Table 1. Measured cadmium concentrations and AVS in spiked sediments.

Nominal Measured
Concentration Concentration % of SEM AVS
(mg/kg dw) (mg/kg dw) SD Nominal (umol/g) (umol/g) SEM/AVS
25 2.6 0.5 104 0.021 0.02 1.1
10.0 9.5 0.3 95 0.077 0.019 44
40.0 37.4 46 91 0.315 0.024 13.1
160.0 185.7 28 116 1.247 0.028 44.5

Table 2. Measured DDT and TOC in spiked sediments.

Nominal Measured DDT
Concentration Concentration % of % Concentration
(mg/kg dw) (mg/kg dw) SD Nominal TOC (mg/g OC)
0.64 0.33 0.20 52 0.7 0.09
1.60 1.53 1.34 96 0.7 0.23
4.00 2.58 0.61 65 0.7 0.57
10.00 5.93 0.69 59 0.7 1.43

Table 3. Measured fluoranthene concentrations and TOC in spiked sediments.

Nominal Measured Fluoranthene
Concentration Concentration % of Concentration
(mg/kg dw) (mg/kg dw) sD Nominal % TOC (mg/g OC)
0.78 0.67 0.10 85 0.48 0.16
3.12 2.84 0.39 91 0.48 0.65
12.50 9.79 1.67 78 0.48 2.60

50.00 42.01 4.55 84 0.48 10.42

Table 4. Summary results from aqueous assays with cadmium.

Mean
Exposure LC50/EC50 Statistical Pooled Sensitivity 95% Cl
Species Period (mg/L) SD Comparisons’ LC50 (mg/L) Ranking (mg/L)
M. mercenaria 24h 0.4 0.1 A 0.4 4 04-05
A. tenuiremis 24h 1.5 0.3 B 1.6 2 1.4-1.7
A. abdita 24h 5.7 1.0 Cc 5.8 3 5.2-6.5
A. verrilli 24h 6.0 1.5 Cc 5.6 3 5.3 -6.0
M. bahia th 34.8 10.7 D 34.2 4 26.6 - 43.9
P. pugio 24h 29.9 8.1 D 31.7 4 23.6 - 42.6
V. fisheri(microtox) 15min 24.9 3.5 D NC 4 NC
B. plicatilis 24h 75.0 3.6 E 74.8 5 71.3 - 78.5

TMean LC50s/EC50s for species sharing the same letter are not significantly different at a = 0.05.

14



Appendix B.

Results obtained from Cadmium aqueous and sediment bioassays

35


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
Mean (-)2.Stand (+) 2 Stand SDS

Test Date Comments LCgo LCey Dev Dev Pass/Fail
10:22 AM baseline 16.5 16.5 NC NC

3/16/95 baseline 22.8 19.6 10.7 28.5

3/16/95 baseline 22.6 20.6 13.4 27.8

3/16/95 baseline 18.8 20.1 14.0 26.3

3/16/95 baseline 17.4 19.6 13.8 25.4

3/28/95 Cd aqueous 20.5 19.8 14.5 250 . Pass
3/28/95 Cd aqueous 21.6 20.0 15.0 25.0 Pass
5/2/95 Cd sediment 21.6 20.2 15.4 25.0 Pass
5/2/95 Cd sediment 19.5 20.1 15.6 24.6 Pass
6/26/95 DDT aqueous 25.2 20.7 15.3 26.0 Pass
6/26/95 DDT aqueous 20.5 20.6 15.6 25.7 Pass
10/2/95 Cd sediment 19.5 20.5 15.7 25.4 Pass
12/11/95 DDT range finder 21.6 20.6 15.9 25.3 Pass
12/18/95 DDT aqueous 25.2 21.0 15.8 26.1 . Pass
128/95 DDT aqueous 24.0 21.2 16.0 26.3 Pass
1/23/96 DDT sediment 17.6 20.9 15.6 26.2 Pass
1/23/96 DDT sediment 21.6 21.0 15.8 26.1 Pass
2/26/96 Fluoranthene aqueous 17.6 20.8 15.6 26.0 Pass
2/26/96 Fluoranthene aqueous 19.4 20.7 15.6 25.8 Pass
3/26/96 Fluoranthene sediment 20.6 20.7. 15.7 25.7 Pass
3/26/96 Fluoranthene sediment 20.6 20.7 15.8 25.6 Pass

Final Average 20.7

31



Toxicant: | Cadmium (mg/L)
Species: Vibrio fischeri

Microtox Solvent Extract EC,.s for Spiked Sediments

Cadmium
Concentration 5 min ECey 15 min ECgy
[mg dw/ml] [mg dw/ml]
0.0 >58 75.8
250 >5.7 >5.7
10.00 >57 257

40.00 >5.7 >5.7
160.00 > 5.7 > 5.7

Microtox Solid Phase EC,,s for Spiked
Sediment

Cadmium
Concentration 5 min EC,
[mg/kg dw] [mg dw/ml]

0.0 66.6 (11.2)
25 75.6 (11.6)
10.0 64.4 (10.5)
40.0 52.3 (4.9)
160.0 44.4 (7.1)

*Significantly different from control at « = 0.05

Mutatox Results for Spiked Sediment Extracts
Direct Assay Time S-9 Assay Time

Concentra
Tmgkgdwy. = 14—«16:—« 20 HCHBCCO

0.0 -



and detector temperatures were 250°C and 320°C,
respectively. The sample was injected (1 wl) using a
splitless Grob technique (1 min split time). The initial
oven temperature was 50°C with a one-minute hold,
followed by an increase to 170°C at 4°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
Sediments

Fluoranthene-spiked sediments were prepared and
dried as described above. The dried sample was
then transferred toa Pyrex Soxhlet thimble, and the
internal standards D,-naphthalene (200 ng), Djo-
acenapthalene (200 ng), D,.-phenanthrene (502 ng),
D,o-fluoranthene (497 ng), D,.-perylene (102 ng),
dibromooctafluorobiphenyl (PCB-103; 20 ng), and
2,2',3,3',4,5,5'6-octachlorobiphenyl (PCB-198; 20ng)
were added. The sample was then extracted ina
Soxhlet apparatus with 250 ml of CH,Cl, 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
chromatograph 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
CH,Cl, at a flow rate of 7 ml/min. A 400-mi 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

in Spiked

beginning 1 minute before the retention time of
DBOFBP and ending 2 minutes after perylene. The
tesulting fraction was reduced in volume as above.
The CH,Cl, was replaced with hexane and concen-
trated to a final volume of about 0.5 mi. 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 yl
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 ul) 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-um
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



