Toxicological and chemical screening of Antarctica sediments: Use of whole sediment toxicity tests, microtox, mutatox and semipermeable membrane devices (SPMDs)

Pergamon Pll: S0025-326X(96)00088--4

Marine Pollution Bulletin, Vol. 34, No. 3, pp. 194-202, 1997 Published by Elsevier Science Ltd Printed in Great Britain 0025-326X/97 $17.00 + 0.00

Toxicological and Chemical Screening of Antarctica Sediments: Use of Whole Sediment Toxicity Tests, Microtox, Mutatox and Semipermeable Membrane Devices (SPMDs) LAVERNE CLEVELAND*, EDWARD E. LITTLE*, J I M M I E D . PETTY*, B. THOMAS JOHNSON*, JON A. LEBO*, CARL E. ORAZIO*, JANE D I O N N E t and ALAN CROCKETT:I * USDL NBS, Midwest Science Center, 4200 New Haven Road, Columbia, MO 65201, USA tNational Science Foundation, Office of Polar Programs, Washington, DC, USA ~Idaho National Engineering Laboratory, Idaho Falls, ID, USA

Eight whole sediment samples from Antarctica (four from fully characterized, but petroleum hydrocarbons, PCBs Winter Quarters Bay and four from McMurdo Sound) and metals are among the contaminants present, were toxicologically and chemically evaluated. Also, the particularly in Winter Quarters Bay where hydrocarbon influence of ultraviolet radiation on the toxicity and concentrations in sediments are as high as 4500 pgg-1 bioavaiinbillty of contaminants associated with the (Lenihan et al., 1990; Risebrough et al., 1990). Lenihan sediment samples was assessed. The evaluations were (1993) showed that benthic invertebrate communities accomplished by use of a 10-day whole sediment test with changed dramatically along contamination gradients in Leptocheirus plumulosus, Microtox ®, Mutatox ® and Winter Quarters Bay. The contaminant problems semipermeable membrane devices (SPMDs). Winter around McMurdo Station will likely persist due to Quarters Bay sediments contained about 250ngg -1 limitations that hydrological flow patterns pose as well (dry weight) total PCBs and 20ltgg -1 total PAHs. as environmental factors that govern the degradation These sediments elicited toxicity in the Microtox test and and removal of anthropogenic pollutants from the avoidance and inhibited burrowing in the L. plumulosus environment. test. The McMurdo Sound sediment samples contained The polar ozone hole and increased incidence of only trace amounts of PCBs and no PAHs, and were less ultraviolet radiation (Smith et al., 1992) are environtoxic in both the L. plumulosus and Microtox tests mental factors that can influence the biological effects of compared to the Winter Quarters Bay sediments. The contamination in Antarctic waters. For example, recent sediments from MeMurdo Sound apparently contained research on PAHs has focused on their modification to some unidentified substance which was photolytically more toxic compounds upon exposure to ultraviolet modified to a more toxic form. The photolytic modifica- radiation. Photolytically modified PAHs have been tion of sediment-associated contaminants, coupled with shown to be more toxic than the parent compounds in the polar ozone hole and increased incidence of ultraviolet fish (Bowling et al., 1983; Oris & Giesy, 1985, 1987), radiation could significantly increase hazards to Antarctic daphnids (AUred & Giesy, 1985; Newsted & Giesy, marine life. Published by Elsevier Science Ltd 1987), algae (Cody et al., 1984; Gala & Giesy, 1992), and duckweed (Ren et al., 1994). Ren et al. (1994) Keywords: Antarctica; sediments; toxicity; SPMDs; uv demonstrated that the toxic potency of PAHs were radiation; PAHs; PCBs. correlated with their rates of photomodification as well as their photosensitization activity as parent compounds. Research on the potential of sedimentThe United States manages operations at three main associated PAHs to be photolyticaUy modified and the Antarctic facilities: McMurdo; Palmer; and South Pole resultant effects on benthic organisms is extremely stations. These facilities are occupied continuously and sparse (e.g. Ankley et al., 1994; Monson et al., 1995). provide accommodation and support for research and The present study was conducted as an initial chemical general personnel. Over the past thirty years near-shore and toxicological screen of sediment samples collected sediments around Antarctic facilities have been con- from selected sites in Antarctica. The objectives of this taminated with high levels of hydrocarbons, PCBs and study were: 1. to assess the acute toxicity and genometals (Lenihan et al., 1990; Risebrough et al., 1990). toxicity of three different phases of the Antarctic Contamination around McMurdo Station has not been sediments (whole sediments, organic solvent extracts

