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

-------

-------
                                                           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

-------
                                   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.

-------
                                   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

-------
                                     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

-------
                                          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.

-------

-------
                                          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.

-------
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

-------
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

-------
 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.

-------
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

-------
 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

-------
                                         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

-------
    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.
  (Species profiles: Life histories and environmental
  requirements of coastal fishes and invertebrates
  (Gulf of Mexico) Grass Shrimp.) U.S.  Fish Wildl.
  Serv. Biol. Rep. 82 (11.35), U.S. Army Corps of
  Engineers, TR EL-82-4. 19 pp.
ASTM.  1993. ASTM standards on Aquatic Toxi-
  cology and Hazard Evaluation Sponsored by ASTM
  Committee  E-47   on  Biological  Effects and
  Environmental  Fate.   ASTM Publication Code
  Number (PCN): 03-547093-16. 538p.
Aqua Survey. 1994.  Mysid  IQ Toxicity Test Kit
  Instructions. Aqua Survey, Inc., Flemington, New
  Jersey.
Bousfield, E.L. 1973. Shallow-water Gammaridean
  Amphipoda of  New England.  Cornell University
  Press, Ithaca, New York. "312p.
Burton,  D. and D. Fisher. 1990. Acute toxicity of
  cadmium,  copper, zinc, ammonia, 3,3'-dichloro-
  benzidine,  2,6-dichloro-4-nitroaniline,  methylene
  chloride and 2,4,6-trichlorophenol to juvenile grass
  shrimp and killifish. Bull. Environ. Contam.  Toxicol.
  44: 776-783.
Calabrese, A., J.R. Maclnnes, D.A. Nelson and J.E.
  Miller. 1977. Survival and growth of bivalve larvae
  under heavy-metal Stress. Mar. Biol. 41:179-184.
Champlin, D.M.  and S.L. Poucher.  1991.   Acute
  toxicity of  fluoranthene to various marine organ-
  isms.  Personal Communication to D.J. Hansen.
  U.S. EPA.  Narragansett, Rl.  (Unpublished).
Chandler, G.T.  1986.  High density  culture  of
  meiobenthic harpacticaid copepods within a muddy
  sediment substrate. Canadian Journal of Fisheries
  and Aquatic Sciences 43:53-59.
Chandler,  G.T.  1990.  Effects of sediment bound
  residues of the pyrethroid insecticide, fenvalerate,
  on survival and reproduction in meiobenthic cope-
  pods.  Marine Environmental Research 29:65-76.
Chandler,  G.T.  and G.I. Scott.  1991.  .Effects of
  sediment-bound  endosulfan on  survival, repro-
  duction  and  larval  settlement  of  meiobenthic
  polychaetes and copepods. Environmental Toxico-
  logy and C/7e/7?/sf/y 10:375-385.
Cripe, G.M.  1994. Comparative  acute toxicities of
  several pesticides and metals to Mysidopsis bahia
  and postlarval Penaeus duorarum. Environmental
  Toxicology and Chemistry 13(11): 1867-1872.
DeWitt,  T.H., R.J. Ozretich, R.  C. Swartz,  J.O.
  Lamberson, D.W. Schults, G.R. Ditsworth, J.K.P.
  Jones, L. Hoselton and  L.M.  Smith. 1992. The
  effect  of organic matter quality on the toxicity and
  partitioning of sediment-associated fluoranthene to
  the  infaunal  marine  amphipod,  Rhepoxynius
  abronius. Environmental Toxicology and Chemistry
  11:197-208.
Dillon, R.T. Jr. and J.J. Manzi. 1989.  Genetic and
  shell morphology in a  hybrid zone between  the
  hard   clams  Mercenaria  mercenaria  and   M.
  campechiensis.  Mar. Biol. 100:217-222.
DiToro,  D.M., J.D.  Mahony, D.J. Hansen, K.J. Scott,
  M.B. Hicks, S.M. Mayrand M.S. Redmond.  1990.
  Toxicity of Camiun in Sediments:  The Role of Acid
  Volatile Sulfide.   Environmental Toxicology and
  Chemistry 9:1487-1502.
Eisler, R.  1969. Acute toxicities of insecticides to
  marine  decapod  crustaceans.    Crustaceana
  16(3):302-310.
Eisler, R.  1971. Cadmium poisoning in  Fundulus
  heteroclitus (Pisces:Cyprinodontidae) and other
  marine organisms.  J.  Fish.  Res. Board Can.
  28(9):  1225-1234.
EPA. 1978. in-depth studies on health and environ-
  mental  impacts  of  selected  water pollutants.
  EG&G, Bionomics, Wareham,  MA to U.S. EPA,
                                               25