Species: Vibrio fischeri

(Microtox)

Replicate

5 min EC50

15 min EC50

A
B
Cc

D
MEAN
sD

> 1100
1057

> 1100

> 1100
NC
NC

> 1100
> 1100
> 1100
> 1100
NC
NC

Toxicant: Fluoranthene (mg/kg)
Matrix: Sediment

Species: © Palaemonetes pugio

% Mortality

Duration Replicate

Control 0.78

3.12 12.50

50.00

10 Day A

Cc
D
E

20
0
10
0

30
12

10
20
0

10
ie)
8

10 410
20 20
0 10
10
i)
10

Species: Ampelisca abdita
Duration Replicate

% Mortality

Control 0.78

3.13 12.50

10 Day A

Cc

D

E
Mean

sD
Pooled

0 55
4 9

)

0 50
te) ie)
5 i)
3 21

5
25
5
45
0
16

Species: Ampelisca verrilli

% Mortality

Duration Replicate Control 0.78

3.12 12.50

50.00

40 Day A

Cc
D
E

15

5
ie)



Toxicant: Fluoranthene(ug/L)
Matrix: | Aqueous

Species: Palaemonetes pugio % Mortality

Duration Replicate Control 104

96h A 10
B 10
Cc 10
D 41
E
Mean
D

Ss
Pooled
95% Cl

Species: Ampelisca abdita % Mortality

Duration Replicate Control 38.4 . 108

96h A 0 100
B 10 90
Cc 0 100
D 90

E 100
Mean 96

sD
Pooled
95% Cl

Species: Ampelisca verrilli % Mortality

Duration Replicate Control 38.9 64.8

96h A 10 20
B ie)

0)

Cc 10

D 0

E 10

Mean 8

SD

Pooled
95% Cl

07:7

107.
(97.3, 1

19.2)

Species: Amphiascus tenuiremis % Mortality

Duration Replicate Control 100 290

24h A 15
B i)
Cc

D
E



United States

Environmental Protection Agency

Center for Environmental Research Information
Cincinnati, OH 45268

Official Business
Penalty for Private Use
$300

EPA/600/R-99/085

Please make all necassary changes on the below label,
detach or copy, and return to the address in the upper
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If you do not wish to receive these reports CHECK HERE [1];
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PERMIT No. G-35



Toxicant: DDT
Species: Vibrio fischeri

Microtox Solvent Extract EC.9s for Spiked Sediments

DDT
Concentration 5 min EC, 15 min EC,,
[mg/kg dw] Img dwiml] (8D) [mg dwimi

0.36
0.15
3

2:
19)
15,

Microtox Solid Phase EC,)S for Spiked Sediments

DDT
Concentration 5 min EC,
Img/kg dw] Img dwimi]($D)
9.9 ;

1.7)
. 2.0,
. 3.8
- 0.1
2.1

i)

0.64 1

1.60 1
4.00

10.00

Mutatox Results for Spiked Sediment Extracts
Direct Assay

Concentration Time
Img/kg dw] 14 16 20 24



Table 9. Sensitivity of selected invertebrate species to DDT in water column exposures.

Species Duration Lifestage' LCzp (ug/L) Reference

Daphnia magna — 48h U 0.4 Frear and Boyd, 1967

Gammarus fasciatus 96h U 0.8 Sanders, 1972

Gammarus lacustris 96h U 4 Sanders, 1969

Palaemonetes vulgaris 96h U 2 Eisler, 1969

Asselus brevicaudus 96h U 4 Sanders, 1972

Daphnia magna 24h U 44 Sanders and Cope, 1966

Mysidopsis bahia 1h J 5.9? This study

Palaemonetes pugio 24h A 9.5 This study

Callinectes sapidus 96h U 19 Mahood et al., 1970

Ampelisca verrilli 24h J 39.8 This study

Mercenaria mercenaria 24h J 612 This study

Ampelisca abdita 24h J >10,000 This study

Brachionus plicatus 24h J >10,000 This study

Amphiascus tenuiremis 24h A >10,000 This study

1 J =juvenile, L = larvae, U = unknown, A = adult, E = embryo

'2 EC., for fluorescence reduction
Table 10. Summary of results from sediment assays with DDT.

Pooled
Exposure Mean LCg/EC5y Statistical LC50_— Sensitivity 95% Cl
Species Period (mg/kg) SD Comparisons! (mg/kg) Ranking (mg/kg)

A. tenuiremis 10d 1.0? — — 1 —
P. pugio 10d 45 0.5 A 45 2 3.6 - 5.6
M, mercenaria 10d 5.8 44 _ 6.3 2 48-83
A. abdita 10d 8.2 0.8 B 8.5 3 7.2- 10.0
A. verrilli 10d 8.3 0.9 B 8.5 3 7.2- 10.0
A. tenuiremis 410d > 10.0 — _ > 10.0 4 _
B. plicatilis 24h > 10.0 — > 10.0 4 —
V. fisheri (microtox) | 5 min > 10.0 — > 10.0 4 _
V. fisheri (microtox) — > 10.0 — — _ — —

T Mean LC50s/EC50s for species sharing the same letter are not significantly different at a = 0.05.

2 Concentration whith caused significant reduction in clutch size.

18



Toxicant: Cadmium (mg/L
Matrix: Aqueous ( )

Species:  Palaemonetes pugio % Mortality
Duration _ Replicate Control 48
24h A
B

Cc

D

E
Mean .

SD a
Pooled 67
95% Cl (23.57, 42.56)

Species: Ampelisca abdita % Mortality
Duration Replicate Control 1.4

24h A 30

B 20

Cc 0

D ie)

E 0

Mean 10

sD .

Pooled
95% Cl

£

Ra _anaaan
QO NYooOror

G
2

Species: | Ampelisca verrilli % Mortality

Duration Replicate Control 1.9
24h A 10
B 0

fo)
g

PRONOROZ
@BYWNAD20

Mean
SD
Pooled
95% Cl

fo

bas
oon

Species: Amphiascus tenuiremis % Mortality

Duration Replicate Control 0.6

24h A
B
Cc
D
E
Mean
sD

oO
8

Onasann
ROWNWHDOMS

Pooled
95% Cl

Bo



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 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 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 wm). Field studies in South Carolina have indicated that sites
with high sédiment 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. .