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Volume 34/Number 3/March 1997 and semipermeable membrane device (SPMD) dialysates); 2. to use SPMDs to evaluate the bioavailability of contaminants associated with the sediments; and 3. to determine the influence of UV radiation on the bioavailability and toxicity of contaminants associated with the sediments. The present study was conducted with temperate species under temperate conditions, thus, caution should be exercised in the application of the results of this study to Antarctic conditions and biota.

Materials and Methods Test sediments Eight 1-1 whole sediment samples were collected through the ice cover from two different sites in Antarctica by National Science Foundation support personnel. Four sample replicates were collected from McMurdo Sound at Sea Water Intake on December 3, 1993 and four sample replicates were collected from Winter Quarters Bay on December 5, 1993. The frozen samples were shipped by air to the Midwest Science Center (MSC), Columbia, Missouri where they were stored frozen (-20°C). Four liters of a control sediment, used by the US EPA to culture L. plumulosus, were collected from Eckman Slough, Walport, Oregon on February 3, 1994 by US EPA personnel and shipped to the MSC by air. The control sediment was stored at 4°C. The sediments were homogenized in the shipping containers by stirring with a glass rod for about 15 rain before they were placed in the test chambers. UV irradiation UV irradiation of the sediments was conducted for 24h before the test was started after the sediment samples (2 cm) and overlying water (800 ml) were placed in the test chambers. The samples were irradiated in a solar simulator (0.61m wide by 1.83m long) that contained ten 160-watt cool white lamps (General Electric Co., East Cleveland, OH), four 160-watt UVB313 Lamps (National Biological Corp., Twinsburg, OH), eight 160-watt UVA365 lamps (National Biological Corp., Twinsburg, OH), two 20-watt cool white lamps (Osram Sylvania, Danvers, MA), two 20-watt SF20 sun lamps (Philips Lighting, Somerset, NJ), and eight 75-watt halogen incandescent flood lamps (General Electric Co., East Cleveland, OH). The simulator was suspended over a water bath of similar dimensions and was enclosed with reflective specular aluminum. The UVB lamps were controlled by a recycling 24-h timer to operate for 5 h, simulating a midday exposure. The cool white and UVA fluorescent lamps were controlled by a second timer to operate for a 16-h period, simulating a midsummer photoperiod. Spectral frequencies and irradiance production of the solar simulator were measured with an optronics Model 752 Scanning Spectroradiometer which was calibrated with a NIST traceable standard lamp. Scans were conducted at 1 nm intervals. Test organisms Leptocheirus plumulosus, an infaunal amphipod distributed subtidally along the east coast of the

United States, were exposed to the whole sedimenl samples in a 10-day static toxicity test. Leptocheiru~ plumulosus was chosen as a surrogate species to conducl the test because it tolerates broad ranges of salinity and sediment physical characteristics (e.g. sediment particle sizes). Pre-reproductive life-stage L. plumulosus were obtained from Chesapeake Cultures, Gloucester Point, Virginia. The amphipods were shipped to the MSC overnight in plastic bags containing culture water and overlying oxygen. Upon arrival, the organisms were transferred from the bags to 1-I beakers and placed in a temperature-controlled waterbath at 18°C (shipping temperature). The amphipods were slowly acclimated to the test temperature of 25°C by increasing the waterbath temperature by 2°Cday -1, and they were acclimated from culture water to the exposure water by gradually increasing the proportion of exposure water to culture water (e.g. a 50:50 mixture of culture water:exposure water for 24h followed by a 25:75 mixture of culture water:exposure water for 24 h). The 20 ppt overlying exposure water was reconstituted by blending reverse osmosis water and Instant Ocean ® sea salts. The exposure water was vigorously aerated and aged for about three weeks before it was used for testing.

