphipod Mortality Test 14
Neanthes Biomass Test 22
Microtox Test 26
Echinoderm Embryo Abnormality Test 30
REFERENCES 33
APPENDIX A - Detailed Results of Chemical Analyses and Bioassay Evaluations
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LIST OF FIGURES
Page
Figure 1. Location of sediment collection sites 6
Figure 2. Comparisons of mean percent mortality and holding time for the
amphipod mortality bioassay 16
Figure 3. Comparisons of mean percent total effective mortality and
holding time for the amphipod mortality bioassay 18
Figure 4. Comparisons of coefficient of variation and holding time for the
mortality and total effective mortality endpoints of the amphipod
mortality bioassay 19
Figure 5. Comparisons of LC^ values and holding time for the positive control
samples (reference toxicant = NaPCP) evaluated for the amphipod
mortality test 20
Figure 6. Comparisons of mean total biomass and holding time for the Neanthes
biomass test 23
Figure 7. Comparisons of mean average biomass and holding time for the
Neanthes biomass test 24
Figure 8. Comparisons of coefficient of variation and holding time for the
total and average biomass endpoints of the Neanthes biomass test 25
Figure 9. Comparisons of mean decrease in luminescence and holding time for
the Microtox bioassay 28
Figure 10. Comparisons of coefficient of variation and holding time for the
luminescence endpoint of the Microtox bioassay 29
Figure 11. Comparison of LC^ values and holding time for the positive control
samples (reference toxicant = phenol) evaluated for the Microtox bioassay 31
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LIST OF TABLES
Page
Table 1. Bioassay and chemical sample holding conditions 7
Table 2. Chemicals analyzed in test sediments 9
Table 3. Sediment holding times evaluated for the four sediment bioassays 13
Table 4. Chemical contaminants in Elliott Bay sediment exceeding 1988
bioassay AET values 15
Table 5. Comparisons of observed responses of the amphipod mortality test
between Stations CR and EB 21
Table 6. Comparisons of observed responses of the Neanthes bioassay between
Stations CR and EB 27
Table 7. Comparisons of observed responses of the Microtox bioassay between
Stations CR and EB 32
IV
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LIST OF ACRONYMS
ABN acid/base/neutral
AET apparent effects threshold
CLP Contract Laboratory Program
EPA U.S. Environmental Protection Agency
GC/ECD gas chromatography/electron capture detection
PAH polycyclic aromatic hydrocarbons
PCB polychlorinated biphenyl
PSDDA Puget Sound Dredged Disposal Analysis
PSEP Puget Sound Estuary Program
TOC total organic carbon
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EXECUTIVE SUMMARY
Four of the sediment bioassays commonly used to assess the toxicity of Puget Sound
sediments were used to evaluate the influence of sample holding time on the toxicity of
sediment samples collected from a highly contaminated site (i.e., Station EB) and a reference
area (Station CR) in the sound. Sediments were initially homogenized in the laboratory and
distributed for bioassay testing within 3-5 days after field collection. All subsequent holding
times were evaluated relative to the time elapsed from initial sample homogenization. The
initial holding time for each bioassay ranged from 1 to 2 weeks and was used as the basis
of comparison for all longer holding times (maximum = 16 weeks for all bioassays). Two
kinds of evaluations were made. In the first evaluation, the influence of holding time on the
absolute bioassay responses at each station was determined. In the second evaluation, the
influence of holding time on the relative differences of bioassay responses between the two
stations was determined. The four sediment bioassays evaluated included the following:
• 10-day amphipod mortality test
• 20-day Neanthes biomass test
• 15-minute Microtox test (saline extract)
• 48-hour echinoderm embryo abnormality test.
The results of the echinoderm embryo abnormality test were not evaluated because the
results for the initial sediment holding period did not satisfy quality assurance and quality
control specifications. Larval abnormality in the negative seawater control was 15.9 percent,
which exceeded the maximum allowable value of 10 percent.
The results of the 10-day amphipod mortality test for both Stations CR and EB suggest
that sediment holding times longer than 6 weeks may result in bioassay responses at
individual stations that are substantially different from those observed after a 2-week holding
time. The results for Station CR suggest that holding times of 5.5 and 6 weeks may also
influence sediment toxicity, compared to the results obtained after a 2-week holding time.
The differences observed among the various holding times were not substantially influenced
by changes in the sensitivity of the test organisms or changes in the variability of the
bioassay responses. Patterns based on between-station differences in the results of the
amphipod test suggest that holding times of 5.5 weeks or longer may influence the results
of such comparisons.
The results of the 20-day Neanthes biomass test suggest that sediment holding times
of 6 weeks or longer may result in bioassay responses at individual stations that are different
from those observed after a 1-week holding time. The differences observed among the
various holding times were not substantially affected by changes in the variability of the
bioassay responses. Biomass changes with increasing holding time were relatively small
compared with the differences observed between the two stations. Therefore, the observed
biomass differences between Stations CR and EB were relatively consistent among all
holding times (i.e., differences between Stations CR and EB were significant for all holding
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times). This consistency between stations was likely the result of the relatively high
sensitivity and precision of the Neanthes biomass test.
The results of the Microtox test suggest that sediment holding times of 4 weeks or longer
may result in bioassay responses at individual stations that are substantially different from
those observed after a 2-week holding time. The differences observed among the various
holding times were not substantially influenced by changes in the sensitivity of the test
organisms or variability of the bioassay responses. Patterns based on between-station
differences for various holding times exhibited a high degree of inconsistency and suggest
that holding times of 4 weeks or longer may influence the results of such comparisons.
In summary, the results of this study suggest that sediment holding time can influence
the results of at least three of the sediment bioassays commonly used to assess sediment
toxicity in Puget Sound.
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INTRODUCTION
BACKGROUND
In most studies of contaminated sediments in Puget Sound, sediment samples are
stored for various periods of time after field collection and prior to laboratory toxicity testing.
This storage period is termed holding time, and its influence on sediment toxicity is largely
unknown. To provide accurate estimates of the toxicity of field-collected sediments, it is
essential that toxicity not be substantially altered while samples are stored prior to laboratory
analysis. If the toxicity of the sediments changes during storage, erroneous conclusions
could be reached regarding the toxicity of those sediments in the environment.
At present, the Puget Sound Estuary Program (PSEP) recommends that sediment
holding time not exceed 2 weeks for sediments that are stored at 4°C. Sediments for most
of the bioassays conducted in Puget Sound are stored at that temperature. The PSEP
maximum holding time represents the consensus of regional experts (PSEP 1986a) and is
based largely on best professional judgment rather than conclusive empirical data.
To meet program-specific needs, Puget Sound Dredged Disposal Analysis (PSDDA) has
specified that sediments can be held at 4°C for as long as 6 weeks prior to bioassay
evaluations (PSDDA 1989). The PSDDA recommendation is based largely on the use of a
tiered toxicity evaluation approach, which calls for initial chemical analyses, and, if necessary
or desired, subsequent bioassay evaluations. The PSDDA maximum holding time of 6 weeks
is also not based on conclusive empirical data.
STUDY OBJECTIVE
The objective of the present study was to evaluate the effects of sediment holding time
on sediment toxicity, as estimated by four of the sediment bioassays commonly used in
Puget Sound. The evaluation was conducted using sediments from a highly contaminated
area and from a reference area to bracket the approximate range of sediment contamination
found in the sound. The initial holding time evaluated for each bioassay (i.e., 1-2 weeks) was
used as the basis for comparison with all longer holding times.
The relationship between sediment toxicity and sediment holding time was evaluated for
each bioassay by testing the following two null hypotheses:
• The mean response of each bioassay did not differ between the initial holding
time and each longer holding time
• The outcome of statistical comparisons of each bioassay response between the
contaminated and reference stations did not differ between the initial holding
time and each longer holding time.