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 B. plicatilis

29


Table 5. Sensitivity of selected invertebrate species to cadmium in water column exposures.

Species Duration Life Stage’ LC50 (ug/L) Reference
Mysidopsis bahia 96h J 12 Cripe, 1994
Palaemonetes pugio 96h A 40 Sunda et al., 1978
Homarus americanus 96h L 80 Johnson, 1979
Rhepoxynius abronius 96h U 147 Hong and Reish, 1987
Penaeus duorarum 96h J 312 Cripe, 1994
Callinectes sapidus 96h A 320 Frank and Robertson, 1979
Crangon septemspinosa 96h A 320 Eisler, 1971
Crassostrea gigas 48h E 375 Martin et al., 1981
Palaemonetes vulgaris 96h A 420 Eisler, 1971
Mercenaria mercenaria 24h J 420 This study
Corophium insidiosum 96h U 779 Hong and Reish, 1987
Argopecten irradians 96h J 908 Nelson et al., 1976
Amphiascus tenuiremis 24h A 1,500 This study
Mya arenaria 96h A 2,200 Eisler, 1971
Ampelisca abdita 24h J 5,700 This study
Ampelisca verrilli 24h Ald 6,000 This study
Palaemonetes pugio 48h 13,000 Burton and Fisher, 1990
Palaemonetes pugio 24h A 29,900 This study
Mysidopsis bahia th J 34,800 7 This study
Brachionus plicatilis 24h J 75000 This study

* J =juvenile, L
? EC5O for fluorescence reduction in Mysid 1Q® 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 24h
B. plicatilis sediment porewater assay and the

15

= larvae, U = unknown, A = adult, E = embryo

MicrotoxTM and MutatoxTM bioassays are provided in
Appendix Band 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 umol/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


Table 13. Sensitivity of selected invertebrate species to fluoranthene in water column exposures.

Species Duration stage’ LC50 (ug/L) Reference
Mysidopsis bahia 96h J 40 EPA, 1978
Ampelisca abdita 96h J 60.5 This study
Ampelisca abdita 96h J 66.9 Champlin and Poucher, 1991
Mercenaria mercenaria 96h J < 104 This study
Ampelisca verrili 96h J 112.7 This study
Palaemonetes pugio 96h L 122 Frasca, 1995
Palaemonetes pugio . 96h J 142.5 Champlin and Poucher, 1991
Neanthes arenaceodentata 96h 500 Rossi and Neff, 1978
Brachionus plicatus 48h L >500 This study
Palaemonetes pugio 96h A 594.6 This study
Mercenaria mercenaria 24h J 652 This study
Amphiascus tenuiremis 96h A >1600 This study
Mulinia lateralis 96h J 10710 Champlin and Poucher, 1991

‘J = juvenile, L = larvae, U = unknown, A = adult, E = embryo

Table 14. Summary of results from sediment assays with fluoranthene.

Mean
Exposure LC50/EC50 Statistical Pooled = Sensitivity 95% Cl
Species Period (mg/kg) SD Comparisons LC50 Ranking (mg/kg)
(mg/kg)
M. mercenaria 10d <08 — — <08 41 ae
V. fisheri (mutatox) —_ 3-50! — — 2 —
A. abdita 10d > 50? — >50 2 -
A. tenuiremis 10d >50 — >50 3 a
A. verrilli 10d > 50 >50 3 —
B. plicatilis 24h > 50 —_ — >50 3 —
P. pugio 10d > 50 — — >50 3 =
V. fisheri (microtox) 5 min > 50 — —— > 50 3 —

‘Assay 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 7. Comparison of the relative toxicity of test species to cadmium in sediment
exposures versus other measures of cadmium toxicity.

Significant Effects Concentration

Species _ mg/kg dry wt. Effects Code Source
TEL 0.7 MacDonald, 1994
ER-L 1.2 Long et al., 1995
Mercenaria mercenaria <2.5 A This Study
PEL 42 MacDonald, 1994
Ampelisca verrilli 48 A This Study
ER-M 9.6 Long et al., 1995
Rhepoxynius abronius 9.8 A Mearns et al, 1986
Ampelisca abdita 12.0 A This Study
Palaemonetes pugio 18.2 A This Study
Brachionus plicatilis 41.5 B This Study
Brachionus plicatilis 56.8 Cc Snell and Persoone,1989
Vibrio fischeri (Microtox) 160 D This Study
Amphiascus tenuiremis >45.0 A This Study
Ampelisca abdita 2600 A DiToro et al., 1990

A=LC850 in 10 day sediment exposure

LC50 in 24 hr exposure to sediment porewater extract

LC50 in 24 hr exposure to 30ppt seawater

D = Lowest concentration to elicit significant reduction in fluorescence compared with
controls.

Table 8. Summary of results from aqueous assays with DDT. .

Mean Pooled
Exposure LCS50/EC50 ~ Statistical LC50 Sensitivity 95% Cl
Species Period (ugiL) SD Comparisons’ (ug/L) Ranking (ug/L)
M. bahia Th 5.9 17 A 54 1 39-66
P. pugio 24h 9.5 2.3 A 8.9 4 7.5 - 10.5
A.verrilli 24h 39.8 27.6 B 38.3 2 32.4 -45.3
M. mercenaria 24h 612 135 c 615 3 358 - 1057
A. abdita 24h >10,000 _ _ _ 4 _
A.tenuiremis 24h => 10,000 —_ _ 4
B. plicatilis 24h => 10,000 — — 4
— 4

V. fisheri (microtox) 15min > 10,000 —

* Mean LC50s/EC50s for species sharing the same letter are not significantly different at « = 0.05.