Amphipod exposure procedures In general, the 10-day acute toxicity test with L. plumulosus was conducted according to procedures described in ASTM Guide E-1367-92, 'Standard Guide for Conducting Static Sediment Toxicity Tests with Marine and Estuarine Amphipods' (ASTM, 1993). Exceptions were that the overlying water was continually aerated rather than renewed and the test temperature was 25 rather than 20°C (US EPA, 1994). Twenty acclimated amphipods about 3-5 mm long were exposed to four replicates (80 animals per treatment) of the irradiated and non-irradiated Antarctica and control sediments in a randomized block design. The amphipods were exposed in 1-1 test chambers (beakers) that contained about a 4:1 (v/v) water to sediment ratio and the depth of test and control sediment in each chamber was at least 2 cm. The test was conducted under a 16:8 (light:darkness) photoperiod that provided a light intensity of about 50 footcandles. The amphipods were not fed during the 10-day test. Temperature, aeration, photoperiod and water circulation in the waterbath were checked daily to ensure that the test system functioned properly. On 0, 6 and 10 day dissolved oxygen (DO), pH and salinity were measured (APHA, 1975) in the overlying water of test chambers containing amphipods. Total ammonia was measured (APHA, 1975) at 6 and 10 day. The pH and temperature corrections of Thurston et al. (1977) and temperature-salinity interaction of Spotte & Adams (1983) were used to calculate unionized ammonia concentrations. The burrowing behavior of the amphipods was monitored daily in each test chamber and organisms observed on the sediment surface or in the water column were counted. At the end of the 10-day test surviving 195

Marine PollutionBulletin amphipods were sieved from the test sediments with stacked 0.3, 0.5 and 1 mm mesh screens. Amphipods collected on each screen were washed into sorting trays with temperature-adjusted 20 ppt saline water, counted and recorded. Surviving amphipods from two replicates of the control and McMurdo Sound sediments were placed in exposure chambers containing control sediment to evaluate potential delayed effects of the exposure on their burrowing behavior. Surviving amphipods from the remaining two replicates of the control and McMurdo Sound sediments were exposed to increased UV radiation for 48 h in the reconstituted exposure water to evaluate potential in vivo photoenhancement of bioaccumulated substances. Unexposed amphipods from a laboratory culture were exposed to overlying water collected from the Winter Quarters Bay and control sediment treatments. These amphipods were exposed to the overlying water in exposure chambers that were unshielded or shielded from UV radiation to assess potential photoenhanced toxicity. S P M D exposure procedure The semipermeable membrane devices (SPMDs) described by Huckins et al. (1990, 1993) consist of a thin film or narrow column of lipid (usually triolein) enclosed in layflat or capillary tubing. The SPMDs are designed to mimic the bioconcentration process of many aquatic organisms in which triolein is a major neutral lipid. The transport corridors of the polymeric films are about 10 A in cross-sectional diameter, thus allowing only dissolved or bioavailable organic substances to permeate through the membrane to the enclosed triolein. SPMDs (2.54cm wide and 10.2cm long) containing a thin film of triolein were placed between 1 cm layers of each irradiated and non-irradiated sample and then the overlying water was added to the chambers. No amphipods were placed in the test chambers with the SPMDs and the overlying water in these chambers was not aerated during the 10-day test. At 10 days the SPMDs were removed from the test chambers, sealed in amber jars, and frozen (-20°C) for later analyses of the sequestered contaminants.

In vitro bioluminescent toxicity systems Two in vitro bioluminescent tests, Microtox and Mutatox, were conducted to evaluate the acute toxicity and genotoxicity of organic solvent extracts of the sediments and dialysates from SPMDs exposed to the sediments. The Microtox test was conducted on all sediment hexane extracts and SPMD dialysates (also, see chemical analysis section below) according to procedures described by Microbics (1992) and Johnson (1993) (effective concentration). Light readings were taken for each test vial before and 5 rain after addition of the sediment sample extracts and dialysates. Toxicity was reported as an ECso value. Procedures described by Ulitzur et al. (1980) and Johnson (1992a,b, 1993) were used to conduct the Mutatox assay. DNA-damaging substances were detected by measuring the ability of sediment extracts and SPMD dialysates to restore the luminescent state in the bacterial cells. Phenol and benzo(a)pyrene were used as positive controls in both 196

the Microtox and Mutatox tests. The DMSO carrier solvent for each sediment extract and SPMD dialysate was used as a negative control. A hexane-dichloro-. methane extract of a fine silt/clay-sized particle soil obtained from Florissant, MO (Ingersoll & Nelson, 1990) was used as a procedural control to compare the relative toxicity of sediment samples.

