x>EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/R-99/085
August 1999
Comparative Toxicity
Testing of Selected
Benthic and Epibenthic
Organisms for the
Development of Sediment
Quality Test Protocols
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EPA/600/R-99/085
August 1999
Comparative Toxicity Testing of Selected
Benthic and Epibenthic Organisms for the
Development of Sediment Quality Test Protocols
By
Drs. Michael H. Fulton, Geoffrey I. Scott, and Peter B. Key
National Ocean Service
Center for Coastal Environmental Health and Biomolecular Research
219 Fort Johnson Rd
Charleston, SC 29412-9110
Dr. G. Tom Chandler
School of Public Health
University of South Carolina
Columbia, SC 29208
Dr. Robert F. Van Dolah and Phillip P. Maier
Marine Resources Research Institute
South Carolina Department of Natural Resources
P.O. Box12559
Charleston, SC 29422
DW 13936613-01-0
Project Officer
Dr. Michael A. Lewis
U.S. Environmental Protection Agency
National Health and Environmental Effects Research Laboratory
Gulf Ecology Division
1 Sabine Island Drive, Gulf Breeze, FL 32561-5299
U.S. Environmental Protection Agency
Office of Research and Development
401 M Street, S.W.
Washington, DC 20460
Printed on Recycled Paper
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Notice
The U.S. Environmental Protection Agency through its Office of Research and
Development (funded and managed the research described here under DW13936613-
01-0) to (U.S. National Marine Fisheries Service, Southeast Fisheries Science Center).
It has been subjected to Agency's peer and administrative review and has been
approved for publication as an EPA document.
Disclaimer
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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ABSTRACT
Sediment contamination has resulted in the need to develop an appropriate suite
of toxicity tests to assess ecotoxicological impacts on estuarine ecosystems. Existing
Environmental Protection Agency (EPA) protocols recommend a number of test
organisms, including amphipods, polychaetes, molluscs, crustaceans and fish for use in
sediment toxicity tests. While this suite of test animals represents a diverse group of
fauna, many of the species recommended by the EPA are not indigenous to all
geographic regions of the United States, particularly the Gulf of Mexico and South
Atlantic. As a result, environmental risk assessment based on these organisms may not
adequately protect ecosystem health in the Gulf of Mexico. Ideally, appropriate test
organisms to evaluate sediment toxicity should include species indigenous to the Gulf of
Mexico that are representative of a variety of faunal classes and feeding types.
Additionally, the toxicity test endpoints should include both lethal (mortality) and
sublethal (reproduction, growth, physiological impairment) effects and they should be
sensitive to either porewater and/or whole sediment exposures for all major classes of
chemical contaminants (trace metals, polycyclic aromatic hydrocarbons (PAHs),
pesticides). Finally, test species should be easy to collect and maintain in the
laboratory. This study examined the relative sensitivity of a variety of test organisms,
broadly distributed throughout the southeastern United States and the Gulf of Mexico to
several classes of chemical contaminants in both whole sediment and aqueous/
porewater exposures. Additionally, several rapid screening assays were compared with
these more traditional toxicity evaluations. The three model contaminants selected for
study were cadmium (an inorganic toxicant), DDT (a persistent organochlorine pesticide)
and fluoranthene (a polycyclic aromatic hydrocarbon [PAH]). These compounds
.represent contaminants frequently measured in sediments throughout the Gulf of
Mexico.
Overall, the juvenile clam was the most sensitive species tested in this study from
an acute toxicity standpoint. The grass shrimp and the two amphipod species were
generally similar in sensitivity to each of the three compounds. The copepod assay,
although relatively insensitive in terms of adult mortality, was capable of detecting
sublethal effects at contaminant concentrations below those which caused mortality in
the other more sensitive species. Both the juvenile clam assay and the copepod partial
life cycle test have the potential to serve as sensitive indicators of potential sediment-
associated toxicity which might not be detected using standard acute toxicity bioassays.
The differing species sensitivities observed with the different classes of chemical
contaminants in this study suggest that a multiple species approach may be more
appropriate for a holistic ecological risk assessment of sediment contamination. The
"Crustacean Triad" (copepods, amphipods and grass shrimp) provide a battery of tests
which predict toxicity to epibenthic and behthic crustaceans with known sensitivity to a
variety of chemical contaminants and represent the base of the food chain for most
recreationally and commercially important finfish species that utilize estuarine nursery
grounds. The addition of the juvenile clam assay provides a herbivorous filter feeder
with the ability to bioconcentrate pollutants and which is extremely sensitive in the size
range tested (>212<350 /^m). Field studies in South Carolina have indicated that sites
with high sediment contaminant levels have degraded benthos, with significant effects
observed in crustaceans and molluscs. These findings support our laboratory results
and suggest that an integrated battery of assays may be most appropriate for estimating
field effects.
in
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CONTENTS
Notice ii
Abstract iii
Chapter 1. Introduction 1
Chapter 2. Methods 3
Description of Species Tested 3
Microtox and Mutatox 5
Collection and Holding of Test Organisms 5
Reference Toxicant Tests 6
Analytical Chemistry 6
Aqueous Contaminant Bioassay Protocol 9
Sediment Bioassay Protocol 10
Microtox Assay Protocol 12
Mutatox Bioassay 12
Chapter 3. Results and Discussion 13
Analytical Chemistry 13
Reference Toxicant Tests 13
Chapter 4. Summary and Conclusions 23
Chapter 5. References 25
Appendices
A. Summary of results obtained from the SDS reference toxicant tests using
P. pugio, A. vem'lli, A. Abdita, M. mercenaria, A. tenuiremis and from the
potassium dichromate reference toxicant tests using B. plicatilis 29
B. Results obtained from Cadmium aqueous and sediment bioassays 35
C. Results obtained from DDT aqueous and sediment bioassays 41
D. Results obtained from Fluoranthene aqueous and sediment bioassays. . . 47
IV
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Chapter 1
Introduction
Anthropogenically-induced chemical contamination of
sediments has resulted in the need to develop an
appropriate suite of toxicity tests to holistically assess
ecotoxicological impacts on estuarine ecosystems.
Existing Environmental Protection Agency (EPA)
protocols recommend a number of test organisms,
including amphipods, polychaetes, molluscs,
crustaceans and fish for use in sediment toxicity tests
(EPA, 1991). While this suite of test animals
represents a diverse group of fauna, many of the
species recommended by the EPA are not
indigenous to all geographic regions of the United
States, particularly the Gulf of Mexico and South
Atlantic. As a result, environmental risk assessment
based on these organisms may not adequately
protect ecosystem health in the Gulf of Mexico.
Ideally, appropriate test organisms to evaluate
sediment toxicity should include species indigenous
to the Gulf of Mexico that are representative of a
variety of faunal classes and feeding types.
Additionally, the toxicity test endpoints should include
both lethal (mortality) and sublethal (reproduction,
growth, physiological impairment) effects and they
should be sensitive to either porewater and/or whole
sediment exposures for all major classes of chemical
contaminants (trace metals, polycyclic aromatic
hydrocarbons (PAHs), pesticides). Finally, test
species should be easy to collect and maintain in the
laboratory.
The purpose of this study was to evaluate the relative
sensitivity of a variety of test organisms, broadly
distributed throughout the southeastern United States
and the Gulf of Mexico to several classes of chemical
contaminants in both whole sediment and
aqueous/porewater exposures. Additionally, several
rapid screening assays were compared with these
more traditional toxicity evaluations. The three model
contaminants selected for study were cadmium (an
inorganic toxicant), DDT (a persistent organochlorine
pesticide) and fluoranthene (a polycyclic aromatic
hydrocarbon [PAH]). These compounds represent
contaminants frequently measured in sediments
throughout the Gulf of Mexico. MacDonald (1994)
reported that the percentage of stations on the
Atlantic Coast of Florida where sediment con-
centrations exceeded the Threshold Effects Level
(TEL) was 15.2 % for Cd, 42.2 % for fluoranthene
and 1.2-1.8 % for DDT and related metabolites. On
the Gulf Coast the % of TEL exceedances was 12.6
% for Cd, 20.9 % for fluoranthene and 2.5 % for DDT
and related metabolites.
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Chapter 2
Methods
Description of Species Tested
Palaemonetes pugio
The grass shrimp, Palaemonetes pugio, is a common
shrimp species found in tidal marsh systems along
the east coast of the U.S. and Gulf of Mexico. These
shrimp are a major force in accelerating the
breakdown of detritus in the estuary and are
important dietary components for many fish species
(Wood, 1967). P. pugio can be found in salinities
ranging from 2 to 36 %o and are most abundant in
vegetated habitats (Anderson, 1985). In South
Carolina estuaries, P. pugio occur year round at
densities ranging from <1000/50m of stream in winter
to 28,000/50m of stream in summer. P. pugio
comprise 56 % of the total stream density on an
annual basis, therefore any significant reductions in
grass shrimp populations would greatly affect the
entire estuarine ecosystem (Scott et al., 1994).
These factors have led to P. pugio being used as a
representative nontarget species in many insecticide
toxicity tests (e.g., Mayer, 1987).
Ampelisca abdita
Ampelisca abdita is a tube-dwelling, infaunal
amphipod (Bousfield, 1973). This species has a
cosmopolitan distribution in coastal waters of the
United States ranging from Maine to Florida on the
eastern seaboard, throughout the Gulf of Mexico, and
in San Francisco Bay (Mills, 1967; Bousfield, 1973;
Nichols and Thompson, 1985; Summers, unpubli-
shed). \t is generally found in sediments consisting
of fine sand to mud and it has been reported to range
in depth from the intertidal zone to 60 m. Although A.
abdita has been found during all seasons in several
studies conducted in South Carolina, it is generally
most abundant during the fall, winter and spring
months and is usually only found in areas with
salinities greater than 20 ppt (Van Dolah et al., 1990,
1994; Wendt et al., 1990). A. abdita is both a deposit
and filter feeder and generally constructs a well-
developed tube.
Ampelisca verrilli
Ampelisca verrilli is also an infaunal, tube dwelling
amphipod that has a cosmopolitan distribution
throughout the East coast of the U.S. Extensive
surveys of benthic fauna in subtidal portions of the
Gulf of Mexico estuaries have not collected A. verrilli,
but a very closely related species, A. holmesi (Mills,
1967), is widely distributed in that region (Summers,
unpublished EMAP Louisianian Province data).
Additionally, A. verrilli has been collected from the
west coast of Florida. In South Carolina, A. verrilli
appears to be most abundant in muddy sand flats
near high-salinity inlets at the mouth of estuaries.
We have observed dense populations in fine to
medium sands from high in the intertidal zone to
shallow subtidal bottoms. Bousfield (1973) reports its
distribution to extend from the low intertidal zone to
50 m in depth. Although A. verrilli can be collected
during all seasons, it appears to be more abundant
during the warmer months (Wendt et al., 1990).
Assays conducted in our laboratory to evaluate the
salinity tolerance of A. verrilli indicate that it survives
well above 20 ppt. It also survives well in all non-
toxic sediments that we have tested, ranging from
unconsolidated muds with very little sand to very
sandy sediments with little or no mud. This species is
both a deposit and filter feeder and the tube it
constructs is generally not very well developed
compared with A. abdita tubes, which may increase
its exposure to contaminants. In some sediments, it
does not appear to construct a tube.
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Amphiascus tenuiremis
Amphiascus tenuiremis is an easily-cultured,
diosaccid harpacticoid copepod, collected originally
In 1988 from North Inlet, SC (Chandler, 1986). This
species is more abundant at higher latitudes than SC
but is amphi-Atlantic in distribution ranging from the
North Sea/Baltic intertidal to the southern Gulf of
Mexico. Diosaccid copepods are the most abundant,
diverse and widely-distributed family of sediment-
dwelling copepods. Amphiascus tenuiremis has a
generation time of 21 days at 20°C, and is capable of
multiple clutches in short periods of time (i.e., 10-14
d post-insemination). This species has been shown
to be sensitive to pesticides (Chandler, 1990;
Chandler and Scott, 1991), PAHs (Fulton et al.,
1997) and trace metals (Green et al., 1993) in acute,
chronic and multi-generational bioassays.
Mercenaria mercenaria
Mercenaria mercenaria is a marine filter-feeding,
infaunal mollusk in the family Veneridae
(Pechenik,1991). These bivalves are commonly
known as "northern quahogs," "cherrystones,"
littlenecks" or "hard-shell clams." Among bivalves,
this clam is second only to oysters in commercial
importance in the United States, partly due to its
ability to remain tightly closed and live for weeks out
of water if refrigerated. Mercenaria mercenaria
occurs along the East coast of the United States from
the Gulf of St. Lawrence to the central Florida coast,
with a subspecies, M. mercenaria texana, occurring
in the northern Gulf of Mexico (Menzel,1988; Dillon
and Manzi,1989). Throughout its range, M.
mercenaria primarily inhabits intertidal to shallow
subtidal estuarine areas and filter feeds on detritus
and phytoplankton, such as Isochrysis galbana.
Mercenaria mercenaria have demonstrated
sensitivity to anthropogenic contaminants. Calabrese
et al. (1977) determined that some clams exposed to
heavy metals exhibited retardation of growth. Such
growth retardation may prolong the pelagic life stage
of the larvae which may ultimately lead to increased
predation, thus decreasing the larval survival rate.
Ultimately this results in a lower recruitment into the
population and reduced commercial harvest for
human consumption.
Brachionus plicatilis
Brachionus plicatilis is a rotifer species in the family
Brachionidae, which has a global distribution. This
species has been collected from estuarine habitats in
both the southeastern U.S. and the Gulf of Mexico. It
has also been found on six continents, and several
strains are being cultured worldwide (Snell and
Persoone, 1989). As with other rotifer species, B.
plicatilis filter feeds on phytoplankton and bacteria,
has a rapid reproductive cycle and short generation
time, and can be grown from dormant eggs (cysts)
that can be stored for long periods of time. Snell
and Persoone (1989) developed an acute toxicity
bioassay using this rotifer species for brackish and
marine environments, and have shown that it is
sensitive to several contaminants. This assay has
been further developed by Creasel, Ltd. (Deinze,
Belgium) as the Rotoxkit M® toxicity test kit. Since
the test can be done rapidly (24 hr exposure),
requires relatively little aqueous solution, and is
inexpensive to conduct, it was considered to be a
worthwhile test protocol for comparison with the other
bioassay protocols and species.