17


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 Baseline 140.20 140.20 NC NC
10:22 AM Baseline 108.30 124.25 76.60 171.90
10:22 AM Baseline 154.90 134.47 91.01 177.92
10:22 AM Baseline 115.10 129.63 91.51 167.74
10:22 AM Baseline 136.40 130.98 95.10 166.86
10:22 AM Baseline 117.30 128.70 90.58 166.82
10:22 AM Cd Aqueous (defin.) 102.90 125.01 71.29 178.74 Pass
10:22 AM Cd Trial Sediment 182.30 132.18 81.61 182.74 Fail
10:22 AM Cd Trial Sediment 140.50 133.10 69.36 196.84 Pass
10:22 AM DDT Aq. Rangefinder 200.00 139.79 77.72 201.86 Fail
10:22 AM DDT Aq. Rangefinder 163.00 141.90 82.25 201.55 Pass
10:22 AM DDT Aq. Rangefinder 154.90 142.98 85.86 200.11 Pass
10:22 AM DDT Aqueous (defin.) 145.30 143.16 86.47 199.85 Pass
10:22 AM Cd Sediment (defin.) 116.60 141.26 86.55 195.98 Pass
10:22 AM DDT Sediment (defin.) 147.20 141.66 88.39 194.93 Pass
10:22 AM Fluor Aqueous (defin.) 154.90 142.49 90.51 194.46 Pass
10:22 AM Fluor Sediment (defin.) 129.30 141.71 89.74 193.69 Pass

Final Average 141.71

30



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 mi of 20 um filtered
seawater. The beakers were covered with solvent-
rinsed aluminum foil through which a 1 m¢_ pipette

was inserted and placed in a Revco Environmental
Chamber with airlines attached to the 1 mé 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 ym filtered seawater),
20°C, and a 12-h light :12-h dark cycle. Clams in
each beaker were fed 5 ml of /sochrysis 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

MicrotoxTMassays were performed generally following
protocols from Microbics Corporations’ MicrotoxTM
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,, for each dilution
series at both time points using a log-linear
regression model. Five replicate assays were
performed for each contaminant.

For sediment MicrotoxTM assays, sediments were
collected and spiked with the model contaminants

12

(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 EC,, for
each spiked sediment sample. A total of three
replicate assays were performed for each of the
sediment spikes.

Mutatox Bioassay

The MutatoxTM genotoxicity bioassay was performed
as described in Microbics Corporations’ MutatoxTM
manual using the same DMSO solvent extracts that
were prepared for the Microtoxorganic extract assay
(Microbics Corporation, 1992). The MutatoxTM 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’
MutatoxTM 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.



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 /sochrysis galbana.

Mercenaria mercenaria have demonstrated
sensitivity to anthropogenic contaminants. Calabrese
etal. (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 bea |
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 %o. 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 TestTM) that utilizes
a sublethal toxic response in M. bahia. The IQ testis
based on the ability of a healthy, unstressed
organism to ingest and metabolize a fluorigenic
substrate, 4-methyllumbelliferyl-B-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



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 II], 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. Macinnes
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, MJ. 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.

27

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. Chesler, 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. |. A test from brackish
and marine environments with Brachionus plicatilis.
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
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Estuaries. Volume1. Biological and Physical
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- Health and Environmental Effects Research
Laboratory, Atlantic Ecology Division, Narragansett,
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Strobel, C.J, H.W. Buffum, S.J. Benyi, E.A. Petrocelli,
D.R. Reifsteck, And D.J, Kieth. 1995. Statistical
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Atlantic Ecology: Division, Narragansett, R.I.
EPA/620/R-94/026. 99p.


values for Cd and fluoranthene. 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<350 wm). 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.



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.



Abstract

CONTENTS

Chapter 1. Introduction

Chapter 2. Methods

Description of Species Tested

Microtox and Mutatox

Collection and Holding of Test Organisms
Reference Toxicant Tests

Analytical Chemistry

Aqueous Contaminant Bioassay Protocol
Sediment Bioassay Protocol

Microtox Assay Protocol

Mutatox Bioassay

Chapter 3. Results and Discussion

Analytical Chemistry
Reference Toxicant Tests

Chapter 4. Summary and Conclusions

Chapter 5. References

Appendices

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 B. plicatilis.

Results obtained from Cadmium aqueous and sediment bioassays.
Results obtained from DDT aqueous and sediment bioassays.

Results obtained from Fluoranthene aqueous and sediment bioassays. . .



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
umol/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

13

toxicant tests for the definitive assays using the other
test species ( A. abdita, B. plicatilis, M. mercenaria
and A. tenuiremis) provided LC,, 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 LC, or EC,, estimate
for that replicate series. Therefore, both the mean
LC,, (based on only the replicate series which
provided an LC,, estimate) and a pooled LC,,

‘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. fenuiremis,
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 1QTM or MicrotoxTM 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



Chapter 2

Methods

Description of Species Tested

Palaemonetes pugio

The grass shrimp, Palaemonetes pugio, is acommon
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).

Ampelisea 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). 4t 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.

Ampelisea 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.



Table 11. Comparison of the relative toxicity of test species to DDT in sediment exposures versus other

measures of DDT toxicity.

Significant Effects Concentration

Species mg/kg dry wt.

mg/g OC Effects Code

Source

ER-L

TEL

Crangon septemspinosa
ER-M 0.0461
PEL * 0.0517
Amphiiascus tenuiremis 1
Palaemonetes pugio 45
Mercenaria mercenaria 5.8
Ampelisca verrilli 83
Ampelisca abdita 8.2
Rhepoxynius abronius
Eohaustorius estuarius
Hyalella azteca
Brachionus plicatilis
Vibrio fischeri (Microtox)
Amphiascus tenuiremis
Nereis virens

0.0016
0.0028
0.0310

>10.0
>10.0
>10.0
>16.5

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

TrTOamMosagagnAIAVIAAD

A=LC850 in.96 hr sediment exposure

Li in 10 d sediment exposure

Significant decrease in.clutch size from control after 10 d exposure
LC650 in 24 hr exposure to sediment porewater extract

owest concentration to elicit significant reduction in fluorescence compared with controls.