Chemical analyses Antarctic and control sediments, both non-irradiated and irradiated, were analysed for polyaromatic hydrocarbons (PAHs) and halogenated organic contaminants. Contaminant analysis consisted of sediment dehydration, extraction, contaminant enrichment and chromatographic analysis. Prior to dehydration, sample integrity was maintained by storing the sediment and associated seawater in sealed glass containers at 4°C. Initially, the sediments were poured into glass pans and placed in a fumehood until the overlying water had evaporated. Then the wet sediment was dehydrated with anhydrous sodium sulphate (4:1, wt/wt sodium sulphate to sediment). The sediment-sodium sulphate mixture was extracted with methylene chloride. After reduction of the extract volume by rotary evaporation, large biogenic components were removed from the opaque greenish-brown extracts using a gravity-flow gel permeation chromatographic column packed with 10 g of S-X3 Bio-Beads (Bio-Rad, Hercules, CA). The eluant was 100 ml of a 80:20 mixture (v/v) of hexane/methylene chloride and the appropriate fraction (27-100ml) was collected. Dissolved elemental sulphur was reduced by treating the purified extracts with acid-washed copper metal. About 10% of each sediment extract was used for chromatographic analysis and the remainder was used in the Microtox and Mutatox tests. The aliquots of extracts analysed by gas chromatography were further purified by a chromatographic clean-up using potassium silicate. The aliquots used for Microtox and Mutatox testing were prepared by adding 2 ml of DMSO to the purified extract followed by evaporation of the hexane under a gentle stream of high-purity nitrogen. Analyses of the sediment extracts were performed with a Hewlett-Packard Model 5890 gas chromatograph (Folsom, CA) equipped with 63Ni electron capture (ECD) and 9.5 ev photoionization detectors (PID). Aliquots of l p.1 were injected by an HP 7673A autosampler using the cool on-column technique. A 30 m x 0.25 mm I.D. dimethylpolysiloxane DB-1 capillary column with ECD and a 30mx0.25mm I.D. (5%phenyl) methylpolysiloxane DB-5 capillary column with PID were used for analyses. The analytical columns were preceded by 1-m deactivated fused silica retention gaps. Response factors generated from standard mixtures of EPA priority pollutant PAHs and Aroclors® were used to estimate concentrations in the 0.5ml sample extracts. Procedural recoveries were confirmed by analysis of a Missouri freshwater pond sediment spiked with ~4C-phenanthrene and 14C-2,5,2',5'-PCB: recoveries were 92% and 97%, respectively. Antarctic sediment duplicates and spikes were not available, thus

Volume 34/Number 3/March 1997 corrections for recoveries were not applied, and the reported concentrations are approximate. Contaminants sequestered by the SPMDs were recovered and analysed using procedures developed at the MSC (Lebo et al., 1992; Huckins et al., 1993; Meadows et al., 1993; Petty et al., 1993, 1995). The SPMDs were dialysed in hexane for 48h and the dialysates were purified using a 60cm × 7,8 mm Phenogel liquid chromatographic column (100 ,~, pores, 10 la particles; Phenomenex, Torrance, CA). The dialysates were further enriched with a potassium silicate adsorptive clean-up, copper-treated and analysed chromatographically as described earlier. Purified extracts were prepared for Microtox and Mutatox tests using the procedure described above. Statistical analyses Analysis of variance was used to test for significant differences in amphipod percent survival (arcsinetransformed values). Statistical analyses were performed with Statistical Analysis System (SAS Institute, 1985) computer programs. Differences between pairs of means were determined with Fisher's Least Significant Difference (LSD) test. The 5-min ECs0s were determined for each test sediment and control extract using a Microtox data reduction software package (Microbics).