Mysidopsis bahia
The mysid, Mysidopsis bahia, is a crustacean found
in the estuarine waters of the northern Gulf of Mexico
from southwestern Florida to Mexico. These
crustaceans are ecologically important as food for
many fish species and are also important in detritus
breakdown. Mysids inhabit shallow water grass flats
with salinities ranging from 9 to 29 %0. Of the mysid
species, M. bahia has been the most extensively
studied. The USEPA, other US government agencies
and private laboratories have selected this
crustacean as a toxicity model for many of their
assessment programs (EPA, 1980, 1994; Nimmo et
al., 1977). Aqua Survey, Inc. has developed a rapid
toxicity screening test (Mysid IQ Test™) that utilizes
a sublethal toxic response in M. bahia. The IQ test is
based on the ability of a healthy, unstressed
organism to ingest and metabolize a fluorigenic
substrate, 4-methyllumbelliferyl-3-D-galactoside
(MUG). Non-affected organisms are able to cleave
galactose from MUG, forming 4-umbelliferone which
is strongly fluorescent under longwave UV light.
Impacted organisms display reduced fluorescence
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which can be quantified relative to control organisms.
Since this test can be done rapidly (1 h exposure)
and requires little test solution, it was included
among the suite of bioassay protocols evaluated.
Microtox and Mutatox
The Microtox and Mutatox screening assays were
evaluated to determine their relative sensitivity to the
three model toxicants. The Microtox assay utilizes
the photoluminescent bacterium, Vibrio fischeri, to
provide a sublethal toxicity measure which is based
on the attenuation of light production by the bacterial
cells due to toxicant exposure. The Mutatox assay
utilizes a dark strain of Vibrio fischeri which reverts
to the bioluminescent strain when exposed to
mutagenic substances.
Collection and Holding of Test Organisms
Palaemonetes pugio
Adult grass shrimp, P. pugio, were collected from
Leadenwah Creek, a tidal tributary of North Edisto
River estuary, located on Wadmalaw Island, SC.
Seawater used for holding and exposures was
collected from Bohicket Creek, another tributary of
North Edisto River. Adult shrimp (20-35 mm) were
acclimated in 76-L tanks at 20°C, 30 %o salinity and
12-h light: 12-h dark cycle. Shrimp were fed daily with
Tetramin Fish Flakes and newly hatched Artemia.
Shrimp were collected five to seven days prior to
testing.
Ampelisca abdita
Since local populations of A. abdita were not
available for use in the bioassays, all A. abdita were
obtained either from Science Applications
International in Narragansett, Rhode Island, or from
East Coast Amphipods, Inc. in Kingston, Rhode
Island. Both facilities collected their amphipods from
tidal flats in the Pettaquamscutt River which flows
into Narragansett Bay, Rhode Island. The majority of
specimens tested in our assays ranged in size from
3 to 5 mm. Bousfield (1973) reported this size to be
in the juvenile to early adult size range. Prior to
testing, A abdita were acclimated in the laboratory to
the testing temperature (20°C) for a period of 2-6
days, with daily feeding using Phaeodactylum
tricornutumorChaetocerussp.
Ampelisca verrilli
The A. verrilli used in this study were collected from
intertidal flats in a relatively pristine, undeveloped
section of the Folly River near Charleston, South
Carolina. The majority of organisms tested in our
assays ranged in size from 5 to10 mm, which is
considered to be in the juvenile to adult size range
(Mills, 1967). Prior to testing, all A. verrilli were
acclimated in the laboratory and fed using the same
protocols described for A. abdita.
Amphiascus tenuiremis
The A. tenuiremis used in this study were obtained
from laboratory stock cultures established in 1988
from North Inlet, SC brood lines. Stock populations
were continuously cultured underflow in clean sedi-
ments at 30 %0 salinity, 21 °C and 12:12 L:D photo-
period. Populations were fed twice weekly with
concentrated (>106 cells/ml) phytoplankton. In
preparation for each experiment, sediment was
removed from the copepod culture system and rinsed
with clean seawater on a 125 //m sieve to collect the
copepods. Copepods were gently washed into
plastic petri dishes where they were sorted by sex.
Copepods were kept in clean seawater in petri dishes
at 21 °C up to 48 h prior to the initiation of an
experiment.
Mercenaria mercenaria
Juvenile clams, M. mercenaria, were acquired from
Atlantic Littleneck Clam Farm located on James
Island, SC. Seawater used for holding and
exposures was collected from Bohicket Creek, a tidal
tributary of North Edisto River estuary. Juvenile
clams (>212<350 /^m) were acclimated for 24 to 48h
in 16 oz precleaned glass jars at 20°C, 30 %o salinity
and 12-h light: 12-h dark cycle. Clams were fed daily
Isochrysis galbana obtained from the stock culture at
the clam farm.
Brachionus plicatilis
The B. plicatilis used in this study were supplied as
dried cysts in the Rotoxkit M® test kits. The cysts
were hatched by incubating them in 10 ml of 20 %o
artificial seawater under continuous lighting at 25 °C
for a period of approximately 28-30 hrs just prior to
testing.
Mysidopsis bahia
The M. bahia used in the tests were shipped
overnight from Flemington, New Jersey to the
National Ocean Service laboratory at Charleston,
South Carolina. One-day-old mysids placed in
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artificial seawater (Forty Fathoms™) containing food
were shipped overnight in insulated containers, along
with a container of dilution water. The dilution water
was the same solution of Forty Fathoms™ seawater
mix but without added food. Upon arrival at the
laboratory, mysids and their dilution water (28 ± 2 %0)
were transferred to small aquaria (2.5 gallons). An
airstone was placed in the aquaria to maintain
adequate dissolved oxygen levels. Mysids were fed
Artemia nauplii larvae. Prior to dosing, the test
organisms were held overnight in order to acclimate
them to the laboratory conditions, as well as to clear
their intestinal tracts prior to toxicity testing.
Reference Toxicant Tests
Sodium dodecyl sulfate (SDS) was used as reference
toxicant for most test species to ensure that each
batch of organisms used in the contaminant
bioassays were of comparable sensitivity. The grass
shrimp, amphipods, copepods and clams were tested
by exposing 10-20 organisms to various
concentrations of SDS for a 24 h period at 20 °C and
30 %o salinity (20 /urn filtered seawater). Exposure
media volumes were 2 L for grass shrimp, 0.8 L for
amphipods, 0.050 L for copepods and 0.5 L for the
clams. Dose ranges varied for each species,
depending on their sensitivity to the toxicant. To
develop baseline data, at least five reference toxicant
tests were completed prior to conducting any of the
model toxicant bioassays. Subsequent SDS
bioassays conducted for each definitive test were
added to the database to create a running mean. The
acceptance criteria for a given batch of animals was
defined as the mean LC^ for all previous SDS tests
± two standard deviations. Reference toxicant tests
for B. plicatilis were conducted using serial dilutions
of the toxicant, potassium dichromate, which was
provided with the test kits. The exposure protocol
was the same as described above, except that 30
rotifers were exposed at each potassium dichromate
concentration. Percent survival was assessed after
24 hours and compared with the acceptable limits
(95% CI) provided by the manufacturer for that batch
of animals. A phenol standard was used as a positive
control in all microtox assays. Acceptance criteria
were provided by the manufacturer. No reference
toxicant tests were conducted with the mysid tests.
Analytical Chemistry
Contaminant Stock Solutions
Spiking solutions were prepared by dissolving the
toxicant in deionized water (cadmium) or acetone
(DDT and fluoranthene). Stock solutions were
quantified using either inductively coupled plasma
spectroscopy (ICP) (cadmium), gas chromatography
with electron capture detection (GC-ECD) (DDT) or
high performance liquid chromatography (HPLC) with
fluorescence detection (fluoranthene). Stocks were
then distributed to each laboratory for use in the
aqueous assays which required various dose levels
dependent upon the species and/or protocol being
tested. All contaminant-spiked sediments were also
prepared using these stocks. Specific sediment
spiking protocols are described in the sections
describing bioassay protocols.
Quantification of Cadmium in Spiked Sediments
Twenty gram aliquots were taken from each batch of
cadmium-spiked sediment for analysis. Each aliquot
was transferred to a 30-ml acid-washed plastic
sample cup. The sample was then covered and dried
at 70°C for 24 hours. After drying, the sample was
reweighed to determine moisture content. The dried
sediment was then ground with a mortar and pestle
and transferred to a 20-ml plastic screw-top
container.
Ground samples were extracted using a closed-
vessel, concentrated acid microwave digestion
technique. A 0.5-g subsample of the ground
sediment was weighed (0.0001 g) into a Teflon-lined
digestion vessel, and 10 ml of concentrated HNO3
(Instra-analyzed) plus 0.5-ml deionized water was
added. The sample was then microwaved using a
well ventilated, 600 watt corrosion-resistant digestion
microwave (CEM Model MDS-2000) for 2 hours at
full power and 120 psi. The sample was allowed to
cool, then 2.0 ml of 30% H2O2 was added. The vessel
was then microwaved for an additional 10 minutes at
full power and 80 psi. After cooling, the digestate
was filtered (#41 filter paper) into a 50-ml volumetric
flask and brought to volume with deionized water.
The sample was then transferred by pouring into a
50-ml polypropylene conical centrifuge tube for
analysis.
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Cadmium-spiked samples were analyzed by induc-
tively coupled plasma spectroscopy (ICP). The
instrument (Perkin Elmer Plasma 1000 with auto-
sampler) was calibrated by developing a standard
curve. The response factor was determined as the
slope of the standard curve line (absorbance/mg
cadmium). Sample extracts were analyzed in dupli-
cate and the results averaged and reported as mg/Kg
dry weight (dw)
Quantification of Acid Volatile Sulfides (AVS) and
Simultaneously Extractable Metals (SEM) in
Cadmium-Spiked Sediments
The general procedure for measuring AVS and SEMs
was based on Allen et al. (1991) with modifications
as described below. Sulfide and metals were
released from sediment using a N2 gas supply
system and a reaction/trap system by placing about
5 g of wet homogenized sediment into each of six
500-ml round-bottom flasks. Deionized water (80 ml)
was then added to each flask together with a small
Teflon-coated stir bar, and the injection ports were
sealed with rubber septums. The sediment-
deionized water mixtures were then purged with
nitrogen for 10 minutes to remove residual oxygen.
After the nitrogen flow was stopped, 20 ml of 6 M HCl
was added to each flask. The HCl was added
through the rubber septum using a syringe to
volatilize the sulfides and metals in the sediment
sample. The samples were stirred with the magnetic
stirrers and the volatilization reaction allowed to
proceed for 1.5 hours. Each boiling flask was then
filtered through a 0.45-,am membrane filter. The flask
was rinsed several times with deionized water, with
the rinses added to the filtrate. The volume of the
filtrate was measured, and a 50.0-mf aliquot removed
for SEM analysis.
Impingers contained 0.5 M NaOH to capture H2S
retained from the boiling flask. The NaOH traps were
developed by adding 10 ml of a mixed diamine.
reagent (Allen et al., 1991) and allowing the mixture
to react for 30 minutes. The solution was
quantitatively transferred to a 100-ml volumetric flask
and brought to volume. Approximately 2 ml of
solution were transferred to a cuvette, and the
absorbance at 670 nm read using a spectra-
photometer (Milton Roy Spectronic Model 601).
A standard sulfite solution was prepared by weighing
12 g of Na2S.9H2O into 1.0 L of deionized water. The
solution was standardized by the sodium thiosulfate
titration procedure described in Allen et al. (1991)
using a starch indicator. From the standardized
solution, 0 (blank) to 10 ml were pipetted in 1-ml
increments, transferred to 100-ml volumetric flasks
and developed using the mixed diamine reagent.
Absorbance was measured at 670 nm and used with
solutions of known concentration to construct a
standard curve.
Simultaneously extracted metals (SEMs) were
measured in the 50.0-ml aliquot removed from the
sediment extract. The acid treatment removes
metals which are weakly associated with the
sediments and not incorporated in crystalline
matrices. Samples were analyzed by ICP for
cadmium using the methods previously described.
Quantification of DDT in Spiked Sediments
The methods for extraction and sample preparation
for organic contaminants in sediments were similar to
those of Krahn et al. (1985) with a few modifications.
In preparation for analysis, sediment samples were
thawed and allowed to reach room temperature.
Visible detritus was removed from the sample, and
the sediment thoroughly stirred with a stainless steel
spatula. A portion of the sediment was transferred to
a beaker and placed on a top-loading balance, where
about 8.5 g of sediment was accurately weighed by
difference (0.01 g) and placed in a Pyrex mortar.
The sediment was then dried by mixing with 100 g of
Na2SO4 which had been ashed for 16 h at 700°C.
The dried sample was transferred to a Pyrex Soxhlet
thimble. The sample was then extracted in a Soxhlet
apparatus with 250 ml of CH2CI2for 18 hrs. Sample
extracts were reduced in volume by a stream of
purified nitrogen using: a nitrogen blow-down
concentrator (Turbo Vap, Zymark Instruments) to
about 0.5 ml. The CH2CI2 was replaced with
isooctane and concentrated to a final volume of
about 1.0 ml and transferred to an autosampler vial
for analysis by gas chromatography (GC) with
electron capture detection (ECD).