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The first hypothesis addressed whether variable holding times influenced the absolute
response of each bioassay, and considered each station independently. The second
hypothesis evaluated whether variable holding times affected the relative responses of each
bioassay between the contaminated and reference stations.
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METHODS
FIELD COLLECTION
Sediment samples were collected at two stations in Puget Sound in May 1989 aboard
the RV Kittiwake (Figure 1). The two stations represented a contaminated area and a
reference area. Bioassay responses were evaluated independently for each station and were
also compared between the two stations.
Station CR was sampled at a depth of 50 meters in Carr Inlet on 5 May 1989 and was
used to represent a Puget Sound reference area. This station has been used previously by
PSDDA as a Puget Sound reference station (PTI 1988, 1989). Station EB was sampled at a
depth of 10 meters in Elliott Bay on 8 May 1989 and was used to represent a highly
contaminated area. This station is located off a major industrialized area on Harbor Island,
which has been sampled during several previous studies (Gamponia et. al. 1986; Seller et.
al. 1988; Johns 1988; Pastorok and Becker 1989).
At each station, approximately 20 liters of sediment was collected using a 0.1-m2 van
Veen bottom grab. After any overlying water was drained from each grab sample, the entire
sediment sample was transferred to a 20-liter plastic bucket. Sediments were later
homogenized in the laboratory. Samples were rejected if they were greatly disturbed or
winnowed during collection. After the required amount of sediment was collected at each
station, the 20-liter bucket was sealed tightly, transferred to the laboratory, and held at 4°C
in the dark.
LABORATORY ANALYSIS
Sample Homogenization and Storage
Sediments were homogenized on 10 May 1989. For each station, all of the field-
collected sediment was combined and thoroughly mixed in plastic buckets using plastic
utensils. Mixing was considered complete when the sediment exhibited no visible
heterogeneity in color or texture.
After the sediment from each station was thoroughly mixed, aliquots were taken at
random, distributed to containers, and stored for chemical, physical, and bioassay analyses
as described in Table 1. For each bioassay, sediments were stored in multiple containers,
so that a separate container could be opened for the evaluation of each holding time. Any
remaining sediment was then discarded. This procedure ensured that the results of each
evaluation were not affected by sample disturbance caused by an earlier evaluation.
After sediment homogenization, every effort was made to minimize sample
contamination. All subsequent chemical and bioassay analyses should therefore be
considered representative of the sediment samples at the time of homogenization, rather
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^
•'jf^OLYMPI*
Figure 1. Location of sediment collection sites
6
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TABLE 1. BIOASSAY AND CHEMICAL SAMPLE HOLDING CONDITIONS
Analysis
Bioassays
Semivolatile organic compounds
Metals
Grain size
Total organic carbon
Container*
Q
G
G
P
G
Preservative
4° C in nitrogen atmosphere6
Freeze
Freeze
4°C
Freeze
a G - chemically cleaned glass; P - plastic.
b Each jar was filled to within 1 cm of the top, and the remaining headspace was filled with nitrogen gas
before the jar was capped.
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than at the time of field collection. May 10 should therefore be considered the starting time
for all of the holding time experiments, and all references to sediment holding time in this
report relate to the time elapsed from this initial sediment homogenization. This starting time
was 5 days after sample collection at Station OR and 2 days after sample collection at
Station EB. This approach was similar to that used for the PSEP bioassay comparison study
(Pastorok and Becker 1989).
Chemical Analyses
Chemical analyses of sediments from Stations CR and EB were conducted for the metals
and organic compounds listed in Table 2. The particle-size distribution and total organic
carbon (TOC) content of the sediments were also evaluated.
Concentrations of organic compounds were determined using protocols modified from
those of the U.S. Environmental Protection Agency (EPA) Contract Laboratory Program (CLP)
(U.S. EPA 1986). The analyses of semivolatile compounds [including acid/base/neutral
(ABN) extractables, polychlorinated biphenyls (PCBs), and pesticides] followed modified EPA
CLP procedures that were consistent with the relatively low detection limits recommended
by PSEP (1986b). Separate sediment subsamples were used for ABN and pesticide/PCB
extraction. Ultrasonic extraction was conducted using CLP procedures. Gel permeation
chromatography was conducted for all ABN extracts to reduce interference and attain the
^recommended detection limits. Pesticide/PCB analyses were conducted using a modified
version of the EPA CLP procedure. These analyses included extract cleanup by alumina
column chromatography and, when necessary, elemental sulfur cleanup, followed by gas
chromatography/electron capture detection (GC/ECD) analysis. GC/ECD quantification and
confirmation analyses were conducted with fused silica capillary columns rather than the
packed columns commonly used in CLP procedures.
Concentrations of metals were determined by initial digestion of sediment samples using
the strong-acid technique specified in EPA CLP procedures (U.S. EPA 1986). Metals
concentrations in the digestates were then determined by graphite furnace atomic absorption
or by direct-flame atomic absorption spectrometry (except for mercury, which was
determined using cold vapor atomic absorption spectrometry).
Sediment particle-size distribution and TOC content were determined using the
procedures recommended by PSEP (1986c). Particle-size distribution was determined using
standard sieve and pipette techniques. TOC was determined by an elemental analyzer
following sample combustion.
Bioassay Analyses
Four of the sediment bioassays commonly used in Puget Sound were used to evaluate
sediment toxicity. They included the following tests:
• Amphipod mortality test
• Neanthes biomass test
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TABLE 2. CHEMICALS ANALYZED
IN TEST SEDIMENTS
MGtdlS
antimony copper nickel
arsenic lead silver
cadmium mercury zinc
Phenols and Substituted Phenols
phenol 2,4-dimethylphenol
2-methylphenol pentachlorophenol
4-methylphenol
Low Molecular Weight Pdycycfic Aromatic Hydrocarbons (LPAH)
naphthalene phenanthrene
acenaphthylene anthracene
acenaphthene 2-methylnaphthalene
fluorene
High Molecular Weight Pdycycfic Aromatic Hydrocarbons (HPAH)
fluoranthene benzo(a)pyrene
pyrene indeno(1,2,3-c,d)pyrene
benz(a)anthracene dibenzo(a,h)anthracene
chrysene benzo(g,h,i)perylene
benzofluoranthenes
Chlorinated Aromatic Hydrocarbons
1,2-dichlorobenzene 1,2,4-trichlorobenzene
1,3-dichlorobenzene hexachlorobenzene (HCB)
1,4-dichlorobenzene
Polycnkxinated Biphenyts
total PCB (mono- through decachlorobiphenyls)
Chlorinated Aliphatic Hydrocarbons
hexachlorobutadiene hexachloroethane
Phthalate Esters
dimethyl phthalate butyl benzyl phthalate
diethyl phthalate bis(2-ethylhexyO phthalate
di-n-butyl phthalate di-n-octyl phthalate
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TABLE 2. (Continued)
Miscellaneous Oxygenated Compounds
benzyl alcohol benzole acid
dibenzofuran
Organonfrogen Compounds
N-nitrosodiphenylamine
Pesticides
total DDT (p,p') aldrin
heptachlor dieldrin
o-chlordane y-HCH (lindane)
10
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• Microtox test (saline extract)
• Echinoderm embryo abnormality test.
