EPA 910/9-90-005
Puget Sound Estuary Program
DEVELOPMENT OF A
NEANTHES SEDIMENT BIOASSAY
FOR USE IN PUGET SOUND
March 1990

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PTI Environmental Services
15375 SE 30th Place
Suite 250
Beilevue, Washington 98007
DEVELOPMENT OF A NEANTHES SEDIMENT
BIOASSAY FOR USE IN PUGET SOUND
By
D. Michael Johns 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
March 1990

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CONTENTS
Page
LIST OF FIGURES	iv
LIST OF TABLES	v
ACKNOWLEDGMENT	vii
INTRODUCTION	1
METHODS	3
GENERAL EXPERIMENTAL DESIGN	3
SEDIMENT COLLECTION	3
Elliott Bay and Carr Inlet Sediments	8
West Beach Sediment	8
Duckabush River Sediment	8
SEDIMENT CHEMISTRY	9
BIOLOGICAL TESTING	9
Worm Density	13
Food Ration	13
Static vs. Static Renewal Exposure System	14
Test Duration	14
Salinity Tolerance	17
Sensitivity to Sediment Grain Size	17
Sensitivity of Neanthes to a Reference Toxicant	18
DATA ANALYSIS	18
RESULTS AND DISCUSSION	20
SEDIMENT CHEMISTRY	20
BIOLOGICAL TESTING	20
Worm Density	20
Food Ration	27
Static vs. Static Renewal Exposure System	34
Test Duration	41
Salinity Tolerance	46
Sensitivity to Sediment Grain Size	50
Sensitivity of Neanthes to a Reference Toxicant	52
Response of the Biomass Endpoint and Statistical Power	52
ii

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SUMMARY
REFERENCES
APPENDIX A - SEDIMENT CHEMISTRY DATA
APPENDIX B - BIOASSAY TEST DATA
APPENDIX C ¦ WATER QUALITY MONITORING DATA
iii

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LIST OF FIGURES
Figure 1. General experimental design showing testing sequence	4
Figure 2. Location of sediment collection sites	7
Figure 3. Static exposure system used for the Neanthes sublethal bioassay	12
Figure 4. Survival and biomass of juvenile worms from the worm
density experiment	23
Figure 5. Statistical power vs. minimum detectable difference for
total biomass in the worm density experiment	28
Figure 6. Survival, biomass, and individual biomass of juvenile worms
from the food ration experiment	30
Figure 7. Survival and biomass of juvenile worms from the exposure
system experiment	36
Figure 8. Survival, total biomass, and individual biomass of juvenile worms from
the test duration experiment	43
Figure 9. Statistical power vs. minimum detectable difference for total
biomass in the test duration experiment	45
Figure 10. Statistical power vs. minimum detectable difference for total
biomass in the test duration experiment calculated using average
mean square error	47
IV

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LIST OF TABLES
Page
Table
1.
Experimental design for sediment exposures
5
Table
2.
Chemicals analyzed in test sediments
*
10
Table
3.
Sampling schedule for water quality analysis
15
Table
4.
Polycyclic aromatic hydrocarbons measured in water samples taken from
the static and static renewal exposure systems containing Elliott Bay
sediments
16
Table
5.
Chemical contaminants in Elliott Bay sediment exceeding 1988 bioassay AET
21
Table
6.
Survival, total biomass, and average individual biomass data for the
worm density experiment
22
Table
7.
Pairwise statistical comparison of Neanthes bioassay responses for the
worm density experiment
25
Table
8.
Results of one-way analysis of variance for biomass data from the worm
density experiment
26
Table
9.
Survival, total biomass, and average individual biomass data for the
food ration experiment
29
Table
10.
Pairwise statistical comparison of Neanthes bioassay responses between
Elliott Bay and Carr Inlet treatments for the food ration experiment
32
Table
11.
Results of one-way analysis of variance for biomass data from the food
ration experiment
33
Table
12.
Survival, total biomass, and average individual biomass data for the
exposure system experiment
35
Table
13.
Pairwise statistical comparison of Neanthes bioassay responses for the
exposure system experiment
37
Table
14.
Ammonia concentration from static and static renewal exposure systems
during a 20-day exposure period
38
Table
15.
Contaminant concentrations from static and static renewal exposure systems
containing Elliott Bay sediment
39
Table
16.
Mass loss of contaminants in the static renewal exposure system containing
Elliott Bay sediment
40
Table
17.
Survival, total biomass, and average individual biomass data for the test
duration experiment
42

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Page
Table 18. Pairwise statistical comparison of Neanthes bioassay responses for
the test duration experiment	44
Table 19. Survival data for the 96-hour salinity tolerance experiment	48
Table 20. Survival, total biomass, and average individual biomass data for the Neamhes
salinity tolerance experiment	49
Table 21. Survival, total biomass, and average individual biomass for the sediment
grain-size experiment	51
Table 22. Results of one-way analysis of variance for biomass data from the grain-size
experiments	53
Table 23. Results of statistical comparisons and percent differences between test
sites for Neamhes bioassays	54
vi

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ACKNOWLEDGMENT
This document was prepared by PTI Environmental Services under the direction of Dr. Thomas
C. Ginn for U.S. Environmental Protection Agency (EPA) Region 10, Office of Puget Sound, under
EPA Contract No. 68-D8-0085. Ms. Catherine Krueger and Dr. Jack Gakstatter served as
Technical Monitors for EPA. This project was funded by the EPA National Estuary Program.
Research presented in this report was conducted at the E.V.S. Consultants' (E.V.S.) laboratory
in North Vancouver, British Columbia, with the assistance of Dr. Peter Chapman and his staff.
Technical support throughout the entire project was provided by Ms. Rita Ciammaichella. Neanthes
juveniles used in this work were provided by Dr. Donald Reish (California State University, Long
Beach). Ms. Lorraine Read was responsible for data management, computer applications in data
analysis, and graphics. The drawing of Neanthes that appears on the cover was done by Lisa
Reish. Written review comments on the draft report were provided by the following individuals:
Messrs. Brett Betts, Keith Phillips, and Dave Smith (Washington Department of Ecology), Dr. Paul
Dinnel (University of Washington), Ms. Carol Pesch and Dr. Richard Swartz (EPA), Ms. Rita
Ciammaichella (E.V.S.), and Dr. Usha Varanasi (National Oceanic and Atmospheric Administration).
The following experts participated in the Washington Department of Ecology workshop that
resulted in the general experimental design used in the program:
Individual
Dr. Peter Chapman
Dr. Thomas Ginn
Dr. D. Michael Johns
Mr. Philip Oshida
Ms. Carol Pesch
Dr. Donald Reish
Dr. Richard Swartz
Mr. Jack Word
Dr. Tom Wright
Affiliation
E.V.S. Consultants, North Vancouver, British Columbia
PTI Environmental Services, Bellevue, Washington
PTI Environmental Services, Bellevue, Washington
U.S. Environmental Protection Agency, Region 9
San Francisco, California
U.S. Environmental Protection Agency, Environmental
Research Laboratory, Narragansett, Rhode Island
California State University, Long Beach, California
U.S. Environmental Protection Agency, Newport, Rhode Island
Battelle Northwest Laboratory, Sequim, Washington
U.S. Army Corps of Engineers, Waterways Experiment Station,
Vicksburg, Mississippi
vn

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INTRODUCTION
A draft protocol for a sublethal sediment bioassay using the juvenile stage of Neanthes sp.
(polychaete) was developed as part of a bioassay test demonstration study conducted for the Seattle
District of the U.S. Army Corps of Engineers and the Puget Sound Dredged Disposal Analysis
(PSDDA) study (Johns 1988). The study was undertaken to evaluate options in incorporating
sublethal tests into the PSDDA dredged material decision-making framework.
The draft protocol for the Neanthes sediment bioassay involved measuring survival and change
in biomass (total and individual) following exposure to test sediments. Test conditions for the
interim bioassay included static renewal of seawater during a 20-day exposure. The five test
organisms in each exposure chamber were fed during the bioassay. The results of the test
demonstration study suggested the Neanthes sublethal bioassay would be a useful test for
characterizing sediment quality because of the following factors:
¦	Neanthes juveniles appeared to be sensitive to changes in sediment quality
¦	Neanthes juveniles exhibited consistent responses to a series of sediment exposures
¦	Neanthes sublethal bioassay response criteria (i.e., survival and biomass) are relevant
factors in evaluating the potential impacts of contaminated sediments on benthic
organisms
¦	Neanthes are easily cultured, and a ready supply of test organisms from a standard
stock could be available throughout the year
¦	Neanthes sublethal bioassays can be conducted using a relatively simple static renewal
exposure system.
Following completion of the test demonstration study and development of a draft protocol for
conducting the Neanthes sublethal bioassay, the Washington Department of Ecology conducted an
experts workshop on the development of the bioassay to be used as part of the state's marine
sediment management program. The general objectives of the workshop were to evaluate the draft
protocol for conducting sublethal sediment bioassays using Neanthes and to determine the
information and research that may be needed to further refine the test development.
As part of the workshop, the experts were asked to categorize and rank research needs
obtained during review of the draft protocol to provide guidance on suggested changes to the draft
protocol. The workshop resulted in development of an interim protocol (Johns et al. 1989) based
on recommended changes that could be made without further testing. The workshop also resulted
in the development of research needs for future refinement of the test (Johns et al. 1989).
Research topics identified by the experts were not considered critical to conducting the
Neanthes bioassay or to establishing an interim protocol, but the experts recommended that results
from the research be incorporated into the final protocol to further enhance and extend the
usefulness of the test. Research topics identified at the workshop include:
1

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¦	Determine the optimal number of Neanthes that should be placed in each exposure
chamber
¦	Determine the level of food ration that should be provided to each exposure chamber
¦	Determine if the test can be conducted using a static exposure system
¦	Determine if the length of the exposure period can be shortened from the current
20 days
¦	Determine the salinity tolerance limits of the test endpoints
¦	Determine if sediment grain size has an effect on increases in worm biomass during
the exposure period.
Following development of these research recommendations, the workshop participants developed
a general experimental approach for each research topic. The research presented in this report
results directly from implementation of the recommended studies.
The purpose of this study is to address the research topics identified at the experts workshop
and to develop a final Neanthes protocol based on the research findings. The final protocol will
be published as a separate document and will be included in EPA's Recommended Protocols for
Measuring Selected Environmental Variables in Puget Sound.
2

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METHODS
GENERAL EXPERIMENTAL DESIGN
This study was designed to address the research topics recommended by the experts workshop.
The workshop resulted in discrete work elements that were translated into individual experiments
as part of the overall study design. However, because the individual experiments do not represent
independent study elements, the experiments were not conducted simultaneously. The tests were
conducted in a sequential manner so that the results of a particular experiment would be used to
modify subsequent experiments, as appropriate. The sequential framework for the six primary
experiments is shown in Figure 1. In this series of experiments, the test conditions were
maintained according to the interim protocol unless experimental results demonstrated a definite
advantage in implementing a modified technique. Experiments 1 through 4 were designed to
evaluate the four primary exposure conditions recommended for testing:
¦	Number of test organisms
¦	Food ration
¦	Exposure condition
¦	Test duration.
If a modified technique was selected in experiments 1 through 4 (Figure 1), the new technique
was used in all subsequent experiments.
The six experiments involving sediment exposures were established according to a factorial
design involving sediment type and each experimental variable (Table 1). The overall objective
of the first four experiments was to evaluate each experiment individually using statistical
comparisons to determine whether a modification in the protocol would result in one or more of
the following benefits:
1.	Increased sensitivity to detect sediment toxicity
2.	Simplification of testing procedures
3.	Reduced costs.
In experiments 5 and 6, the objective was to determine the applicability of the Neanthes bioassay
to conditions of reduced salinity or of very fine sediments. An additional test, experiment 7, was
used to establish sensitivity of the test organism to a reference toxicant.
SEDIMENT COLLECTION
Sediments used in this study were collected from three sites in Puget Sound and one location
in Hood Canal (Figure 2). Within Puget Sound, sampling sites were located in a contaminated
embayment (Elliott Bay) and in two relatively uncontaminated areas (Carr Inlet and West Beach).
Sediments from Hood Canal were collected from the mouth of the Duckabush River.
3

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SELECT
FOOD
RATION
STATIC OR
STATIC
RENEWAL
SELECT \
APPROPRIATE
V NUMBER >
r SELECT
TEST
DURATION
SALINITY
TOLERANCE
PARTICLE
SIZE
REFERENCE
TOXICANT
EXPOSURE
CONDITION
FOOD
RATION
TEST
DURATION
1 NUMBER OF
TEST ORGANISMS
Figure 1. General experimental design showing testing sequence
4

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TABLE 1. EXPERIMENTAL DESIGN FOR SEDIMENT EXPOSURES
Experiment No. 1 - Worm Density
Sediment
Organisms/Exposure Chamber
5 10 15 20
Elliott Bay

¦x^
X
X
X
Carr Inlet


x
X
X
West Beach





Experiment No. 2 - Food Ration
Sediment
Food Ration (mg)/2 days
0 20 40 60 80
Elliott Bay
X
X

x
X
Carr Inlet
X
X
iiixi:!
X
X
West Beach
X

;;:x;i;

X
Experiment No. 3 - Exposure Conditions
Sediment
Exposure Condition
Static Static Renewal
Elliott Bay
X





Carr Inlet
X


liXi;


Experiment No. 4 - Test Duration
Sediment
Test Duration (days)
10 15 20
Elliott Bay
X
X



Carr Inlet
X
X


xiiiiiiiiii
West Beach
X
X



Experiment No. 5 - Salinity Tolerance
Sediment
Salinity (ppt)
30 28 25 22 19
Duckabush River
X


Xi:

X
X
X
Carr Inlet



xi:




West Beach



xi;:




5

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TABLE 1. (Continued)
Experiment No. 6 - Sediment Grain Size
Sediment
Percent Fines (silt/clay)
2 88-89 93 97
Carr Inlet (CR01)

X


Carr Inlet (CR02)

X


Carr Inlet (CR03)



X
Carr Inlet (CR04)

X


Carr Inlet (CR05)


X

West Beach
X



~ Shading represents exposure condition in interim protocol.
6

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7

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Elliott Bay and Carr Inlet Sediments
In Elliott Bay, sediments were collected from a station in Elliott Bay at the north end of
Harbor Island (Figure 2). Pastorok and Becker (1989) and Beller et al. (1988) showed that this
area of Elliott Bay is contaminated by a complex mixture of chemicals including organic compounds
[e.g., polycyclic aromatic hydrocarbons (PAH), pentachlorophenol (PCP), and polychlorinated
biphenyls (PCB)] and metals (e.g., arsenic, copper, and mercury). Sediments collected from this
station were used as one of the test sediments for the Neanthes test demonstration study (Johns
1988) and in a recent bioassay comparison study conducted by Pastorok and Becker (1989).
Of the uncontaminated sediments collected from two stations sampled in Carr Inlet (Figure 2),
sediment from station CR01 was used as the reference material for all tests conducted as part of
this study. Carr Inlet sediments have been found to be relatively free of contaminants and have
been used previously as a reference sediment for bioassays (PTI 1988, 1989). Therefore, sediment
from Carr Inlet could serve as a suitable reference sediment for conducting bioassays. Sediment
obtained from the second station in Carr Inlet (CR02) was used in a test designed to evaluate the
effect of grain size on the survival and growth of Neanthes juveniles during a 20-day exposure
period.
At both the Elliott Bay and the Carr Inlet stations, approximately 24 liters of sediment were
collected using a 0.1 -m2 van Veen bottom grab sampler. To obtain the desired amount of sediment,
multiple casts of the sampler were required. All samples from a single station were placed in a
plastic bucket and later homogenized in the laboratory. Prior to homogenization, the sediments
were press-sieved through a 1.0-mm screen to remove all large infauna and debris.
Following homogenization, sediment from each station was placed in 1-L jars. To preserve
the quality of the sediment during the study period, each jar was filled to capacity with sediment
and nitrogen was passed over the sediment surface prior to sealing the jar. Sediment samples were
maintained at 4° C until needed for a test. Sediment used in each test originated from a full,
sealed jar. Jars with unused portions of sediment samples were discarded.
West Beach Sediment
Sediment collected from West Beach on Whidbey Island (Figure 2) was used as the control
sediment for all tests. West Beach sediment was found to be a suitable substrate for Neanthes
juvenile survival and growth by Johns (1988). West Beach sediment was collected within 1 week
of beginning each test. Test sediment was obtained from subtidal areas using an epibenthic dredge.
In the laboratory, the sediment was sieved through a 0.5-mm screen and stored in plastic containers
until needed.
Duckabush River Sediment
Four sediment samples were also collected along a salinity gradient at the mouth of the
Duckabush River, which is located south of Brinnon on the western shore of Hood Canal
(Figure 2). Prior to obtaining a sediment sample, the pore-water salinity was determined using a
portable refractometer. Pore water was obtained by removing a small amount of water-saturated
sediment from an intertidal area along the river, then allowing water to collect into the hole. Once
8