The instrument (GC-ECD; Hewett-Packard 5890
series II) was configured with one column, a 30-m x
0.25-mm i.d. (0.25-mm film thickness) DB-5 (5%
phenyl; J&W Scientific). The initial carrier gas
constant average linear velocity was 33 cm/sec. The
carrier and detector makeup gasses were helium
and nitrogen (95%:5%), respectively. The injector
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and detector temperatures were 250 °C and 320 °C,
respectively. The sample was injected (1 yul) using a
splitless Grab technique (1 min split time). The initial
oven temperature was 50°C with a one-minute hold,
followed by an increase to 170°C at4°C/min, then to
210°C at 1°C/minute, and finally to 310°C at
4°C/min with a 10 min hold. The detector signal was
digitized and processed using the Windows-based
EZChrom software (Scientific Software Inc.).
The instrument was calibrated using a mixed
standard of the target analytes (chlorinated
pesticides, NIST SRM 2261). The slope of the
response curve with respect to the internal standards
was used to quantify the concentrations of the
analytes in the unknown (i.e., test) sample. The
calibration curve was verified at the beginning of
each sample set by injecting the mid-level, con-
tinuing calibration, which is a check standard which
was required to be with ± 20% of the known value for
each analyte; otherwise, the instrument was
recalibrated.
Quantification of Fluoranthene in Spiked
Sediments
Fluoranthene-spiked sediments were prepared and
dried as described above. The dried sample was
then transferred to a PyrexSoxhlet thimble, and the
internal standards D8-naphthaIene (200 ng), D10-
acenapthalene (200 ng), D10-phenanthrene (502 ng),
D,0-fluoranthene (497 ng), D12-perylene (102 ng),
dibromooctafluorobiphenyl (PCB-103; 20 ng), and
2,2'13,3l,4>5,5I6-octachlorobiphenyl (PCB-198; 20ng)
were added. The sample was then extracted in a
Soxhlet apparatus with 250 ml of CH2CI2 for 18
hours. Sample extracts were reduced in volume by
a stream of purified nitrogen using a nitrogen blow-
down concentrator (Turbo Vap, Zymark Instruments)
to about 0.5 ml. Lipids and other high molecular
weight compounds were removed from the sample
by gel permeation chromatography. The liquid
cnromatograph consisted of an autosampler (Gilson
Model 231), a Waters HPLC pump (Model 501), two
22.5-mm x 500-mm gel permeation columns in series
(Phenomenex Phenogel, 100 A pore size), a UV
detector (Linear Model U-106), and a fraction
collector (Gilson Model 201). The mobile phase was
CH2CI2 at a flow rate of 7 ml/min. A 400-ml sample
was injected into the system. Lipids and other high
molecular weight compounds were eluted in the first
14 minutes. The fraction of interest was collected
beginning 1 minute before the retention time of
DBOFBP and ending 2 minutes after perylene. The
resulting fraction was reduced in volume as above.
The CH2CI2 was replaced with hexane and concen-
trated to a final volume of about 0.5 ml. At this point,
elemental sulfur was removed from the sample by
treatment with activated copper. To remove
remaining polar interferences, the sample was
transferred to a 6-g cyanopropyl solid-phase
extraction cartridge (Varian, pre-rinsed with 6 ml of
hexane) and eluted with 12 ml of hexane. The eluent
was reduced in volume to about 0.5 ml, and 200 [A
were transferred to an autosampler vial for analysis
by gas chromatography (GC) with electron capture
detection (ECD) and GC with ion trap mass
spectrometry (GC-ITMS) detection analysis (see
below). The hexane in the remainder of the sample
(about 200 jj\) was replaced with acetonitrile for
analysis by high performance liquid chromatography
with fluorescence detection (HPLC-fluorescence).
PAHs were additionally quantified using HPLC with
fluorescence detection utilizing a method similar to
Wise et al. (1988) and Schantz et al. (1990). The
instrument consisted of two HPLC pumps (Waters
6000A), a 680 gradient controller (Waters Model
680), and an autosampler (Waters WISP). The
column dimensions were 6 mm x 25 cm, with a 5-^m
particle size (Supelco LC-PAH). The column was
heated to 30 ° (Fiaton TC-50 column heater controller
and a CH-30 column heater). The solvent was
pumped at a constant flow rate of 1.5 ml/min with a
gradient program that started with a two-minute hold
at 60% water: 40% acetonitrile followed by a linear
increase to 55% water: 45% acetonitrile in 15
minutes and a final increase to 0% water: 100%
acetonitrile in 35 minutes with a 10-minute hold.
Fluorescence was monitored with two fluorescence
detectors (Perkin Elmer LC-240 and LS-4) connected
in series at wavelengths specific to individual PAHs
(Appendix B - Table B-1). The separation between
deuterated and nondeuterated PAHs was 0.44,0.40,
and 0.41 minutes for phenanthrene, fluoranthene,
and perylene, respectively.
Data collection was accomplished using Perkin Elmer
Omega II personal computer-based software. A
NIST certified PAH standard solution and the
deuterated PAH internal standards were used to
calibrate the instrument. Sample peaks were
identified by retention times and fluorescence at
8
-------
specific wavelengths. TOC was measured using a
Perkin Elmer Model 2400 Series IICHNS/O Analyzer
on three replicate 15 g sediment samples.
Aqueous Contaminant Bioassay Protocol
All 24 h aqueous contaminant bioassays were
conducted at 20 °C and 30±2%o salinity. The rationale
for the aqueous bioassays was to determine the
inherent sensitivity of the test species in seawater
exposures where organisms were directly exposed to
a given toxicant. Often in sediment exposures, the
interaction of the contaminant with the sediment may
reduce or prevent exposure of the organism. Thus,
the aqueous exposures were used to assess the
overall sensitivity of each species which could then
be compared with the sediment toxicity test results.
Deionized water was the carrier for the cadmium
bioassays while acetone was used for the DDT and
fluoranthene tests. All test concentrations and the
controls received the same carrier concentration. For
all tests, the trimmed Spearman-Karber method was
used to calculate the LC50 for each replicate based
on nominal doses, whenever at least one dose
produced mortality > 50%. Comparisons of LC50
estimates among species were performed using
ANOVAon log (x+1) transformed data. Bonferroni's
test was used to identify specific group differences.
Aqueous bioassays • with grass shrimp were
conducted in 4-L wide-mouth glass jars with five
replicate chambers for each test concentration and
the control. Two liters of test solution were added to
each jar. Six serial dilutions (60%) were used for
cadmium and DDT and five were used for
fluoranthene. Ten grass shrimp were added to each
jar and all jars were then placed in a Revco
Environmental Chamber. Tests were run under a 12-
h light: 12-h dark cycle. Shrimp were not fed during
the test. Temperature, dissolved oxygen, salinity and
pH were recorded from each control replicate at the
beginning and end of the exposure period. Shrimp
mortality at the end of the test was recorded from
each jar based on lack of movement and the failure
to respond to tactile stimulation.
Aqueous bioassays using the two amphipod species
involved exposure of 10 amphipods/ replicate 1-liter
beaker in 700 ml of toxicant solution under constant
aeration and a 12L12D light cycle. Five replicate
groups of amphipods were used for each treatment.
Each contaminant test consisted of six treatments
containing seawater spiked with the varying doses of
the toxicant (60% dilution series) and a control
containing seawater with an equivalent carrier dose.
Usually, both species were tested at the same time
with the same stock solution and all treatment groups
randomized with respect to location on the water
table. Amphipod mortality at the end of the test was
recorded from each jar based on lack of movement
and the failure to respond to tactile stimulation.
Copepod bioassays were run in five replicate, 50-ml
glass crystallizing dishes containing 45 ml of the
appropriate test solution. There were five replicates
for each of five toxicant concentrations and the
control. Twenty copepods were used per replicate.
Bioassays were run in total darkness. After 24 hours,
each dish was sieved on a 63 /^m sieve and the
number of live and dead copepods determined.
Temperature, salinity, dissolved oxygen and pH were
measured at beginning and end of each bioassay.
Clam bioassays were run under constant aeration
and a 12L12D light cycle in 600 ml Pyrex glass
beakers containing 500 ml of test solution, with five
replicates for each test concentration and the control.
Ten clams were added to each beaker and the
beakers were placed in a Revco Environmental
Chamber. Clams were not fed during the 24-h test
periods. Temperature, dissolved oxygen, salinity and
pH were recorded from each control replicate at the
beginning and end of the test. At the end of the
bioassay, clam mortality from each beaker was
recorded. Clam mortality was determined using an
Olympus SZH10 Microscope under 7.0 x magnifica-
tion and Mocha Image Analysis (Jandel Scientific) to
capture images of the clams. Clams were
determined to be alive if locomotion was exhibited
following placement in the petri dish. Some clams
which remained closed for several minutes were
gently moved by tapping the petri dish or moving the
clam onto its umbo to ensure they were alive. Dead
organisms were assessed based on a gaping shell
and/or no response to tactile stimulation.
Both the aqueous and sediment pore-water assays
using B. plicatilis were performed using procedures
similar to those described by Snell and Persoone
(1989), with some modifications. Toxicant doses for
the rotifers were mixed from natural seawater (30 %o)
and the same contaminant stocks used for the
-------
amphipod assays. Sediment porewater was
obtained from each dose of the spiked sediments
used for the amphipod assays by centrifuging
approximately 50 ml of sediment at 10,000 RPM for
10 minutes. Rotifers were exposed in multi-well test
plates which had one large well for hatching the cysts
and six series of additional wells that included one
0.7 ml rinsing well and six 0.3 ml exposure
wells/series. Approximately 50 neonates were
transferred from the hatching well into each rinsing
well which contained one of the toxicant doses or
control water. From there, 30 neonates were
transferred to six test wells (5 neonates/well) which
contained the same dose of toxicant or control water.
The plates were then covered and incubated in a
12L12D light cycle at 25 °C. The number of alive
versus dead rotifers were counted after 24 h to
determine percent survival for each toxicant dose,
and an LC^ was computed using the trimmed
Spearman Karber method whenever at least one
dose resulted in >50% mortality. A spiked-sediment
porewater concentration was considered to be toxic
if its survival was statistically lower (p<0.05) and less
than 80% of control survival.
Mysid IQ™ assays were performed following
protocols described by the manufacturer (Aqua
Survey, 1994). Test organisms were exposed in
ultraviolet transmissible acrylic plates (12cm x 8.5cm
x 1cm) containing six shallow cylindrical chambers
(1cm deep, 3.5cm diameter). Three of the chambers
(exposure chambers) were filled with five ml of test
solution, and the other three chambers (response
chambers) were filled with five ml of Aqua Survey
Dilution Water (ASDW). Using a wide bore pipette,
six mysids were added to each of the exposure
chambers where they were kept for one hour. Next,
0.25 ml of the fluorigenic substrate, 4-methyl-
umbelliferyl-B-D-galactoside (MUG), was added to
the exposure chambers. After 20 min, mysids were
transferred to the response chambers using great
care to minimize the amount of substrate entering the
chambers. After the mysids were placed in the
response chambers, the lights in the room were
turned off, and the mysids were placed over a
longwave UV lamp. The number of fluorescing
mysids were recorded. The proportion of mysids not
fluorescing in each treatment was calculated. An
EC50 was then calculated using the trimmed
Speamnan-Karber method.
Sediment Bioassay Protocol
Sediment was collected from a "relatively pristine"
site located on the tidally influenced Folly River, SC.
Sediment was press-sieved through a 1 mm mesh
screen into 5-gallon plastic buckets at the collection
site, then transported to the laboratory and stored at
3°C until used in the bioassays (<, seven days).
Sediment for the grass shrimp, amphipod , Microtox,
Mutatox and rotifer assays was spiked in acid
washed 4-L wide-mouth plastic jars 24 h before the
start of the bioassay, then rolled on a jar mill until
thorough mixing had occurred (~2 h). Contaminant
spikes were based on the estimated dry weight of the
sediment. Deionized water was the carrier for
cadmium while DDT and fluoranthene were dissolved
in acetone. All test concentrations and the controls
received the same carrier concentration.
For the copepod bioassays, sediment was wet-
sieved on a 212 ^m sieve and collected in a clean 4
L beaker. The sediment slurry was allowed to settle
for 24 h at 4°C. Supernatant was removed from the
slurry by aspiration. The sediment slurry was
transferred to a clean 1 L beaker and homogenized
for 30 min. by continuous stirring with a 3" stirring
bar. Aliquots of 75-200 ml of homogenized sediment
slurry were distributed to clean 250 ml glass beakers
and stirred. Appropriate amounts of contaminant
were added to stirring sediment slurry. The slurry
was homogenized for 2 h before placing 10 ml of
each concentration in each of five replicate test
chambers containing 20 ml of filtered artificial
seawater.
Sediment for the clam bioassays was press-sieved
through a 2*\2^m mesh screen for M. mercenaria
bioassays. Sediment was spiked in acid-cleaned
1000 ml Pyrex beakers with the appropriate amount
of testing compound 24 h before the start of the
bioassay, then stirred vigorously by hand (~5 mins)
until thorough mixing had occurred.
All bioassays were run at 30 %o salinity and 20°C.
Each day, temperature, dissolved oxygen, salinity
and pH were recorded from each control replicate.
On days 0,2 and 8, ammonia was measured in three
randomly selected control replicates and one
randomly selected treatment replicate. When
10
-------
possible the trimmed Spearman-Karber method was
used to calculate LC50 values as previously
described. In those cases when LC50s could not be
calculated, mortality in treatment groups was com-
pared with that in the controls using either a t-test or
ANOVA on the transformed percentage (arcsine sq.
rt. ofP).
Grass shrimp bioassays were run in 4-L wide-mouth
glass jars. There were five replicates for each
concentration and the control. The sieved sediments
were allowed to warm to room temperature and then
rolled on a jar mill for approximately 30 minutes,
since separation into liquid and solid phases may
have occurred. Approximately 300 ml of sieved
sediment was placed into each 4-L jar, then 1700 ml
of 20 /^m filtered seawater was added. The jars were
capped with Teflon-lined plastic caps through which
a 1 m£ pipette was inserted into a pre-drilled hole.
The jars were placed in a Revco Environmental
Chamber with airlines attached to the 1 me pipettes.