The amphipod mortality test evaluated mortality of adult amphipods (Rhepoxynius
abronius) following a 10-day exposure to bedded test sediments. The primary endpoints
were percent mortality and percent total effective mortality. The latter endpoint was
represented by the number of amphipods that died combined with the number of survivors
that failed to rebury in clean sediment after the 10-day exposure period. It was assumed that
failure to rebury represented effective mortality, as the affected individuals would have been
rapidly consumed by predators in the environment. The methods for this test are described
by Swartz et al. (1985) and PSEP (1986a). Five replicate laboratory analyses were conducted
for each field sample. The sensitivity of the test organisms was evaluated using sodium
pentachlorophenate (NaPCP) as the reference toxicant for positive control samples.
The Neanthes biomass test evaluated growth of juvenile polychaetes (Neanthes
arenaceodentata) following a 20-day exposure to bedded test sediments. The primary
endpoints were total and average biomass. Total biomass represented the pooled dry weight
of surviving individuals, and thereby incorporated mortality. Average biomass represented
the mean dry weight of individual survivors and did not incorporate mortality. The methods
for this test are described in Johns et al. (1989). Five replicate laboratory analyses were
conducted for each field sample. Positive controls were not analyzed for all holding times
because this test was in the developmental stage and a separate experiment was conducted
to develop appropriate positive control conditions. Therefore, potential differences among
the various holding times in the sensitivity of the test organisms could not be evaluated for
this test.
The Microtox test evaluated luminescence of bioluminescent bacteria (Photobacterium
phosphoreum) following a 15-minute exposure to a saline sediment extract. The primary
endpoint was percent decrease in luminescence, which represented changes in cellular
metabolic state. The methods for this test are described by Beckman Instruments (1982),
PSEP (1986a), and Williams et al. (1986). In the present study, two kinds of analyses were
conducted for the Microtox test. In the first analysis, samples were evaluated using the
dilution series recommended by Williams et al. (1986). In the second analysis, evaluations
were made using four replicate samples of the highest sample dilution used in the first
analysis (i.e., the 50 percent dilution). The second analysis was implemented for sediment
holding times longer than 2 weeks when it was found that the test organisms were respond-
ing weakly to extracts from Station EB, and the maximum extract dilution resulted in only a
15-16 percent reduction in luminescence. It was therefore not possible to calculate EC^
values for statistical comparisons. The second analysis was conducted at the same time and
using the same sample extract as the first analysis. For the 2-week holding period, the
results based on the 50 percent dilution (n=2) for the first analysis were used as the basis
for comparison with the results of the second analysis for all longer holding times. For each
sediment holding time, the sensitivity of the test organisms was determined using phenol as
the reference toxicant for positive control samples.
The echinoderm embryo abnormality test evaluated mortality and abnormality in larval
sand dollars (Dendraster excentricus) following a 48-hour exposure to bedded test sediment.
The primary endpoints were percent mortality and percent abnormality. Larval abnormality
was defined as failure to develop to the normal pluteus stage after the 48-hour exposure
11
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period. The methods for this test are described by Dinnel and Stober (1985). Five replicate
laboratory analyses were conducted for each field sample. For each holding time, the
sensitivity of the test organisms was determined using sodium dodecyl sulfate as the
reference toxicant for positive control samples.
The sediment holding times evaluated for each of the four bioassays are presented in
Table 3. For each test, the maximum holding time recommended by PSDDA (i.e., 6 weeks)
was evaluated. The maximum holding time recommended by PSEP (i.e., 2 weeks) was
evaluated for all of the bioassays except the Neanthes biomass test. That bioassay was
evaluated after a holding time of 1 week, which is within the PSEP guidelines and therefore
is considered appropriate as the basis for evaluating the longer holding times.
DATA ANALYSIS
To test the first null hypothesis regarding the influence of variable sediment holding time
on the absolute response of each bioassay, the mean response observed for each initial
holding time was compared with the mean response for each longer holding time. Pairwise
comparisons were made between the results for the initial holding time and each additional
holding time using the Student's Mest and a comparisonwise, two-tailed error rate of 0.05.
Corrections to the error rate for multiple comparisons were not made because each pairwise
comparison was considered a test of an independent null hypothesis. Before each Mest was
made, heterogeneity of variances was tested using the F^ test (Sokal and Rohlf 1981). If
heterogeneous variances were found, the pairwise comparison was made using the approxi-
mate Mest (Sokal and Rohlf 1981). For the Microtox test, pairwise comparisons were made
using replicated data (n=2 for the 2-week holding time, n=4 for holding times longer than
2 weeks) for the maximum sample dilutions. Statistical comparisons were not made using
the information on dilution series because EC^ values could not be calculated. For some
samples, results of all replicates of the Microtox test were zero percent, so no standard
deviation could be determined and a Mest could not be conducted. In such cases,
comparisons between samples were made using the nonparametric Mann-Whitney LMest.
To test the second null hypothesis regarding the influence of variable sediment holding
time on the relative responses of each bioassay between Stations CR and EB, the mean
responses observed at the two stations for each holding time were compared using the same
statistical techniques described for testing the first null hypothesis. The results of the
pairwise comparisons were then examined to determine if the statistical outcome of between-
station comparisons varied as a result of different holding times.
The variability of the responses of each bioassay for the various holding times was
evaluated to determine whether response variability was affected by sediment holding time
to the extent that it could influence the statistical comparisons. Response variability was
estimated using the coefficient of variation [i.e., [(standard deviation + mean) x 100].
Potential variability among the various holding times in the sensitivity of the test
organisms was evaluated by examining the LCX values observed for the positive control
samples.
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TABLE 3. SEDIMENT HOLDING TIMES EVALUATED
FOR THE FOUR SEDIMENT BIOASSAYS
Bioassay
Holding Times
(Weeks from Initial Sediment Homogenization)8
Amphipod mortality test
Neanthes biomass test
Microtox test
Echinoderm embryo abnormality test
2.0, 5.5, 6.0, 11.0, 12.5, 16.0
1.0, 6.0, 11.0, 16.0
2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0
2.0, 6.0, 11.0, 16.0
8 Initial sediment homogenization occurred on 10 May 1989. This date was 5 days after sample
collection at Station CR and 2 days after sample collection at Station EB.
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RESULTS AND DISCUSSION
CHEMICAL ANALYSES
Results of all chemical analyses are presented in Appendix A (Table A-1). In this
section, chemical concentrations are discussed relative to the 1988 Puget Sound apparent
effects threshold (AET) values for bioassays (Barrick et al. 1988). These AET values are
based on three sediment bioassays [i.e., the amphipod mortality, oyster larvae abnormality,
and Microtox (saline extract) tests]. AET values provide an estimate of the concentration of
each chemical above which adverse biological effects are always predicted in Puget Sound.
Sediment from Station CR was relatively fine-grained (i.e., 52.8 percent fine-grained
material), with a moderate level of TOO content (i.e., 1.3 percent). Sediment from Station EB
was coarser than sediment from Station CR (i.e., 47.2 percent fine-grained sediment), but
had a higher TOC content (i.e., 2.1 percent).
Sediment from Station CR was relatively uncontaminated. No metal or organic
compound exceeded any of its 1988 Puget Sound bioassay AET values. Although several
polycyclic aromatic hydrocarbons (PAH) were detected (i.e., phenanthrene, anthracene, and
chrysene), concentrations of these compounds were all less than 100 parts per billion (ppb).
Sediment from Station EB was highly contaminated with both metals and organic compounds
(Table 4). Four metals exceeded at least one 1988 Puget Sound bioassay AET value, and
copper and mercury exceeded all three bioassay AET values. Nineteen organic compounds
exceeded at least one bioassay AET value, and six of these compounds exceeded all three
values. The six organic compounds exceeding all three bioassay AET values included four
PAH compounds [i.e., benzofluoranthenes, benzo(a)pyrene, dibenzo(a,h)anthracene, and
benzo(g,h,i)perylene] and two phenols (2-methylphenol and pentachlorophenol).