-------
Criteria Branch, Washington, DC under contract No.
68-01-4646.
EPA. 1980. Methods for the analysis of pesticides
  in (issue, sediment, and water. USEPA, Research
  Triangle Park, NC, USEPA Report No. EPA-600/8-
  80-038. 602p.
EPA.   1991.    Evaluation of Dredged  Material
  Proposed for Ocean Disposal (Testing Manual).
  Report prepared by U.S. Environmental Protection
  Agency and  the U.S. Army Corps of Engineers,
  EPA-503/8-91/001.
EPA. 1993. Sediment Quality Criteria for the Protect-
  ion of Benthic Organisms: Fluoranthene. EPA-822-
  R-93-012. U.S. EPA Office of Water and Office of
  Research and Development, Washington, D.C.
EPA. 1994. Methods for Assessing the Toxicity of
  Sediment-Associated Contaminants with Estuarine
  and  Marine  Amphipods.  EPA  600/R-94/025.
  Office   of  Research   and   Development,
  Environmental  Protection Agency,  Narragansett,
  Rl.
Frank,  P. and  Robertson.  1979. The influence of
  salinity on toxicity of cadmium and chromium to
  blue  crab, Callinectes sapidus.   Bull. Environ.
  Contam. Toxicol. 21(1 -2):74-78.
Frasca, A. A.  1995. A method to assess the risk of
  acute arid chronic toxicity to estuarine systems
  from  non-point  source urban  runoff.   Master's
  Thesis. University of South Carolina, Columbia,
  SC.
Frear, D.E. and J.E. Boyd. 1967. Use of Daphnia
  magna   for microbioassay  of  pesticides.   I.
  Development of standardized techniques  for
  rearing daphnia and  preparation  of dosage-
  mortality curves for pesticides. J. Econ. Entomol.
  60(5):1228-1236.
Fulton, M.H., G.T. Chandler and G.I. Scott. 1997.
  Urbanization effects on the fauna of a Southeastern
  U.S.A  bar-built estuary,   p.477-504.   In:  F.
  Vernberg, W. Vernberg and  T. Siewicki (eds.),
  Sustainable  Development in the  Southeastern
  Coastal Zone.  The Belle W. Baruch Library in
  Marine Science (number 20), Columbia, SC.
Green, A.S., G.T. Chandler and E.R. Blood. 1993.
  Aqueous-, pore-water and sediment-phase toxicity
  relationships fora meiobenthic copepod. Environ-
  mental Toxicology and Chemistry 12:1497-1506.
Hong, J.S. and D.J. Reish.  1987.  Acute toxicity of
  cadmium to eight species of marine amphipod and
  isopod crustaceans from Southern California. Bull.
  Environ. Contam. Toxicol. 39(5):884-888.
Johnson, M.W. 1979. Acute toxicity of cadmium,
  copper and mercury to larval  American Loster,
  Homarus americanus.  Bull.  Environ.  Contam.
  Toxicol. 22:258-264.
Krahn, M.M., C.A. Wigner, R.W. Pearce, L.K. Moore,
  R.G. Bogar, W.D.Macleod, Jr., S.L.Chan and D.W.
  Brown. 1985.  New HPLC cleanup and revised
  extraction procedures for organic contaminants.
  NOAA Technical Memorandum.  NMFS F/NWC-
  153.
Long, E.R. and R. Markel.  1992.  An evaluation of
  the  extent and magnitude of biological effects
  associated with chemical contaminants in  San
  Francisco  Bay, California.    NOAA  Technical
  Memorandum. NOS ORCA 64.  86pp.
Long, E.R., D.D.  MacDonald, S.L. Smith, and  F.D.
  Calder.  1995.   Incidence of adverse biological
  effects within ranges of chemical concentrations in
  marine and  estuarine sediments.  Environmental
  Management 19(1): 81-97.
MacDonald, D. 1994. Approach to the assessment of
  sediment quality  in  Florida   coastal waters.
  Volumel.  "Development  and  Evaluation  of
  Sediment  Quality  Assessment   Guidelines."
  Prepared for Florida Department of Environmental
  Protection, Tallahassee, FL. 126 p.
Mahood, R., M. McKenzie, D. Middaugh, S. Bollar, J.
  Davis and D. Spitzbergen. 1970.  "A report of the
  cooperative blue crab study-South Atlantic States."
  U.S. Department  of  the  Interior, Bureau of
  Commercial Fisheries.
Martin, M., K.  Osborn, P. Billig and N. Glickstein.
  1981. Toxicities of ten metals to Crassostrea gigas
  and Mytilus edulis  embryos and Cancer magister
  larvae. Mar. Poll. Bull. 12: 305-308.
Mayer,  F.  1987.   Acute toxicity handbook of
  chemicals to estuarine organisms. U.S. EPA Office
  of  Research and  Development,  Environmental
  Research Laboratory, Gulf Breeze, FL EPA/600/8-
  87/017. 274p
McLeese, D. and C. Metcalfe. 1980. Toxicities of
  eight organochlorine  compounds in sediment and
  seawater  to Crangon  septemspinosa.    Bull.
  Environ. Contam. Toxicol. 25:921-928.
McLeese, D., L. Burridge and J.  Van Dinter. 1982.
  Toxicities of  five  organochlorine compounds in
  water and sediment to Nereis virens. Bull. Environ.
  Contam. Toxicol. 28:216-220.
Mearns, A.J.,  R.C.  Swartz, J.M. Cummins,  P.A.
  Dinnel, P. Plesha, and P.M. Chapman. 1986. Inter-
  laboratory comparison of a sediment toxicity test
                                              26