The sediment was allowed to settle under aeration in
the bioassay jars for 24-h before the addition of the
grass shrimp. After 24-h, ten grass shrimp were
added to each jar and the jars returned to the
environmental chambers. Bioassays were conducted
using 12-h light: 12-h dark cycle. Shrimp in each jar
were fed Tetramin fish flakes every 48 h in order to
reduce mortality from cannibalism. Mortality was
determined on day 10.
The sediment bioassays for both amphipod species
involved a 10-day static whole sediment assay using
procedures similar to those described by Swartz et
al., (1985) and ASTM (1993). Test chambers were
1-liter pyrex beakers filled with 200 ml of sediment
and 800 ml of seawater and covered with an inverted
glass dish. The sediment and seawater were added
to the beakers approximately 24 hr prior to
inoculating the sediments with amphipods using the
procedures described above.
Five replicate series of beakers were tested, with
each series consisting of four doses containing
sediment spiked with varying concentrations of
contaminants as described above and one control
containing sediment with an equivalent concentration
of either distilled water or acetone only. All tests
were conducted under constant lighting to inhibit
amphipod emergence from the sediment. Air was
provided using oil-free pumps and glass pipettes
inserted into the test chambers. For most tests, both
species were tested at the same time and all
treatment groups were randomly located on the water
table.
At the end of each assay, the test chambers were
sieved through a 0.5 mm mesh screen and the
number of animals alive, dead, or missing was
recorded. Sediment test results were considered
valid if the overall survival was >85% in the control
group and no replicate fell below 80% survival. All
beakers were also inspected daily to record the
number of animals that were found either dead or
alive on the surface of the sediments. The size (total
length) of the amphipods used in each assay was
measured by selecting 10 amphipods from one
randomly-selected beaker representing each
treatment dose.
Test chambers for the copepod bioassays consisted
of 50 ml Teflon® Erlenmeyer flasks fitted with outflow
ports covered with 63 ^m Nitex mesh. Fifteen A.
tenuiremis adult males and 1-5 non-gravid females
were added to each test chamber without disturbing
the sediment. Chambers were placed in an incubator
under flow at 20°C, 30 %o salinity and 12:12 L:D
cycle for 10 days. Physical parameters were
monitored at the beginning and end of an
experiment.
Upon completion of an experiment, test chambers
were removed from the incubator. Contents of the
chamber were washed onto a 63 ^.m sieve with
filtered artificial seawater and rinsed into a plastic
petri dish. The contents of each dish were stained
with Rose Bengal and preserved with formalin to a
final concentration of 5%. The dishes were
refrigerated until their contents could be counted
under stereo dissection microscope.
Female and male mortality was assessed. In
addition, the reproductive endpoints of copepodite,
nauplii and clutch size were also examined. In cases
where mortality was significant, an LC50 value was
computed as previously described. In cases where
mortality was minimal, reproductive endpoints were
compared in a general linear model (GLM) procedure
using SAS® statistical software. Significant
differences among treatments were detected using
11
-------
Tukey's Studentized T-Test and differences between
test concentrations and the control were assessed
using Dunnett's test.
The clam bioassays were run in 600 ml Pyrex glass
beakers, with five replicates for each contaminant
concentration and the control. The sieved sediments
were allowed to warm to room temperature and then
stirred vigorously by hand, since separation of liquid
and solid phases may have occurred. Approximately
100 ml of spiked sediment was placed into each 600
ml beaker, followed by 300 ml of 20 ^m filtered
seawater. The beakers were covered with solvent-
rinsed aluminum foil through which a 1 me pipette
was Inserted and placed in a Revco Environmental
Chamber with airlines attached to the 1 me pipettes.
The sediment was allowed to settle under aeration
for 24-h before the addition of the clams. After 24-h,
50 clams were added to each beaker. Bioassays
were run at 30 %o salinity (20 ^m filtered seawater),
20°C, and a 12-h light :12-h dark cycle. Clams in
each beaker were fed 5 ml of Isochrysis galbana
every 48 hours. At the end of ten days, clam
mortality was determined from each jar and recorded.
Comparisons of LC50 estimates among species were
performed using ANOVA on log (x+1) transformed
data. Bonferroni's test was used to identify specific
group differences.
Microtox Assay Protocols
Microtox™assays were performed generally following
protocols from Microbics Corporations' Microtox™
Manual (Microbics Corporation, 1992). A phenol
standard solution was used as positive control with
each microtox bioassay.
For aqueous bioassays, serial dilutions of each
contaminant were prepared in a 2% saline diluent. A
reagent solution which contained the bacteria was
then added to each dilution. Light emission readings
were taken after 5 and 15 minutes. The percent
decrease in bioluminescence relative to the reagent
blank was used to calculate an EC^or each dilution
series at both time points using a log-linear
regression model. Five replicate assays were
performed for each contaminant.
For sediment Microtox™ assays, sediments were
collected and spiked with the model contaminants
(cadmium, DDT and fluoranthene) as previously
described. Spiked sediments were analyzed using
established protocols (Microbics Corporation, 1992;
Long and Markel, 1992). Samples were evaluated
using both the organic extract and solid phase
protocols.
The percent decrease in bioluminescence relative to
the reagent blank was used to calculate an EC50 for
each spiked sediment sample. A total of three
replicate assays were performed for each of the
sediment spikes.
Mutatox Bioassay
The Mutatox™ genotoxicity bioassay was performed
as described in Microbics Corporations' Mutatox™
manual using the same DMSO solvent extracts that
were prepared for the Microtoxorganic extract assay
(Microbics Corporation, 1992). The Mutatox™ test
uses a dark strain of the bioluminescent bacteria,
Vibrio fischeri, which will revert back to
bioluminescent strain if exposed to mutagenic
substance. Two assay protocols were utilized. The
first, the S-9 assay, utilizes media which contain
mammalian hepatic enzymes which can metabolize
promutagenic compounds and thus can be used to
screen sediments for mutagens which require
metabolic activation. The second assay, the direct
assay, uses media which contains no mammalian
enzymes and thus can be used to screen for
mutagens which do not require activation. The
mutagenic potential of samples was evaluated using
the criteria described in the Microbics Corporations'
Mutatox™ Manual. A total of three replicate assays
were performed for each spiked sediment. A spiked
sediment was considered to be mutagenic if all three
replicates met the criteria for mutagenicity.
12
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Chapter 3
Results and Discussion
Analytical Chemistry
Measured concentrations of the contaminant stock
solutions were generally similar to nominal values.
Measured concentrations were 91.6 ± 3.7% of
nominal for cadmium, 107.8 ± 7.2% for DDT and
102.1 ± 2.8% for fluoranthene. Cadmium concen-
trations measured in spiked sediments used in
definitive bioassays were generally quite similar to
nominal values, with recoveries ranging from 91-
116% of nominal estimates (Table 1). Acid Volatile
Sulfide (AVS) levels in these cadmium spiked
sediments were low and ranged from 0.019-0.028
/^mol/g.
Measured DDT concentrations ranged from 52-96 %
of the nominal estimates (Table 2). The mean TOC
concentration in these spiked sediments was 0.70%.
Measured fluoranthene concentrations ranged from
78-91% of the nominal values (Table 3). The mean
TOC in these spiked sediments was 0.48%. Except
where noted, all subsequent discussions of
contaminant concentrations will be based on nominal
concentrations.
Reference Toxicant Tests
Results obtained from all the reference toxicant tests
conducted in conjunction with the Gulf of Mexico
Project contaminant bioassays are provided in
Appendix A. Only two batches of P. pugio failed to
meet acceptance criteria. Neither of these tests was
associated with a definitive contaminant bioassay.
Only one assay using the amphipod, A. verrilli, failed
to pass acceptance criteria. This assay was
repeated with a new batch of animals which passed
the reference toxicant criteria limits. All the reference
toxicant tests for the definitive assays using the other
test species (A. abdita, B.,plicatilis, M. mercenaria
and A.tenuiremis) provided LC50 estimates that met
acceptance criteria.
Cadmium
Results obtained for the aqueous cadmium assays
for each species are provided in Appendix B and
summarized in Table 4. Although a minimum of five
replicate cadmium exposures were conducted for
each species, some of the test series resulted in
either insufficient or excessive responses which
precluded computation of an LC50or EC50 estimate
for that replicate series. Therefore, both the mean
LC50 (based on only the replicate series which
provided an LC50 estimate) and a pooled LC50
estimate (all replicates combined) are presented. The
results using each approach were quite similar in all
cases.
Comparison of the results obtained from these
aqueous bioassays (Table 4) indicated significant
differences among the eight organisms tested (p <
0.0001, ANOVA). Based on pairwise multiple
comparisons among the species using the Bonferroni
test, the juvenile clam, M. mercenaria, was the most
sensitive species and the copepod, A. tenuiremis,
was the next most sensitive species. The two
amphipod species (A. abdita and A. verrilli) showed
comparable sensitivity to this toxicant and both
species were significantly more sensitive to cadmium
than the P. pugio, Mysid IQ™ or Microtox™ assays.
The rotifer, B. plicatilis, was the least sensitive
species tested.
Table 5 provides a comparison of the 24 h aqueous
LC50 values obtained in this study with literature
13
-------
Table 1. Measured cadmium concentrations and AVS in spiked sediments.
Nominal
Concentration
(mg/kg dw)
Measured
Concentration
(mg/kg dw)
% of SEM AVS
SD Nominal (/umol/g) (Mmol/g) SEM/AVS
2.5
10.0
40.0
160.0
2.6
9.5
37.4
185.7
0.5
0.3
4.6
28
104
95
91
116
0.021
0.077
0.315
1.247
0.02
0.019
0.024
0.028
1.1
4.1
13.1
44.5
Table 2. Measured DDT and TOC in spiked sediments.
Nominal
Concentration
(mg/kg dw)
Measured
Concentration
(mg/kg dw)
SD
%of
Nominal
TOC
DDT
Concentration
(mg/g OC)
0.64
1.60
4.00
10.00
0.33
1.53
2.58
5.93
0.20
1.34
0.61
0.69
52
96
65
59
0.7
0.7
0.7
0.7
0.09
0.23
0.57
1.43
Table 3. Measured fluoranthene concentrations and TOC in spiked sediments.
Nominal
Concentration
(mg/kg dw)
Measured
Concentration
(mg/kg dw)
SD
%of
Nominal
% TOC
Fluoranthene
Concentration
(mg/g OC)
0.78
3.12
12.50
50.00
0.67
2.84
9.79
42.01
0.10
0.39
1.67
4.55
85
91
78
84
0.48
0.48
0.48
0.48
0.16
0.65
2.60
10.42
Table 4. Summary results from aqueous assays with cadmium.
Exposure
Species Period
M. mercenaria
A. tenuiremis
A. abdita
A. verrilli
M. bahla
P. pug/o
V. fisheri (microtox)
B. plicatills
24 h
24 h
24 h
24 h
1 h
24 h
15min
24 h
Mean
LC50/EC50 Statistical Pooled
(mg/L) SD Comparisons1 LC50 (mg/L)
0.4
1.5
5.7
6.0
34.8
29.9
24.9
75.0
0.1
0.3
1.0
1.5
10.7
8.1
3.5
3.6
A
B
C
C
D
D
D
E
0.4
1.6
5.8
5.6
34.2
31.7
NC
74.8
Sensitivity 95% Cl
Ranking (mg/L)
1
2
3
3
4
4
4
5
0.4 - 0.5
1.4-1.7
5.2 - 6.5
5.3 - 6.0
26.6 - 43.9
23.6-42.6
NC
71.3-78.5
Mean LC50s/EC50s for species sharing the same letter are not significantly different at a = 0.05.
14
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Table 5. Sensitivity of selected invertebrate species to cadmium in water
Species Duration Life Stage1 LC50
column exposures.
Reference
Mysidopsis bahia
Palaemonetes pugio
Homarus americanus
Rhepoxynius abronius
Penaeus duorarum
Callinectes sapidus
Crangon septemspinosa
Crassostrea gigas
Palaemonetes vulgaris
Mercenaria mercenaria
Corophium insidiosum
Argopecten irradians
Amphiascus tenuiremis
Mya arenaria
Ampelisca abdita
Ampelisca verrilli
Palaemonetes pugio
Palaemonetes pugio
Mysidopsis bahia
Brachionus plicatilis
96 h
96 h
96 h
96 h
96 h
96 h
96 h
48 h
96 h
24 h
96 h
96 h
24 h
96 h
24 h
24 h
48 h
24 h
1 h
24 h
J
A
L
U
J
A
A
E
A
J
U
J
A
A
J
A/J
A
J
J
12
40
80
147
312
320
320
375
420
420
779
908
1,500
2,200
5,700
6,000
13,000
29,900
34,800 2
75000
Gripe, 1994
Sundaetal., 1978
Johnson, 1979
Hong and Reish, 1987
Gripe, 1994
Frank and Robertson, 1979
Eisler, 1971
Martin etal., 1981
Eisler, 1971
This study
Hong and Reish, 1987
Nelson etal., 1976
This study
Eisler, 1971
This study
This study
Burton and Fisher, 1990
This study
This study
This study
1 J = juvenile, L = larvae, U = unknown, A = adult, E = embryo
2 EC50 for fluorescence reduction in Mysid IQ® test
values for other invertebrate species. Little
comparable data was available to assess the relative
assay and this species was not retested. The value
for this species shown in Table 6 is from the muddy
sediment bioassay. M. mercenaria was the most
sensitive species to cadmium-spiked sediments with
sensitivity of the species used in this study with
others cited in the literature, since most of the
published values were for longer duration exposures.
As stated previously, the main purpose of the short-
term aqueous bioassays was to provide a basis for
comparing the inherent sensitivity of each species
used in this study and to relate this inherent
sensitivity to the results obtained from the sediment
bioassays. Results obtained from the definitive 10-
day sediment bioassays (P. pugio, A. verrilli, A.
abdita, M. mercenaria and A. tenuiremis ), the 24 h
B. plicatilis sediment porewater assay and the
Microtox™ and Mutatox™ bioassays are provided in
Appendix B and the results for all species are
summarized in Table 6. Preliminary 10-day spiked-
sediment bioassays with P. pugio, A. abdita , A.
verrilli and A. tenuiremis were conducted with a
muddy sediment collected from North Inlet, South
Carolina. No significant contaminant-related mortality
was observed in any of the test species at
concentrations as high as 36 mg/Kg dw (P. pugio, A.
abdita, A. verrilli) and 45 mg/Kg dw (A. tenuiremis).