SEDIMENT BIOASSAYS
Detailed results of all four sediment bioassays are presented in Appendix A (Tables A-2
to A-8). This section summarizes and discusses those results relative to the various
sediment holding times. The results for each kind of bioassay are discussed separately.
Amphipod Mortality Test
Wrthin-Station Comparisons—For Station CR, 10-day amphipod mortality for the 2-week
sediment holding time was 3 percent (Figure 2). Mortality then increased to 12 percent after
a 5.5-week holding time, but was not significantly different (P>0.05) from the value observed
for the 2-week holding time. The elevated mortality and relatively high variability observed
for the 5.5-week holding time was due primarily to a single replicate exhibiting a mortality of
40 percent, whereas mortality in the remaining four replicates ranged from 0 to 10 percent.
After a 6-week holding time, mortality was 11 percent and was significantly different (PsO.05)
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TABLE 4. CHEMICAL CONTAMINANTS IN ELLIOTT BAY
SEDIMENT EXCEEDING 1988 BIOASSAY AET VALUES
Chemical
Concentration
at Station EB*
AET
Exceedancesb
Metals
Arsenic
Copper
Mercury
Zinc
Organic Compounds
Low molecular weight polycylic
aromatic hydrocarbons (LPAH)
112
1,490
3.5
1,010
A
A,M,O
A.M.O
A
Total LPAH
Acenaphthene
Fluorene
Phenanthrene
Anthracene
High molecular weight polycyclic
aromatic hydrocarbons (HPAH)
Total HPAH
Fluoranthene
Pyrene
Benz(a)anthracene
Chrysene
Benzofluoranthenes
Benzo(a)pyrene
lndeno(1 ,2,3-c,d)pyrene
Dibenzo(a,h)anthracene
Ben2o(g,h,i)perylene
Phthalates
Dimethyl phthalate
Butyl benzyl phthalate
Bis(2-ethylhexyOphthalate
Phenols
2-methylphenol
Pentachlorophenol
Total PCBs
9,400
780
790
4,800
1,900
52,000
8,100
12,000
4,000
3,300
10,000
8,900
1,600
710
3,600
110
320
6,100
78
1,900
1,460
A,M,O
M,O
M,O
M,O
M.O
A.M.O
M,O
M.O
M,O
M,O
A.M.O
A.M.O
M,O
A.M.O
A.M.O
M
M
M,O
A.M.O
A.M.O
M.O
* Metals concentrations are reported in mg/kg dry weight. Concentrations of organic
compounds are reported in |ig/kg dry weight.
b A - amphipod mortality test
M - Microtox test (saline extract)
O - oyster larvae abnormality.
15
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100
80
60-
40-
Percent Mortality
20
Station CR
2.0 5.5 6.0 11.0 12.5 16.0
Weeks From Initial Sample Homogenization
100
Percent Mortality
80-
60
40
20
Station EB
2.0 5.5 6.0 11.0 12.5 16.0
Weeks From Initial Sample Homogenization
* Significantly different (P<0.05) from the value observed for 2.0 weeks.
Figure 2. Comparisons of mean percent mortality and holding time for the amphipod mortality
bioassay (bars represent standard deviations)
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from the value observed for the 2-week holding time. Unlike the results for the 5.5-week
holding time, the results for the 6-week holding time exhibited relatively low variability, as
mortality in all five replicates ranged from 10 to 15 percent. For holding times of 11, 12.5,
and 16 weeks, mortality increased to 16 and 32 percent, and then declined to 19 percent.
All three of these values were significantly different (PsO.05) from the value observed for the
2-week holding time.
For Station EB, amphipod mortality for the 2-week sediment holding time was 14 percent
(Figure 2). Mortality remained relatively constant at 13 and 14 percent for holding times of
5.5 and 6 weeks, respectively. Both of these values were not significantly different (P>0.05)
from the value observed for the 2-week holding time. Mortality peaked at 54 percent for the
11-week holding time, and then declined to 40 and 37 percent for holding times of 12.5 and
16 weeks. The values observed for holding times of 11 and 12.5 weeks were significantly
different (PsO.05) from the value observed for the 2-week holding time, whereas the value
observed for the 16-week holding time was not significantly different (P>0.05) from the value
observed for the 2-week holding time.
For both Stations CR and EB, total effective mortality of amphipods exhibited patterns
identical to those described for mortality (Figure 3).
Although the coefficients of variation differed among the various sediment holding times
for both bioassay endpoints at both Stations CR and EB (Figure 4), there did not appear to
be a consistent relationship between response variability and holding time.
The results of the positive controls for the various sediment holding times are presented
in Figure 5. As indicated by the observed LC^ values, the sensitivity of the test organisms
was relatively consistent for all holding times except 12.5 weeks. Organism sensitivity
appeared to be considerably lower for the 12.5-week holding time. However, this apparent
reduced sensitivity did not prevent the observed bioassay response from being among the
highest observed during the study. This pattern suggests that variations in organism
sensitivity did not substantially influence the differences in bioassay responses observed
among the various holding times.
Between-Station Comparisons—The results of comparisons of mortality and total
effective mortality between Stations CR and EB for each sediment holding time are presented
in Table 5. For both endpoints, differences between the two stations were significant
(PsO.05) for holding times of 2 and 11 weeks and were not significant (P>0.05) for holding
times of 5.5, 6, 12.5, and 16 weeks.
Summary—The results of the amphipod mortality test suggest that sediment holding
times longer than 6 weeks may result in bioassay responses that are substantially different
from those observed after a 2-week holding time. For both Stations CR and EB, most
bioassay responses for holding times greater than 6 weeks were significantly different
(PsO.05) from the responses observed after the 2-week holding time. Patterns based on
between-station differences for holding times longer than 6 weeks were not as consistent as
absolute bioassay responses. For both bioassay endpoints, between-station differences
were significant (PiO.05) for the 2-week holding time, but not significant (P>0.05) for two of
the three holding times greater than 6 weeks.
17
-------
100
80
60 H
Percent Total Effective Mortality
Station CR
2.0 5.5 6.0 11.0 12.5 16.0
Weeks From Initial Sample Homogenization
100
80
60
40
20
Percent Total Effective Mortality
Station EB
2.0 5.5 6.0 11.0 12.5 16.0
Weeks From Initial Sample Homogenization
* Significantly different (P<0.05) from the value observed for 2.0 weeks.
Figure 3. Comparisons of mean percent total effective mortality and holding time for the
amphipod mortality bioassay (bars represent standard deviations)
18
-------
180
Coefficient of Variation (%)
140-
120-
100-
80-
60
40
20-
2.0 6.5 6.0 11.0 12.6 16.0
Weeks From Initial Sample Homogenization
160
Coefficient of Variation (%)
140-
120-
100-
80
60-
40-
20-
2.0 6.6 6.0 11.0 12.6 16.0
Weeks From Initial Sample Homogenization
Station CR
Station EB
Figure 4. Comparisons of coefficient of variation and holding time for the mortality (above)
and total effective mortality (below) endpoints of the amphipod mortality bioassay
19
-------
1200
1000
800
600
400
200
Concentration (ppb v/v)
2.0 5.5 6.0 11.0 12.5 16.0
Weeks From initial Sample Homogenization
Figure 5. Comparison of LC^ values and holding time for the positive control samples
(reference toxicant = NaPCP) evaluated for the amphipod mortality test
(bars represent 95 percent confidence limits)
20
-------
TABLE 5. COMPARISONS OF OBSERVED RESPONSES OF THE
AMPHIPOD MORTALITY TEST BETWEEN STATIONS CR and EB"
Difference Between Stations CR and EBC
Date6
May 24
June 16
July 20
June 25
August 5
August 30
Weeks from
Initial Sediment Homogenization
2.0
5.5
6.0
11.0
12.5
16.0
Percent
Mortality
11*
1 ns
4 ns
38*
8 ns
18ns
Percent Total
Effective Mortality
17*
0 ns
5 ns
40*
7 ns
20ns
* Comparisons were made using a f-test.
b Date bioassay
0 * - PsO.05
ns - P>0.05.
was initiated. All tests were conducted
in 1989.