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a sampling site was identified as having the appropriate sediment based on pore-water salinity,
surficial sediment (i.e., upper 2 cm) was collected using a polypropylene scoop. Sediment collected
from each station was placed in a stainless steel bowl, homogenized using the scoop, and placed in
1-L jars. Two liters of sediment were collected at each station. The four Duckabush River stations
that were sampled represent sediments with interstitial salinities of 19, 22, 25, and 30 parts per
thousand (ppt).
SEDIMENT CHEMISTRY
Chemical analyses of sediment samples collected from Carr Inlet and Elliott Bay were
performed for both organic compounds and metals (Table 2). Conventional sediment variables
were also measured, including grain size and total organic carbon (TOC) (PSEP 1986). Concentra-
tions of organic compounds were determined following modified U.S. Environmental Protection
Agency (EPA) Contract Laboratory Program (CLP) protocols (U.S. EPA 1986a). The analysis of
semivolatile compounds, including acid/base/neutral (ABN) extractables, PCBs, and pesticides,
followed modified EPA CLP protocols that were consistent with PSEP recommendations for analyses
with relatively low detection limits. In particular, modifications included larger sample size
(typically 50-100 grams dry weight) and smaller final extract volume for gas chromatography/mass
spectroscopy (GC/MS) analyses. Separate sediment subsamples were used for ABN and pesticide/
PCB extractions. Ultrasonic extraction was carried out as described by the CLP procedure. Gel
permeation chromatography (GPC), an optional cleanup step under CLP, was performed for all
sediment ABN extracts to reduce interferences and attain necessary detection limits. Pesticide/
PCB analyses were conducted with a slightly modified version of the EPA CLP method. 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 routinely used in CLP.
For metals, samples were first digested using the total acid digestion technique in the EPA
CLP program (U.S. EPA 1986a). Metals in sediment digestates were 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).
BIOLOGICAL TESTING
All tests were conducted using juvenile laboratory-cultured Neanthes obtained from Dr.
Donald Reish at California State University, Long Beach. The bioassay approach used in this study
was based on the interim protocol for conducting a sublethal bioassay with juvenile Neanthes sp.
described by Johns et al. (1989).
The interim protocol calls for the Neanthes bioassay to be conducted using a static renewal
exposure system (Figure 3). Each exposure system consists of a 1-L jar containing 2 cm of
sediment and seawater (at salinities between 28 and 30 ppt). Worms in each exposure chamber
are provided with 40 mg of food (TetraMarin®) every second day during the exposure period.
Every third day, one-third of the water volume is exchanged with fresh seawater. Measure-
ments of dissolved oxygen, pH, salinity, and temperature are made prior to each seawater exchange.
9

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TABLE 2. CHEMICALS ANALYZED
IN TEST SEDIMENTS
Metals
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 Polycyclic Aromatic Hydrocarbons
naphthalene	phenanthrene
acenaphthylene	anthracene
acenaphthene	2-methylnaphthalene
fluorene
High Molecular Weight Polycyclic Aromatic Hydrocarbons
fluoranthene	benzo(a)pyrene
pyrene	indeno(l,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
Polychlorinated Biphenyls
total PCB (mono- through decachlorobiphenyls)
Chlorinated Aliphatic Hydrocarbons
hexachlorobutadiene	hexachloroethane
Phthalate Esters
dimethyl phthalate	butyl benzyl phthalate
diethyl phthalate	bis(2-ethylhexyl)phthalate
di-n-butyl phthalate	di-n-octyl phthalate
10

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TABLE 2. (Continued)
Miscellaneous Oxygenated Compounds
benzyl alcohol	benzoic acid
dibenzofuran
Organooitrogen Compounds
N-nitrosodiphenylamine
Pesticides
total DDTs (p,p')	aldrin
heptachlor	dieldrin
a-chlordane	^f-HCH (lindane)
11

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Air Supply
Water
Test Sediment
Figure 3. Static exposure system used for the Neanthes sublethal bioassay

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Following a 20-day exposure period, all surviving worms from each chamber are dried to a constant
weight and their biomass is determined. To determine biomass, the worms are quickly rinsed in
distilled water, dried at 50° C for 24 hours, and then weighed to the nearest 0.1 mg. Three
response criteria are examined: survival, total biomass (as dry weight), and average individual
biomass (i.e., total biomass divided by the number of surviving worms).
Because the main focus of this study was to experimentally evaluate recommended changes in
the protocol, modifications to the interim test protocol were required. However, in all test series,
at least one treatment followed the interim protocol. The following experiments were conducted
as part of this study to evaluate recommended changes to the interim protocol.
Worm Density
An experiment was conducted to determine if the number of organisms placed in each
exposure chamber should be increased from the presently recommended number of 5 worms per
chamber. The test was conducted with four densities of worms: 5, 10, 15, and 20 worms per
chamber. Sediments used in the test included samples collected from Elliott Bay, Carr Inlet, and
West Beach. All four worm density treatments were tested with the Elliott Bay and Carr Inlet
sediments, while only the lowest density treatment was tested with the West Beach sediment
(Table 1). There were five replicates of each density/sediment combination.
All treatments were given the same food ration (40 mg of TetraMarin® every other day),
regardless of worm density. Other bioassay conditions were held constant for all treatments based
on the interim protocol. Following the 20-day exposure period, surviving worms from each
treatment were collected and dried to a constant weight and total biomass was determined.
Food Ration
The amount of food that should be provided to each chamber during the exposure period was
examined by conducting a food ration experiment. The interim protocol calls for 40 mg of food
every second day of exposure. A total of five food ration treatments were tested, ranging from no
food to 80 mg (dry weight) of TetraMarin® (i.e., 0, 20, 40, 60, or 80 mg/48 hours). The
appropriate food ration was provided to each exposure chamber every second day. The number
of worms placed in each exposure chamber was based on test results from the previously described
experiment.
Sediments used in this test included samples collected from Elliott Bay, Carr Inlet, and West
Beach. All five food ration levels were used with Elliott Bay and Carr Inlet sediments, while only
three of the five ration levels (i.e., 0, 40, and 80 mg/48 hours) were used with sediment collected
from West Beach (Table 1).
Other bioassay conditions were held constant based on the interim protocol. Following the 20-
day exposure period, surviving worms from each treatment were collected and dried to a constant
weight and total biomass was determined.
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Static vs. Static Renewal Exposure System
An experiment was conducted to determine whether the Neanthes sublethal bioassay could be
conducted under static exposure conditions. Current protocol recommends the use of a static
renewal exposure system in which one-third of the exposure water is exchanged with fresh seawater
every third day during the exposure period. A side-by-side comparison of the static and static
renewal exposure systems was used to evaluate the need for the static renewal exposure system.
The test was conducted with sediments collected from Elliott Bay, Carr Inlet, and West Beach
(Table 1).
The number of worms placed in each container and the amount of food provided during the
exposure period was based on the results of the previous two experiments. Other bioassay
conditions were held constant for all treatments based on the interim protocol. Following the 20-
day period, surviving worms from each treatment were collected and dried to a constant weight and
total biomass was determined.
During the exposure period, dissolved oxygen, pH, salinity, and temperature were measured
every third day just prior to exchanging water in the static renewal exposure system. In addition
to measuring the above parameters, water samples were taken periodically to determine the
concentration of ammonia and selected contaminants present in the static and static renewal
exposure systems. The sampling schedule for water quality is presented in Table 3. Ammonia
concentrations were determined for both the static and static renewal exposure systems containing
Elliott Bay and Carr Inlet sediments following standard methods published by AMPHA (1985).
Contaminants assayed included PAH (Table 4), copper, and mercury. Both dissolved and total
concentrations of copper and mercury were determined. Contaminant concentrations were
determined for both the static and static renewal exposure systems, but only for treatments
containing Elliott Bay sediment. Water samples for chemical analysis of the selected contaminants
were obtained from extra exposure chambers having the same sediment volume and number of
juvenile Neanthes as all other exposure chambers used in the test. Thirty minutes prior to collecting
a water sample, aeration to the exposure chamber was stopped to allow particle settling.
Concentrations of PAH were analyzed by U.S. EPA SW-846 Method 8100 (U.S. EPA 1986b),
which employs dual column gas chromatography with flame ionization detection. Total copper was
digested by U.S. EPA SW-846 Method 3020 (U.S. EPA 1986b) and analyzed by Method 6010 using
furnace atomic absorption spectroscopy. Dissolved copper was determined using Method 6010 after
the sample was filtered through a 0.45-^m filter. The concentration of total mercury was
determined by Method 7470 using cold vapor atomic absorption spectrometry (U.S. EPA 1986b).
Dissolved mercury was determined using Method 7470 after the sample was filtered through a
0.45-#im filter.
Test Duration
An experiment was conducted to determine the optimal exposure period that can be used
with the Neanthes sublethal bioassay to detect a statistically significant level of organism response.
Exposure periods evaluated were durations of 10, 15, and 20 days. Sediments used in this test were
collected from Elliott Bay, Carr Inlet, and West Beach (Table 1). Five replicates were used for
each treatment and exposure period combination.
14

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TABLE 3. SAMPLING SCHEDULE FOR
WATER QUALITY ANALYSIS
Sampling Date


Water Quality
Parameter

(Days)

Sediment
Exposure System
3
6
12
20
Carr Inlet
Static
Ammonia

X
X
X

Static renewal
Ammonia
X
X
X
X
Elliott Bay
Static
Ammonia
X
X
X
X


PAHb
X
X
X
X


Metals
X
X
X
X

Static renewal
Ammonia

X
X
X


PAH

X
X
X


Metals
	a
X
X
X
* Sample not taken for this exposure condition at these sampling dates.
b PAH - polycyclic aromatic hydrocarbons.
15

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TABLE 4. POLYCYCLIC AROMATIC HYDROCARBONS
MEASURED IN WATER SAMPLES TAKEN FROM THE STATIC
AND STATIC RENEWAL EXPOSURE SYSTEMS CONTAINING
ELLIOTT BAY SEDIMENTS
Low Molecular Weight Polycyclic Aromatic Hydrocarbons
naphthalene	phenanthrene
acenaphthylene	anthracene
acenaphthene	2-methylnaphthalene
fluorene
High Molecular Weight Polycyclic Aromatic Hydrocarbons
fluoranthene	benzo(a)pyrene
pyrene	indeno(l,2,3-c,d)pyrene
benz(a)anthracene	dibenzo(a,h)anthracene
chrysene	benzo(g,h,i)perylene
benzofluoranthenes
16

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The test was conducted using the number of organisms, food ration, and exposure system
identified as the most appropriate based upon the results of the previously described experiments.
Other bioassay conditions were held constant for all treatments based on the interim protocol.
Following exposure periods of 10, 15, and 20 days, surviving worms from the five replicates of
each treatment were collected and dried to a constant weight and total biomass was determined.
Salinity Tolerance
Two experiments were conducted to determine the salinity tolerance of Neanthes. The first
test was an acute bioassay to determine the 96-hour tolerance of juvenile Neanthes to low-salinity
water. Salinities tested were 10, 15, 20, 25, and 28 ppt.
Water of the appropriate test salinity was prepared by taking field-collected seawater at 28 ppt
and diluting samples of the water with distilled water until the desired salinity was attained.
Salinity was determined using a hand-held refractometer. The test was conducted without sediment.
The worms were not fed during the 96-hour exposure period.
Five worms were placed in 1-L beakers containing 500 mL of water of the appropriate
salinity. Water quality conditions (i.e., dissolved oxygen, pH, temperature, and salinity) and the
number of surviving worms were determined on a daily basis. Two replicates of each salinity
treatment were used in this experiment.
The second salinity tolerance experiment was conducted using sediment samples collected along
a salinity gradient on the Duckabush River. Interstitial salinities of the four sediments collected
were 30, 25, 22, and 19 ppt. Sediments from Carr Inlet and West Beach were also included in this
salinity tolerance experiment. Interstitial salinity of the Carr Inlet and West Beach sediments was
between 28 and 30 ppt. The test was conducted following the interim protocol. In all treatments,
water overlying the sediment was 28 ppt.
To reduce the amount of initial mixing between the interstitial water and the overlying water,
water placed in each exposure chamber was allowed to slowly flow down the inside of the exposure
chamber. Following the 20-day exposure period, surviving worms from each treatment were
collected and dried to a constant weight and total biomass was determined.
Sensitivity to Sediment Grain Size
The influence of sediment grain size on juvenile Neanthes survival and growth was determined
following a 20-day exposure to sediments having differing granulometry (expressed as a percentage
of the silt/clay fraction in the sediment). In particular, the test was designed to evaluate the
potential effects on survival and growth of sediments containing high silt/clay fractions (>88
percent). Six sediment treatments were used in this test, including three field-collected sediments
(i.e., two stations in Carr Inlet, stations CR01 and CR02, and one from West Beach). A fourth
treatment, CR03, was prepared by collecting that portion of Carr Inlet sediment from station CR02
that passed through a 125-pm screen. The fifth and sixth sediment treatments, designated CR04
and CR05, were prepared by mixing equal weights of sediments from CR01 and CR02, and CR02
and CR03, respectively.
17