Subsequent AVS analysis revealed extremely high
AVS levels (>7 /miol/g) in these sediments which
explained the lack of cadmium toxicity. All
subsequent spiked-sediment bioassays were
conducted with a much sandier sediment collected
from Folly Beach, South Carolina which was
autoclaved prior to spiking. The AVS levels in this
autoclaved sediment were much lower (<0.03
15
-------
Table 6. Summary of results from sediment assays with cadmium.
Species
M. mercenaria
A. verrilli
A. abdita
P. pugio
B. plicatilis
A. tenuiremls
V, fisheri (microtox)
V. fisheri (mutatox)
Exposure
Period
10d
10d
10d
10 d
24 h
10 d
5min
Mean
LC50/EC50
(mg/kg)
<2.5
4.8
12
18.2
41.5
>45
16021603
SD
—
0.4
4.3
0.2
11.7
—
—
—
Statistical
Comparisons1
—
A
A, B
A,B
B
—
—
—
Pooled
LC50
(mg/kg)
4.5
11.8
17.9
41.9
—
—
Sensitivity
Ranking
1
2
2
2
3
4
44
95% Cl
(mg/kg)
-—
3.9-5.1
9.9-14.2
16.2-19.9
35.8-49.1
—
—
1 Mean LC50s/EC50s for species sharing the same letter are not significantly different at a = 0.05.
2 Lowest sediment cadmium concentration which caused a significant reduction in bioluminescence relative to
control in the microtox solid phase bioassay. This was the most sensitive microtox endpoint evaluated.
3 Lowest sediment cadmium concentration which gave a positive response in the mutatox screening assay.
jumol/g). Unfortunately, the sandy nature of this
sediment made it unsuitable for the A. tenuiremis
assay and this species was not retested. The value
for this species shown in Table 6 is from the muddy
sediment bioassay.
M, mercenaria was the most sensitive species to
cadmium-spiked sediments with 100% mortality at
2.5 mg/kg dw, the lowest concentration tested. A.
verrilli, A. abdita and P. pugio were comparable in
sensitivity and were the next most sensitive group.
The B. plicatilis porewater assay was less sensitive
than A. verrilli, but was not different from the A.
abdita and P. pugio assays. The Microtox™ and
Mutatox™ bioassays were the least sensitive of the
assays conducted with comparable sediments.
Comparisons of aqueous versus sediment toxicity
testing indicated generally similar relative sensitivities
to cadmium. The copepod, A. tenuiremis, was an
exception since its relative sensitivity was greater in
the aqueous exposure (Tables 4 and 6).
Table 7 provides a comparison of the sensitivity of
the species used in this study with other measures of
cadmium toxicity from the literature. M. mercenaria
was the only species tested which showed significant
toxicity near the reported TEL and ER-L levels of 0.7
mg/Kg dw and 1.2 mg/Kg dw, respectively. Both
amphipod species and the grass shrimp were
sensitive to cadmium-spiked sediments at
concentrations near the ER-M of 9.6 mg/Kg dw (Long
et al., 1995). The remainder of the species tested in
this study were only sensitive to cadmium-spiked
sediments at concentrations which exceeded the
ER-M. As was noted earlier, AVS levels were quite
low in the cadmium-spiked sediments used in this
study. The SEM/AVS ratio was >1.0 at all cadmium
spike levels (Table 1). DiToro et al. (1990) has
reported that an SEM/AVS ratio > 1.0 is necessary
for the manifestation of cadmium-induced toxicity
from sediments. The lowest cadmium spike level
(2.5 mg/Kg dw) which caused toxicity in M.
mercenaria had an SEM/AVS ratio (1.1) which was
very near this reported minimum threshold for toxic
effects. This suggests that this species is one of the
most sensitive organisms tested.
DDT
Results obtained for the 24 h aqueous DDT assays
for each of the test species are provided in Appendix
C and summarized in Table 8. Results indicated
that the P. pugio and the Mysid IQ™ tests were the
most sensitive endpoints evaluated. A. verrilliwas the
next most sensitive species, with M. mercenaria
being the third most sensitive species to DDT. The
remaining four species were insensitive to DDT at
the highest concentration tested (10,000 ,ug/l). The
large differences in apparent sensitivity may have
resulted, in part, due to the limited solubility of DDT
in water. The reported solubility of DDT in water
is~35 /j,g/\, thus much of the DDT may have been
unavailable at the higher exposure concentrations.
Table 9 provides a comparison of the 24 h aqueous
LC50 values obtained in this study with literature
16
-------
Table 7. Comparison of the relative toxicity of test species to cadmium in sediment
exposures versus other measures of cadmium toxicity.
Significant Effects
Species mg/kgdrywt.
TEL 0.7
ER-L 1.2
Mercenaria mercenaria <2.5
PEL 4.2
Ampelisca verrilli 4.8
ER-M 9.6
Rhepoxynius abronius 9.8
Ampelisca abdita 12.0
Palaemonetes pugio 18.2
Brachionus plicatilis 41 .5
Brachionus plicatilis 56.8
Vibrio fischeri (M icrotox) 160
Amphiascus tenuiremis >45.0
Ampelisca abdita 2600
Concentration
Effects Code
A
A
A
A
A
B
C
D
A
A
Source
MacDonald, 1994
Longetal., 1995
This Study
MacDonald, 1994
This Study
Long et al., 1995
Mearnsetal, 1986
This Study
This Study
This Study
Snell and Persoone,
This Study
This Study
DiToro et al.., 1990
1989
A = LC50 in 1 0 day sediment exposure
B = LC50 in 24 hr exposure to sediment porewater extract
C = LC50 in 24 hr exposure to SOppt seawater
D = Lowest concentration to elicit significant reduction in fluorescence compared with
controls.
Table 8. Summary of results from aqueous assays with DDT.
Mean
Exposure LC50/EC50
Species Period (/^g/L) SD
M.bahia 1 h 5.9 1.7
P. pugio 24 h 9.5 2.3
A.verrilli 24 h 39.8 27.6
M. mercenaria 24 h 612 135
A. abdita 24 h >1 0,000
A.tenuiremis 24 h > 10,000
B. plicatilis 24 h > 10,000
V. fisheri (microtox) 15min > 10,000
Statistical
Comparisons1
A
A
B
C
Pooled
LC50 Sensitivity
Cwg/L) Ranking
5.1 1
8.9 1
38.3 2
615 3
4
A
4
• - ' 4
95% Cl
6"g/L)
3.9 - 6.6
7.5-10.5
32.4 - 45.3
358-1057
1 Mean LC50s/EC50s for species sharing the same letter are not significantly different at a = 0.05.
17
-------
Table 9. Sensitivity of selected invertebrate species to DDT in water column exposures.
Species
Duration Lifestage1 LC
SO
Reference
Daphnia magna
Gammarus fasciatus
Gammarus lacustris
Palaemonetes vulgaris
Asselus brevicaudus
Daphnia magna
Mysidopsis bahia
Palaemonetes pugio
Callinectes sapidus
Ampelisca verrilli
Mercenaria mercenaria
Ampelisca abdita
Brachionus plicatus
Amphiascus tenuiremis
48 h
96 h
96 h
96 h
96 h
24 h
1 h
24 h
96 h
24 h
24 h
24 h
24 h
24 h
U
U
U
U
U
U
J
A
U
J
J
J
J
A
0.4
0.8
1
2
4
4.4
5.9 2
9.5
19
39.8
612
>1 0,000
>1 0,000
>1 0,000
Frearand Boyd, 1967
Sanders, 1972
Sanders, 1969
Eisler, 1969
Sanders, 1972
Sanders and Cope,
This study
This study
Mahood et al., 1970
This study
This study
This study
This study
This study
1966
1 J = juvenile, L = larvae, U = unknown, A = adult, E = embryo
2 ECso for fluorescence reduction
Table 10. Summary of results from sediment assays with DDT.
Exposure
Species Period
A. tenuiremis
P. pugio
M. mercenaria
A. abdita
A. verrilli
A. tenuiremis
B. plicatilis
V. fisheri (microtox)
V. fisheri (microtox)
10d
10d
10d
10d
10d
10d
24 h
5 min
—
Mean LC50/EC
(mg/kg)
1.0 2
4.5
5.8
8.2
8.3
>10.0
>10.0
>10.0
>10.0
Pooled
so Statistical LCSO Sensitivity 95% Cl
SD Comparisons1 (mg/kg) Ranking (mg/kg)
—
0.5
1.1
0.8
0.9
—
—
—
—
A 4.5
6.3
B 8.5
B 8.5
> 10.0
> 10.0
> 10.0
____. ____
1
2
2
3
3
4
4
4
— —
—
3.6 - 5.6
4.8-8.3
7.2-10.0
7.2 -mo
—
—
—
•"**""
1 Mean LC50s/EC50s for species sharing the same letter are not significantly different at a = 0
2 Concentration which caused significant reduction in clutch size.
05.
18
-------
7aJb/e 11. Comparison of the relative toxicity of test species to DDT in sediment exposures versus other
measures of DDT toxicfty.
Species
Significant Effects Concentration
mg/kg dry wt. mg/g OC Effects Code
Source
ER-L
TEL
Crangon septemspinosa
ER-M
PEL
Amphiiascus tenuiremis
Palaemonetes pugio
Mercenaria mercenaria
Ampelisca verrilli
Ampelisca abdita
Rhepoxynius abronius
Eohaustorius estuarius
Hyalella azteca
Brachionus plicatilis
Vibrio fischeri (Microtox)
Amphiascus tenuiremis
Nereis virens
0.0016
0.0028
0.0310
0.0461
0.0517
1
4.5 0.6
5.8 0.8
8.3 1.2
8.2 1.2
1.0
2.5
2.6
>10.0 >1.4
>10.0 >1.4
>10.0 >1.4
>16.5
A
B
C
C
C
C
C
C
C
D
E
C
F
Long et al., 1995
MacDonald, 1994
McLeese and Metcalfe, 1980
Long et al., 1995
MacDonald, 1994
^This Study
This Study
This Study
This Study
This Study
Swartz et al., 1994
Swartz et al., 1994
Swartz et al, 1994
This Study
This Study
This Study
McLeese et al, 1982
A = LC50 in 96 hr sediment exposure
B = Significant decrease in clutch size from control after 10 d exposure
C = LC50 in 10 d sediment exposure
D = LC50 in 24 hr exposure to sediment porewater extract
E = Lowest concentration to elicit significant reduction in fluorescence compared with controls.
F = LC50 in 12 d sediment exposure
values for other invertebrate species. Although little
comparable data was available for this exposure
period, the 24 h LC50 (4.4 ^g/l) reported for D.
magna was similar t the values obtained for P. pugio
(9.5 ;ug/l) and M. bahia (5.9 ^g/l) "in this study.
The results from the sediment DDT assays
(Appendix C, Table 10) indicated that M. mercenaria
and P. pugio were the two most sensitive species
tested based on mortality endpoints and both were
more sensitive than the two amphipod species. A.
tenuiremis was insensitive to DDT-induced mortality
at the highest concentration tested (10 mg/kg
dw);however, clutch size was reduced in this species
at concentrations as low as 1 mg/kg dw (Table 10).
Both the B. plicatilis porewater assay and the
Microtox™ bioassay were insensitive to DDT at the
highest concentration tested. In general, all of the
LC50 and EC50 values obtained for the species used
in this study were higher than the reported TEL, ER-
L, ER-M and PEL values (Table 11, MacDonald,
1994; Longetal., 1995).
Fluoranthene
Results obtained for the individual aqueous
fluoranthene assays are provided in Appendix D and
mortality was observed in most species after 24 h of
exposure at the highest concentration tested (800
summarized in Table 12. Due to the fact that <50%
Mg/l) which was at the limit of solubility, the duration
of these tests was extended to 96 h. Both 24 h and
96 h data are presented for M. mercenaria. A. abdita
and M. mercenaria were the two most sensitive
species tested and both were slightly more sensitive
19
-------
than A. verrilli. P. pugio was the next most sensitive
organism. The remaining four organisms were
insensitive to fluoranthene at the highest
concentrations tested. In the Mysid IQ™ test, the
organisms exposed to higher fluoranthene
concentrations exhibited greater fluorescence than
the control organisms. This may have been due to an
artifact of the assay protocol since fluoranthene also
fluoresces under UV light and uptake of this
contaminant by the mysids may have masked any
stress-induced reduction in fluorescence. In general,
the aqueous LCSOs determined for the species used
in this study were similar to those reported for other
invertebrates (Table 13).
Individual sediment fluoranthene assay results are
shown in Appendix D and the results for all species
are summarized in Table 14. M. mercenaria was by
far the most sensitive species to fluoranthene,
with > 50% mortality at the lowest concentration
tested (0.78 mg/Kg dw). Comparison of aqueous
and sediment bioassays with fluoranthene indicated
generally similar relative sensitivities for the test
species in both exposure matrices (Table 12 and
14). The estimated LC50 (<0.11 mg/g OC/O.8
mg/Kg dw) for juvenile M. mercenaria was lower
than the EPA SQC (0.3 mg/g OC) and was similar to
the ER-L (0.6 mg/Kg dw) reported by Long et al.
(1995). This suggests t hat this is one of the
most sensitive species tested and that these criteria
may not adequately protect this organism. The next
most sensitive species was A. abdita which
experienced significant mortality (45% at 50 mg/Kg
dw). The remaining species were insensitive to
fluoranthene at the highest concentration tested (50
mg/Kg dw) which exceeded the ER-M of 5.1 mg/Kg
dw reported by Long et al. (1995) (Table 15).