21
-------
For sediment holding times of 5.5 and 6 weeks, the absolute bioassay responses for
Station EB differed little from those found after the 2-week holding time. By contrast, the 5.5-
and 6-week responses for Station CR increased by approximately 10 percent above the
relatively low mortality found for the 2-week holding time. However, only the responses
observed for the 6-week holding time were significantly different (P*0.05) from the value
observed for the 2-week holding time.
Patterns based on between-station differences for sediment holding times of 5.5 and
6 weeks were consistently different from the results found for the 2-week holding time. For
both endpoints, differences between stations were significant (PsO.05) for the 2-week holding
time, but not significant (P>0.05) for holding times of 5.5 and 6 weeks. However, it should
be noted that the difference between stations observed for the mortality endpoint after the
2-week holding time (i.e., 11 percent) was relatively small.
Neanthes Biomass Test
Within-Station Comparisons—Neither initial total biomass nor initial average biomass
exhibited significant differences (P>0.05; analysis of variance) among the four sediment
holding times evaluated for the Neanthes test. Comparisons of final biomass values among
the different holding times were therefore not biased by different initial biomass values.
After the 20-day exposure period, total biomass for Station CR for the 1-week sediment
holding time was 79.9 mg (Figure 6). Total biomass peaked at a value of 112.5 mg for the
6-week holding time, and then steadily declined to 105.8 and 67.8 mg for holding times of
11 and 16 weeks. Only the values observed for holding times of 6 and 11 weeks were
significantly different (PsO.05) from the value observed for the 1-week holding time.
For Station EB, total biomass exhibited a pattern similar to that found for Station CR.
Total biomass for the 1-week sediment holding time was 17.7 mg (Figure 6). Total biomass
peaked at a value of 18.1 mg for the 6-week holding time, and then steadily declined to 11.1
and 4.5 mg for holding times of 11 and 16 weeks. Only the value observed for the 16-week
holding time was significantly different (P*0.05) from the value observed for the 1-week
holding time.
For both Stations CR and EB, average biomass exhibited patterns identical to those
described for total biomass (Figure 7). This similarity between endpoints was partly the result
of the relatively low mortality observed for most sediment holding times. For Station CR,
mortality values for holding times of 1, 6, 11, and 16 weeks were 4, 4, 0, and 0 percent,
respectively. For Station EB, mortality values for the four holding times were 0, 24, 8, and
16 percent, respectively.
The coefficients of variation exhibited relatively small differences among the various
sediment holding times for both bioassay endpoints at both Stations CR and EB (Figure 8).
However, a general negative relationship between response variability and holding time was
evident.
22
-------
175
Total Biomass (mg dry weight)
150 H
125
100
75-
50-
25
0 5 10 15 20
Weeks From Initial Sample Homogenization
Station CR
Station EB
* Significantly different (P<0.05) from the value observed for 1.0 week.
Figure 6. Comparisons of mean total biomass and holding time for the Neanthes
biomasstest (bars represent standard deviations)
23
-------
35
Average Biomass (mg dry weight)
30-
25-
20-
15-
10
0 5 10 15 20
Weeks From initial Sample Homogenization
Station CR
Station EB
* Significantly different (P<0.05) from the value observed for 1.0 week.
Figure 7. Comparisons of mean average biomass and holding time for the Neanthes
biomasstest (bars represent standard deviations)
24
-------
60
Coefficient of Variation (%)
50-
40
30-
20
10
i e 11 16
Weeks From Initial Sample Homogenlzation
60
Coefficient of Variation (%)
50-
40
30-
20-
10-
1 6 11 16
Weeks From Initial Sample Homogenization
Station CR
Station EB
Figure 8. Comparisons of coefficient of variation and holding time for the mortality (above)
and average (below) biomass endpoints of the Neanthes biomass test
25
-------
Between-Station Comparisons—The results of comparisons of total and average
biomass between Stations CR and EB for each sediment holding time are presented in
Table 6. For both endpoints, differences between the two stations were significant (PiO.05)
for all holding times.
Summary—The results of the 20-day Neanthes biomass test suggest that sediment
holding times of 6 weeks or longer may result in bioassay responses at individual stations
that are different from those observed after a 1 -week holding time. For Station CR, Neanthes
biomass for holding times of 6 and 11 weeks was significantly different (PiO.05) from the
biomass observed after the 1-week holding time. For Station EB, Neanthes biomass for the
16-week holding time was significantly different (PiO.05) from the biomass observed for the
1-week holding time.
Patterns based on between-station differences in Neanthes biomass were consistent for
all of the sediment holding times evaluated. In all cases, differences between stations were
significant (PsO.05). This consistency is likely the result of both the high sensitivity (i.e., large
differences between responses for Stations CR and EB) and the precision (i.e., relatively low
standard deviations) of the test. These results suggest that although absolute bioassay
responses may vary with holding times, between-station differences may not be affected if
the magnitude of bioassay responses at the test site is considerably higher than the magni-
tude of responses found at the reference site. However, if response magnitudes do not differ
substantially between test and reference sites (as was the case for the amphipod mortality
test), variability of absolute responses as a result of different holding times could influence
the statistical significance of between-station differences in sediment toxicity.
Microtox Test
Wrthin-Station Comparisons—After the 15-minute exposure period, decrease in lumines-
cence for Station CR for the 2-week sediment holding time was 9.3 percent (Figure 9). The
bioassay response declined to 0.7 percent for the 4-week holding time and then increased
steadily to 10.7, 12.8, and 13.6 percent for holding times of 6, 8, and 10 weeks. The
response then declined to 1.5, 0, and 0 percent for holding times of 12,14, and 16 weeks.
The responses for holding times of 4 weeks and 10-16 weeks were significantly different
(P*0.05) from the response observed for the 2-week holding period.
For Station EB, decrease in luminescence for the 2-week sediment holding time was
15.9 percent (Figure 9). The bioassay response then exhibited a somewhat erratic pattern.
Relatively high values of 30.1 and 42.8 percent were found for holding times of 4 and 10
weeks, respectively, whereas moderate values of 12.1,16.0, and 13.4 percent were found for
holding times of 6, 8, and 12 weeks, respectively. Finally, low values of 0 and 0.8 percent
were found for holding times of 14 and 16 weeks, respectively. The values observed for
holding times of 4-6 weeks and 10-16 weeks were significantly different (PsO.05) from the
value observed for the 2-week holding time.
Although the coefficients of variation differed among the various sediment holding times
for both Stations CR and EB (Figure 10), there did not appear to be a consistent relationship
between response variability and holding time.
26
-------
TABLE 6. COMPARISONS OF OBSERVED RESPONSES OF
THE NEANTHES BIOASSAY BETWEEN STATIONS CR AND EB*
Date*
May 18
June 23
July 28
August 29
Weeks from
Initial Sediment Homogenization
1.0
6.0
11.0
16.0
Difference Between
Total Biomass
(mg dry weight)
62.2*
94.4*
94.7*
63.3*
Stations CR and EB°
Average Biomass
(mg dry weight)
13.2*
18.4*
18.7*
15.5*
* Comparisons were made using a f-test.
b Date bioassay was initiated. All tests were conducted in 1989.
c * - P*0.05.