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The test was conducted using the interim protocol. Following the 20-day exposure period,
surviving worms from each treatment were collected and dried to a constant weight and total
biomass was determined.
Sensitivity of Neanthes to a Reference Toxicant
Two experiments were conducted to determine the sensitivity of Neanthes juveniles to a
reference toxicant, cadmium chloride (CdCl2). The first test was a 96-hour toxicity test to
determine the range of Cd concentrations to which Neanthes is acutely responsive. Concentrations
used in the test were I, 10, 100, and 1,000 parts per million (ppm) Cd.
Based on the results of the first test, a second 96-hour toxicity test was conducted to further
define the 96-hour LCJ0 values for Neanthes juveniles exposed to Cd as CdCl2 . Concentrations
used in this test were 10, 18, 32, 56, and 100 ppm Cd. LCJ0 values were determined using probit
analyses (Peltier and Weber 1985).
In both experiments, five worms were placed in 1-L jars containing 500 mL of seawater at
the appropriate Cd concentration. Three replicates of each Cd concentration were used. The tests
were conducted without sediment and the worms were not fed during the 96-hour exposure period.
Water quality conditions (i.e., dissolved oxygen, pH, temperature, and salinity) were measured on
a daily basis. The number of surviving worms was determined at the end of the 96-hour period.
DATA ANALYSIS
Pairwise statistical comparisons were used to evaluate statistical differences. This approach
to statistical testing is typically used to analyze sediment bioassays conducted for environmental
regulatory programs and involves pairwise testing of a potentially contaminated sample against a
reference sample. To evaluate the recommended changes in the bioassay protocol, pairwise
comparisons were made between the relatively uncontaminated Carr Inlet and the contaminated
Elliott Bay sediment treatments having similar exposure conditions.
Prior to conducting the pairwise comparisons, treatment pairs were tested for homogeneity of
variances using a Cochran's C-test. In several treatment pairs, variances were found to be
heterogeneous (P<0.05). In some cases, variance homogeneity could not be tested because no
variance estimate could be made for one or both of the data pairs. This situation arose with
survival data when the survival in all treatment replicates was 100 percent.
Approximately 30 percent of the pairwise comparisons evaluated for this study failed to meet
the variance assumption required when using the more common parametric tests [e.g., t-test and
one-way analysis of variance (ANOYA)J. Therefore, all pairwise comparisons were made using
a nonparametric test, the Mann-Whitney C/-test.
In some experiments, additional statistical evaluations of the test data were conducted using
ANOVA across all treatments having the same sediment. If significant differences were found, a
Student-Newman-Keuls procedure was conducted to identify nonsignificant subsets of treatments.
Statistical power analysis was used to evaluate the experimental results for the number of test
organisms and test duration. Statistical power analyses were conducted using a one-way ANOVA
18

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model (Scheffe 1959; Cohen 1977). For these analyses, the within-group mean square for the
ANOVA was used as an estimate of residual variance. The results of comparisons of contaminated
vs. reference sediments among different numbers of test organisms and exposure times were
evaluated as minimum detectable difference (MDD) and corresponding power for a fixed design
[i.e., replicate number, treatment number, and significance level (a)]. The predicted MDD between
the mean response to a contaminated sample and the mean response to a reference sample at a
specified power was used as a measure of the bioassay performance among alternative numbers of
organisms and alternative exposure times.
19

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RESULTS AND DISCUSSION
SEDIMENT CHEMISTRY
This section provides an overview of chemical concentrations found in each sediment and of
general sediment quality based on comparisons to 1988 Puget Sound apparent effects threshold
(AET) values normalized to sediment dry weight (Barrick et al. 1988). AET values provide an
estimate of the concentration of each chemical that may be associated with adverse effects in Puget
Sound. Data on the chemical concentrations and conventional variables measured in sediments
collected from both Carr Inlet stations (i.e., stations CR01 and CR02) and Elliott Bay are presented
in Appendix A,
Sediments from both Carr Inlet stations are relatively fine-grained (>88 percent silt/clay) and
contain levels of TOC (1.3 and 0.93 percent for Carr Inlet stations CR01 and CR02, respectively)
commonly found in Puget Sound sediments.
Sediments from Carr Inlet contain relatively low concentrations of organic compounds and
metals. Several PAH compounds were detected at Carr Inlet, including phenanthrene, anthracene,
and chrysene, but all concentrations were below 100 parts per billion (ppb). None of the organic
contaminants detected in sediments collected from Carr Inlet exceeded any of the 1988 bioassay
AET values (i.e., amphipod bioassay, oyster larvae bioassay, and Microtox). Arsenic, cadmium,
copper, lead, and mercury were detected in both Carr Inlet sediments; however, none of the con-
centrations exceeded the 1988 bioassay AET values.
Sediment from the Elliott Bay station is moderately fine-grained (> 47 percent silt/clay) and
has a TOC content of 2.1 percent. This sediment was highly contaminated with several organic
compounds and metals. Organic contaminants exceeding one or more of the 1988 bioassay AET
values include low molecular weight PAH, high molecular weight PAH, phthalates, 2-methyl-
phenol, PCP, and PCBs (Table 5). Copper and mercury exceeded all three bioassay AET values
(i.e., amphipod bioassay, oyster larvae bioassay, and Microtox). Zinc and arsenic exceeded only
the amphipod AET.
BIOLOGICAL TESTING
Worm Density
Mean survival of Neanthes juveniles following a 20-day exposure to Elliott Bay, Carr Inlet,
and West Beach sediments ranged from a high of 100 percent to a low of 36 percent (Table 6;
Figure 4). Survival was greater than 95 percent for all treatments containing Carr Inlet and West
Beach sediments, regardless of density, and for Elliott Bay sediment where the density was five
worms per chamber. The coefficient of variation (mean value divided by the standard deviation)
for the survival data for these treatments was low, ranging from 0 to 9 percent.

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TABLE 5. CHEMICAL CONTAMINANTS IN ELLIOTT BAY
SEDIMENT EXCEEDING 1988 BIOASSAY AET


Elliott Bay


Sediment
AET
Chemical
Concentration®
Exceedancesb
Metals


Arsenic
112
A
Copper
1,490
A,M,0
Mercury
3.5
A,M,0
Zinc
1,010
A
Organic Compounds


LPAH


Acenaphthene
780
M,0
Fluorene
790
M,0
Phenanthrene
4,800
M,0
Anthracene
1,900
M.O
HPAH


Fluoranthene
8,100
M,0
Pyrene
12,000
M,0
Benz(a)anthracene
4,000
M,0
Chrysene
3,300
M,0
Benzofluoranthenes
10,000
A,M,0
Benzo(a)pyrene
8,900
A,M,0
Indeno( 1,2,3 -c,d)pyrene
1,600
M,0
Dibenzo(a,h)anthracene
710
A,M,0
Benzo(g,h,i)perylene
3,600
A,M,0
Phthalates


Dimethyl phthalate
110
M
Butyl benzyl phthalate
320
M
Bis(2-ethylhexyl)phthalate
6,100
M.O
Phenols


2-methylphenol
78
A,M,0
Pentachlorophenol
1,900
A,M,0
Total PCB
1,460
M,0
* Metals concentrations are reported in mg/kg dry weight. Concentrations of
organic compounds are reported in fig/kg dry weight.
b A - amphipod mortality test
M - Microtox test (saline extract)
O - oyster larvae abnormality.
21

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TABLE 6. SURVIVAL, TOTAL BIOMASS, AND
AVERAGE INDIVIDUAL BIOMASS DATA FOR
THE WORM DENSITY EXPERIMENT
Worm Density	Average
(no. worms,/	Total Biomass" Individual Biomass
Sediment
chamber)
Survival"
(mg dry weight)
(mg dry weight)
West Beach
5
95.0 ± 4.5
22.6 ± 6.2
4.6 ± 0.7
Carr Inlet
5
96.0 ± 4.0
79.9 ± 6.1
16.7 ± 1.2

10
98.0 ± 2.0
111.6 ± 7.1
11.9 ± 0.8

15
98.6 ± 1.4
134.4 ± 10.2
9.0 ± 0.6

20
97.0 ± 2.0
147.5 ± 9.5
7.6 ± 0.6
Elliott Bay
5
100 ± 0.0
17.7 ± 4.4
3.5 ± 0.9

10
72.0 ± 9.2
24.5 ± 6.4
3.3 ± 0.6

15
41.2 ± 14.3
17.4 ± 8.9
1.9 ± 0.7

20
36 ± 12.1
21.8 ± 10.0
2.1 ± 0.7
' Value reported as mean ± standard error.
b Biomass of worms at test initiation was 1.0 ± 0.2 mg (dry weight).
22

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100
60
60
40
5 Worms/Chambar
Percent Survival	Biomass (tng dry weight)
10 Worms/Chamber
20-
i-.SvT
I
1
it
I
I
160 100
- 120
•80
40
Percent Survival
Biomass 
-------
In contrast to the Carr Inlet sediment exposures, survival was dependent upon worm density
in the Elliott Bay sediments. The lowest survival rates were found in Elliott Bay treatments
containing 10, 15, and 20 worms per exposure chamber. In all cases, survival was not greater than
72 percent and decreased with increasing worm density (Table 6). In contrast to the treatments
exhibiting high survival rates, the coefficient of variation for treatments with low survival rates
was high, ranging from 21 to 38 percent.
A pairwise comparison of survival rates between Carr Inlet and Elliott Bay treatments with
the same worm densities showed that significant differences in survival rate existed for some
treatment pairs. No significant differences were found in survival between the Carr Inlet and
Elliott Bay sediments having a worm density of five worms per chamber. However, significant
differences were noted at all other worm densities (Table 7).
For both the Carr Inlet and Elliott Bay sediment treatments, the total biomass of tissue
collected from each treatment increased with the number of worms remaining in the replicate
chamber (Table 6; Figure 4). Highest total biomass values at a given worm density were found for
worms collected from the Carr Inlet sediment. Significant differences were noted in total biomass
for all Carr Inlet and Elliott Bay treatment pairs having the same worm density (Table 7). In all
cases, Carr Inlet treatments exhibited significantly higher total biomass than did Elliott Bay
treatments.
An ANOVA of the data for treatments containing Carr Inlet sediment indicates significant
differences in total biomass among the worm density treatments. An a posteriori analysis showed
that total biomass was lowest at a density of five worms, with other worm densities forming two
overlapping, nonsignificant subsets (Table 8). A similar comparison for the Elliott Bay sediment
indicated that no significant differences were found in total biomass among the worm density
treatments.
Average individual biomass (total biomass divided by the number of surviving worms per
exposure chamber) decreased with the number of surviving worms in each exposure chamber
(Table 6). Highest average individual biomass values were found for worms collected from the
Carr Inlet sediment. Significant differences were noted in average individual biomass for all Carr
Inlet and Elliott Bay treatment pairs having the same worm density (Table 7). In all cases, Carr
Inlet treatments exhibited significantly higher average individual biomass than the Elliott Bay
treatments.
The greatest numerical difference between treatment pairs occurred at a density of five
worms. At this density, average worm growth in Elliott Bay sediment was only about 21 percent
of the growth in Carr Inlet sediments. This results from two factors. First, average worm growth
in Carr Inlet sediment decreased with increasing worm density, probably resulting from food
limitation and possibly from aggressive interaction among worms. Second, the average individual
biomass in contaminated Elliott Bay sediments was consistently low and did not change as a
function of worm density.
An ANOVA of the individual biomass data for treatments containing Carr Inlet sediment
indicates significant differences among the treatments (Table 8). An a posteriori analyses indicated
that higher individual biomass was measured at a density of 5 worms. Lowest individual biomass
occurred at densities of 15 and 20 worms and these data were not distinguishable from each other.
No significant differences were noted in average individual biomass among the worm density
treatments in the Elliott Bay sediment.
24

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TABLE 7. PAIRWISE STATISTICAL COMPARISON
OF NEANTHES BIOASSAY RESPONSES FOR
THE WORM DENSITY EXPERIMENT

Worm Density
(no. worms/
chamber)4
Treatment
Comparison6
Response
Comparison
Result'
5
EB vs. CI
Percent survival
Total biomass
Individual biomass
ns
~
*
'10
EB vs. CI
Percent survival
Total biomass
Individual biomass
*
*
*
15
EB vs. CI
Percent survival
Total biomass
Individual biomass
~
*
*
20
EB vs. CI
Percent survival
Total biomass
Individual biomass
*
~
*
* Worm density treatment based on the initial number of worms placed in
each exposure chamber.
b EB- Elliott Bay
CI - Carr Inlet.
c * - significant, P<0.05
ns - not significant, P>0.05.
25

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TABLE 8. RESULTS OF ONE-WAY ANALYSIS
OF VARIANCE FOR BIOMASS DATA FROM
THE WORM DENSITY EXPERIMENT
Sediment Response	ANOVA
Type Parameter	Results8 Treatment Groupingb
Carr Inlet Total biomass	* CI5 CI10 CI 1S CI2Q
Individual biomass	* CI5 CI10 CI15 CI20
Elliott Bay Total biomass	ns
Individual biomass	ns
a * - significant, P<0.05
ns - not significant, P>0.05.
b Treatments grouped by the same line are	not significantly different based on an a posteriori
analysis.
26

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A power analysis model was used to estimate the ability to statistically discriminate differences
in average individual biomass between treatments containing Carr Inlet and Elliott Bay sediments.
At all four worm densities, the ability to detect significant differences between the two sediments
increases as the difference in average biomass increases (Figure 5). Treatments containing 5, 10,
or 20 worms per chamber displayed similar levels of statistical power to detect a given difference
in biomass between sediment types. For example, at three densities (5, 10, and 20) there is a power
of about 0.8 to detect a 40-50 percent difference in the overall mean. At a density of 15 worms,
the power was considerably less, resulting in a power of only 0.4 to detect a 50 percent difference
in overall mean individual biomass.
Based on the results of this experiment, it was concluded that the Neanthes sublethal bioassay
can be successfully conducted with worm densities ranging from 5 to 20 worms per exposure
chamber. Worms survived in all treatment combinations, and sufficient tissue was available at the
end of the 20-day exposure period to determine total and individual biomass.
Because the primary response criterion for the Neanthes sublethal bioassay is a change in
biomass, the recommended worm density at which the bioassay should be conducted should be one
at which worm survival is maximized. Maximization of survival will ensure the presence of
sufficient tissue to determine biomass at the conclusion of the exposure period. Although survival
was high at all worm densities for treatments containing West Beach and Carr Inlet sediments,
survival decreased as worm density increased in the contaminated sediment from Elliott Bay. Only
at a worm density of five worms per chamber was the survival in the Elliott Bay sediments greater
than 80 percent.
Results of the power analysis indicate that at the lower worm density, relatively small changes
in biomass can be statistically detected. Increasing the number of worms per exposure chamber
results in no increased ability to detect significant effects. Due to the observed high survival rate,
the cost savings using only five worms, and because sufficient statistical power can be achieved at
the density of five worms per chamber, it is recommended that all Nemthes sublethal bioassays be
conducted using this density. Therefore, all subsequent experiments in this study were conducted
using five worms per exposure chamber.
Food Ration
Mean survival of Neanthes juveniles following the 20-day exposure to Elliott Bay, Carr Inlet,
and West Beach sediments ranged from a high of 100 percent to a low of 0 percent (Table 9;
Figure 6). Survival was consistently high (>92 percent) for all treatments containing Carr Inlet
and West Beach sediments and for the Elliott Bay sediment in which the food ration was 80 mg/
48 hours. The coefficient of variation for the survival data for these treatments was low to
moderate, ranging from 0 to 18 percent.
The lowest survival rates were found in treatments containing Elliott Bay sediment and a food
ration of less than 80 mg/48 hours. In all cases, survival was lower than 76 percent, with mortality
increasing as food rations decreased (Table 9). Unlike the data for treatments exhibiting high
survival rates, the coefficient of variation for treatments with low survival rates was moderate to
high, ranging from 17 to 43 percent.
27

-------
Power
1
0.8
0.6
0.4
0.2
0
0
. ^ V'%0'p
¦'A f B
// -0

A
/ *
x
/ / 0

0
-/- r ^
A / t-A
4 /r^
-r > 0
/ / 0
V
4 / 0
^/ pi
#/<0
/ / 0
A/0
/#
?-/\Z
44*
0\ ].
\ipK
y T>
*
:L^'
tM
M

_,-

**¦

5 Worms
10 Worms
15 Worms
20 Worms
20	40	60	80
Minimum Detectable Difference (% mean)
*
100

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TABLE 9. SURVIVAL, TOTAL BIOMASS, AND
AVERAGE INDIVIDUAL BIOMASS DATA FOR
THE FOOD RATION EXPERIMENT
Sediment
Food Ration4
Survivalb
Total Biomass6
(mg dry weight)
Average
Individual Biomass1
(mg dry weight)
West Beach
0
100 ± 0.0
6.5 ± 0.6
1.3 ± 0.1