F =LC850 in 12 d sediment exposure

values for other invertebrate species. Although little
comparable data was available for this exposure
period,.the 24h LC50 (4.4 ug/l) reported for D.
magna was similar t the values obtained for P. pugio
(9.5 ug/l) and M. bahia (5.9 yg/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 spécies. 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 8B. plicatilis porewater assay and the
MicrotoxTM 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; Long et al., 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%
ugh) 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



w

EPA

United States Office of Research and . EPA/600/R-99/085
Environmental Protection Development August 1999
Agency Washington DC 20460

Comparative Toxicity
Testing of Selected
Benthic and Epibenthic
Organisms for the
Development of Sediment
Quality Test Protocols



DDT (ug/L)
Aqueous

Toxicant:
Matrix:

Species: Palaemonetes pugio

% Mortality

Duration Replicate

Control

3.90 6.50

10.80 18.00

ie}
g

24h A
B

Cc
D
E
Mean
D

Ss!
Pooled

20
lo)
0

40

30
18

30 70
50
20
40
40

36

50

LL ONOOBNAS

OWNAMROOHA

o

95% Cl

% Mortality
1300 2200 3600

Species: Ampelisca abdita

Duration Replicate Control 800 6000 10000

24h A
B

Cc
D

E
Mean
S)

iD
Pooled

% Mortality
6.5 108

Species: Ampelisca verrilli

18.0 30.0 50.0 83.3

@

Control
410

Duration
24h A
B

Replicate

“on
| lososo

O000000005,

Mean
D

Pooled

38.3
95% Cl (32.4, 45.3)

% Mortality
Duration 0.6 1.25 2.5
24h 5 i) 0 5

48h i) 0 0 20
96h 0 10 25

Amphiascus tenuiremis
Control

Species:



artificial seawater (Forty FathomsTM) containing food
were shipped overnight in insulated containers, along
with a container of dilution water. The dilution water
was the same solution of Forty FathomsTM seawater
mix but without added food. Upon arrival at the
laboratory, mysids and their dilution water (28 + 2 %o)
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 \arvae. 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 um 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% Cl) 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 HNO,
(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% H,O, 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.



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. Aduit 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 5mm. 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
tricornutum or Chaetocerus sp. .

Ampelisea 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.

Ampbhiascus 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 under flow in clean sedi-
ments at 30 %o salinity, 21°C and 12:12 L:D photo-
period. Populations were fed twice weekly with
concentrated (>10° cells/ml) phytoplankton. In
preparation for each experiment, sediment was
removed from the copepod culture system and rinsed
with clean seawater on a 125 jum 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 zm) 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



Toxicant: Cadmium k
Matrix: Sediment (maika)

Species: Palaemonetes pugio % Mortality
Duration Replicate Control 2.5 10
10 Day A 0 20 20
B 10 10 10
Cc 0 20 0

D 20 20
20
18

E i)
Mean 6
SD

Pooled
95% Cl

Species: Ampelisca abdita % Mortality
Duration Replicate Control 2.5
10 Da A
¥ B

Cc

dD

E
Mean

SD
Pooled
95% Cl

Species: Ampelisca verrilli % Mortality

Duration Replicate Control
10 Day A 50
B

Cc

D

E
Mean

SD
Pooled
95% Cl

Species: Amphiascus tenuiremis % Mortality
Duration Replicate Control 9 18

40 Day 23 10 17
B 30 0 7
Cc 17 3 23
D 33 50 3
E 23 40 3
Mean 25 21 11
sD
Pooled
95% Cl



Criteria Branch, Washington, DC under contract No.

68-01-4646.

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in tissue, sediment, and water. USEPA, Research
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EPA. 1993. Sediment Quality Criteria for the Protect-
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EPA. 1994. Methods for Assessing the Toxicity of
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RI.

Frank, P. and Robertson. 1979. The influence of
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Hong, J.S. and D.J. Reish. 1987. Acute toxicity of
cadmium to eight species of marine amphipod and
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Environ. Contam. Toxicol. 39(5):884-888.

26

Johnson, M.W. 1979. Acute toxicity of cadmium,
copper and mercury to larval American Loster,
Homarus americanus. Bull. Environ. Contam.
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Krahn, M.M., C.A. Wigner, R.W. Pearce, L.K. Moore,
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Long, E.R. and R. Markel. 1992. An evaluation of
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Mahood, R., M. McKenzie, D. Middaugh, S. Bollar, J.
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Martin, M., K. Osborn, P. Billig and N. Glickstein.
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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
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87/017. 274p

McLeese, D. and C. Metcalfe. 1980. Toxicities of
eight organochlorine compounds in sediment and
seawater to Crangon septemspinosa. Bull.
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McLeese, D., L. Burridge and J. Van Dinter. 1982.
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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



Toxicant:

DDT (ug/L)
Matrix: Gah)

Aqueous

Species: Mercenaria mercenaria

% Mortality

Duration

Replicate Control

313 625 1250

2500

5000

10000

24h A
B

Cc
D

E
Mean
SD

Pooled
95% Cl

80
80
100
100

90
90

Species: Brachionus plicatilis

% Mortality

Duration

Replicate Control

1300 2200 3600

6000 10000

24h A
B
Cc
D
E
Mean
SD
Pooled

3
0

Species: Mysidopsis bahia

% Not Fluorescing

Duration Replicate

Control

12

Lo)
8

th A
B

Mean
sD
Pooled
95% Cl

17

50
67
100
100
83

100
83

BS
©

AAA, RMD
INO HWAHO
a

Species:

Vibrio fischeri (microtox)

- Replicate

5 min EC50 15 min EC50

A
B
Cc
D
MEAN
sD

> 10,000 > 10,000
> 20,000 > 20,000
> 27,000 > 27,000
> 50,000 > 50,000
NC NC

NC NC



References

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Table 6. Summary of results from sediment assays with cadmium.

Mean
Exposure LC50/EC50
Period (mg/kg) SD

Species

Comparisons' (mg/kg)

Pooled

Statistical LCso 95% Cl

Sensitivity
(mg/kg)

Ranking

10d <2.5 —
10d 48 0.4
10d 12 43
10d 18.2 0.2
24h 415 11.7
A. tenuiremis 10d >45 —

V. fisheri (microtox) 5 min 16021603
V. fisheri (mutatox) —

M. mercenaria
A. verrilli

A. abdita

P. pugio

B. plicatilis

4.5 2 3.9-5.1
11.8 9.9-14.2
17.9 16.2 - 19.9
41.9 35.8 - 49.1

' Mean LC50s/EC50s for species sharing the same letter are not significantly different at « = 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.

umol/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 MicrotoxTM and
MutatoxTM 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
etal., 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 IQTM 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 g/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 ug/l, 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





Table A-4. SDS reference toxicant bioassays for the Gulf of Mexico Project using Amphiascus tenuiremis.