27
-------
50
Percent Luminescence Decrease
40
30
20
10-
Station CR
2 4 6 8 10 12 14 16
Weeks From Initial Sample Homogenization
Percent Luminescence Decrease
2 4 6 8 10 12 14 16
Weeks From Initial Sample Homogenization
r Significantly different (P<0.05) from the value observed for 2.0 weeks.
Figure 9. Comparisons of mean decrease in luminescence and holding time for the
Neanthes biomass test (bars represent standard deviations)
28
-------
140
Coefficient of Variation (%)
40
20-
2 4 6 8 10 12 14 16
Weeks From Initial Sample Homogenization
Station CR
Station EB
Note: Coefficients of variation could not be determined
for weeks 14 and 16 at Station CRand for week 14 at
Station EB because the standard deviation was zero.
Figure 10. Comparisons of coefficient of variation and holding time for the luminescence
endpoint of the Microtox bioassay
29
-------
The results of the positive controls for the various sediment holding times are presented
in Figure 11. As indicated by the observed LC^ values, the sensitivity of the test organisms
was relatively consistent for all holding times. This pattern suggests that variations in
organism sensitivity did not substantially influence the differences in bioassay responses
observed among the various holding times.
Between-Station Comparisons—Results of comparisons of decrease in luminescence
between Stations CR and EB for each sediment holding time are presented in Table 7.
Differences between the two stations were significant (P*0.05) for holding times of 4,10,12,
and 16 weeks, and were not significant (P>0.05) for holding times of 2, 6, 8, and 14 weeks.
Summary—The results of the Microtox test suggest that sediment holding times of
4 weeks or longer may result in bioassay responses that are substantially different from those
observed after a 2-week holding time. For both Stations CR and EB, bioassay responses for
most (i.e., 11 of 14 cases) holding times of 4-12 weeks were significantly different (PsO.05)
from the response observed for the 2-week holding time.
Patterns based on between-station differences for various sediment holding times exhi-
bited a high degree of inconsistency. Differences between Stations CR and EB were not sig-
nificant (P>0.05) for the 2-week holding time. By contrast, differences between stations were
significant (PsO.05) for holding times of 4,10,12, and 16 weeks. These results suggest that
holding times of *4 weeks may influence between-station differences in sediment toxicity.
Echinoderm Embryo Abnormality Test
The results of the echinoderm embryo abnormality test were not considered appropriate
for statistical analysis. Larval abnormality in the negative seawater control for the 2-week
sediment holding time was 15.9 percent, which exceeded the maximum allowable level of
10 percent. Therefore, results for that holding time could not be considered reliable.
Because the 2-week holding time was the basis of comparison for all longer holding times,
quantitative evaluations of the longer holding times could not be made.
A qualitative evaluation of the results of the echinoderm embryo abnormality test showed
that embryo mortality for Station CR was considerably higher after a 6-week sediment holding
time (76.0 percent) than the value observed for the 2-week holding time (18.8 percent; Table
A-8). By contrast, abnormality for Station CR was similar between holding times of 2 weeks
(14.1 percent) and 6 weeks (10.3 percent).
For Station EB, embryo mortality was at or close to 100 percent for all sediment holding
times evaluated (i.e., 2-16 weeks). Because of the low number of surviving embryos at
Station EB, the abnormality endpoint could only be evaluated for the 16-week holding time.
Between-station differences could only be evaluated for the mortality endpoint because
the number of surviving embryos was too low to estimate percent abnormality at Station EB
during the 2- and 6-week sediment holding times. For the 2-week holding time, embryo mor-
tality at Station EB exceeded the value at Station CR by 81.2 percent. For the 6-week hold-
ing time, mortality at Station EB exceeded the value observed at Station CR by 20.8 percent.
30
-------
30
Concentration (ppm v/v)
25-
20
15
10
2 4 6 8 10 12 14 16
Weeks From Initial Sample Homogenization
Figure 11. Comparison of LC^ values and holding time for the positive control samples
(reference toxicant = phenol) evaluated for the Microtox bioassay
31
-------
TABLE 7. COMPARISONS OF OBSERVED RESPONSES OF
THE MICROTOX BIOASSAY BETWEEN STATIONS CR AND EB*
Dateb
May 26
June 8
June 21
July 6
July 20
August 3
August 18
August 31
Weeks from
Initial Sediment Homogenization
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
Percent Decrease
in Luminescence0
6.6 ns
29.4*
1.4 ns
3.2ns
29.2*
11.9*
0 ns
0.8*
8 Comparisons were made using a f-test.
b Date bioassay was initiated. All tests were conducted in 1989.
0 * - PsO.05
ns - P>0.05.
32
-------
REFERENCES
Barrick, R.B., S. Becker, L Brown, H. Seller, and R.A. Pastorok. 1988. Sediment quality
values refinement: 1988 update and evaluation of Puget Sound AET. Volume 1. Final
Report. Prepared for Tetra Tech, Inc., Bellevue, WA, and U.S. Environmental Protection
Agency Region 10, Office of Puget Sound, Seattle, WA. PTI Environmental Services,
Bellevue, WA. 74 pp. + appendices.
Beckman Instruments. 1982. Microtox system operating manual. Beckman Publication No.
015-555879. Beckman Instruments, Inc., Carlsbad, CA.
Seller, H.R., R.A. Pastorok, D.S. Becker, G. Braun, Q. Bilyard, and P. Chapman. 1988. Elliott
Bay Action Program: analysis of toxic problem areas. Final Report. Prepared for U.S.
Environmental Protection Agency Region 10, Office of Puget Sound, Seattle, WA. Tetra
Tech, Inc., Bellevue, WA, and PTI Environmental Services, Bellevue, WA.
Dinnel, P.A., and Q.J. Stober. 1985. Methodology and analysis of sea urchin embryo
bioassays. Circular No. 85-3. University of Washington, Fisheries Research Institute, Seattle,
WA. 19pp.
Gamponia, V., T. Hubbard, P. Romberg, T. Sample, and R. Swartz. 1986. Identifying hot
spots in the lower Duwamish River using sediment chemistry and distribution patterns.
Municipality of Metropolitan Seattle, Seattle, WA.
Johns, D.M. 1988. Puget Sound dredged disposal analysis sublethal test demonstration.
Prepared for U.S. Army Corps of Engineers, Seattle District. PTI Environmental Services,
Bellevue, WA. 94 pp. + appendix.
Johns, D.M., T.C. Ginn, and D.J. Reish. 1989. Interim protocol for juvenile Neanthes
bioassay. Prepared for U.S. Environmental Protection Agency Region 10, Office of Puget
Sound, Seattle, WA. PTI Environmental Services, Bellevue, WA.
Pastorok, R.A., and D.S. Becker. 1989. Comparison of bioassays for assessing toxicity in
Puget Sound. Prepared for U.S. Environmental Protection Agency Region 10, Office of Puget
Sound, Seattle, WA. PTI Environmental Services, Bellevue, WA. 85 pp. + appendices.
PSDDA. 1989. Management plan report—unconfined open-water disposal of dredged
material, Phase II (north and south Puget Sound). Draft Report. Puget Sound Dredged
Disposal Analysis, Seattle, WA.
PSEP. 1986a. Recommended protocols for conducting laboratory bioassays on Puget
Sound sediments. Final Report. Prepared for U.S. Environmental Protection Agency. Tetra
Tech, Inc., Bellevue, WA, and E.V.S. Consultants Ltd., Bellevue, WA. 55 pp.
33
-------
PSEP. 1986b. Recommended protocols for measuring organic compounds in Puget Sound
sediments and tissue samples. Prepared for U.S. Environmental Protection Agency. Tetra
Tech, Inc., Bellevue, WA. 65 pp. + appendices.