40
96.0 ± 4.0
106.1 ± 5.9
22.1 ± 1.7

80
92.0 ± 8.0
109.8 ± 9.5
23.0 ± 2.0
Carr Inlet
0
92.0 ± 4.9
2.8 ± 0.3
0.6 ± 0.1

20
88.0 ± 4.9
65.1 ± 8.9
14.6 ± 1.3

40
96.0 ± 4.0
112.5 ± 10.9
23.2 ± 1.6

60
92.0 ± 4.9
124.5 ± 19.2
27.2 ± 4.6

80
96.0 ± 4.0
163.8 ± 8.4
34.4 ± 2.3
Elliott Bay
0
0.0 ± 0.0
0.0 ± 0.0
	d

20
16.0 t 7.5
2.5 ± 1.7
4.1 ± 2.6

40
76.0 ±11.7
18.1 ± 3.7
4.8 ± 0.7

60
76.0 ± 19.4
27.6 ± 7.7
7.3 ± 0.8

80
92.0 ± 8.0
46.5 ±11.7
9.8 ± 2.1
a Value presented as mg of food provided every 48 hours.
b Value reported as mean ± standard error.
c Biomass of worms at test initiation was 0.8 ±0.1 mg (dry weight).
d Individual biomass could not be determined.
29

-------
Percent Survival
140,	
120 -
0	20	40	60	60
Total Biomass (mg dry weight)
200 r	—
150 f™
100
so
so
0
20
40
80
Individual Biomass (mg dry weight)
80 i	
40
30
—1 (
20
10
40
80
0
20
eo
Food Ration (mg/48 hours)
i
x West Beach —Carr Inlet A Elliott Bay
Figure 6. Survival (a), biomass (b), and individual biomass (c) of juvenile worms from the
food ration experiment. Bars represent standard error.
30

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The addition of food appeared to ameliorate the lethal effects of Elliott Bay sediments. With
no added food, there was a 100 percent mortality of test organisms in Elliott Bay sediments. The
addition of increasing amounts of food resulted in corresponding increases in the survival of test
organisms. A pairwise comparison of survival rates between Carr Inlet and Elliott Bay treatments
having a similar food ration showed that significant differences in survival rate existed only at the
lower food levels (i.e., no food and 20 mg/48 hours) (Table 10).
For both Carr Inlet and Elliott Bay sediment treatments, the total biomass of tissue collected
from each treatment increased with the number of worms remaining in each replicate chamber
(Table 9; Figure 6). Highest total biomass values were found for worms collected from the Carr
Inlet sediment. Significant differences were noted in total biomass for all Carr Inlet and Elliott
Bay treatment pairs having the same worm density (Table 10). In all cases, Carr Inlet treatments
exhibited significantly higher biomass values than did Elliott Bay treatments.
An ANOVA of the total biomass data for treatments containing Carr Inlet sediment indicates
significant differences among the treatments (Table 11). The treatment with the highest food
ration (i.e., 80 mg/48 hours) exhibited a higher total biomass than all other treatments. Significant
differences in total biomass were also noted between the other treatments. An ANOVA of the total
biomass data for the Elliott Bay sediment also indicates significant differences among the treatments
(Table 11). Differences are due to the higher food ration treatments (i.e., 60 and 80 mg/48 hours)
having a higher total biomass than treatments provided with lower food rations.
Average individual biomass increased as food rations increased (Table 9; Figure 6). Highest
individual biomass values were found for worms collected from the Carr Inlet sediment. Lowest
individual biomass values were observed for worms recovered from the Elliott Bay sediment.
For both biomass endpoints (total and individual), growth appeared to increase as food rations
increased up to the maximum feeding rate (Figure 6). With the exception of individual biomass
in the Elliott Bay treatment, the statistical results do not indicate the existence of a food saturation
level up to a ration of 80 mg/48 hours. However, the relative increases in biomass with increasing
food ration were generally lower from 40-80 mg/48 hours than from 0-40 mg/48 hours.
Although biomass increased as food rations increased for all three sediments tested, the Elliott
Bay treatments with food rations of 60 and 80 mg/48 hours had excess food and fungal growth on
the sediment surface of several exposure chambers. Excess food was also observed at the sediment
surface of one replicate of the 80 mg/48 hours Carr Inlet treatments.
Because of lower survival rates in toxic sediments at food rations below 40 mg/48 hours and
the fact that excess food and fungal growth were observed in treatments containing greater than
60 mg of food every 48 hours, it is recommended that a ration of 40 mg TetraMarin® be provided
every 48 hours to each exposure chamber when conducting the Neanthes sublethal bioassay.
Although the addition of no food during testing would result in a cost savings, it is not
recommended because of the very low growth rate observed in all sediments. It should be noted
that TOC could be used as a food source by Neanthes if the carbon is in a labile form and is
present in high concentrations. Worms did not appear to obtain any significant nutritional value
from the sediments used in this study (based on the results from the treatments containing no
added food source).
31

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TABLE 10. PAIRWISE STATISTICAL COMPARISON
OF NEANTHES BIOASSAY RESPONSES BETWEEN
ELLIOTT BAY AND CARR INLET TREATMENTS
FOR THE FOOD RATION EXPERIMENT

Food Ration*
Treatment
Comparison6
Response
Comparison
Result'
No food
EB vs. CI
Percent survival
4


Total biomass
*


Individual biomass
*
20 mg/48 hours
EB vs. CI
Percent survival
~


Total biomass
*


Individual biomass
~
40 mg/48 hours
EB vs. CI
Percent survival
ns


Total biomass
*


Individual biomass
*
60 mg/48 hours
EB vs. CI
Percent survival
ns


Total biomass
*


Individual biomass
*
80 mg/48 hours
EB vs. CI
Percent survival
ns


Total biomass
*


Individual biomass
*
* Neanthes were provided a level of TetraMarin® as a food source during the
20-day exposure period.
b EB - Elliott Bay
CI - Carr Inlet.
c * - significant, P<0.05
ns - not significant, P>0.05.
32

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TABLE 11. RESULTS OF ONE-WAY ANALYSIS
OF VARIANCE FOR BIOMASS DATA FROM
THE FOOD RATION EXPERIMENT

Sediment
Response
ANOVA


Type
Parameter
Results8

Treatment Grouping15
Carr Inlet
Total biomass
*
CIO
CI20 CI40 CI60 CI80

Individual biomass
*
CIO
CI20 CI40 CI60 CI80
Elliott Bay
Total biomass
»
EBO
EB20 EB40 EB60 EB80

Individual biomass
ns


5 * - significant, P<0.05
ns - not significant, P>0.05.
b Treatments grouped by the same line are not significantly different based on an a posteriori
analysis.
33

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Static vs. Static Renewal Exposure System
With the exception of the Elliott Bay treatment employing the static exposure system, mean
survival in all treatments was greater than 92 percent (Table 12; Figure 7). In the Elliott Bay
treatment employing the static system, mean survival of the five replicates was 60 percent. The
coefficient of variation for all treatments exhibiting high survival was low, ranging from 0 to 11
percent. The coefficient of variation in the one treatment with a low survival rate was 37 percent,
resulting from a range of 20 to 100 percent survival in the individual replicates. A pairwise
comparison of survival data between the static and static renewal exposure systems for Elliott Bay
and Carr Inlet treatments showed that no significant differences (P>0.05) in survival rate existed
(Table 13).
For each sediment type, worm growth was similar under static and static renewal conditions.
No significant differences in total biomass were detected between worms exposed in the static and
in the static renewal exposure systems for a given sediment type (Table 13). Significant differences
in total biomass were detected, however, between worms exposed to the different sediments.
Regardless of the type of exposure system employed, the total biomass of worms exposed to the
Elliott Bay sediment was significantly lower than the total biomass of worms exposed to either the
Carr Inlet or the West Beach sediments (Table 12).
As observed with total biomass, no significant differences in average individual biomass were
detected within each sediment between worms exposed in the static and the static renewal exposure
systems (Table 13). Significant differences in individual biomass were detected, however, between
worms exposed to the different sediments (Table 12). The individual biomass of worms exposed
to the Elliott Bay sediment in both exposure systems was significantly lower than the individual
biomass of worms exposed to either the Carr Inlet or the West Beach sediments.
Monitoring of water quality during the test identified differences between the static and static
renewal exposure systems. For both the Carr Inlet and Elliott Bay sediments, ammonia levels
tended to be higher in exposure chambers using the static exposure system than chambers having
the static renewal exposure system (Table 14).
For treatments containing Elliott Bay sediment, concentrations of copper were similar in water
samples taken from the static and static renewal systems (Table 15). Total copper concentrations
in the static exposure system ranged from 30 to 53 ng/L during the exposure period, with dis-
solved copper concentrations ranging from 18 to 41 fig/L. Overall, the dissolved fraction of copper
accounted for 60-93 percent of the concentration of copper in the static exposure system water.
Total copper concentrations in the static renewal exposure system ranged from 35 to 61 pg/L
during the exposure period. Dissolved copper, which accounted for 61 to 94 percent of the copper
concentration, ranged from 22 to 39 ^g/L.
Mercury was not detected (detection limit of 0.1 pg/L) in any of the water samples taken from
treatments for either the static or the static renewal exposure systems. PAH was also not detected
(detection limit of 1 Mg/L) in any of the water samples taken from the static and static renewal
exposure systems.
Contaminants present in the water were periodically removed from the exposure chamber
when using the static renewal system. However, analysis of the contaminant mass that was removed
relative to the mass present in the sediment indicates that the loss was minimal (Table 16). For
copper, the only contaminant consistently detected in the water samples, less than 1 percent
34

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TABLE 12. SURVIVAL, TOTAL BIOMASS, AND
AVERAGE INDIVIDUAL BIOMASS DATA FOR
THE EXPOSURE SYSTEM EXPERIMENT

Sediment
Exposure System
Survival®
Total Biomass*
(mg dry weight)
Average
Individual Biomassa,b
(mg dry weight)
West Beach
Static
100 ± 0.0
118.9 ± 7.9
23.8 ± 1.6

Static renewal
100 ± 0.0
112.9 ± 10.9
22.6 ± 2.2
Carr Inlet
Static
96.0 ± 4.0
90.8 ± 14.6
18.5 ± 2.6

Static renewal
100 ± 0.0
105.8 ± 6.8
21.2 ± 1.3
Elliott Bay
Static
60.0 ± 16.7
11.5 ± 5.3
3.6 ± 1.1

Static renewal
92.0 ± 4.9
11.1 ± 1.8
2.5 ± 0.4
8 Value reported as mean ± standard error.
b Biomass of worms at test initiation was 1.0 + 0.1 mg (dry weight).

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STATIC
Percent Survival	Biomass (mg dry weight)
Survival	Total Biomass Individual Biomass
STATIC RENEWAL
Percent Survival	Biomass (mg dry weight)
Survival	Total Biomass Individual Biomass
West Beach
MM Carr In 1 e 1
Elliott Bay
Figure 7. Survival and biomass of juvenile worms from the exposure system experiment
36

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TABLE 13. PAIRWISE STATISTICAL COMPARISON
OF NEANTHES BIOASSAY RESPONSES FOR
THE EXPOSURE SYSTEM EXPERIMENT


Treatment

Comparison
Exposure System
Comparison*
Response
Result*
Static'
EB vs. CI
Percent survival
ns


Total biomass
*


Percent biomass
*
Static renewald
EB vs. CI
Percent survival
ns


Total biomass
*


Percent biomass
*
a EB- Elliott Bay
CI - Carr Inlet.
b * - significant, F<0.05
ns - not significant, P>0.05.
c In the static exposure system, no water changes were made during the 20-
day exposure period.
d In the static renewal exposure system, one-third of the water volume was
exchanged with fresh seawater every third day.
37

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TABLE 14. AMMONIA CONCENTRATIONS
FROM STATIC AND STATIC RENEWAL EXPOSURE
SYSTEMS DURING A 20-DAY EXPOSURE PERIOD
Ammonia Concentration
(mg/L)
Sediment
Exposure System
Day 3
Day 6
Day 12
Day 20
Carr Inlet
Static
a
3.20
0.62
0.12

Static renewal
1.20
2.90
0.33
0.11
Elliott Bay
Static
1.60
4.48
4.63
0.29

Static renewal

3.51
3.04
0.08
a Sample not collected for this treatment.
38

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TABLE 15. CONTAMINANT CONCENTRATIONS FROM
STATIC AND STATIC RENEWAL EXPOSURE SYSTEMS
CONTAINING ELLIOTT BAY SEDIMENT
Contaminant Concentration4
(Mg/L)
Exposure system
Contaminant
Day 3
Day 6
Day 12
Day 20
Static
PAHb
1U
1U
1U
1U

Total copper
30
53Z
43Z
44Z

Dissolved copper
18
32Z
33Z
41Z

Total mercury
0.1U
0.1U
0.1U
0.1U

Dissolved mercury
0.1U
0.1U
0.1U
0.1U
Static renewal
PAH
__c
1U
1U
1U

Total copper
--
61Z
35Z
35Z

Dissolved copper
--
39Z
22Z
32Z

Total mercury
--
0.1U
0.1U
0.1U

Dissolved mercury
—
0.1U
0.1U
0.1U
a Qualifier codes:
U - undetected at detection limit shown
Z - blank-corrected, still above detection limit.
b Polycyclic aromatic hydrocarbons (PAH) analyzed as part of this study are reported in Table 4.
c Sample not collected.
39

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TABLE 16. MASS LOSS OF CONTAMINANTS IN THE STATIC RENEWAL
EXPOSURE SYSTEM CONTAINING ELLIOTT BAY SEDIMENT

Compound
Sediment
Mass (Mg)
Water
Concentration
(Mg/L)
Mass Load Removed with
with Wuer Exchange0 (fig)
Percent Loss
of Contaminant''
Percent
Contaminant Remaining
in Sediment
PAH
2-Methylnaphthalene
30
<1
<1.8
<6.0
>94
Pyrcne
1,392
<1
<1.8
<0.1
>99.9
Copper
17,284
44
79.2
0.5
99.5
Mercury
406
<0.1
<0.2
<0.05
>99.95
a Mass determined as the product of average concentration of chemical and total water volume removed during the entire exposure period.
b Percentage loss determined by dividing chemical mass removed by the sediment concentration of the contaminant.