-------
using the marine amphipod, Rhepoxynius abronius.
Marine Environmental Research 19:13-37.
Menzel, R.W.   1988.  The  biology,  fishery,  and
  culture of quahog clams, Mercenaria. In: Manzi, J.,
  Castagna, M. (eds.)  Clam mariculture in North
  America.  Elsevier Publishing Co., Amsterdam.
Microbics Corporation.  1992. Microtox Manual: A
  toxicity testing handbook. Volume III, Condensed
  Protocols, pp.227-232.
Mills, E.L.   1967.  The biology of an amphipod
  crustacean  sibling  species  pair.   Journal  of
  Fisheries Research Board of Canada 24:305-355.
Nelson, D., A. Calabrese,  B. Nelson, J.  Maclnnes
  and D. Wenzloff. 1976. Biological effects of heavy
  metals on  juvenile  bay  scallops,  Argopecten
  irradians, in short-term exposures.  Bull. Environ.
  Contam. Toxicol. 16(3):275-282.
Nichols, F.H. and J.K. Thompson.  1985. Persist-
  ence of an introduced mudflat community in south
  San  Francisco Bay, California.  Marine Ecology
  Progress Series 24:83-97.
Nimmo,  D.R.,  L.H.  Bahner, R.A.  Rigby, J.M.
  Sheppard and A.J. Wilson,  Jr. 1977. Mysidopsis
  bahia: An estuarine species suitable for life cycle
  toxicity tests to determine the effects of a pollutant.
  Aquatic Toxicology and Hazard Evaluation.  ASTM
  STP 634, FL. Mayer and J.L.  Hamelink (eds.)
  American Society for Testing and Materials. 109-
  116.
Otis, M.J., S. Andon, and R. Bellmer. 1990. New
  Bedford Harbor Superfund Pilot Study: Evaluation
  of dredging and dredged materials disposal. U.S.
  Army Corps of Engineers, New England Division,
  Walthan, MA. 1 v. (various pagings).
Pechenik, J.A. 1991. Biology of the Invertebrates.
  Wm. C. Brown Publishers: Dubuque. 567p.
Pequegnat, W.E., L.H. Pequegnat,  B.M James, E.A.
  Kennedy, R.R. Fay,  A.D.  Fredericks.   1981.
  Procedural guide for designation surveys of ocean
  dredged  material  disposal sites.   Final  Report
  prepared by TerEce Corp. for U.S. Army Engineer
  Waterways Experiment Station, Technical  Report
  EL-81-1,268p.
Rossi,  S.S.  and J.M.  Neff.  1978.   Toxicity  of
  polynuclear   aromatic  hydrocarbons to   the
  polycheate Neanthes arenaceodentata.   Marine
  Pollution Bulletin 9:220-223.
Sanders,  H. 1969. Toxicity of pesticides  to  the
  crustacean, Gammarus lacustris.  Bureau of Sport
  Fisheries and  Wildlife,  Technical Paper  25,
  Government Printing Office, Washington, D.C.
Sanders, H. 1972. The toxicities of some insecticides