PSEP. 1986C. Recommended protocols for measuring sediment conventional variables in
Puget Sound. Final Report. Prepared for U.S. Environmental Protection Agency Region 10,
Office of Puget Sound. Tetra Tech, Inc., Bellevue, WA. 46 pp.
PTI. 1988. Baseline survey of phase I disposal sites. Prepared for Washington Department
of Ecology, Olympia, WA. PTI Environmental Services, Bellevue, WA.
PTI. 1989. Baseline survey of phase II disposal sites. Prepared for Washington Department
of Ecology, Olympia, WA. PTI Environmental Services, Bellevue, WA.
Sokal, R.R., and F.J. Rohlf. 1981. Biometry. 2nd ed. W.H. Freeman and Co., San
Francisco, CA. 859 pp.
Swartz, R.C., W.A. DeBen, J.K. Phillips, J.O. Lamberson, and F.A. Cole. 1985.
Phoxocephalid amphipod bioassay for marine sediment toxicity. pp. 284-307. In: Aquatic
Toxicology and Hazard Assessment: Seventh Symposium. R.D. Cardwell, R. Purdy, and R.
Bahner (eds). ASTM STP 854. American Society for Testing and Materials, Philadelphia, PA.
U.S. EPA. 1986. Test methods for evaluating solid waste. U.S. Environmental Protection
Agency, Office of Solid Waste and Emergency Response.
Williams, L.G., P.M. Chapman, and T.C. Ginn. 1986. A comparative evaluation of sediment
toxicity using bacterial luminescence, oyster embryo, and amphipod sediment bioassays.
Mar. Environ. Res. 19:225-249.
34
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APPENDIX A
DETAILED RESULTS OF CHEMICAL ANALYSES
AND BIOASSAY EVALUATIONS
-------
TABLE A-1. CONCENTRATIONS OF CHEMICALS OF CONCERN
IN CARR INLET AND ELLIOTT BAY SEDIMENTS
Compound
METALS (mg/kg dry weight; ppm)
Antimony
Arsenic
Cadmium
Copper
Lead
Mercury
Nickel
Silver
Zinc
ORGANICS (ug/kg dry weight; ppb)
1 d^u ll«->l«->fM Aftm- llfiiJiaJ J n«-»fc ««-• «<-Jmr>
LOW Molecular weight Porycycnc
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
2-Methylnaphthalene
High Molecular Weight Porycydc
Fluoranthene
Pyrene
Benz (a) anthracene
Chrysene
Benzofluoranthenes
Benzo(a)pyrene
lndeno(1 ,2,3,-c,d)pyrene
Dibenzo(a,h)anthracene
Benzo(g,h,0perylene
Chlorinated Hydrocarbons
1 ,3-Dichlorobenzene
1 ,4-Dichlorobenzene
1 ,2-Dichlorobenzene
Hexachlorobenzene
Carr Inlet
(Station CR)a
1.4G
18.1
0.8E
62.3
37.5
0.14
36.7
0.39E
111
Aromatic nyarocaroons (LPAH)
14U
14U
14U
14U
100
38
14U
Aromatic Hydrocarbons (HPAH)
170E
170E
83E
100
150E
90E
50E
14U
56E
14U
14U
14U
14U
Elliott Bay
(Station EB)a
10.3G
112
1.4E
1,490
384
3.5
50.5
1.2E
1,010
390
440
780
790
4,800
1,900
260
8,100
12,000
4.000E
3,300
10,000
8,900
1,600
910
3,600
13U
14U
13U
13U
A-1
-------
TABLE A-1. (Continued)
Carr Inlet Elliott Bay
Compound (Station CR)* (Station EB)a
Phthalates
Dimethyl phthalate 14U 110
Diethyl phthalate 14U 13U
Di-n-butyl phthalate 14U 140
Butyl benzyl phthalate 14U 320E
Bis(2-ethylhexyl)phthalate 64 6100
Di-n-octyl phthalate 14U 130U
Polychlorinated Biphenyts
Total PCBs 8.2K 1460
Phenols
Phenol 27U 170
2-Methylphenol 14U 78E
4-Methylphend 14U 180E
2,4-Dimethylphenol 33U 31U
Pentachlorophenol 22U 1,900
Misceflaneous Extractabtes
Benzyl alcohol 68U 64U
Benzoic acid 140U 128U
Dibenzofuran 14U 110
Hexachloroethane 41U 39U
Hexachlorobutadiene 14U 13U
N-Nitrosodiphenylamine 14U 13U
Pesticides
Total DDT
Aldrin
Chlordane
Dieldrin
Heptachlor
LJndane
2U
1U
1.5U
2U
1U
1U
34
1U
1.5U
2U
1U
1U
a Qualifier codes used:
U - Undetected at detection limit shown
E - Estimate
G - Estimate is greater than value shown
K - Detected at less than detection limit shown.
A-2
-------
TABLE A-2. RESULTS OF THE AMPHIPOD MORTALITY
BIOASSAY FOR THE MORTALITY ENDPOINT
Weeks from
Dateb Start Date
May 24 2.0
June 16 5.5
June 20 6.0
July 25 11.0
August 5 12.5
August 30 16.0
Replicate
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Station CR
Number Percent
Dead Mortality6
0 3.0 ± 4.5
0
0
1
2
2 12.0 ± 16.0
0
1
8
1
2 11.0 ± 2.2
3
2
2
2
4 16.0 ± 8.2
5
2
1
4
10 32.0 ± 14.4
2
6
7
7
5 19.0 ± 9.6
6
3
1
4
Station
Number
Dead
3
2
3
4
2
1
2
5
2
3
4
3
5
3
0
12
10
13
15
4
6
9
8
9
8
12
2
6
11
6
EB
Percent
Mortality6
14.0 ± 4.2
13.0 ± 7.6
15.0 ± 9.4
54.0 ± 21JO
40.0 ± 6.1
37.0 ± 205
a Values of mean mortality in the negative controls for tests run on May 23, June 16, June 20, July 25,
August 5, and August 30 were 3, 4, 5, 1, 8, and 3 percent, respectively. All of these values are less
than the maximum allowable value of 10 percent (PSEP 1986).
b Date bioassay was initiated in 1989.
0 Mean mortality for the five replicates ± standard deviation.
A-3
-------
TABLE A-3. RESULTS OF THE AMPHIPOD MORTALITY
BIOASSAY FOR THE ENDPOINT BASED ON TOTAL
EFFECTIVE MORTALITY"
Weeks from
Date* Start Date
May 24 2.0
June 16 5.5
June 20 6.0
July 25 11.0
August 5 12.5
August 30 16.0
Replicate
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Station CR
Number Percent Total
Not Effective
Reburying Mortality0
0 3.0 ± 4.5
0
0
1
2
2 14.0 ± 17.8
0
1
9
2
2 11.0 ± 2.2
3
2
2
2
4 16.0 ± 8.2
5
2
1
4
10 33.0 ± 12.5
3
6
7
7
5 19.0 ± 9.6
6
3
1
4
Station EB
Number
Not
Reburying
5 20.0
2
5
4
4
1
2
6
2
3
4
4
5
3
0
12
11
13
15
5
6
9
8
9
8
13
2
7
11
6
Percent Total
Effective
Mortality0
± 6.1
14.0 ± 9.6
16.0 ± 9.6
56.0 ± 18.8
40.0 ± 6.1
39.0 ± 21.6
a Total effective mortality = number dead + number of survivors that fail to rebury.
b Date bioassay was initiated in 1989.