-------
of the mass present in the sediment was removed during the 20-day exposure period. Although
mercury and PAH were not detected in the water samples, the maximum potential loss of these
compounds can be approximated by assuming the concentrations were at the detection limits of
0.1 pg/L and 1 isg/L for mercury and PAH, respectively. For mercury, the maximum potential loss
relative to the sediment mass was less than i percent. For PAH, the maximum potential loss
ranged from less than 1 percent for pyrene to 6 percent for 2-methylnaphthalene.
Although there are potential cost savings in using a static exposure system, it was concluded
that the Neanthes sublethal bioassay should continue to be conducted with the static renewal
exposure system. Overall water quality appeared to be better in the static renewal system than in
the static system. Survival of worms to 20 days was high in all sediment types when the static
renewal system was employed. In contrast, low survival rates were observed in the static exposure
system for worms exposed to Elliott Bay sediment. Elliott Bay sediment has a higher organic
content (2.1 percent TOC) than the other test sediments. Furthermore, use of the static renewal
exposure system does not appear to result in significant loss of contaminants from the exposure
system.
Test Duration
The survival rate at the end of all three exposure periods was greater than 76 percent in all
three sediment treatments (Table 17; Figure 8). No mortality was observed in the West Beach
control, while the lowest survival rate observed in the Carr Inlet sediment was 96 percent for the
10-day exposure period. Survival in the Elliott Bay sediment was lower than in the other sediments
and ranged from 76 to 84 percent, A pairwise comparison of survival rates between Carr Inlet and
Elliott Bay treatments for the three exposure times showed no significant difference in survival
rates (Table 18). Worms maintained in the West Beach and Carr Inlet sediments exhibited
increases in total and average individual biomass with increasing exposure periods (Figure 8).
Growth curves for worms from both the Carr Inlet and the West Beach sediments indicated that
a significant proportion of the biomass gained during the 20-day exposure period occurred between
days 10 and 20. Total biomass increases between test initiation and day 10 ranged from 20 to 24
mg (dry weight). Total biomass increases between day 10 and day 20 ranged from 44 to 55 mg
(dry weight) (Table 17).
Worms maintained in the Elliott Bay sediments exhibited little change in total biomass and
average individual biomass between the 10-day and the 20-day exposure periods (Table 17). For
these worms, average biomass of individual worms ranged from 1.1 to 1.4 mg (dry weight) per
worm. Total biomass was also relatively stable during the 20-day exposure period. During this
same period, the biomass of individual worms maintained in West Beach and Carr Inlet sediments
increased by approximately 2.9 times.
A pairwise comparison of the biomass data for worms from the Elliott Bay sediment to worms
from the Carr Inlet sediment indicated that significant differences in both total biomass and average
individual biomass existed at all three exposure periods (Table 18).
A power analysis model was used to determine the change in the ability to detect a statistically
significant difference in total biomass between worms maintained in Elliott Bay sediment and
those maintained in Carr Inlet sediment with increasing exposure periods. At all three exposure
periods, the ability to discriminate between the Elliott Bay and the Carr Inlet sediments increases
as the difference in mean biomass increases (Figure 9). However, much smaller differences in
41

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TABLE 17. SURVIVAL, TOTAL BIOMASS, AND
AVERAGE INDIVIDUAL BIOMASS DATA FOR
THE TEST DURATION EXPERIMENT
Average
Exposure Period	Total Biomass" Individual Biomassi'b
Sediment	(days)	Survival® (mg dry weight) (mg dry weight)
West Beach
10
100 ±
o
o
23.9
+
4.7
4.8
± 1.0

15
100 ±
0.0
39.9
±
1.5
8.0
± 0.4

20
100 ±
0.0
67.8
±
4.9
13.5
± 1.0
Carr Inlet
10
96.0 ±
4.0
27.9
±
4.0
5.7
± 0.7

15
100 ±
0.0
55.1
±
6.7
11.0
± 1.4

20
100 ±
0.0
83.0
±
2.8
16.6
± 0.6
Elliott Bay
10
00
O
o
H-
6.3
5.8
±
1.0
1.4
± 0.2

15
76.0 ±
11.7
4.2
±
0.9
1.1
t 0.1

20
84.0 ±
7.5
4.5
±
0.8
1.1
± 0.1
a Value reported as mean ± standard error.
b Biomass of worms at test initiation was 0.8 ± 0.1 mg (dry weight).
42

-------
Percent Survival
60 •
a
401~
20 -
0	1			1	L-
0	6	10	15	20
Total Blomaas (ng dry weight)
100 -
Individual Biomass (mg dry weight)
20
10
20
0
5
IS
Exposure Period (Days)
West Beach	$ Carr Inlet	Elliott Bay
Figure 8. Survival (a), total biomass (b), and individual biomass (c) of juvenile worms from
the test duration experiment. Bars represent standard error.
43

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TABLE 18. PAIRWISE STATISTICAL COMPARISON
OF NEANTHES BIOASSAY RESPONSES FOR
THE TEST DURATION EXPERIMENT

Exposure



Period
Treatment

Comparison
(days)
Comparison®
Response
Result6
10
EB vs. CI
Percent survival
ns


Total biomass
~


Individual biomass
*
15
EB vs. CI
Percent survival
ns


Total biomass
*


Individual biomass
*
20
EB vs. CI
Percent survival
ns


Total biomass
*


Individual biomass
*
8 EB - Elliott Bay
CI - Carr Inlet.
b * - significant, P<0.05
ns - not significant, P>0.05.
44

-------
Power
0.8
?r
0.6
0.4
0.2
Exposure Duration
—10 Day	15 Day ^ 20 Day
80
0
20
40
60
100
120
140
Minimum Detectable Difference (% mean)
Figure 9. Statistical power vs. minimum detectable difference for total biomass in the test duration experiment

-------
mean values can be discriminated following a 20-day exposure than following 10- and 15-day ex-
posure periods. For example, at a power of 0.8, the MDD in mean values that can be detected
following 20 days is 21 percent, while the MDD is 78 and 74 percent following 10- and 15-day
exposure periods, respectively. Therefore, although statistical differences were detected between
Carr Inlet and Elliott Bay treatments for all three exposure periods, there is a substantially higher
probability of detecting effects at the longer exposure period.
The growth characteristics of Neanthes juveniles appear to follow the pattern generally
observed in marine invertebrates in which the life cycle is characterized by periods of rapid growth
(Rossi and Anderson 1976; Petrich and Reish 1979). Typically, these periods of rapid growth occur
prior to attaining sexual maturity. In Neanthes, development time to sexual maturity is
approximately 5-6 weeks. Thus, the 20-day exposure period originally used with the Neanthes
sublethal bioassay was designed to incorporate the effects of being exposed during a significant
portion of the growth-critical juvenile life stage. The 20-day exposure period accounts for between
48 to 57 percent of the juvenile life stage in Neanthes (Reish 1980).
The results of this experiment indicate that statistical differences between growth patterns in
worms exposed to clean and contaminated sediments can be detected following exposure periods
of 10 or 15 days. As exposure time increases to 20 days, however, the power to detect statistical
differences increases substantially. Of the three exposure periods examined in this experiment,
statistical power is the greatest following a 20-day exposure period. In this experiment, statistical
power was similar for the 10- and 15-day exposures. The similarity of these results was caused
by the relatively high variance associated with the 15-day exposure. To reduce the influence of
this possible experimental artifact, the power analyses were recalculated using an average of the
within-group mean squares for the individual ANOVAs. This value was used as an estimate of the
overall average residual variance and is independent of any specific results associated with
individual experiments. Power analyses using this average error estimate (Figure 10) show that a
15-day exposure would result in a statistical power intermediate between the 10- and 20-day
exposures. The results of the statistical power analysis of the biomass data indicate that the 20-
day exposure period recommended in the interim protocol is the most suitable period for evaluating
the effects of exposure to contaminated sediments on the growth of juvenile Neanthes.
Although a potential cost savings could be achieved by using a shorter exposure period, it is
recommended that the exposure period for the Neanthes sublethal bioassay remain at 20 days.
Because a 20-day exposure period covers a significant portion of the juvenile life stage, care must
be taken to ensure that the worms are only 2-3 weeks postemergence at the initiation of the
bioassay (Johns et al. 1989). If worms older than 3 weeks are used, test data may not be valid
because of the marked decrease in tissue production observed as Neanthes reach sexual maturity
(Johns and Ginn 1990).
Salinity Tolerance
Neanthes juveniles were acutely sensitive to low salinity waters based on a 96-hour exposure
to salinities ranging from 10 to 28 ppt (Table 19). No mortalities were observed at salinities above
20 ppt. Below 20 ppt, however, mortality rate increases as salinity decreases, with the LCJ0 value
approximated at 15 ppt.
In the second salinity experiment, mean survival rates were high in all sediment treatments,
ranging from 88 to 100 percent (Table 20). Neanthes growth was similar in all sediment types in
46

-------
I
Power
0.8
0.6
0.4
0.2 \-
***
+ n 111: n i-f-i -f-
-¥¦

f-
4-

/
/ /
f /
/
/
-f
/
J V
*
-f~
/'
/*
y
,/


o
L.
S
/
/
/
Exposure Duration
10 day I" 15 day
_L
0
20 day
20 40 60 80 100 120 140
Minimum Detectable Difference (% mean)
160
Figure 10. Statistical power vs. minimum detectable difference for total biomass in the test duration experiment calculated using
average mean square error

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TABLE 19. SURVIVAL DATA FOR THE
96-HOUR SALINITY TOLERANCE EXPERIMENT
Salinity
(ppt)	Percent Survival"
28	100 ± 0.0
25	100 ± 0.0
20	100 ± 0.0
15	50 ± 10
10	0.0 ± 0.0
4 Value reported as mean ± standard error.
48

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TABLE 20. SURVIVAL, TOTAL BIOMASS, AND
AVERAGE INDIVIDUAL BIOMASS DATA FOR THE
NEANTHES SALINITY TOLERANCE EXPERIMENT
Sediment
Initial
Interstitial
Salinity
(Ppt)
Survival®
Total
Biomass
(mg dry weight)"
Average
Individual
Biomass
(mg dry weight)**
West Beach
28
96.0±4.0
47.715.8
9.9+1.0
Carr Inlet
28
100.0+0.0
51.313.8
10.310.8
Duckabush River
30
100.010.0
49.915.3
10.012.4

25
88.014.9
39.017.4
9.012.1

22
100.010.0
59.9115.4
12.013.1

19
100.010.0
42.717.7
8.510.4
* Value reported as mean ± standard error.
b Biomass of worms at test initiation was 0.4 ±0.1 mg (dry weight).

-------
this experiment (Table 20). No significant differences (P>0.05) in totai biomass or average
individual biomass were detected among the treatments.
Although no significant differences in worm response were noted following a 20-day exposure
to sediments having initially low salinity interstitial water, it should be noted that Neanthes is
acutely sensitive to salinity exposures below 20 ppt. Thus, caution should be used when performing
and interpreting the results of Neanthes bioassays conducted with sediments collected from low
salinity areas. It should be recognized that this experimental design did not allow evaluation of
worm response to interstitial waters below a salinity of 19 ppt. Further testing may be required
to define the effects of low interstitial salinity on worm survival and growth.
Differences between the results observed in the 96-hour seawater test and the 20-day sediment
test may be attributable to behavioral modifications and to the effects of overlying water in the
exposure chamber. In the sediment exposure experiment, worms placed in treatments containing
low salinity sediments could effectively avoid the low salinity by remaining close to the sediment
surface where the overlying salinity was 28 ppt. Burrowing activity could also facilitate the mixing
of interstitial water with overlying water, thus increasing the interstitial salinity. Based on the test
data for the two salinity experiments, Neanthes bioassays should be conducted with interstitial
salinities greater than 20 ppt when possible. If the bioassay is conducted with salinities lower than
20 ppt, care should be taken in ascribing changes in Neanthes response solely to factors other than
salinity.
Sensitivity to Sediment Grain Size
Mean survival was consistently high (96-100 percent) for Neanthes juveniles exposed to
uncontaminated sediments having silt/clay fractions between 53 and 97 percent (Table 21). In
addition, no mortality was observed in the West Beach control, which is primarily sand (silt/clay
fraction = 2 percent). A pairwise comparison of survival data for the various grain-size treatments
to data for Carr Inlet showed that no significant differences in survival rate exist.
Neanthes growth was also very similar among the ranges of particle sizes tested (Table 21).
Total biomass at 20 days ranged from 65.7 to 72.9 mg over the range of sediment types tested.
Statistical analysis by ANOVA revealed no significant difference (P>0.05) in total or individual
biomass.
In this experiment, the response criteria used in the Neanthes sublethal bioassay (survival, total
biomass, and average individual biomass) do not appear to be sensitive to changes in sediment grain
size, even when it ranges from very coarse-grained sands to silty material. During sieving procedu-
res conducted at the end of the experiment, it appeared that surviving worms were able to construct
and maintain tubes in all sediment types. In their natural habitat, Neanthes live in subsurface
tubes constructed of mucus and material collected from the surrounding environment. If not
offered sediment in the laboratory, Neanthes will build a mucus tube on the container surface
incorporating food particles, fecal material, and available debris into the tube matrix.
The results of this experiment indicate that Neanthes is able to survive and grow in a wide
range of sediment types. Sediment type, based on the silt and clay composition, should not affect
the suitability of the Neanthes sublethal bioassay for assessing most marine sediments. It should
be noted, however, that statistical differences in worm growth were noted between worms exposed
to West Beach and Carr Inlet sediments in three of the experiments (i.e., worm density, test
50

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TABLE 21. SURVIVAL, TOTAL BIOMASS, AND
AVERAGE INDIVIDUAL BIOMASS FOR THE
SEDIMENT GRAIN-SIZE EXPERIMENT
Total Biomass
Average
Individual
Biomass
Sediment Type
Grain Size"
Survival
(mg dry weight)b
West Beach
2
100+0.0
65.7±6.4
CR01
53
100±0.0
73.2±7.1
CR04
71 .
100±0.0
65.9±5.3
CR02
89
96.0±4.0
75.4±9.7
CR05
93
100±0.0
62.7±6.8
CR03
97
96.0+4.0
72.9±7.1
sb.c
13.2±1.3
14.6+1.4
13.2± 1.1
15.5±1.6
12.5± 1.4
15.5+2.1
a Grain size reported as percent silt/clay.
b Value reported as mean ± standard error.
c Biomass of worms at test initiation was 0.5 ± 0.2 mg (dry weight).
5J

-------
duration, and food ration at 80 mg/48 hours) conducted as part of this study. Because of the
potential for statistically detecting differences in growth for worms exposed to clean sediment of
widely differing grain size, the reference sediment used in Neanthes bioassays should have a similar
grain size to the test sediments to avoid potential differences in organism response related to the
physical characteristics of the sediment.
Sensitivity of Neanthes to a Reference Toxicant
Neanthes juveniles were found to be acutely sensitive to cadmium exposure. The 96-hour
LC50 value for cadmium is 22 ppm (Table 22). This value is similar to the LCJ0 value (12 ppm)
reported by Reish (1984) for adult Neanthes.
It is recommended that a reference toxicant control using CdCI2 be required when any
Neanthes sublethal bioassay is performed for an environmental regulatory program. Data from the
toxicant control can be used to determine the relative sensitivity of the particular test organisms
in each bioassay series to ensure comparability of test results.
Response of the Biomass Endpoint and Statistical Power
A comparison of the relative percent reduction in biomass between worms maintained in Can-
Inlet and Elliott Bay sediments was made to evaluate the consistency of the biomass response
endpoint. Data for all treatment conditions from the six experiments in which Carr Inlet, West
Beach, and Elliott Bay sediments were tested are included in this comparison. For the interim
protocol exposure conditions, the range in percent reduction (Elliott Bay vs. Carr Inlet) observed
for the total biomass endpoint was from 78 to 95 percent (Table 23). The total biomass response
for Elliott Bay and Carr Inlet treatments was also relatively consistent among the different exposure
conditions evaluated in this study. For the total range of test conditions examined, the percent
reduction in total biomass in Elliott Bay exposures ranged from 72 to 96 percent (Table 23). These
results show a relatively high degree of consistency in the response of Neanthes when exposed to
the same sediments over a period of 4 months.
Table 23 also contains a compilation of all two-sample comparisons of the total biomass in
Elliott Bay and West Beach exposures with the total biomass in reference sediment exposures from
Carr Inlet. Two measures of percent difference are compiled: the percent change from reference
biomass and the percent change from the overall mean biomass. The two-sample statistical results
conducted for this study show the lowest differences that were found to be statistically signifi-
cant (P<0.05) were 18 and 10 percent relative to the reference and mean values, respectively.
Differences greater than 33 percent (from the reference) and 20 percent (from the mean) were
always significant in the test results.
The actual statistical results presented in Table 23 agree well with the power analysis results
presented in Figures 5, 9, and 10. Depending on individual experimental variances, the power
analyses predict a moderate (~ 50 percent) to high (- 90 percent) statistical probability of detecting
a 30 percent change relative to the overall mean. The power analyses indicate that there would be
a low probability of detecting differences of less than 10 percent from the mean biomass. In the
test results, all differences from the mean biomass of less than 10 percent were nonsignificant
(P>0.05).
52