Table 12. Summary of results from aqueous assays with fluoranthene.
Mean Pooled
Exposure LC50/EC50 Statistical LC50 Sensitivity 95% Cl
Species Period (M9/L) SD Comparisons1 (p.g/L) Ranking 0«g/L)
A. abdita
M. mercenaria
A.verrilli
P. pugio
A.tenuiremls
B. pticatilis
M. bahia
V. fisheri (microtox)
M. mercenaria
96 h
96 h
96 h
96 h
96 h
48 h
1 h
5min
24 h
60.5
<104
113
595
>1600
>500
>800
>1100
652
12
—
30
175
—
120
A 59 1
-j
B 108 2
C 565 3
4
4
4
4
735
55-63
—
97-119
411 -776
-— -
441 - 1225
'Mean LC50s/EC50s for species sharing the same letter are not significantly different at a = 0.05.
20
-------
Table 13. Sensitivity of selected invertebrate species to fluoranthene in water column exposures.
Species
Mysidopsis bahia
Ampelisca abdita
Ampelisca abdita
Mercenaria mercenaria
Ampelisca verrili
Palaemonetes pugio
Palaemonetes pugio
Neanthes arenaceodentata
Brachionus plicatus
Palaemonetes pugio
Mercenaria mercenaria
Amphiascus tenuiremis
Mulinia lateralis
Duration
96 h
96 h
96 h
96 h
96 h
96 h
. 96 h
96 h
48 h
96 h
24 h
96 h
96 h
Life
Stage*
J
J
J
J
J
L
J
L
A
J
A
J
LC50 (//g/L)
40
60.5
66.9
<104
112.7
122
142.5
500
>500
594.6
652
>1600
10710
Reference
EPA, 1978
This study
Champlin and Poucher,
This study
This study
Frasca, 1995
Champlin and Poucher,
Rossi and Neff, 1 978
This study
This study
This study
This study
Champlin and Poucher,
1991
1991
1991
J = juvenile, L = larvae, U = unknown, A = adult, E = embryo
Table 14. Summary of results from sediment assays with fluoranthene.
Species
M. mercenaria
V. fisheri (mutatox)
A. abdita
A. tenuiremis
A. verrilli
B. plicatilis
P. pugio
V. fisheri (microtox)
Exposure
Period
10d
—
10d
10d
10d
24 h
10d
5 min
Mean
LC50/EC50
(mg/kg)
<0.8
3-501
>502
>50
>50
>50
>50
>50
Statistical Pooled
SD Comparisons LC50
(mg/kg)
<0.8
— — —
>50
> 50
> 50
> 50
> 50
> 50
Sensitivity
Ranking
1
2
2
3
3
3
3
3
95% Cl
(mg/kg)
—
—
—
—
—
—
—
—
1Assay screened positive for mutagenicity in sediment samples spiked with fluoranthene at 3.12 and 50
ppm but not at 12.5 ppm.
Significant mortality (45%)
21
-------
Table 15. Comparison of the relative toxicity of test species to fluoranthene in sediment
exposures versus other measures of fluoranthene toxicity
Species
TEL
ER-L
Mercenaria mercenaria
EPA SQC
PEL
Hyalella azteca
Rhepoxynius abronlus
ER-M
Rhepoxinius abronius
Ampelisca abdita
Ampelisca verrilli
Palaemonetes pugio
Brachionus plicatilis
Vibrio fischeri (Microtox)
Amphiascus tenuiremis
Significant Effects Concentration
Effects
mg/kg dry wt. mg/g OC Code
0.1
0.6
<0.8
1.5
2.3 - 7.4
3.4-10.7
5.1
8.7-19.1
50
>50.0
>50
>50.0
>50
>50.0
<0.11
0.3
0.5-1.5
1 .9 - 2.2
1.4-4.4
10.4
>10.4
>10.4
>10.4
>10.4
>10.4
A
A
A
A
B
B
B
B
C
B
Source
MacDonald,
Long et al.,
This Study
EPA, 1993
MacDonald,
Suedel et ai
Swartz et al
Long et al.,
DeWitt et al
This Study
This Study
This Study
This Study
This Study
This Study
1994
1995
, 1994
., 1993
., 1990
1995
., 1992
A = LC50 in 10 day sediment exposure
B = Significant mortality < 80% of control survival
C = LC50 in 24 hr exposure to sediment porewater extract
D = Lowest concentration to elicit significant reduction in fluorescence compared with controls
22
-------
Chapter 4
Summary and Conclusions
The juvenile clam was the most sensitive species to
cadmium in both aqueous and sediment exposures.
The sensitivities of the two amphipod species and the
grass shrimp to cadmium were similar in both water
and sediment exposures, while the rotifer assay was
generally less sensitive. The Microtox™ assay was
relatively sensitive to cadmium in the aqueous assay,
but insensitive to sediment-associated cadmium. The
copepod assay was sensitive to cadmium in the
aqueous assay; however, its sensitivity to sediment-
associated cadmium could not be compared with the
other test species. For the most part, the relative
sensitivity of the test organisms to sediment-
associated cadmium paralleled their sensitivity in the
aqueous tests. Only the clam assay was sensitive to
sediment-associated cadmium at concentrations near
the ER-L (1.2 mg/Kg dw) and TEL (0.7 mg/Kg dw)
values. The remaining species were only sensitive at
concentrations ;> ERM (9.6 mg/Kg dw) and PEL (4.2
mg/Kg dw) levels.
The grass shrimp and Mysid IQ™ assays were most
sensitive to DDT in aqueous exposures, with A.
verrilli being the next most sensitive species. M.
mercenaria was ~10x less sensitive than A. verrilli.
The remaining assays were ^ 10x less sensitive than
M. mercenaria. These apparent large differences
may have been due, in part, to the limited solubility
of DDT in water (-35 ^g/L). DDT concentrations
which exceeded the solubility would be mostly
unavailable for uptake. The differences of the test
species to sediment-associated DDT were less
dramatic than those observed in the aqueous tests.
The most sensitive species (P. pugio and M.
mercenaria) were only slightly more sensitive than
the two amphipods. The remaining species were
insensitive to DDT at the highest concentration tested
(10 mg/kg dw). Survival of adult copepods was not
affected at 10 mg/Kg dw; however, reproductive
output was depressed at DDT concentrations as low
as 1 mg/kg dw. These findings suggest that DDT may
cause sublethal effects in many species at
concentrations well below those producing acute
toxicity. None of the species tested in this study were
sensitive to DDT at concentrations near the ER-L
(0.0016 mg/Kg dw) or ER-M (0.0461 mg/Kg dw).
The juvenile clam was the most sensitive species to
fluoranthene in both aqueous and sediment
exposures and was sensitive to sediment-associated
fluoranthene at concentrations at or below the ER-L
of 0.6 mg/Kg dw and the EPA sediment quality
criterion of 0.3 mg/g OC. The remaining species
tested were generally only sensitive to fluoranthene
at concentrations z 50 mg/Kg dw.
Overall, the juvenile clam was the most sensitive
species tested in this study from an acute toxicity
standpoint. The grass shrimp and the two amphipod
species were generally similar in sensitivity to each of
the three compounds. The copepod assay, although
relatively insensitive in terms of adult mortality, was
capable of detecting sublethal effects at contaminant
concentrations below those which caused mortality in
the other more sensitive species. Both the juvenile
clam assay and the copepod partial life cycle test
have the potential to serve as sensitive indicators of
potential sediment-associated toxicity which might
not be detected using standard acute toxicity
bioassays.
Comparisons of ERL/TEL and ERM/PEL sediment
quality guidelines generally indicated that the most
sensitive species tested (e.g., Cd-clam, DDT-
copepod reproduction and fluoranthene-clam) were
sensitive at concentrations at or just above the ERL
23
-------
values for Cd and fiuoranthene. The remaining test
species were sensitive to these compounds at
concentrations just below or above the ERM. The
lack of sensitivity in our suite of bioassays to DDT
suggests that existing ERL/ERM and TEL/PEL
guidelines may be overly protective. Our most
sensitive species value based on copepod reproduct-
ion is nearly two orders of magnitude higher than the
ERM/PEL guidelines. In sediments where DDT is the
only contaminant, our findings suggest that these
guidelines may overestimate potential toxicity.
The differing species sensitivities observed with the
different classes of chemical contaminants in this
study suggest that a multiple species approach may
be more appropriate for a holistic ecological risk
assessment of sediment contamination. The
"Crustacean Triad" (copepods, amphipods and grass
shrimp) provide a battery of tests which predict
toxicity to epibenthic and benthic crustaceans with
known sensitivity to a variety of chemical
contaminants and represent the base of the food
chain for most recreationally and commercially
important finfish species which utilize estuarine
nursery grounds. The addition of the juvenile clam
assay provides a herbivorous filter feeder with the
ability to bioconcentrate pollutants and which is
extremely sensitive in the size range tested
(>212<350ywm). Field studies in South Carolina
have indicated that sites with high sediment
contaminant levels have degraded benthos, with
significant effects observed in crustaceans and
molluscs (F. Holland, South Carolina Department of
Natural Resources, personal communication). These
findings support our laboratory results and suggest
that an integrated battery of assays may be most
appropriate for estimating field effects.
24
-------
References
Allen, H.E., G. Fu, W. Boothman, D.M. DiToro and
J.D. Mahony. 1991. Determination of acid volatile
sulfide and selected simultaneously extractable
metals in sediment. EPA/821/12-91/100. U.S.
EPA, Environmental Research Laboratory,
Narragansett, Rl. 22p .
Anderson, G., L. Shanks and J. Parsons. 1985.
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Appendix A
Summary of results obtained from the SDS reference toxicant tests using P. pugio, A. verrilli,
A. abdita, M. mercenaria, A. tenuiremis and from the potassium dichromate reference toxicant
tests using 6. plicatilis
29
-------
Table A-1. SDS reference toxicant bioassay results for the Gulf of Mexico Project using P. pugio.
All concentrations are in mg/L.
Acceptable Test Criteria
Average (-) 2 Stand. (+) 2 Stand. SDS
Test Date Comments LC50 LC50 Dev Dev Pass/Fail
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
Baseline
Baseline
Baseline
Baseline
Baseline
Baseline
Cd Aqueous (defin.)
Cd Trial Sediment
Cd Trial Sediment
DDT Aq. Rangefinder
DDT Aq. Rangefinder
DDT Aq. Rangefinder
DDT Aqueous (defin.)
Cd Sediment (defin.)
DDT Sediment (defin.)
Fluor Aqueous (defin.)
Fluor Sediment (defin.)
Final Average
140.20
108.30
154.90
115.10
136.40
117.30
1 02.90
182.30
140.50
200.00
163.00
154.90
145.30
116.60
147.20
154.90
129.30
140.20
124.25
134.47
129.63
130.98
128.70
125.01
132.18
133.10
139.79
141.90
142.98
143.16
141.26
141.66
142.49
141.71
141.71
NC
76.60
91.01
91.51
95.10
90.58
71.29
81.61
69.36
77.72
82.25
85.86
86.47
86.55
88.39
90.51
89.74
NC
171.90
177.92
167.74
166.86
166.82
178.74
182.74
196.84
201.86
201.55
200.11
199.85
195.98
194.93
194.46
193.69
Pass
Fail
Pass
Fail
Pass
Pass
Pass
Pass
Pass
Pass
Pass
30
-------
Table A-2. SDS reference toxicant bioassay results for the Gulf of Mexico Project using the amphipod,
Ampelisca abdita. All concentrations are in mg/l.
Acceptable Test Criteria
Test Date
10:22 AM
3/16/95
3/16/95
3/16/95
3/16/95
3/28/95
3/28/95
5/2/95
5/2/95
6/26/95
6/26/95
10/2/95
12/11/95
12/18/95
12/18/95
1/23/96
1/23/96
2/26/96
2/26/96
3/26/96
3/26/96
Comments
baseline
baseline
baseline
baseline
baseline
Cd aqueous
Cd aqueous
Cd sediment
Cd sediment
DDT aqueous
DDT aqueous
Cd sediment
DDT range finder
DDT aqueous
DDT aqueous
DDT sediment
DDT sediment
Fluoranthene aqueous
Fluoranthene aqueous
Fluoranthene sediment
Fluoranthene sediment
Final Average
•LCm
16.5
22.8
22.6
18.8
17.4
20.5
21.6
21.6
19.5
25.2
20.5
19.5
21.6
25.2
24.0
17.6
21.6
17.6
19.4
20.6
20.6
Mean
LCsn
16.5
19.6
20.6
20.1
19.6
19.8
20.0
20.2
20.1
20.7
20.6
20.5
20.6
21.0
21.2
20.9
21.0
20.8
20.7
20.7
20.7
20.7
(-) 2 Stand
Dev
NC
10.7
13.4
14.0
13.8
14.5
15.0
15.4
15.6
15.3
15.6
15.7
15.9
15.8
16.0
15.6
15.8
15.6
15.6
15.7
15.8
(+) 2 Stand
Dev
NC
28.5
27.8
26.3
25.4
25.0
25.0
25.0
24.6
26.0
25.7
25.4
25.3
26.1
26.3
26.2
26.1
26.0
25.8
25.7
25.6
SDS
Pass/Fail
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
31
-------
Table A-3. SDS reference toxicant bioassay results for the Gulf of Mexico Project using the amphipod,
Ampellsca verrflli. All concentrations are in mg/l.