Acceptable Test Criteria

SDS Average (-)2Stand (+) 2 Stand SDS
Test Date Comments LC50 LC50 Dev Dev Pass/Fail
10:22 AM 12.13 12.13 NC NC
10:22 AM Baseline 10.52 11.33 9.05 13.60
10:22 AM Baseline 12.36 11.67 9.66 * 13.68
10:22 AM Baseline 12.99 12.00 9.90 14.10”
10:22 AM Baseline ~ 16.86 12.97 8.26 17.69
10:22 AM Cd Aqueous 14.76 13.27 8.81 17.73 Pass
10:22 AM Cd Sediment 14.24 13.41 9.27 17.55 Pass
10:22 AM DDT (aqueous and sediment) 13.43 13.41 9.58 17.24 Pass
10:22 AM Fluor (aqueous and sediment) 13.97 13.47 9.87 17.08 Pass

Final Average 13.47

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

Test Date Comments LC50 LC50 Dev Dev Pass/Fail
6/22/95 Baseline 6.29 6.29 NC NC
6/22/95(B) Baseline 6.04 6.17 5.81 6.52
6/28/95(A) Baseline 8.27 6.87 4.43 9.30
6/28/95(B) Baseline 6.13 6.68 4.56 8.80
6/28/95(C) Baseline 7.87 6.92 4.80 9.04
8/31/95 DDT Aq. Rangefinder 8.26 7.14 4.95 9.33 Pass
10/4/95 Cd Aqueous (defin.) 5.98 6.98 4.79 9.16 Pass
2/21/96 DDT Sediment (defin.) 7.92 7.09, 4.97 9.22 Pass
3/7/96 DDT Aqueous (defin.) 6.13 6.99 4.90 9.08 Pass
3/19/96 Fluor Aqueous (defin.) 7.74 7.06 5.03 9.09 Pass
3/30/96 Fluor Sediment (defin.) 7.80 7.13 5.15 9.41 Pass
5/20/96 Cd Sediment (defin.) 743 7.16 5.26 9.05 Pass
Final Average 7.16

33 7


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 LCs Batch LC50 lower Cl upper Cl

1/18/95 baseline 304.2 323.0 226.0 420.0
10:22 AM baseline 261.8 323.0 226.0 420.0
10:22 AM Cd aqueous 303.6 323.0 226.0 420.0
10:22 AM Cd porewater (lower) 324.7 323.0 226.0 420.0
10:22 AM Cd porewater (higher) 339.1 323.0 226.0 420.0
10:22 AM DDT aqueous 278.4 323.0 226.0 420.0
10:22 AM DDT porewater 301.2 323.0 226.0 420.0
10:22 AM Fluoranthene aqueous 314.6 323.0 226.0 420.0
10:22 AM__Fluoranthene sediment__ 315.4 323.0 226.0 420.0



Toxicant: Fluoranthene (ug/L)
Matrix: Aqueous

Species: Mercenaria mercenaria % Mortality
Duration Replicate Control 104 173 288 480 800 LCgo
24h A (0) 10 10 30 “50 60 512
B 0) 20 30 40 40 50 800
Cc 0 20 30 30 50 50 620
D iY) 20 20 10 30 60 675
E 10 10 20 40 30 40 >800
Mean 652
SD -
Pooled 735
95% Cl (441, 1225)
Species: Mercenaria mercenaria % Mortality
Duration Replicate Control 104 173 288 480 800 LC.
96h A 10 70 70 100 100 100 <104
B 10 80 90 100 100 100 < 104
Cc 0) 80 100 100 100 100 < 104
D 0 60 100 100 100 100 < 104
E 10 60 100 100 100 100 <104
Mgan 6 70 92 100 100 100 < 104
Pooled < 104
Spécies: Brachionus plicatilis % Mortality
Duration Replicate Control 38.4 64.8 108 180 300 500 LCs
24h A i (0) i) (0) 4 0 0) NC
B 0 ie) 0 ie) (0) 0 i) NC
Cc 3 i) 0) 0 4 0 i) NC
D 0 i 3 0 3 ie) 0 NC
~ E 3 8 0 0 i) ie) i NC
Mean 1 2 1 0 2 0 0 NC
sD NC
Pooled NC
Species: Mysidopsis bahia % Not Fluorescing
Duration Replicate Control 104 173 288 480 800 EC50
1h A 17 17 33 50 33 33 NC
a B 17 (0) 40 40 33 33 NC
Cc 17 0 0 33 33 i) NC
D 0 0 0 0 i) oO NC
E 0 Oo (0) i) i) 0 NC
F (0) i} 0 0 0 0 NC
Mean 3 12 20 17 1 NC
sD NC
Pooled NC

49



Table 15. Comparison of the relative toxicity of test species to fluoranthene in sediment
exposures versus other measures of fluoranthene toxicity
Significant Effects Concentration

Effects
Species mg/kg dry wt. mg/g OC Code Source

TEL 0.1 MacDonald, 1994
ER-L 0.6 Long et al., 1995
Mercenaria mercenaria <0.8 This Study

EPA SQC . EPA, 1993

PEL 1.5 MacDonald, 1994
Hyalella azteca 2.3-7.4 Suedel et al., 1993
Rhepoxynius abronius 3.4-10.7 Swartz et al., 1990
ER-M 5.1 Long et al., 1995
Rhepoxinius abronius 8.7 -19.1 DeWitt et al., 1992
Ampelisca abdita 50 . This Study
Ampelisca verrilli >50.0 This Study
Palaemonetes pugio >50 This Study
Brachionus plicatilis >50.0 This Study

Vibrio fischeri (Microtox) >50 This Study
Amphiascus tenuiremis >50.0 This Study

A=LC50 in 10 day sediment exposure
ignificant mortality < 80% of control survival
C=LCS50 in 24 hr exposure to sediment porewater extract
D = Lowest concentration to elicit significant reduction in fluorescence compared with controls



Toxicant: Fluoranthene
Species: Vibrio fischeri

Microtox Solvent Extract EC,,s for Spiked Sediments

Fluoranthene
Concentration 5 min EC, 15 min EC,
[mg/kg dw] [mg dw/ml] (Sb) [mg dw/ml] ($8)

te) 0.83 0.23; 0.81
0.78 “ . 2.11
3.12 af . 3.38
12.50 . 0.18) 1.41
50.0 : (1.35, 1.34

*Significantly different from control a = 0.05

Microtox Solid Phase EC,s for Spiked Sediments

Fluoranthene
Concentration 5 min EC,
Img/kg dw] [mg dwimi] (8D)

iF

1.6,
(0.2.
(2.6,

Mutatox Results for Spiked Sediments

Direct Assay 15 min EC,
Concentration Time [mg dwiml] (SD)
[mg/kg dw] 16 20 24 14 16 20 24



Notice

The U.S. Environmental Protection Agency through its Office of Research and
Development (funded and managed the research described here under DW 13936613-
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.