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TABLE 22. SURVIVAL DATA FOR 96-HOUR LCM
TEST CONDUCTED WITH NEANTHES JUVENILES
USING CADMIUM CHLORIDE
Cadmium
Concentration
Test Series
(ppm)
Survival"
Test I
0
100 ± 0.0

1
100 ± 0.0

10
100 ± 0.0

100
0.0 ± 0.0

1,000
0.0 ± 0.0
Test II
0
100 ± 0.0

10
100 ± 0.0

18
80 ± 0.0

32
0.0 ± 0.0

56
0.0 ± 0.0

100
0.0 ± 0.0
• Value reported as mean ± standard error.
53

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TABLE 23. RESULTS OF STATISTICAL COMPARISONS
AND PERCENT DIFFERENCES BETWEEN TEST SITES
FOR NEANTHES BIOASSAYS
	Percent Difference	
Comparison	Statistical	From	From
Test8	(test, reference)1"	Results6	Reference®1	Overall Mean6
WD-5
EB,
CI
•
78
64
WD-5
WB,
CI
*
72
56
WD-10
EB,
CI
*
78
64
WD-15
EB,
CI
*
87
77
WD-20
EB,
CI
*
85
74
FR-20
EB,
CI
*
96
93
FR-40
EB,
CI
*
84
72
FR-40
WB,
CI
ns
6
3
FR-60
EB,
CI
*
78
64
FR-80
EB,
CI
*
72
56
FR-80
WB,
CI
*
33
20
S
EB,
CI
*
87
78
S
WB,
CI
ns
31
13
SR
EB,
CI
*
90
81
SR
WB,
CI
ns
7
3
TD-20
EB,
CI
~
95
90
TD-20
WB,
CI
*
18
10
TD-15
EB,
CI
*
92
86
TD-15
WB,
CI
ns
28
16
TD-10
EB,
CI
*
79
66
TD-10
WB,
CI
ns
14
8
GS
3=
CO
CI(01)
ns
11
5
S
WB,
CI
ns
7
4
* WD
- worm density (organisms per exposure chamber)
FR
- food ration (mg/48 hours)
S
- static exposure
SR
- static renewal exposure
TD
- test duration (days)
GS
- grain size
S
- salinity.
b EB
- Elliott Bay
CI
- Carr Inlet
WB
- West Beach.
c *
- significant, P<0.05
ns
- not significant, P>0.05.
100 (l test
reference
100
f ( 	test	\1
|^1 - ^ test+reference J J
2
54

-------
SUMMARY
Based on the results presented in this report, the interim protocol for conducting the sublethal
Neanthes bioassay described by Johns et al. (1989) can be finalized with little revision. The results
of experiments indicate that the test parameters identified in the interim protocol were suitable for
test refinement. Specifically, the experiments showed that:
¦	A worm density of five worms per exposure chamber results in sufficient biomass
to statistically detect differences among treatments. Increasing worm density did not
result in an increased ability to detect significant effects.
¦	A food ration of 40 mg/48 hours is sufficient to promote adequate growth in
Neanthes juveniles during a 20-day exposure period. Although the experimental
results did not indicate the existence of a food saturation level up to a ration of 80
mg/48 hours, increasing the food ration above 40 mg/48 hours resulted in excess
food and fungal growth on the sediment surface in some exposure conditions. Food
rations below 40 mg/48 hours resulted in lowered survival rates in the exposure
chambers containing contaminated sediment.
¦	The static renewal exposure system should be employed in all Neanthes sublethal
bioassays conducted with sediment. Use of the static renewal exposure system
resulted in high survival in all sediment types, and overall water quality appeared
to be better in the static renewal system than in the static exposure system. In
addition, use of the static renewal exposure system does not appear to result in
significant loss of contaminants.
¦	Test duration for the Neanthes sublethal bioassay should remain at 20 days.
Although significant differences in growth between treatments can be detected at
exposure times of less than 20 days, the experiment results indicated that statistical
power is greatest following a 20-day exposure period.
¦	Based on the test data for the two salinity experiments, Neanthes bioassays should
be conducted with interstitial salinities greater than 20 ppt. If the bioassay is going
to be conducted with sediment containing interstitial salinities lower than 20 ppt,
care should be taken in ascribing changes in Neanthes response to factors other than
salinity. In all cases, the salinity of the overlying water in the exposure chamber
should be 28 ppt.
¦	Sediment grain size does not appear to affect the suitability of the Neanthes sublethal
bioassay for assessing marine sediments. Because differences in growth could occur
due to sediment grain size or TOC, the reference sediment should be similar to the
test sediment for these two factors.
¦	Comparisons of the relative percent reduction in biomass between worms maintained
in Carr Inlet and in Elliott Bay sediments show a high degree of consistency in the
response of Neanthes.
55

-------
REFERENCES
AMPHA. 1985. Standard methods for the examination of water and wastewater. American Public
Health Association. American Water Works Association, and Water Pollution Control Federation,
Washington, DC.
Barrick, R., S. Becker, L. Brown, H. Beller, and R. Pastorok. 1988. Sediment quality values
refinement 1988 update and evaluation of Puget Sound AET. Volume 1. Final Report. Prepared
for Tetra Tech, Inc. and U.S. Environmental Protection Agency Region 10, Office of Puget Sound,
Seattle, WA. PTI Environmental Services, Bellevue, WA. 74 pp. + appendices.
Beller, H.R., R.A. Pastorok, D.S. Becker, G. Braun, G. 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.
Cohen, J. 1977. Statistical power analysis for the behavioral sciences. Academic Press, New York,
NY.
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.
Johns, D.M., and T.C. Ginn. 1990. Neanthes long-term exposure experiment: the relationship
between juvenile growth and reproductive success. Prepared for U.S. Environmental Protection
Agency Region 10, Office of Puget Sound, Seattle, WA. PTI Environmental Services, Bellevue,
WA.
Johns, D.M., T.C. Ginn, and D.J. Reish. 1989. Interim protocol for juvenile Neanthes bioassay.
Draft Report. Prepared for Washington Department of Ecology, Olympia, 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.
Peltier, W.H., and C.I. Weber. 1985. Methods for measuring acute toxicity of effluents to
freshwater and marine organism. EPA/600/4-85/013. U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH.
Petrich, S.M., and D.J. Reish. 1979. Effects of aluminum and nickel on survival and reproduction
in polychaetous annelids. Bull. Environ. Contam. Toxicol. 23:698-702.
PSEP. 1986. 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.
56

-------
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.
Reish, D.J. 1980. The effect of different pollutants on ecologically important polychaete worms.
EPA Res. Rep. Ser. (Ecol. Res.). EPA 600/3-80-053. U.S. Environmental Protection Agency,
Washington, DC. 138 pp.
Reish, D.J. 1984. Marine ecotoxicological tests with polychaetous annelids. In: Ecotoxicological
Testing for the Marine Environment. G. Persoone, E. Jaspers, and C. Claus (eds). State University
of Ghent, Bredene, Belgium, Vol. I, 427-454.
Rossi, S.S., and J.W. Anderson. 1976. Toxicity of water soluble fractions of no. 2 fuel oil and
south Louisiana crude oil to selected states in the life history of the polychaete, Neanthes
arenaceodentata. Bull. Environ. Contam. Toxicol. 16:18-24.
Scheffe, H. 1959. The analysis of variance. John Wiley & Sons, New York, NY. 477 pp.
U.S. EPA. 1986a. Recommended protocols for measuring selected environmental variables in
Puget Sound. Final Report. Prepared for U.S. Environmental Protection Agency Region 10, Office
of Puget Sound, and U.S. Army Corps of Engineers. Tetra Tech, Inc., Bellevue, WA.
U.S. EPA. 1986b. Test methods for evaluating solid waste. U.S. Environmental Protection
Agency, Office of Solid Waste and Emergency Response.
57

-------
APPENDIX A
Sediment Chemistry Data

-------
TABLE A-l. CONCENTRATIONS OF CHEMICALS OF CONCERN
IN CARR INLET AND ELLIOTT BAY SEDIMENTS


Carr Inlet
Carr Inlet

Compound
(CR01 )a
(CR02)4
Elliott Bay3
METALS (mg/kg dry weight; ppm)



Antimony
1.4G
0.14G
10.3G
Arsenic
18.1
4.4
112
Cadmium
0.8E
0.7E
1.4E
Copper
62.3
28.5
1,490
Lead
37.5
13.3
384
Mercury
0.14
0.05
3.5
Nickel
36.7
35.8
50.5
Silver
0.39E
0.14E
1.2E
Zinc
111
61.6
1,010
ORGANICS (ug/kg dry weight; ppb)



Low Molecular Weight Polycyclic Aromatic Hydrocarbons (PAH)

Naphthalene
14U
11U
390
Acenaphthylene
14U
11U
440
Acenaphthene
14U
. 11U
780
Fluorene
14U
11U
790
Phenanthrene
100
9E
4,800
Anthracene
38
11U
1,900
2-Methylnaphthalene
14U
11U
260
High Molecular Weight PAH



Fluoranthene
170E
32E
8,100
Pyrene
170E
27E
12,000
Benz(a)anthracene
83E
10E
4,000E
Chrysene
100
20
3,300
Benzofluoranthenes
150E
28E
10,000
Benzo(a)pyrene
90E
11U
8,900
Indeno( 1,2,3,-c,d)pyrene
50E
11U
1,600
Dibenzo(a,h)anthracene
14U
11U
910
Benzo(g,h,i)perylene
56E
11U
3,600
Chlorinated Hydrocarbons



1,3-Dichloro benzene
14U
11U
13U
1,4-Dichlorobenzene
14U
11U
14U
1,2-Dichlorobenzene
14U
11U
13U
Hexachlorobenzene
14U
11U
13U
Phthalates



Dimethyl phthalate
14U
11U
110
Diethyl phthalate
14U
11U
13U
Di-n-butyl phthalate
14U
11U
140
Butyl benzyl phthalate
14U
11U
320E
Bis(2-ethylhexyl)phthalate
64
11U
6100
Di-n-octyl phthalate
14U
11U
130U
A-]

-------
TABLE A-l. (Continued)


Carr Inlet
Carr Inlet

Compound
(CROl)'
(CR02)a
Elliott Bay"
Polychlorinated Biphenyls



Total PCB
8.2K
4.2K
1460
Phenols



Phenol
27U
22U
170
2-Methylphenol
14U
11U
78E
4-Methylphenol
14U
11U
180E
2,4-Dimethylphenol
33U
26U
31U
Pentachlorophenol
22U
17U
1,900
Miscellaneous Extractables



Benzyl alcohol
68U
54U
64U
Benzoic acid
140U
108U
128U
Dibenzofuran
14U
11U
110
Hexachloroethane
41U
33U
39U
Hexachlorobutadiene
14U
11U
13U
N-Nitrosodiphenylamine
14U
11U
13U
Pesticides



Total DDT
2U
2U
34
Aldrin
1U
1U
1U
Chlordane
1.5U
1.5U
1.5U
Dieldrin
2U
2U
2U
Heptachlor
1U
1U
1U
Lindane
1U
1U
1U
* 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

-------
APPENDIX B
T~* •	rp .	a
Bioassay Test Data

-------
TABLE B-l. BIO AS SAY TEST DATA FOR
THE WORM DENSITY EXPERIMENT


Number
Total Biomass
Average Biomass
Treatment3
Replicate
Surviving
(mg dry weight)
(mg dry weight)
West Beach-5
1
4
12.8
3.2

->
5
18.9
3.8

3
5
29.9
6.0

4
0
--
--

5
5
28.6
5.7
Carr Inlet-5
1
5
70.2
14.0

2
5
103.5
20.7

3
5
74.4
14.9

4
4
71.8
18.0

5
5
79.6
15.9
Carr Inlet-10
1
10
113.8
11.4

2
10
135.3
13.5

3
10
114.0
14.0

4
10
93.0
9.3

5
9
101.8
11.3
Carr Inlet-15
1
15
160.7
10.7

2
14
108.1
7.7

3
15
144.5
9.6

4
15
146.1
9.7

5
15
112.8
7.5
Carr Inlet-20
1
18
157.7
8.8

2
19
152.3
8.0

3
20
168.8
8.4

4
20
145.9
7.3

5
20
112.7
5.6
Elliott Bay-5
i
5
22.1
4.4

2
5
11.8
2.4

3
5
32.2
6.4

4
5
6.5
1.3

5
5
15.9
3.2
Elliott Bay-10
1
9
46.5
5.2

2
9
29.2
3.2

3
4
13.6
3.4

4
7
21.9
3.1

5
7
11.2
1.6
Elliott Bay-15
1
0
0.0
	

2
5
4.6
0.9

3
5
7.8
1.6

4
13
47.7
3.7

5
8
27.1
3.4
B-l

-------
TABLE B-l.
(Continued)






Number
Total Biomass
Average Biomass
Treatment4
Replicate
Surviving
(mg dry weight)
(mg dry weight)
Elliott Bay-20
1
12
47.6
4.0

2
5
10.9
2.2

3
15
44.3
3.0

4
4
6.1
1.5

5
0
0.0
--
8 5 - 5 worms/chamber
10 = 10 worms/chamber
15 = 15 worms/chamber
20 = 20 worms/chamber.
B-2

-------
TABLE B-2. BIOASSAY TEST DATA
FOR THE FOOD RATION EXPERIMENT


Number
Total Biomass
Average Biomass
Treatment3
Replicate
Surviving
(mg dry weight)
(mg dry weight)
West Beach-0
1
5
6.9
1.4

2
5
6.6
1.3

3
5
6.6
1.3

4
5
4.5
0.9

5
5
8.1
1.6
West Beach-40
1
5
101.0
20.2

2
5
123.6
24.7

3
4
107.3
26.8

4
5
87.9
16.8

5
5
110.7
22.1
West Beach-80
I
5
141.2
28.2

n
L.
4
103.1
25.8

3
5
88.3
17.7

4
5
95.9
19.1

5
5
120.6
24.1
Carr Inlet-0
1
4
2.4
0.6

2
5
2.8
0.6

3
5
3.5
0.7

4
5
3.2
0.6

5
4
2.1
0.5
Carr Inlet-20
1
5
91.1
18.2

2
5
78.6
15.7

3
4
57.0
14.3

4
4
58.5
14.6

5
4
40.3
10.1
Carr Inlet-40
1
5
124.0
24.8

2
5
137.4
27.5

3
5
118.7
23.7

4
5
109.6
21.9

5
4
72.6
18.2
Carr Inlet-60
1
5
160.1
32.0

2
5
103.6
20.7

3
4
168.5
42.1

4
5
127.0
25.4

5
4
63.4
15.9
Carr Inlet-80
1
4
163.0
40.8

2
5
173.6
34.7

3
5
188.5
37.7

4
5
155.1
31.0

5
5
138.7
27.7
B-3

-------
TABLE B-2. (Continued)
Number	Total Biomass	Average Biomass
Treatment3	Replicate	Surviving (mg dry weight) (mg dry weight)
Elliott Bav-0
Elliott Bay-20
Elliott Bay-40
Elliott Bay-60
Elliott Bay-80
1
0
0.0
--
2
0
0.0
--
3
0
0.0
—
4
0
0.0
—
5
0
0.0
—
J
1
9.0
9.0
2
0
0.0
—
3
1
3.2
3.2
4
0
0.0
--
5
2
0.3
0.2
1
4
21.0
5.3