Test Date
1/18/95
1/19/95
1/19/95
10:22 AM
1/19/95
1/19/95
2/2/95
2/2/95
10:22 AM
10:22 AM
10:22 AM
4/17/95
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10/2/95
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
Comments
range finder
baseline data
baseline data
baseline data
baseline data
baseline data
baseline data
baseline data
baseline data
baseline data
baseline data
Cd aqueous
Cd sediment
Cd sediment
DDT aqueous
DDT aqueous
DDT aqueous
DDT aqueous
baseline data
baseline data
baseline data
baseline data
Cd sediment
DDT aqueous
DDT aqueous
DDT sediment
DDT sediment
Fluoranthene aqueous
Fluoranthene aqueous
Fluoranthene sediment
Fluoranthene sediment
Final Average
LC*,
66.5
65.0
61.7
58.5
54.5
58.2
58.2
66.0
65.0
42.3
58.7
53.0
47.9
54.3
33.5
39.7
42.1
45.5
47.9
45.5
45.1
41.1
47.9
58.7
45.5
40.5
45.5
45.5
45.5
55.8
55.1
Acceptable Test Criteria
(-)2 Stand (+)2 Stand
Mean LC™ Dev Dev
66.5
65.8
64.4
62.9
61.2
60.7
60.4
61.1
61.5
59.6
59.5
59.0
58.1
57.8
NC
NC
56.8
56.1
55.6
55.0
54.5
53.8
53.5
53.8
53.4
52.9
52.6
52.3
52.1
52.2
52.3
52.3
NC
63.6
59.5
55.8
51.5
51.6
51.8
52.2
52.8
44.9
45.6
45.2
43.5
43.7
40.9
39.7
39.3
38.5
37.8
36.5
36.4
37.0
36.6
35.6
35.4
35.3
35.1
35.5
35.9
NC
67.9
69.3
70.1
71.0
69.8
68.9
69.9
70.2
74.2
73.4
72.7
72.7
72.0
72.6
72.4
71.9
71.6
71.2
71.2
70.6
70.6
70.2
70.1
69.7
69.3
69.0
68.8
68.7
SDS
Pass/Fail
Pass
Pass
Pass
Fail
Fail
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
32
-------
Table A-4. SDS reference toxicant bioassays for the Gulf of Mexico Project using Amphiascus tenuiremis.
Test Date
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
Comments
Baseline
Baseline
Baseline
Baseline
Cd Aqueous
Cd Sediment
DDT (aqueous and sediment)
Fluor (aqueous and sediment)
Final Average
SDS
LC50
12.13
10.52
12.36
12.99
16.86
14.76
14.24
13.43
13.97
Acceptable Test Criteria
Average (-) 2 Stand (+) 2 Stand
LC50 Dev Dev
12.13
11.33
1 1 .67
12.00
12.97
13.27
13.41
13.41
13.47
13.47
NC
9.05
9.66
9.90
8.26
8.81
9.27
9.58
9.87
NC
13.60
13.68
14.10 '
17.69
17.73
17.55
17.24
17.08
SDS
Pass/Fail
Pass
Pass
Pass .
Pass
Table A-5. SDS reference toxicant bioassay results for the Gulf of Mexico Project using M.
mercenaria. All concentrations are in mg/L.
Acceptable Test Criteria
Average (-) 2 Stand. (+) 2 Stand. SDS
LC50 Dev Dev Pass/Fail
Test Date
Comments
LC50
6/22/95
6/22/95(6)
6/28/95(A)
6/28/95(6)
6/28/95(C)
8/31/95
10/4/95
2/21/96
3/7/96
3/19/96
3/30/96
5/20/96
6aseline
6asel ine
Baseline
Baseline
Baseline
DDTAq. Rangefinder
Cd Aqueous (defin.)
DDT Sediment (defin.)
DDT Aqueous (defin.)
Fluor Aqueous (defin.)
Fluor Sediment (defin.)
Cd Sediment (defin.)
Final Average
6.29
6.04
8.27
6.13
7.87
8.26
5.98
7.92
6.13
7.74
7.80
7.43
6.29
6.17
6.87
6.68
6.92
7.14
6.98
7.09.
6.99
7.06
7.13
7.16
7.16
NC
5.81
4.43
4.56
4.80
4.95
4.79
4.97
4.90
5.03
5.15
5.26
NC
6.52
9.30
8.80
9.04
9.33
9.16
9.22
9.08
9.09
9.11
9.05
Pass
Pass
Pass
Pass
Pass
Pass
Pass
33
-------
Table A-6. Potassium dichromate reference toxicant bioassay results for the Gulf of
Mexico Project using the rotifer, B. plicatilis. All concentrations are in mg/l.
Acceptable Test Criteria
Test Date Comments
LC,
Batch LC50 lower Cl upper Cl
1/18/95
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
10:22 AM
baseline
baseline
Cd aqueous
Cd porewater (lower)
Cd porewater (higher)
DDT aqueous
DDT porewater
Fluoranthene aqueous
Fluoranthene sediment
304.2
261.8
303.6
324.7
339.1
278.4
301.2
314.6
315.4
323.0
323.0
323.0
323.0
323.0
323.0
323.0
323.0
323.0
226.0
226.0
226.0
226.0
226.0
226.0
226.0
226.0
226.0
420.0
420.0
420.0
420.0
420.0
420.0
420.0
420.0
420.0
34
-------
Appendix B.
Results obtained from Cadmium aqueous and sediment bioassays
35
-------
Toxicant: Cadmium (mg/L)
Matrix: Aqueous
Species: Palaemonetes pugio
% Mortality
Duration Replicate Control 4.8
24 h A
B
C
D
E
Mean
SD
Pooled
95% Cl
Species: Ampelisca abdita
Duration Replicate
24 h A
B
C
D
E
Mean
SD
Pooled
95% Cl
Species: Ampelisca verrilli
Duration Replicate
24 h A
B
C
D
E
F
G
H
Mean
SD
Pooled
95% Cl
Species: Amphiascus tenuiremis
Duration Replicate
24 h A
B
C
D
E
Mean
SD
Pooled
95% Cl
0 0
0 20
0 10
10 30
10 30
4 18
8
20
10
20
40
40
26
13
40
10
40
40
10
28
22
20
50
30
80
30
42
36.8 61.3
60 50
60 40
30 80
40 90
50 80
52 68
LC50
33.2
36.8
28.5
16.4
34.5
29.9
8.1
31.67
(23.57, 42.56)
% Mortality
Control
0
0
10
0
0
2
1.4
30
20
0
0
0
10
2.2
10
20
0
10
10
10
3.7
20
60
40
50
20
38
6.2
60
50
40
50
50
50
10.3 17.2
90 100
80 90
80 100
60 80
40 100
70 94
LC50
5.5
4.4
5.8
5.8
7.2
5.7
1
5.8
(5.2, 6.5)
% Mortality
Control
10
0
0
0
0
0
10
10
4
1.9
20
0
30
0
10
0
10
0
9
3.2
20
50
20
30
10
0
10
10
20
5.4
40
0
70
70
60
40
90
80
56
9
50
80
70
60
40
80
90
80
69
15
50
50
80
50
60
100
100
100
74
25
20
70
80
70
60
100
100
100
78
(5
LCso
NC
8.1
4.6
6.1
7.7
6.3
4.2
4.8
6
1.5
5.6
.3, 6.0)
% Mortality
Control
0
0
5
0
5
2
0.6
55
10
10
5
5
17
1.1
45
10
10
30
25
24
1.8
45
55
50
55
95
60
3
80
85
80
95
100
88
5
90
95
80
95
100
92
LC50
1.1
1.8
1.9
1.6
1.3
1.5
0.3
1.6
(1-4,1.7)
36
-------
Toxicant:
Matrix:
Cadmium (mg/L)
Aqueous
Species: Mercenaria mercenaria
% Mortality
Duration Replicate
24 h A
B
C
D
E
Mean
SD
Pooled
95% Cl
Species: Brachionus plicatilis
Duration Replicate
Species:
Duration
1 hr
24 h A
B
C
D
E
Mean
SD
Pooled
95% Cl
Mysidopsis bahia
Replicate Control 8
A 33 33
B 0 33
C 33 17
D 0 -
E 0 -
F 17
Mean 14 28
SD
Pooled
95% Cl
Species: Vibrio fischeri
Replicate
A
B
C
D
E
MEAN
SD
Control
0
10
10
0
0
4
Control
0
3
0
0
3
1
13.2 16
40
0
0
17
33
50
13 33
0.2
0
20
10
10
10
10
12
0
0
0
0
0
0
22.1
33
17
0
17
0.3
20
10
20
60
50
32
20
0
0
0
0
3
1
% Not
26.5
67
67
33
56
(Microtox)
5 min EC50
374.5
304.0
333.3
257.5
154.4
284.7
84.4
0.5 0.9
100 100
30 80
50 100
80 100
100 100
72 96
% Mortality
33 55
3 13
3 13
3 13
3 7
7 20
4 13
Fluorescing
36.8 44.2
33
67
AO
*-^\j — —
83
67
83
47 78
1 5 min EC50
27.7
26.5
26.8
24.4
19.0
24.9
3.5
1.5
100
90
100
90
80
92
92
73
70
70
73
87
75
61.3
60
50
67
59
2.45
100
100
100
100
100
100
(0
LC50
75.6
76.9
76.9
76.9
68.6
75
3.6
74i8
(71.3,78.
73.6 123
50 83
100 83
67 83
72 83
LC50
0.37
0.63
0.48
0.32
0.32
0.42
0.14
0.4
.35, 0.45)
5)
EC50
49.8
33.2
44.6
30.2
20.6
30.1
34.8
10.7
34.2
(26.6,43.9)
37
-------
Toxicant: Cadmium (mg/kg)
Matrix: Sediment
Species: Palaemonetes pugio
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
95% Cl
Species: Ampelisca abdita
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
95% Cl
% Mortality
Control
0
10
0
20
0
6
Control
0
0
5
0
0
1
2.5
20
10
20
20
20
18
%
2.5
20
10
5
20
5
12
10
20
10
10
10
10
12
Mortality
10
55
5
30
45
45
36
40
90
100
100
100
100
98
40
100
100
100
100
100
100
160
100
100
100
100
100
100
160
100
100
100
100
100
100
LC50
18.1
18.5
17.7
17.7
17.7
18.2
0.2
17.9
(16.2,19.9)
LC50
7.8
18.9
13.1
9.7
10.4
12
4.3
11.8
(9.85, 14.2)
Species: Ampelisca verrilli
% Mortality
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
95% Cl
Species: Amphiascus tenuiremis
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
95% Cl
Control
50
5
5
0
5
13
Control
23
30
17
33
23
25
2.5
40
15
10
25
25
23
9
10
0
3
50
40
21
10
90
85
90
75
90
86
Mortality
18
17
7
23
3
3
11
40
100
100
100
100
100
100
36
33
10
20
20
17
20
160
100
100
100
100
100
100
45
17
37
20
17
23
23
LC50
NC
5
5
5
4.3
4.8
0.37
4.5
(3.9,5.1)
LC50
NC
NC
NC
NC
NC
NC
NC
NC
NC
38
-------
Toxicant: Cadmium (mg/kg)
Matrix: Sediment
Species: Mercenaria mercenaria
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
Species: Brachionus plicatilis
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
95% Cl
Control
0
0
0
0
0
0
Control
0
0
0
0
0
0
%
2.5
100
100
100
100
100
100
°A
2.5
0
0
0
3
3
1
Mortality
10
100
100
98
100
98
99
B Mortality
10
0
0
0
0
0
0
40
100
100
100
100
100
100
40
77
50
52
21
48
49
160
100
98
100
100
100
99
160
77
100
100
100
100
95
LC50
<2.5
<2.5
<2.5
<2.5
<2.5
<2.5
<2.5
LC50
27.6
40
39.1
60.2
40.7
41.5
11.7
41.9
(35.8,49.1)
39
-------
Toxicant:
Species:
Cadmium (mg/L)
Vibrio fischen
Microtox Solvent Extract EC50s for Spiked Sediments
Cadmium
Concentration
[mg/kg dw]
0.0
2.50
10.00
40.00
160.00
5 min EC50
[mg dw/ml]
>5.8
>5.7
>5.7
>5.7
>5.7
1 5 min EC50
[mg dw/ml]
>5.8
>5.7
>5.7
>5.7
>5.7
Microtox Solid Phase EC50s for Spiked
Sediment
Cadmium
Concentration
[mg/kg dw]
0.0
2.5
10.0
40.0
160.0
5 min EC
[mg dw/ml]
50
66.6
75.6
64.4
52.3
44.4
(11.2)
(11.6)
(10.5)
(4.9)
(7.1)*
*Significantly different from control at a = 0.05
Mutatox Results for Spiked Sediment Extracts
Direct Assay Time
Concentration
[mg\kg dw]
14 16 20 24
S-9 Assay Time
14 16 20 24
0.0
2.5
10.0
40.0
160.00
40
-------
Appendix C.
Results obtained from DDT aqueous and sediment bioassays.