,  to four species of malocastrean Crustacea.  Fish.
  Pest. Res. Lab., Columbia, MO., Bureau of Sport
  Fish and Wildlife.
Sanders, H.O. and O.B. Cope. 1966. Toxicities of
  several pesticides to two species of cladocerans.
  Trans. Amer. Fish. Soc. 95:165-169.
Schantz, M.M.,  B.A.  Benner,  S.N.  Chester, B.J.
  Koster, K.E. Hehn,  S.F.  Stone, W.R. Kelly,  R.
  Zeisler, and S.A. Wise.  1990.  Preparation and
  analysis of marine sediment reference material for
  the determination of trace organic constituents.
  Fresenius J. Anal. Chem. 338(4):501-514.
Scott, K.J. and M.S. Redmond. 1989. "The Effects of
  a Contaminated Dredged Material on  Laboratory
  Populations of the Tubicolous Amphipod Ampelisca
  abdita." Aquatic Toxicology and Hazard Assess-
  ment: 12th Volume, ASTM STP 1027. U.M.Cowgill
  and  L.R. Williams,  eds., American Society for
  Testing and Materials, Philadelphia, pp 289-303.
Scott, G.I., M.H. Fulton, M.C. Crosby, P.B. Key, J.W.
  Daugomah,  J.T.  Waldren, E.D. Strozier, C.J.
  Louden,  G.T.  Chandler,  T.F.  Bidleman, K.L.
  Jackson, T.W. Hampton, T. Huffman, A. Shulz and
  M. Bradford. 1994.  Agricultural Insecticide Runoff
  Effects  on  Estuarine  Organisms:   Correlating
  Laboratory and Field Toxicity Tests, Ecophysiology
  Bioassays,   and   Ecological  Biomonitoring.
  EPA/600/R-94/004.   U.S. EPA, Environmental
  Research Laboratory, Gulf Breeze, FL. 288 p.
Snell, T.W. and G. Persoone. 1989. Acute toxicity
  bioassays using rotifers.   I.  A test from brackish
  and marine environments with Brachionusplicatilis.
  Aquatic Toxicology 14: 65-80.
Strobel,  C.J.,, D.J.  Klemm, L.B.  Lobring,  J.W.
  Eichelberger  and  A.  Alford-Stevens.     1995.
  Environmental   Monitoring  and  Assessment
  Program  (EMAP) Laboratory Methods Manual:
  Estuaries.  Volumel.   Biological and  Physical
  analyses. EPA/620/R-95-008. U.S. EPA, National
  Health  and  Environmental  Effects  Research
  Laboratory, Atlantic Ecology Division, Narragansett,
  Rl.  127 p.
Strobel, C.J, H.W. Buffum, S.J. Benyi, E.A. Petrocelli,
  D.R. Reifsteck, And D.J. Kieth. 1995. Statistical
  Summary: EMAP - Estuaries Virginian Province -
  1990-1993.    U.S.  EPA  National  Health and
  Environmental  Effects   Research   Laboratory,
  Atlantic  Ecology  Division,  Narragansett,  R.I.
  EPA/620/R-94/026.  99p,        .
                                               27