5
22.9
4.6
3
5
27.0
5.4
4
3
6.5
2.2
5
2
13.2
6.6
1
5
23.8
4.8
2
5
44.3
8.9
3
4
31.4
7.9
4
0
0.0
--
5
5
38.4
7.7
1
5
26.0
5.2
2
5
62.0
12.4
3
5
42.9
8.6
4
5
82.8
16.6
5
3
18.8
6.3
a 0 = no food
20 = 20 mg/48 hours
40 = 40 mg/48 hours
60 = 60 mg/48 hours
80 = 80 mg/48 hours.
B-4

-------
TABLE B-3. BIOASSAY TEST DATA
FOR THE EXPOSURE SYSTEM EXPERIMENT


Number
Total Biomass
Average Biomass
Treatment8
Replicate
Surviving
(mg dry weight)
(mg dry weight)
West Beach - S
1
5
137.2
27.4

2
5
102.4
20.5

3
5
138.8
27.8

4
5
109.2
21.8

5
5
106.7
21.3
West Beach - R
1
5
116.1
23.2

2
5
119.7
23.9

3
5
147.8
29.6

4
5
84.4
16.9

5
5
96.3
19.3
Carr Inlet - S
1
5
123.0
24.6

2
5
96.0
19.2

3
5
105.6
21.1

4
4
36.2
9.1

5
5
93.3
18.7
Carr Inlet - R
1
5
110.6
22.1

2
5
128.5
25.7

3
5
106.1
21.2

4
5
93.4
18.7

5
5
90.4
18.1
Elliott Bay - S
1
1
2.3
2.3

2
1
2.9
2.9

3
4
11.4
2.9

4
4
31.5
7.9

5
5
9.7
1.9
Elliott Bay - R
1
4
12.7
3.2

2
5
16.6
3.3

3
4
10.9
2.7

4
5
5.8
1.2

5
5
9.4
1.9
8 S = static exposure system
R = static renewal exposure system.
B-5

-------
TABLE B-4. BIOASSAY TEST DATA
FOR THE TEST DURATION EXPERIMENT


Number
Total Biomass
Average Biomass
Treatment8
Replicate
Surviving
(mg dry weight)
(mg dry weight)
West Beach-10
1
5
10.4
2.1

2
5
30.9
6.2

3
5
24.2
4.8

4
5
30.1
6.0

5
	b
--
--
West Beach-15
1
5
43.5
8.7

2
5
34.7
6.9

3
5
40.4
8.1

4
5
40.0
8.0

5
5
40.9
8.2
West Beach-20
1
5
56.1
11.2

2
5
80.4
16.1
\
3
5
75.7
15.1

4
5
57.7
11.5

5
5
69.0
13.8
Carr Inlet-10
1
5
27.4
5.5

2
5
30.7
6.1

3
5
37.2
7.4

4
5
31.0
6.2

5
4
13.3
3.3
Carr Inlet-15
1
5
78.9
15.8

2
5
39.5
7.9

3
5
53.8
10.8

4
5
45.6
9.1

5
5
57.5
11.5
Carr Inlet-20
1
5
76.2
15.2

2
5
87.1
17.4

3
5
89.2
17.8

4
5
76.6
15.3

5
5
86.1
17.2
Elliott Bay-10
1
4
7.5
1.9

' 2
4
6.7
1.7

3
5
6.6
1.3

4
3
1.7
0.6

5
4
6.6
1.7
Elliott Bay-15
I
5
7.0
1.4

2
4
5.1
1.3

3
5
4.2
0.8

4
2
1.5
0.8

5
3
3.2
1.1
B-6

-------
TABLE B-4. (Continued)



Number
Total Biomass
Average Biomass
Treatment3
Replicate
Surviving
(mg dry weight)
(mg dry weight)
Elliott Bay-20
1
4
3.5
0.9

">
4
4.8
1.2

3
5
3.3
0.7

4
5
7.6
1.5

5
3
3.1
1.0
a 10 =10 day exposure
15 =15 day exposure
20 = 20 day exposure.
b Replicate dropped in handling.
B-7

-------
TABLE B-5. BIOASSAY TEST DATA
FOR THE SEDIMENT GRAIN-SIZE EXPERIMENT


Number
Total Biomass
Average 1
Treatment3
Replicate
Surviving
(mg dry weight)
(mg dry <
West Beach-1
1
5
81.4
16.3

2
5
60.3
12.1

3
5
80.3
16.1

4
5
51.9
10.4

5
5
54.4
10.9
Carr Inlet-2
1
5
84.7
16.9
(CR01)
2
5
55.3
11.1

3
5
92.0
18.4

4
5
59.4
11.9

5
5
74.5
14.9
Carr Inlet-3
1
5
84.7
16.9
(CR02)
2
5
94.5
18.9

3
4
45.2
11.3

4
5
60.6
12.1

5
5
92.2
18.4
Carr Inlet-4
1
5
64.8
13.0
(CR03)
2
5
87.3
17.5

3
5
67.3
13.5

4
5
53.7
10.7

5
4
91.5
22.8
Carr Inlet-5
1
5
77.7
15.5
(CR04)
2
5
71.3
14.3

3
5
52.1
10.4

4
5
54.4
10.9

5
5
74.1
14.8
Carr Inlet-6
1
5
63.4
12.7
(CR05)
2
5
52.9
10.6

3
5
85.0
9.0

4
5
66.9
17.0

5
5
77.7
13.4
* 1=2 percent fines
2	= 88 percent fines
3	= 89 percent fines
4	= 97 percent fines
5	= 88.5 percent fines
6	= 93 percent fines.
B-8

-------
TABLE B-6. BIOASSAY TEST DATA
FOR TIIE SALINITY EXPERIMENT


Number
Total Biomass
Average Biomass
Treatment3
Replicate
Surviving
(mg dry weight)
(mg dry weight)
West Beach-28
1
5
48.2
9.6

2
5
42.4
8.5

3
5
68.0
13.6

4
5
32.2
8.1

5
4
47.5
9.5
Carr Inlet-28
1
5
39.9
8.0

2
5
57.2
11.4

3
5
47.6
9.5

4
5
61.4
12.3

5
5
50.4
10.1
Duckabush River-30
1
5
68.0
13.6

2
5
35.6
7.1

3
5
47.1
9.4

4
5
46.7
9.3

5
5
52.3
10.5
Duckabush River-25
1
4
39.5
9.9

2
5
38.3
7.7

3
4
28.1
7.0

4
4
48.9
12.2

5
5
40.3
8.1
Duckabush River-22
1
5
76.9
15.4

2
5
39.6
7.9

3
5
49.2
9.8

4
5
70.8
14.2

5
5
62.8
12.6
Duckabush River-15
1
5
43.4
8.7

2
5
39.7
7.9

3
5
42.4
8.5

4
5
33.5
6.7

5
5
54.7
10.9
30
= 30
ppt
salinity
28
= 28
ppt
salinity
25
= 25
ppt
salinity
22
= 22
ppt
salinity
15
= 15
ppt
salinity
B-9

-------
APPENDIX C
Water Quality Monitoring Data

-------
TABLE C-l. WATER QUALITY MONITORING DATA FOR THE
WORM DENSITY EXPERIMENT
Test Date (days)
Treatment3
Replicate
Variable
0
3
6
9
12
15
West Beach-5
2
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
29
30
28
28
31
32


Dissolved oxygen (mg/L)
7.9
7.1
7.1
6.8
7.0
8.2


pH
8.1
8.1
8.0
8.1
8.0
8.0

4
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
30
30
28
28
31
32


Dissolved oxygen (mg/L)
7.9
6.9
6.8
6.9
7.0
8.2


pH
8.1
8.1
7.9
8.1
8.0
8.0
Carr Inlet-5
1
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
29
30
30
28
30
32


Dissolved oxygen (mg/L)
7.7
7.0
7.2
6,6
6.8
8.3


pH
8.1
8.2
8.1
8.0
7.9
8.0

4
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
29
30
30
28
30
32


Dissolved oxygen (mg/L)
7.7
7.1
6.8
7.2
6.8
8.0


pH
8.1
8.0
7.4
8.2
7.9
8.0
Carr Inlet-10
1
Temperature ("C)
18
18
19
20
22
22


Salinity (ppt)
29
28
30
28
30
32


Dissolved oxygen (mg/L)
7.8
6.8
7.6
6.6
7.0
8.4


PH
8.1
8.1
8.1
8.0
8.0
8.1

3
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
29
28
30
28
30
32


Dissolved oxygen (mg/L)
7.7
6.8
7.7
6.9
7.0
8.4


PH
8.1
8.2
8.2
8.0
8.0
8.1
Carr Inlet-15
3
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
29
30
30
28
32
32
_

Dissolved oxygen (mg/L)
7.7
6.9
7.7
7.2
7.2
8.0


PH
8.1
8.2
8.3
8.2
8.1
8.1

5
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
30
30
30
28
32
32


Dissolved oxygen (mg/L)
7.7
7.0
7.8
7.2
7.1
7.9


pH
8.0
8.2
8.1
8.0
8.1
8.1
Carr Inlet-20
3
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
28
30
30
28
30
32


Dissolved oxygen (mg/L)
7.7
7.0
7.7
7.0
7.1
8.0


PH
8.1
8.1
8.1
8.1
7.9
8.1

5
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
28
28
30
28
30
32


Dissolved oxvgen (mg/L)
7.7
7.0
7.8
7.0
7.3
7.9


PH
8.1
8.1
8.2
8.1
7.9
8.1
C-l

-------
TABLE C-l. (Continued)
Test Date (days)
Treatment3
Replicate
Variable
0
3
6
9
12
15
Elliott Bay-5
2
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
30
28
30
28
29
32


Dissolved oxygen (mg/L)
7.7
6.2
7.2
6.8
7.0
8.1


pH
8.0
8.2
8.0
8.1
7.9
8.1

4
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
29
28
30
28
29
32


Dissolved oxygen (mg/L)
7.7
6.4
7.4
6.7
7.1
7.9


pH
8.1
8.0
8.1
8.1
7.9
8.1
Elliott Bay-10
3
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
29
28
30
28
29
32


Dissolved oxygen (mg/L)
7.8
6.4
7.4
6.6
6.8
8.2


pH
8.1
8.1
8.1
8.0
7.9
8.0

5
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
29
28
30
28
29
32


Dissolved oxygen (mg/L)
7.8
6.6
7.1
6.6
6.8
8.0


PH
8.1
8.1
7.9
8.0
7.9
8.0
Elliott Bay-15
2
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
30
28
30
30
28
31


Dissolved oxygen (mg/L)
7.8
6.6
7.1
7.1
6.9
8.0


pH
8.1
81
8.0
8.1
8:1
7.6

4
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
29
28
30
30
3!
32


Dissolved oxygen (mg/L)
7.7
6.5
7.8
7.1
6.9
8.2


pH
8.0
7.9
8.0
8.1
8.1
7.6
Elliott Bay-20
1
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
29
28
30
28
32
32


Dissolved oxygen (mg/L)
7.8
6.3
7.4
6.8
7.0
8.4


PH
8.1
8.0
7.9
7.9
7.9
8.0

4
Temperature (°C)
18
18
19
20
22
22


Salinity (ppt)
28
30
30
28
32
32


Dissolved oxygen (mg/L)
7.7
6.7
7.4
6.7
7.1
8.0


PH
8.1
8.0
8.0
8.0
8.0
8.1
'' 5 = 5 worms/chamber
10= ]0 worms/chamber
15= 15 worms/chamber
20 = 20 worms/chamber.
C-2

-------
TABLE C-2. WATER QUALITY MONITORING DATA
FOR THE FOOD RATION EXPERIMENT
Test Date (days)
Treatment3
Replicate
Variable
2
5
8
11
14
17
20
West Beach-0
2
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.4
8.2
7.6
7.0
7.3
7.6
7.0


pH
8.1
8.1
8.1
8.2
8.1
8.2
8.1

5
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
32
30
30
30


Dissolved oxygen (mg/L)
7.2
8.3
7.4
7.8
7.3
7.5
7.4


pH
7.9
8.1
8.1
8.2
8.2
8.1
8.1
West Beach-40
3
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
29
30
30
30


Dissolved oxygen (mg/L)
7.4
8.1
7.4
6.7
7.5
7.3
7.4


PH
8.1
8.0
8.1
8.1
8.1
7.9
8.0

4
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
29
30
30
30


Dissolved oxygen (mg/L)
7.4
8.3
7.4
7.1
7.3
7.5
7.0


pH
8.1
8.0
8.1
8.2
8.1
8.0
8.0
West Beach-80
2
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
32
30
30
30


Dissolved oxygen (mg/L)
7.4
8.3
7.4
6.8
7.3
7.5
7.0


pH
8.0
8.1
8.1
8.3
8.2
8.2
8.0

4
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.4
8.3
7.6
7.1
7.4
7.6
7.3


pH
8.0
8.1
8.2
8.3
8.2
8.2
8.0
Carr Inlet-0
1
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
32
30
30
30


Dissolved oxygen (mg/L)
7.6
8.3
7.4
7.0
7.4
5.9
7.2


PH
8.1
8.3
8.4
8.5
8.3
8.2
8.2

3
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
32
30
30
30


Dissolved oxygen (mg/L)
7.4
8.3
7.6
7.0
7.4
7.7
7.5


pH
7.9
8.1
8.1
8.6
8.3
8.4
8.2
Carr Inlet-20
4
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
32
30
30
30


Dissolved oxygen (mg/L)
7.4
8.3
7.6
6.9
7.4
7.4
7.7


pH
8.0
8.1
8.3
8.4
8.3
8.3
8.4

5
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
32
30
30
30


Dissolved oxygen (mg/L)
7.4
8.3
7.6
6.8
7.4
7.9
7.5


PH
8.1
8.1
8.3
8.5
8.4
8.4
8.2
C-3

-------
TABLE C-2. (Continued)
Test Date (days)
Treatment3
Replicate
Variable
2
5
8
11
14
17
20
Carr Inlet-40
C
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.6
8.3
7.6
6.8
7.4
7.9
7.5


PH
8.1
8.1
8.3
8.5
8.4
8.4
8.2

5
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.4
8.3
7.6
7.0
7.3
7.9
7.5


PH
8.0
8.1
8.3
8.4
8.3
8.3
8.3
Carr Inlet-60
3
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
32
30
30
30


Dissolved oxygen (mg/L)
7.5
8.2
7.6
7.1
7.3
7.2
7.5


PH
8.1
8.1
8.3
8.4
8.2
8.4
8.2

5
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
32
30
30
30


Dissolved oxygen (mg/L)
7.4
8.3
7.7
7.0
2.7b
7.6
7.7


PH
8.1
8.2
8.3
8.3
8.0
8.2
8.3
Carr Inlet-80
1
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.2
8.4
7.1
7.0
1.0b
7.2
7.4


PH
8.0
8.0
8.2
8.2
7.8
8.2
8.3

5
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.3
8.3
7.4
6.8
6.7
7.3
7.4


PH
8.0
8.0
8.2
8.2
8.1
8.2
8.1
Elliott Bay-0
1
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
28
30
30
30


Dissolved oxygen (mg/L)
7.5
8.1
7.6
6.9
7.3
7.5
7.5


PH
8.1
8.0
8.2
8.3
8.2
8.3
8.2

4
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.4
8.2
7.6
6.9
7.3
7.5
7.7


PH
8.1
8.1
8.2
8.4
8.2
8.4
8.4
Elliott Bay-20
2
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.4
8.1
7.5
7.2
7.1
7.6
7.2