41
-------
Toxicant: DDT Gug/L)
Matrix: Aqueous
Species: Palaemonetespugio
Duration Replicate
24 h A
B
C
D
E
Mean
SD
Pooled
95% Cl
Species: Ampelisca abdita
% Mortality
Control 3.90 6
.50 10.80
0 20 30
0 0 50
0 0 20
0 40 40
0 30 40
0 18 36
50
100
50
40
50
58
18.00
70
100
70
90
70
80
30 50
LC50
100 100 10.5
80 90 6.6
80 100 12.6
100 100 8.4
90 100 9.5
90 98 9.5
2.3
8.9
(7.5, 10.5)
% Mortality
Duration Replicate Control
24 h A
B
c
D
Mean
SD
Pooled
Species: Ampelisca verrilli
0
0
0
0
0
0
800
0
0
0
0
0
0
1300
0
0
0
0
0
0
2200
0
0
0
0
0
0
3600 6000 10000
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
LC50
NC
NC
NC
NC
NC
NC
NC
NC
% Mortality
Duration Replicate Control 2.3
24 h A
B
c
n
P
i
1
Mean
SD
Pooled
95% Cl
Species: Amphiascus
Duration
24 h
48 h
96 h
10 0
10 10
0 0
0 10
0 0
0 -
0 -
0 -
0 -
0 -
0 4
3.9
10
10
0
0
20
_
_
8
6.5
20
20
10
10
20
10
10
0
0
0
10
10.8
30
20
50
10
10
0
?0
?0
10
10
18
18.0
40
40
10
60
50
20
30
30
70
20
37
30.0
80
0
70
60
60
60
70
30
30
60
52
50.0
80
30
70
70
20
30
40
40
40
80
50
83.3
80
20
80
50
80
40
90
50
50
30
57
LC50
18
NC
23.7
16.8
36
NC
30
83.3
83.3
27.3
39.8
27.6
38.3
(32.4, 45.3)
tenuiremis
Control
5
0
0
% Mortality
0.6
0
0
0
1.25
0
0
10
2.5
5
20
25
5
15
10
15
10
10
25
20
LC50
NC
NC
NC
42
-------
Toxicant: DDT (,ug/L)
Matrix: Aqueous
Species: Mercenaria mercenaria
Duration Replicate
24 h
A
B
C
D
E
Mean
SD
Pooled
95% Cl
Control
0
0
0
0
0
0
313
60
50
40
30
30
42
% Mortality
625
30
40
30
60
70
46
1250
80
80
100
100
90
90
2500
100
100
90
100
100
98
5000
100
100
100
100
100
100
10000
100
100
100
100
80
96
LCSO
690
690
743
493
442
612
135
615
(358, 1057)
Species: Brachionus plicatilis
% Mortality
Duration
24 h
Replicate
A
B
C
D
E
Mean
SD
Pooled
Control
0
0
0
0
0
0
1300
0
0
0
0
0
0
2200
0
0
0
0
0
0
3600
0
0
0
0
0
0
6000
3
0
0
3
0
1
10000
40
13
3
7
20
17
LC50
NC
NC
NC
NC
NC
NC
NC
NC
Species: Mysidopsis bahia
% Not Fluorescing
Duration Replicate Control 2.6
1 h A
B
C
D
E
F
Mean
SD
Pooled
95% Cl
Species:
Replicate
A
B
C
D
MEAN
SD
17
0
0
0
33
17
11
29
29
43
17
17
33
28
4 7.2
33 67
33 33
33 50
50 50
67 67
50 100
44 61
12
50
67
100
100
83
100
83
20
83
83
100
100
100
100
94
LC50
6.9
8.6
6.5
5.3
4.3
4
5.9
1.7
5.1
(3.9, 6.6)
Vibrio fischeri (microtox)
5
min EC50
> 10,000
> 20,000
> 27,000
> 50,000
NC
NC
15 min EC50
> 10,000
> 20,000
> 27,000
> 50,000
NC
NC
43
-------
Toxicant: DDT (mg/kg)
Matrix: Sediment
Species: Palaemonetes pugio
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
95% Cl
Species: Ampelisca abdita
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
95% Cl
% Mortality
Control
10
0
0
0
10
4
Control
10
5
10
5
5
7
0.64 1.60
20
10
10
20
30
18
%
0.64
0
0
5
5
5
2
0
10
30
20
30
18
Mortality
1.60
0
5
5
10
10
6
4.00
30
40
40
50
40
40
4.00
15
10
20
15
5
13
10.00
90
90
80
90
70
84
10.00
65
55
55
45
70
58
LC50
5.0
4.5
4.2
3.8
5.0
4.5
0.5
4.5
(3.6, 5.6)
LC50
7.6
9.0
8.8
NC
7.5
8.2
0.8
8.5
(7.2, 10.0)
Species: Ampelisca verrilli
% Mortality
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
95% Cl
Species: Mercenaria mercenaria
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
95% Cl
Control
10
10
10
2
0
10
Control
0
0
0
0
0
0
0.64
5
5
5
15
10
8
%
0.64
6
20
10
0
4
8
1.60
5
0
5
30
15
11
Mortality
1.60
0
10
20
6
20
11
4.00
10
10
10
10
20
12
4.00
50
46
32
26
30
37
10.00
45
70
55
45
75
58
10.00
80
40
70
66
66
64
LCso
NC
7.4
9.0
NC
8.5
8.3
0.88
8.5
(7.2, 10.0)
LC50
4.3
> 10
6.2
6.9
5.8
5.8
1.1
6.3
(4.8, 8.3)
44
-------
Toxicant: DDT (mg/kg)
Matrix: Sediment
Species: Brachionus plicatilis
Duration Replicate
24 h A
B
C
D
E
Mean
SD
Pooled
Species: Amphiascus tenuiremis
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
95% Cl
Control
0
0
0
0
0
0
Control
33
33
13
30
10
24
%
0.64
0
0
0
0
0
0
0.1
43
40
10
0
10
21
Mortality
1.60
0
0
0
0
0
0
% Mortality
1
33
33
0
3
27
19
4.00
0
0
0
0
0
0
10
7
17
17
10
17
13
10.00
3
0
0
0
0
. 1
100
10
43
27
23
7
22
LC50
NC
NC
NC
NC
NC
NC
NC
NC
LC60
NC
NC
NC
NC
NC
NC
NC
NC
NC
Clutch size in Amphiascus tenuriemis exposed to DDT in
sediments for 10 days
DDT
Clutch Size ± SD (eggs/female)
0
0.1
1.0
10.0
100.0
8.83 ± 3.02
7.45 ± 2.87
7.34* ± 3.09
6.65* ± 2.93
6.89* ± 2.98
*significantly different at alpha = 0.05
45
-------
Toxicant:
Species:
DDT
Vibrio fischeri
Microtox Solvent Extract EC50s for Spiked Sediments
DDT
Concentration
[mg/kg dw]
0.00
0.64
1.60
4.00
10.00
5 min ECcn 15 min EC™
[mg dw/ml] (SD) [mg dw/ml]
0.86
0.98
0.79
0.72
0.81
0.36
0.15
0.23
0.19
0.15
0.93
1.00
0.73
0.63
0.78
0.32)
0.15
0.20)
0.15)
0.15)
Microtox Solid Phase ECSOS for Spiked Sediments
DDT
Concentration
[mg/kg dw]
0
0.64
1.60
4.00
10.00
5 min EC50
[mg dw/ml](SD)
9.9
13.0
10.1
7.4
8.7
1.7
2.0
3.8
0.1 ,
2.1
Mutatox Results for Spiked Sediment Extracts
Concentration
[mg/kg dw]
Direct Assay
time
14 16 20 24
14
S-9 Assay
Time
16 20 24
0
0.64
1.60
4.00
10.00
46
-------
Appendix D.
Results obtained from Fluoranthene aqueous and sediment bioassays.
47
-------
Toxicant: Fluoranthene(//g/L)
Matrix: Aqueous
Species: Palaemonetes pugio
% Mortality
Species:
Species:
Species:
Duration Replicate
96 h A
B
C
D
E
Mean
SD
Pooled
95% Cl
Ampelisca abdita
Duration Replicate
96 h A
B
C
D
E
Mean
SD
Pooled
95% Cl
Ampelisca verrilli
Duration Replicate
96 h A
B
C
D
E
Mean
SD
Pooled
95% Cl
Amphiascus tenuiremis
Duration Rpplicate
24 h A
B
C
D
E
Mean
SD
Pooled
Control
0
0
0
10
10
4
104
10
10
10
10
0
8
173
20
10
20
30
10
18
288 480
50
30
40
20
50
38
70
30
40
30
30
40
800
80
50
60
70
60
64
LC50
313.7
800
619.7
619.7
619.7
594.6
175.4
564.6
(410.6, 776.4)
% Mortality
Control
0
10
0
0
20
6
Control
10
0
0
0
0
2
38.4
0
10
0
20
20
10
38.9
20
0
10
0
10
8
64.8
60
20
60
70
100
62
64.8
40
40
20
40
10
30
108
100
90
100
90
100
96
%
108
40
80
30
70
30
50
180
100
100
100
100
100
100
Mortality
180
50
90
60
80
70
70
300
100
100
100
100
100
100
300
90
100
90
100
100
96
500
100
100
100
100
100
100
500
90
80
100
90
90
90
LC50
61.6
78.5
61.6
53.5
47.1
60.5
11.8
59.1
(55.4, 62.0)
LC50
124
75.4
139.4
87.2
137.7
112.7
29.6
107.7
(97.3, 119
.2)
% Mortality
Control
15
0
0
5
0
4
100
25
0
10
0
5
8
170
10
5
10
5
5
7
290
20
5
10
0
10
9
480
5
20
10
10
20
13
800
0
15
15
15
5
10
1600
20
10
20
20
20
18
LC50
NC
NC
NC
NC
NC
NC
NC
NC
48
-------
Toxicant: Fluoranthene
Matrix: Aqueous
Species:
Species:
Species:
Species:
Mercenaria mercenaria
Duration Replicate
24 h A
B
C
D
E
Mean
SD
Pooled
95% Cl
Mercenaria mercenaria
Duration Replicate
96 h A
B
C
D
E
Mean
SD
Pooled
Brachionus plicatilis
Duration Replicate
24 h A
B
C
D
E
Mean
SD
Pooled
Mysidopsis bahia
Duration Replicate
1 h A
B
C
D
E
F
Mean
SD
Pooled
% Mortality
Control
0
0
0
0
10
104
10
20
20
20
10
173
10
30
30
20
20
288
30
40
30
10
40
480
50
40
50
30
30
800
60
50
50
60 ..
40
LC50
512
800
620
675
>800
652
735
(441, 1225)
% Mortality
Control
10
10
0
0
10
6
104
70
80
80
60
60
70
173
70
90
100
100
100
92
288
oooooo
oooooo
480
oooooo
oooooo
800
oooooo
oooooo
LC50
<104
<104
<104
<104
<104
<104
<104
% Mortality
Control
0
0
3
0
3
1
38.4
0
0
0
0
8
2
64.8
0
0
0
3
0
1
108
0
0
0
0
0
0
180 300 500 LC50
4 0
0 0
4 0
3 0
0 0
2 0
0
0
0
0
0
0
zzzzzzzz
oooooooo
% Not Fluorescing
Control
17
17
17
0
0
0
104
17
0
0
0
0
0
3
173
33
40
0
0
0
0
12
288
50
40
33
0
0
0
20
480
33
33
33
0
0
0
17
800
33
33
0
0
0
0
11
EC50
ooooooooo
zzzzzzzzz
49
-------
Species: Vibrio fischeri (Microtox)
Replicate
A
B
C
D
MEAN
SD
5min EC50
>1100
1057
>1100
>1100
NC
NC
1 5 min EC50
>1100
>1100
>1100
>1100
NC
NC
Toxicant: Fluoranthene (mg/kg)
Matrix: Sediment
Species: Palaemonetes pugio
Duration
10 Day
Replicate
A
B
c
D
E
Mean
SD
Pooled
Control
20
0
10
0
30
12
0.78
10
20
0
10
0
8
% Mortality
3.12
10
20
0
10
0
8
12.50
10
20
10
10
0
10
50.00
10
0
10
10
10
8
LC50
>50
>50
>50
>50
>50
>50
-
>50
Species: Ampelisca abdita
% Mortality
Duration Replicate
10 Day A
B
C
D
E
Mean
SD
Pooled
Species: Ampelisca verrilli
Duration Replicate
10 Day A
B
c
D
E
Mean
SD
Pooled
Control
0
10
0
0
5
3
0.78
55
0
50
0
0
21
3.13
5
25
5
45
0
16
12.50
0
0
0
5
0
1
• 50.00
10
25
50
75
65
45
LC50
NC
NC
NC
NC
NC
NC
NC
NC
% Mortality
Control
15
5
10
10
0
8
0.78
0
5
5
5
10
5
3.12
5
0
0
0
0
1
12.50
0
10
5
0
0
3
50.00
5
25
35
5
10
16
LC50
NC
NC
NC
NC
NC
NC
NC
NC
50
-------
Toxicant: Fluoranthene (mg/kg)
Matrix: Sediment
Species: Amphiascus tenuiremis
% Mortality
Species:
Species:
Duration
10 Day
Replicate Control 0.8 3.1 12.5 50
A 0
B 13
C 17
D 0
E
Mean 7
SD
Pooled
95% Cl
Mercenaria mercenaria
17 17 7 0
3 17 10 13
0 27 7 7
17 20 13 0
20 13 33
11 19 8 5
% Mortality
LC50
NC
NC
NC
NC
NC
NC
NC
NC
NC
Duration Replicate Control 0.78 3.12 12.50 50.00 LCSO
24 h
A 0
B 0
C 0
D 0
E 4
Mean 1
SD
Pooled
95% Cl
Brachionus plicatilis
Duration
24 h
Replicate Control
A 0
B 0
C 0
D 0
E 0
Mean 0
SD
Pooled
95% Cl
76 54 80 86
56 78 76 94
70 68 86 86
36 76 76 80
44 78 90 94
56 71 82 88
% Mortality
0.78 3.13 12.50 50.00
00 0 3
0000
0000
0000
00 0 0
000 1
<0.78
<0.78
<0.78
1.27
<0.78
<0 78
LC50
NC
NC
NC
NC
NC
NC
NC
NC
NC
51
-------
Toxicant: Fluoranthene
Species: Vibrio fischeri
Microtox Solvent Extract EC50s for Spiked Sediments
Fluoranthene
Concentration
[mg/kg dw]
5 min EC50
[mgdw/ml] (SD)
15 min EC50
[mgdw/ml] (SD)
0
0.78
3.12
12.50
50.0
0.83
1.41
4.49
0.87
1.83
(0.23
(0.31
(0.82
(0.18
(1.35
0.81
2.11
3.38
1.41
1.31
0.33
1.82
1.93
1.28
0.75
*Significantly different from control a = 0.05
Microtox Solid Phase EC50s for Spiked Sediments
Fluoranthene
Concentration
[mg/kg dw]
0.0
0.78
3.12
12.5
50.0
5 min EC50
[mg dw/ml] (SD)
7.2
7.1
8.8
7.8
9.0
(1.2
(1.0
(1.6
(0.2
(2.6
Mutatox Results for Spiked Sediments
Concentration
[mg/kg dw]
Direct Assay
Time
14 16 20 24
15 min ECSO
[mg dw/ml] (SD)
14 16 20 24
0
0.78
3.12
12.5
50.0
52
-------
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