Appendix C.

Results obtained from DDT aqueous and sediment bioassays.



Toxicant: ~ Cadmium (mg/kg)
Sediment

Matrix:
Species: Mercenaria mercenaria % Mortality
Duration Replicate Control 25 10 40 160 LCs
10 Day A 0 100 100 100 100 <2.5
B 0 100 100 100 98 <2.5
Cc i) 100 98 100 100 <2.5
D 0 100 100 100 100 <2.5
E i) 100 98 100 100 <2.5
Mean 0 100 99 100 99 <2.5
Pooled <2.5
Species: Brachionus plicatilis % Mortality
Duration Replicate Control 2.5 10 40 160 LCxo
10 Day A 0 0 0 77 77 27.6
B 0 0 0 50 100 40
Cc 0 0 0 52 100 39.1
D 0 3 0 21 100 60.2
E 0 3 0 48 100 40.7
Mean 0 41 0 49 95 41.5
SD 11.7
Pooled 41.9
95% Cl (35.8, 49.1)

39



specific wavelengths. TOC was measured using a
Perkin Elmer Model 2400 Series Il CHNS/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 302% 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 LC,, for each replicate based
on nominal doses, whenever at least one dose
produced mortality > 50%. 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.

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 12L:12D 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 um 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 12L:12D 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



Toxicant: Fluoranthene (mg/kg)
Matrix: Sediment

Species: Amphiascus tenuiremis % Mortality
Duration Replicate Control 0.8 3.1 12.5 50 LC.
10 Day A 0 17 17 7 0 NC
B 13 3° #417 10 13 NC
Cc 17 0 27 7 7 NC
D i) 17 20 13 i) NC
E - 20 13 3 3 NC
Mean 7 11 19 8 5 NC
SD NC
Pooled NC
95% Cl NC
Species:  Mercenaria mercenaria % Mortality
Duration Replicate Control 0.78 3.12 12.50 50.00 Lg
24h A 0 76 54 80 86 <0.78
B 0 56 78 76 94 <0.78
c 0 70 68 86 86 <0.78
D Oo 36 76 76 80 1.27
E 4 44 78 90 94 <0.78
Mean 1 56 71 82 88 -
Pooled < 0.78
95% Cl
Species: Brachionus plicatilis % Mortality
Duration Replicate Control 0.78 3.13 12.50 50.00 LC,
24h A 0 0 i) 0 3 NC
B i} 0 0 0 i} NC
Cc 0 i) i) 0 ie) NC
D 0 0 0 0 0 NC
E 0 0 0 (0) i) NC
Mean 0 to) 0 0 1 NC
SD NC
Pooled NC
95% Cl NC

51



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. of P).

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 um filtered seawater was added. The jars were
capped with Teflon-lined plastic caps through which
a 1 mt pipette was inserted into a pre-drilled hole.
The jars were placed in a Revco Environmental
Chamber with airlines attached to the 1 m0 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

1

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 285% 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 um Nitex mesh. Fifteen A.
tenuiremis adult males and 15 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 um 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 LC,, 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



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 |. 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. Box 12559
Charleston, SC 29422

DW 13936613-01-0
Project Officer

Dr. Michael A. Lewis
U.S. Environmental Protection Agency
Nationa! Health and Environmental Effects Research Laboratory
Gulf Ecology Division
4 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



Table A-3. SDS reference toxicant bioassay results for the Gulf of Mexico Project using the amphipod,
Ampelisca verrilli. All concentrations are in mg/l.

Acceptable Test Criteria
()2Stand — (+)2 Stand SDS

TestDate Comments Len Mean LCey Dev Dev Pass/Fail

1/18/95 range finder 66.5 66.5 NC NC

1/19/95 _ baseline data 65.0 65.8 63.6 67.9

1/19/95 baseline data 61.7 64.4 59.5 69.3
10:22 AM __ baseline data 58.5 62.9 55.8 70.1

AN9/95 baseline data 54.5 61.2 51.5 71.0

1/19/95 _ baseline data 58.2 60.7 51.6 69.8

212195 baseline data 58.2 60.4 51.8 68.9

2/2195 baseline data 66.0 61.1 52.2 69.9
10:22 AM baseline data 65.0 61.5 52.8 70.2
10:22 AM baseline data 42.3 59.6 44.9 74.2
10:22 AM __ baseline data 58.7 59.5 45.6 73.4

4117195 Cd aqueous 53.0 59.0 45.2 72.7 Pass
10:22 AM Cd sediment 47.9 58.1 43.5 72.7 Pass
10:22 AM Cd sediment 54.3 57.8 43.7 72.0 Pass
10:22 AM DDT aqueous 33.5 NC Fail
10:22 AM DDT aqueous 39.7 NC Fail
10:22 AM DDT aqueous 42.1 56.8 40.9 72.6 Pass
10:22 AM DDT aqueous 45.5 56.1 39.7 72.4 Pass
10:22 AM baseline data 47.9 55.6 39.3 71.9
10:22 AM baseline data 45.5 55.0 38.5 71.6
10:22 AM baseline data 45.1 54.5 37.8 71.2
10:22 AM __ baseline data 414 53.8 36.5 71.2

10/2/95 Cd sediment 47.9 53.5 36.4 70.6 Pass
10:22 AM DDT aqueous 58.7 53.8 37.0 70.6 Pass
10:22 AM DDT aqueous 45.5 53.4 36.6 70.2 Pass
10:22 AM DDT sediment 40.5 52.9 35.6 70.1 Pass
10:22 AM DDT sediment 45.5 52.6 35.4 69.7 Pass
10:22 AM Fluoranthene aqueous 45.5 52.3 35.3 69.3 Pass
10:22 AM Fluoranthene aqueous 45.5 52.1 35.1 69.0 Pass
10:22 AM Fluoranthene sediment 55.8 52.2 35.5 68.8 Pass
10:22 AM Fluoranthene sediment 55.1 52.3 35.9 68.7 Pass