PH
8.1
8.0
8.2
8.5
8.1
8.4
8.3

3
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxvgen (mg/L)
7.3
8.1
7.5
7.1
6.9
7.7
7.4


pH >
8.1
8.0
8.2
8.1
8.3
8.1
8.2
C-4

-------
TABLE C-2. (Continued)





Test Date (days)


Treatment3
Replicate
Variable
2
5
8
11
14
17
20
Elliott Bay-40
1
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
32
26
26
30
30
30
30


Dissolved oxygen (mg/L)
6.9
7.1
7.5
7.4
8.0
5.1
7.4


pH
8.0
8.0
8.2
8.0
7.4
7.7
7.9

3
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
6.8
7.1
7.5
7.4
7.9
7.3
7.4


pH
8.2
8.0
8.2
8.1
7.9
8.2
8.0
Elliott Bay-60
1
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.2
8.0
7.5
7.2
7.1
7.4
7.2


PH
8.1
8.0
8.2
8.2
8.1
8.1
8.1

5
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
5.4
8.1
7.3
7.4
7.1
7.3
7.5


pH
7.5
8.0
8.1
8.3
8.2
8.2
8.0
Elliott Bay-80
4
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.3
8.2
7.3
7.1
7.3
7.7
7.5


pH
7.9
7.9
8.2
8.2
8.2
8.0
8.1

4
Temperature (°C)
21
19
19.5
20
21
20
19


Salinity (ppt)
30
26
26
30
30
30
30


Dissolved oxygen (mg/L)
7.2
8.2
8.4
6.9
7.3
7.3
7.4


pH
8.1
7.9
8.1
8.2
8.1
8.1
8.0
a 0	= no food
20 = 20 mg/48 hours
40 = 40 mg/48 hours
60 = 60 mg/48 hours
80 = 80 mg/48 hours.
b Aeration to the exposure chamber was not on.
C-5

-------
TABLE C-3. WATER QUALITY MONITORING DATA
FOR THE TEST DURATION EXPERIMENT
Test Date (days)
Treatment
Replicate
Variable
0
3
6
9
12
15
18
20
19
19
20
19




28
28
29
28




7.5
7.5
7.2
7.7




8.1
8.1
8.1
8.1




19
19
20
19




28
28
28
28




7.5
7.5
7.2
7.7




8.1
8.1
8.1
8.1




19
19
20
19
19



28
29
29
29
28



7.5
7.5
7.0
7.5
8.0



8.1
8.0
8.1
8.0
8.0



19
19
20
19
19



28
30
29
28
28
--
__
__
7.5
7.5
7.3
7.7
7.8



8.1
8.1
8.1
8.1
8.1



19
19
20
19
19
19
19
19
28
28
28
28
28
28
28
30
7.5
7.9
7.3
7.7
7.9
7.5
7.3
6.8
8.1
8.0
8.0
8.1
8.1
8.2
8.1
8.2
19
19
20
19
19
18
19
19
28
28
28
28
28
28
28
30
7.5
7.7
7.4
7.7
8.1
7.5
7.1
7.0
8.1
8.2
8.2
8.2
8.2
8.3
8.2
8.2
19
19
20
19




28
28
29
28




7.5
7.7
7.3
7.7




8.1
8.1
8.2
8.2




19
19
20
19




28
28
28
29




7.5
7.7
7.1
7.6




8.1
8.1
8.3
8.3




19
19
20
19
19
18
_ _
_ _
28
30
30
29
28
28
	

7.5
7.7
7.4
7.7
7.9
7.5
--

8.1
8.2
8.1
8.3
8.3
8.3
»-
--
19
19
20
19
19
18

_ _
28
30
29
28
28
28
__
--
7.5
7.7
7.3
7.7
8.0
7.5
	

8.0
8.2
8.3
8.4
8.2
8.1

--
West Beach-10
West Beach-15
West Be3ch-20
Carr Inlet-10
Carr Inlet-15
Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
pH
Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
pH
Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
Temperature (°C)
Salinity (ppt)
Dissolved oxvgen (mg/L)
PH
Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
C-6

-------
TABLE C-3. (Continued)
Treatment'1
Replicate Variable
Test Date (days)
0
3
6
9
12
15
18
20
19
19
20
19
19
18
19
19
28
28
28
28
28
28
29
30
7.5
7.7
7.3
7.7
7.8
7.5
7.2
7.0
8.0
8.0
8.3
8.1
8.2
8.2
8.3
8.3
19
19
20
19
19
18
19
19
28
28
29
28
28
28
29
30
7.5
7.7
7.4
7.7
7.8
7.5
7.3
7.0
8.0
8.1
8.0
8.3
8.3
8.4
8.3
8.4
19
19
20
19




28
28
29
28




7.5
7.9
7.4
7.7
--
__
--
__
8.1
8.2
8.1
8.3




19
19
20
19




28
28
28
28




7.5
7.9
7.3
7.7




8.1
8.2
8.2
8.2
--
--
--
--
19
19
20
19
19
18
	
	
28
28
28
28
28
28
__
--
7.7
7.9
7.4
7.7
7.8
7.5
--
--
8.1
8.1
8.1
8.1
8.1
8.3
--
--
19
19
20
19
19
18
	
	
28
28
28
28
28
28
--
--
7.5
7.7
7.2
7.7
7.7
7.5

--
8.1
8.1
8.1
8.1
8.2
8.3
--
--
19
19
20
19
19
18
19
19
28
28
28
30
28
28
28
28
7.9
7.5
7.2
6.8
7.5
7.7
7.3
7.7
8.2
8.4
8.4
8.3
8.1
8.2
8.4
8.3
19
19
20
19
19
18
19
19
28
22
28
30
28
28
28
28
7.9
7.5
7.2
7.0
7.5
7.7
7.3
7.7
8.0
8.2
8.3
8.2
8.1
8.2
8.2
8.2
Carr Inlet-20
Elliott Bay-10
Elliott Bay-15
Elliott Bay-20
2	Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
3	Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
1 Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
3	Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
4	Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
5	Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
3	Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
pH
4	Temperature (°C)
Salinity (ppt)
Dissolved oxygen (mg/L)
PH
a 10 = 10-day exposure
15 ¦ 15-day exposure
20 = 20-day exposure.
C-7

-------
TABLE C-4. WATER QUALITY MONITORING DATA FOR THE
SEDIMENT GRAIN-SIZE EXPERIMENT
Test Date (days)
Treatment3
Replicate
Variable
0
3
6
9
12
15
18
20
West Beach-1
3
Temperature (°C)
18
24
18
18
20
18
19
19


Salinity (ppt)
30
29
29
29
28
29
30
30


Dissolved oxygen (mg/L)
7.2
6.5
8.3
7.3
7.1
7.8
7.9
7.7


pH
8.2
8.1
8.2
8.0
8.0
8.3
8.2
8.1

5
Temperature (°C)
18
24
18
18
20
18
19
19


Salinity (ppt)
30
29
30
30
30
29
30
30


Dissolved Oxygen (mg/L)
7.2
6.6
8.3
7.6
7.3
7.8
8.0
7.7


pH
8.1
8.1
8.2
8.2
8.0
8.2
8.1
8.0
Carr Inlet-2
1
Temperature (®C)
18
24
18
18
20
18
19
19
(CR01)

Salinity (ppt)
30
29
28
29
31
30
31
31


Dissolved Oxygen (mg/L)
7.2
6.3
8.3
7.5
7.3
7.8
8.0
7.7


pH
8.0
8.1
8.3
8.3
8.2
8.3
8.4
8.4

4
Temperature (°C)
18
24
18
18
20
18
19
19


Salinity (ppt)
30
29
30
29
30
30
32
32


Dissolved Oxygen (mg/L)
7.0
6.5
8.2
7.7
7.5
7.7
7.9
7.7


pH
8.3
8.1
8.3
8.4
8.3
8.5
8.5
8.14
Carr Inlet-3
3
Temperature (°C)
18
24
18
18
20
18
19
19
(CR02)

Salinity (ppt)
30
29
29
29
30
30
30
24


Dissolved oxygen (mg/L)
7.2
6.4
7.9
7.8
7.5
7.9
7.9
7.7


pH
8.2
7.9
8.2
8.2
8.1
8.3
8.2
8.2

4
Temperature (°C)
18
24
18
18
20
18
19
19


Salinity (ppt)
30
29
28
29
29
29
30
28


Dissolved oxygen (mg/L)
7.2
6.6
8.1
7.2
7.5
7.7
7.9
7.6


PH
8.2
8.0
8.1
7.9
7.8
8.0
8.0
8.0
Carr Inlet-4
2
Temperature (°C)
18
24
18
18
20
18
19
19
(CR03)

Salinity (ppt)
30
29
28
29
29
29
30
29


Dissolved oxygen (mg/L)
6.2
6.6
8.3
7.2
6.9
7.7
7.9
7.5


PH
8.1
8.1
8.2
7.9
8.3
8.2
8.2
8.1

4
Temperature (°C)
18
24
18
18
20
18
19
19


Salinity (ppt)
30
29
28
29
28
29
30
28


Dissolved oxygen (mg/L)
6.8
6.7
8.3
7.5
7.3
7.7
7.9
7.5


PH
8.3
8.1
8.1
8.1
8.0
8.2
8.1
8.1
Carr Inlet-5
->
Temperature (°C)
18
24
18
18
20
18
19
19
(CR04)

Salinity (ppt)
30
29
28
29
30
29
30
29


Dissolved oxygen (mg/L)
7.0
6.3
8.3
7.3
7.3
7.7
7.9
7.5


pH
8.2
8.0
8.1
8.1
8.0
8.2
8.2
8.0

3
Temperature (°C)
18
24
18
18
20
18
19
19


Salinity (ppt)
30
29
28
29
28
29
30
29


Dissolved oxygen (mg/L)
7.0
6.5
8.3
7.4
7.3
7.9
8.0
7.7


pH
8.2
8.1
8.2
8.1
8.0
8.3
8.3
8.2
C-8

-------
TABLE C-4. (Continued)
Test Date (days)
Treatment3
Replicate
Variable
0
3
6
9
12
15
18
20
Carr Inlet-6
1
Temperature (°C)
18
24
18
18
20
18
19
19
(CR05)

Salinity (ppt)
30
29
28
29
30
29
30
29


Dissolved oxygen (mg/L)
7.0
6.3
8.3
7.3
7.3
7.7
7.9
7.5


PH
8.2
8.0
8.1
8.1
8.0
8.2
8.0
8.0

3
Temperature (*C)
18
24
18
18
20
18
19
19


Salinity (ppt)
30
29
28
29
28
29
30
29


Dissolved oxygen (mg/L)
7.0
6.6
8.1
7.4
6.6
7.7
7.9
7.5


PH
8.2
8.1
8.1
8.0
8.0
8.3
8.3
8.2
a 1 = 2 percent fines
2	» 88 percent fines
3	= 89 percent fines
4	= 97 percent fines
5	« 88.5 percent fines
6	= 93 percent fines.
C-9

-------
TABLE C-5. WATER QUALITY MONITORING DATA
FOR THE SALINITY EXPERIMENT
Test Date (days)
Treatment3
Replicate
Variable
0
3
6
9
12
15
18
20
West Beach-28
1
Temperature (°C)
18
17.5
20
18
19
19
19
18.5


Salinity (ppt)
26
26
27
28
28
27
28
28


Dissolved oxygen (mg/L)
8.7
7.2
7.1
7.9
7.9
7.3
7.1
7.3


PH
7.8
7.9
7.9
8.3
8.3
8.3
8.1
7.9

2
Temperature (°C)
18
17.5
20
18
19
19
19
18.5


Salinity (ppt)
26
26
27
27
28
28
28
28


Dissolved oxygen (mg/L)
8.7
7.5
7.5
7.9
7.9
7.3
6.9
7.1


PH
8.1
8.1
8.0
8.2
8.2
8.2
7.9
8.0
Carr Inlet-28
1
Temperature (°C)
18
17.5
20
18
19
19
19
18.5


Salinity (ppt)
26
26
29
30
30
30
30
29


Dissolved oxygen (mg/L)
8.7
7.8
7.7
7.9
7.9
7.2
7.2
7.3


pH
8.2
8.2
8.1
8.4
8.4
8.4
8.2
8.3

4
Temperature (°C)
18
17.5
20
18
19
19
19
18.5


Salinity (ppt)
26
26
27
28
28
28
29
29


Dissolved oxygen (mg/L)
8.7
7.1
7.4
7.9
7.9
7.2
7.2
7.3


PH
8.1
8.1
8.0
8.4
8.4
8.4
8.2
8.3
Duckabush
1
Temperature (°C)
18
17.5
20
18
19
19
19
18.5
River-30

Salinity (ppt)
26
26
27
28
28
28
28
26


Dissolved oxygen (mg/L)
7.5
7.7
7.9
7.7
6.9
7.1
7.3
7.4


pH
8.1
8.0
7.9
8.2
8.2
8.1
8.0
8.2

3
Temperature (°C)
18
17.5
20
18
19
19
19
18.5


Salinity (ppt)
26
26
28
28
28
27
28
28


Dissolved oxygen (mg/L)
7.7
7.7
8.5
7.9
7.7
7.1
7.0
7.3


pH
8.1
8.1
8.0
8.2
8.2
8.1
7.7
7.9
Duckabush
1
Temperature (°C)
18
17.5
20
18
19
19
19
18.5
River-25

Salinity (ppt)
26
26
27
27
28
27
28
26


Dissolved oxygen (mg/L)
8.7
6.8
6.4
7.9
7.7
7.1
6.9
7.1


pH
7.9
7.7
7.6
8.2
8.2
8.1
7.9
8.0

5
Temperature (°C)
18
17.5
20
18
19
19
19
18.5


Salinity (ppt)
26
26
27
28
28
27
28
26


Dissolved oxygen (mg/L)
8.7
7.7
7.7
7.9
7.9
7.1
7.6
5.5


PH
8.2
8.1
8.0
8.2
8.2
8.2
7.5
7.6
Duckabush
2
Temperature (°C)
18
17.5
20
18
19
19
19
18.5
River-22

Salinity (ppt)
26
26
28
28
28
27
28
27


Dissolved oxygen (mg/L)
8.7
7.6
7.7
7.9
8.0
7.2
6.9
7.1


PH
8.2
8.1
8.0
8.2
8.2
8.1
7.7
7.9

4
Temperature (°C)
• 18
17.5
20
18
19
19
19
18.5


Salinity (ppt)
26
26
26
26
27
27
27
26


Dissolved oxygen (mg/L)
8.7
7.6
7.7
7.9
8.0
7.2
7.1
7.2


pH
8.1
8.1
8.0
8.1
8.0
7.9
7.7
7.8
C-10

-------
TABLE C-5. (Continued)
Test Date (days)
Treatment3
Replicate
Variable
0
3
6
9
12
15
18
20
Duckabush
3
Temperature (°C)
18
17.5
20
18
19
19
19
18.5
River-15

Salinity (ppt)
26
26
26
27
27
29
28
27


Dissolved oxygen (mg/L)
8.7
7.7
7.7
7.9
7.9
7.3
7.0
7.3


pH
8.2
8.2
8.1
8.4
8.3
8.2
8.0
8.1

5
Temperature (°C)
18
17.5
20
18
19
19
19
18.5


Salinity (ppt)
26
26
26
27
27
27
27
26


Dissolved oxygen (mg/L)
8.7
7.8
7.7
7.9
7.9
7.3
7.1
7.3


pH
8.2
8.2
8.1
8.2
8.1
8.0
7.9
8.0
3 30 = 30 ppt salinity
28 = 28 ppt salinity
25 = 25 ppt salinity
22 = 22 ppt salinity
15 = 15 ppt salinity.
C-ll

-------