-------
Suedel, B.C. J.H. Rodgers, Jr.  and P.A.  Clifford.
  1993. Bioavailability of fluoranthene in freshwater
  sediment toxicity tests. Environmental Toxicology
  and Chemistry 12(1): 155-165.
Summers, J.K.  Unpublished.  EMAP - Estuaries
  Louisianian Province, Benthic Data for 1991 -1994.
  U.S.  EPA Office of Research And Development,
  Environmental Research laboratory, Gulf Breeze,
  FL.
Sunda, W. G. 1978. Effect of chemical speciation on
  the  toxicity   of  cadmium  to  grass  shrimp,
  Palaemonetes pugio: Importance of free cadmium
  ion. Environ. Sci. Technol. 12(4): pp 409.
Swartz, R.C.,  W.A.  DeBen,  J.K.P Jones, J.O.
  Lamberson and  F.A. Cole.  1985.  Phoxocephalid
  amphipod bioassay for marine sediment toxicity.
  Aquatic  Toxicology  and  Hazard  Assessment
  Seventh Symposium,  ASTM   STP  854,  R.D.
  Cardwell,  R.  Purdy,  and  R.C.  Bahner,  eds.,
  American  Society for Testing  and  Materials,
  Philadelphia, pp. 284-307.
Swartz, R.C.,  D.W. Schults, T.H. DeWitt, G.R.
  Ditsworth and J.O. Lamberson. 1990. Toxicity of
  fluoranthene in sediment to marine amphipods: A
  test of the equilibrium partitioning approach to
  sediment quality criteria. Environmental Toxicology
  and Chemistry 9(8):1071-1080.
Swartz, R., F. Cole, J.  Lamberson, F. Ferraro, D.
  Schults, W. DeBen, H. Lee II and R. Ozreich. 1994.
  Sediment toxicity,  contamination and amphipod
  abundance at  a DDT-, and dieldrin-contaminated
  site  in  San  Francisco  Bay.    Environmental
  Toxicology and Chemistry 13(6):949-962.
Van Dolah,  R.F.,  P.M.  Wendt,  and E.L Wenner
  (eds). 1990. A physical and ecological characteri-
  zation of the Charleston Harbor estuarine system.
  Final Report to the South Carolina Coastal Council
  under Grant #NA87AA-D-CC068. 634p.
Van Dolah, R.F., R. M. Martore, A.E. Lynch, M.V.
  Levisen, P.H. Wendt, D.J.  Whitaker, and W.D.
  Anderson.  1994. Environmental Evaluation of the
  Folly Beach Nourishment Project.  Final Report to
  the U.S. Army Corps of Engineers, Charleston
  District, 100p.
Wendt, P.H., R.F. Van Dolah, M.Y.  Bobo, and J.J.
  Manzi.   1990.  Effects of marina proximity on
  certain aspects of the biology of oysters and other
  benthic macrofauna in a South Carolina estuary.
  South  Carolina   Marine   Resources   Center,
  Technical Report No. 74. 50p.
Wise, S.A., B.A. Brenner, G.D. Byrd, S.W. Chesler,
  R.E. Rebbert, and M.M. Schantz. 1988. Determi-
  nation of polycyclic aromatic hydrocarbons in a coal
  tar  standard  reference  material.    Analytical
  Chemistry 60: 887-895.
Wood, C. 1967. Physioecology of the grass shrimp,
  Palaemonetes  pugio  ,in the  Galveston Bay
  estuarine system. Cont. Mar. Sci: 12:54
                                              28

-------
                                    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

-------

-------
£»-o o

§§ 3
o su 2.
  ~£L

  C7?
  -• en
  TI3'
  ^- CD
  < CO
  nj. co
  CD


  en
  CD
o o m c
3" ®  ^ 2

3  CD  O Q.

*°B|
Q1
   3  of CO
   S. —

   3  TJ
O> 3  rn
CX> CD  o
                     33 >
                     CD CO
                     W CD
                     CD 3
                     !—*•


                     O
                    -a
                    m
                    3D
                    nrn,
                    O
   • 3> ~n C/j

       m^!
       coz

       55

       ^§

-------