0 Mean value for the five replicates ± standard deviation.
A-4
-------
TABLE A-4. RESULTS OF THE NEANTHES BIOASSAY FOR
THE ENDPOINT BASED ON TOTAL BIOMASS
Weeks from
Date* Start Date
May 18 1.0
June 23 6.0
July 28 11.0
August 29 16.0
Replicate
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Total Biomass
Station CR
Replicate Mean6
70.2 79.9 ± 13.7
103.5
74.4
71.8
79.6
124.0 112.5 ± 24.4
137.4
118.7
109.6
72.6
110.6 105.8 ± 15.2
128.5
106.1
93.4
90.4
56.1 67.8 ± 10.7
80.4
75.7
57.7
69.0
(gm dry wt)
Station
Replicate
22.1
11.8
32.2
6.5
15.9
21.0
22.9
27.0
6.5
13.2
12.7
16.6
10.9
5.8
9.4
3.5
4.8
3.3
7.6
3.1
EB
Meanb
17.7 ± 9.9
18.1 ± 8.2
11.1 ± 4.0
4.5 ± 1.9
8 Date bioassay was initiated in 1989.
b Mean biomass for the five replicates ± standard deviation.
A-5
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TABLE A-5. RESULTS OF THE NEANTHES BIOASSAY FOR THE
ENDPOINT BASED ON AVERAGE BIOMASS
Weeks from
Date8 Stan Date
May 18 1.0
June 23 6.0
July 28 11.0
August 29 16.0
Replicate
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Average Biomass
Station CR
Replicate Mean*
14.0 16.7 ± 2.7
20.7
14.9
18.0
15.9
24.8 23.2 ± 3.5
27.5
23.7
21.9
18.2
22.1 21.2 ± 3.0
25.7
21.2
18.7
18.1
15.2 16.6 ± 1.2
17.4
17.8
15.3
17.2
(gm dry wt)
Station
Replicate
4.4
2.4
6.4
1.3
3.2
5.3
4.6
5.4
2.2
6.6
3.2
3.3
2.7
1.2
1.9
0.9
1.2
0.7
1.5
1.0
EB
Meanb
3.5 ± 20
4.8 ± 1.6
2.5 ± 0.9
1.1 ± 0.3
a Date bioassay was initiated in 1989.
b Mean biomass for the five replicates ± standard deviation.
A-6
-------
TABLE A-6. RESULTS FOR THE MICROTOX BIOASSAY
BASED ON DILUTION SERIES
Weeks
from
Start
Date8 Date
May 23 2.0
June 8 4.0
June 21 6.0
July 6 8.0
July 20 10.0
August 3 12.0
August 18 14.0
August 31 16.0
Percent Decrease in Luminescence
Concen-
tration
6.25
12.50
25.00
50.00
6.25
12.50
25.00
50.00
6.25
12.50
25.00
50.00
6.25
12.50
25.00
50.00
6.25
12.50
25.00
50.00
6.25
12.50
25.00
50.00
6.25
12.50
25.00
50.00
6.25
12.50
25.00
50.00
Replicate 1
6.6
8.3
11.5
10.9
1.9
1.5
0
0
19.1
19.7
20.4
19.6
4.1
8.0
11.0
14.4
11.9
14.1
9.5
8.6
11.7
10.5
9.9
12.4
0
0
0
0
0
0
1.1
0.8
Station CR
Replicate 2
3.5
7.6
10.7
7.6
1.4
3.2
3.1
0
15.6
19.2
21.6
20.5
9.5
10.4
12.7
15.0
11.3
12.4
7.9
9.4
6.3
8.6
7.3
7.4
0
0
0
0
0
0
2.9
2.9
Mean"
5.1
8.0
11.1
9.3
1.7
2.4
1.6
0
17.4
19.5
21.0
20.0
6.8
9.2
11.9
14.7
11.6
13.3
8.7
9.0
9.0
9.6
8.6
9.9
0
0
0
0
0
0
2.0
1.9
Replicate 1
0
0
5.1
15.4
3.4
5.7
11.9
24.0
0
0
2.9
10.7
0
0
0
12.4
1.5
9.9
24.3
39.4
0
0
0
12.7
7.6
0
0
0
0
0
0
0
Station EB
Replicate 2
0
0
2.2
16.3
5.5
5.1
10.9
23.9
0.3
0
2.4
15.4
0
0
0
13.3
4.1
13.7
27.3
40.9
0
0
0
15.1
0
0
0
0
0
0
0
0.5
Mean6
0
0
3.7
15.9
4.5
5.4
11.4
24.0
0.2
0
2.7
13.1
0
0
0
12.9
2.8
11.8
25.8
40.2
0
0
0
13.9
3.8
0
0
0
0
0
0
0.3
a Date bioassay was initiated in 1989.
b Mean of the two replicate values.
A-7
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TABLE A-7. RESULTS FOR THE MICROTOX BIOASSAY
BASED ON REPLICATE EVALUATIONS OF THE
50 PERCENT SAMPLE DILUTION
Weeks from
Date' Start Date
June 8 4.0
June 21 6.0
July 6 8.0
July 20 10.0
August 3 12.0
August 18 14.0
August 31 16.0
Replicate
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
Percent Decrease
Station CR
Replicate Mean"
0.5 0.70 ± 0.4
1.0
0.2
1.1
12.2 10.7 ± 1.4
11.1
10.8
8.8
1Z3 12.8 ± 2.2
12.9
15.6
10.2
12.9 13.6 ± 1.7
13.6
11.9
15.9
0.3 1.5 ± 0.9
2.0
1.5
2.3
0 0
0
0
0
0 0
0
0
0
in Luminescence
Station
Replicate
27.8
29.2
31.5
32.0
15.7
12.4
10.7
9.7
20.0
14.3
14.3
15.5
43.1
4.25
42.7
42.8
13.6
14.0
12.5
13.5
0
0
0
0
0.8
0.1
0.0
2.2
EB
Mean5
30.1 ± 2.0
12.1 ± 2.6
16.0 ± 27
42.8 ± 0.3
13.4 ± 0.6
0
0.8 ± 1.0
* Date bioassay was initiated in 1989.
b Mean value for the four replicates ± standard deviation.
A-8
-------
TABLE A-8. RESULTS OF THE ECHINODERM EMBRYO BIOASSAY
tA^ALtt
ViroOKB
from
Start Repli-
Date* Date cate
May 23 2.0 1
2
3
4
5
June 20 6.0 1
2
3
4
> 5
(0
July 24 11.0 1
2
3
4
5
August 29 16.0 1
2
3
4
5
Station CR
Total Peroam Abnormality Mean
Larvae Replicate Mean0 Mortality
14 14.3 14.1*14.6 16.6
13 23.1
1 0
1 0
3 33.3
34 5.9 10.3±4.6 76.0
27 7.4
35 17.0
24 6.3
23 13.0
Station CR
was not tested
Station CR
was not tested
Total
Larvae
0
0
0
0
0
9
0
3
6
1
0
1
0
0
0
23
5
22
18
3
Station EB
Percent Abnormality Mean
Replicate Mean6 Mortality
100.0
-
-
.
-
66.9 76.4t29.5 96.8
-
33.3
83.3
100.0
99.9
0
-
-
•
91.3 83.1 1 12.9 99.9
80.0
77.3
100.0
66.7
Control Seawater" Control Sediment
Percent Percent Percent Percent
Abnormality Mortality Abnormality Mortality
153d 9.6 8.3 1.6
4.7 0 8.4 0
1.9 19.8 1.9 0
7.9 9.2 8.3 0
8 Date bioassay was initiated in 1989.
b Maximum allowable abnormality - 10 percent, maximum allowable mortality « 30 percent
c Mean of the five replicates t standard deviation.
d Exceeds maximum allowable control value.
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