Final Average 52.3

32



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 IQTM 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 LC50s 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/<0.8
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 that 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 (ug/L) SD Comparisons’ (ug/L) Ranking (ug/L)

A. abdita 96h 60.5 12 A 59 1 55-63
M. mercenaria 96h < 104 —_— _ — 41 —
A.verrilli 96h 113 B 108 2 97-119
P. pugio 96h 595 c 565 3 411-776
A.tenuiremis 96h  >1600 — —_ 4 —_
B. plicatilis 48h > 500 — _ 4
M. bahia 1h > 800 —_ _ 4
V. fisheri(microtox) 5min > 1100 —_ — — 4 ——
M. mercenaria 24h 652 120 —_ 735, 441-1225

‘Mean LC50s/EC50s for species sharing the same letter are not significantly different at a = 0.05.

20





Toxicant: DDT (mg/k:
Matrix: Soaont a)

Species: Palaemonetes pugio % Mortality

Duration Replicate Control 0.64 1.60 4.00 10.00 LCs

10 Day A 10

B 0)

Cc 0

D i)

E 0

Mean 4
$s

D
Pooled
95% Cl

RORTORRH
AAAAOONUS

2)
2
2

Species: Ampelisca abdita % Mortality
Duration Replicate Control 0.64 1.60 4.00
10 Day A 10 0
B 5 it)
Cc 10
D 5
5
7

a
8

E
Mean

LQODNZOON
AONAKwWOD

Pooled 5.
95% Cl (7.2, 10.0)

Species: Ampelisca verrilli % Mortality
Duration Replicate Control 0.64 1.60 4.00 10.00
10 Day A 10
B 10
Cc 10
D 2
E 1)
Mean 10
sD
Pooled
95% Cl

wOnaZOnZz
Bwndonod

a
bb
so
&

Species: Mercenaria mercenaria % Mortality
Duration Replicate’ Control 0.64 1.60 4.00
10 Da A
y B

plo

6 0
20 10
Cc 10 20
D 6

E 20

Mean 1

SD
Pooled
95% Cl

v
DADADD
DWWABOONDS

&

a
2



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,

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 Il 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.

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(eds). 1990. A physical and ecological characteri-
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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
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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
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Palaemonetes pugio ,in the Galveston Bay
estuarine system. Cont. Mar. Sci: 12:54



Toxicant: © Cadmium (mg/L)
Matrix: Aqueous
Species:  Mercenaria mercenaria % Mortality
Duration Replicate Control 0.2 0.3 0.5 0.9 1.5 2.45 LC.
24h A IY) Qo 20 100 100 100 100 0.37
B 10 20 10 30 80 90 100 0.63
Cc 10 10 20 50 100 100 100 0.48
D i) 10 60 80 100 100 0.32
E 0 10 50 100 100 80 100 0.32
Mean 4 10 32 72 96 92 100 0.42
sD 0.14
Pooled 0.4
95% Cl (0.35, 0.45)
Species: Brachionus plicatilis % Mortality
Duration Replicate Control 12 20 33 55 92 LCe5
24h A ie) ie] i) 3 13 73 75.6
B 3 (0) 0 3 13 70 76.9
c 0 0 0 3 13 70 76.9
D id) 0 i} 3 73 76.9
E 3 (0) 3 7 20 87 68.6
Mean 1 0 1 4 13 75 75,
sD 3.6
Pooled 74.8
95% Cl (71.3, 78.5)
Species: Mysidopsis bahia % Not Fluorescing
Duration Replicate Control 8 132 16 22.1 265 368 442 613 73.6 123 EC50
1 hr A 33 33 40 - 33. - 33 - 60 - - 49.8
B 0 33 0 - 17 - 67 - 50 - - 33.2
Cc 33 17 i) = 0 - 40 = 67 = - 44.6
D 0 - - 47 - 67 = 83 = 50 83 30.2
E ie] - -. 33 - 67 - 67 - 100 83 20.6
F 17 = - 60 - 33 - 83 — 67 83 30.4
Megan 14 28 13 33 17 56 AT 78 59 72 83 348
Pooled : : 34.2
95% Cl (26.6,43.9)
Species: Vibrio fischeri _ (Microtox)
Replicate 5 min EC50 15 min EC50
A 374.5 27.7
B 304.0 26.5
Cc 333.3 26.8
D 257.5 24.4
E 154.4 19.0
MEAN 284.7 24.9
SD 84.4 3.5
37



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 MicrotoxTM 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 |QTM 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 = 10xless sensitive than
M. mercenaria. These apparent large differences

may have been due, in part, to the limited solubility”

of DDT in water (~35 pg/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

23

affected at 10 mg/Kg dw; however, reproductive
output was depressed at DDT concentrations as low
as 1mg/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 2 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


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 N, 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 HCI
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-.m 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-ml aliquot removed
Yor SEM analysis.

Impingers contained 0.5 M NaOH to. capture HS
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 spectro-
photometer (Milton Roy Spectronic Model 601).

Astandard sulfite solution was prepared by weighing
12g of Na,S.9H,O 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-m! 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
Na,SO, 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 CH,Cl, for 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 CH,Cl, 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-mx
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



Toxicant: DDT (mg/kg)
Matrix: Sediment

Species: Brachionus plicatilis % Mortality

Duration Replicate Control 0.64 1.60 4.

24h A
5 B

Cc
D
E
Mean
Ss

D
Pooled

Species: Amphiascus tenuiremis % Mortality

Duration Replicate Control
10 Da A
¥ B

Cc
D
E
Mean
SD

Pooled
95% Cl

Clutch size in Amphiascus tenuriemis exposed to DDT in
sediments for 10 days

DDT Clutch Size + SD (eggs/female)

8.83 + 3.02

1 7.45 + 2.87

0 7.34* + 3.09
10.0 6.65* + 2.93
100.0 6.89% + 2.98

*significantly different at alpha = 0.05



Appendix D.

Results obtained from Fluoranthene aqueous and sediment bioassays.



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
12L:12D 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 IQTM 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 tem) 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
Spearman-Karber method.

10

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 um 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 212um 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