Results The sediment from Winter Quarters Bay had an extremely pungent odour, consisted of black, silty particles, and was almost devoid of biota. The M c M u r d o Sound sediment consisted o f brownish, sand-sized particles, and contained several different species of invertebrates, which were removed during homogenization. The control sediment had been sieved before shipment to the MSC to remove endemic organisms and consisted of particles < 0 . 2 m m in size. Mean values for DO, salinity, p H and ammonia in the overlying water were similar to expected values during the 10-day exposure (Table 1). The solar simulator used to irradiate the sediment samples produced spectral frequencies and irradiances that paralleled natural sunlight during June, 1994 at 38.5°N latitude (Fig. 1).

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TABLE 1

Mean 4- standard deviation with sample size (n) in parentheses for dissolved oxygen, salinity, pH and total and unionized ammonia in the overlyingwater during a 10-dayexposure of Leptocheirus plumulosus to irradiated and non-irradiated sediment samples collectedfrom Winters Quarters Bay and MeMurdo Sound, Antarctica and control sediments. Treatment and sample site

Dissolved oxygen (rag t- i)

Winters Quarters Bay Non-irradiated 7.1 +0.7 (6) Irradiated 7.34-0,1 (6) McMurdo Sound Non-irradiated 6.84-0.8 (7) Irradiated 7,1 4-0,3 (6) Eckman Slough, Walport, Oregon [Control] Non-irradiated 7.74-0.2 (6) Irradiated 7.8+0.1 (6)

Salinity (ppt)

pH

Total ammonia (mg I- ~)

Unionizedammonia (mg 1- =)

22.44-0.6(6) 22.84-0.7(6)

8.0+0.1 (3) 8.14-0.06(3)

5.8 ± 1.5 (2) 5.94- 1.6 (2)

0.3 4-0.02 (2) 0.34-0.04 (2)

22.24-0.6(6) 22.8 4-0,6 (6)

8.04-0.5(3) 8.1 4-0.3 (3)

20.64-2.4(2) 23.4+3.7 (2)

1.64-0.2 (2) 1.84-0.3 (2)

21.04-0.3(6) 21.3 +0.4 (6)

7.84-0.1(3) 7.8±0.2 (3)

0.1 4-0.0 (2) 0.1 4-0.0 (2)

0.003:t:0.001 (2) 0.003 4-0.001 (2) 197

Marine Pollution Bulletin

100

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Fig. 2 Percent survival of Leptocheirusplumulosus exposed in a 10-day static acute toxicity test to non-irradiated and irradiated control sediments and sediments from MeMurdo Sound and Winter Quarters Bay, Antarctica. Vertical lines on bars are standard deviations of the mean and bars with the same letters are not significantly different (p<0.05, LSD test).

Sound and irradiated and non-irradiated control treatments (Fig. 2). Amphipods that were recovered from the control and McMurdo Sound sediments treatments and placed in 250 ml beakers containing control sediment showed no delayed effects on their ability to rebury. Also, no indications of in vivo photoenhancement of accumulated contaminants were observed. All the animals burrowed into the sediment within 30 rain and no mortality was elicited. Response of the Microtox test was consistent with that of the amphipods in the acute toxicity test. Microtox 5-min ECsos (mg equivalents of sediment, dry weight) for whole sediment extracts were 1.4 for Winter Quarters Bay sediments, 5.5 for non-irradiated McMurdo Sound sediments, 0.1 for irradiated McMurdo Sound sediments, and 50 for the control sediments (Table 2). Neither of the Antarctic sediments was genotoxic in the Mutatox test. Dialysates of the SPMD samples from the irradiated and non-irradiated Winter Quarters Bay sediment elicited similar toxicity in the Microtox test (Table 2). SPMD dialysates from the

Chemical analyses Concentrations of PCBs and PAHs in the Winter Quarters Bay sediments were higher than those in McMurdo Sound (Figs 3 and 4). The chromatographic patterns for Winter Quarters Bay and McMurdo Sound irradiated sediments were not detectably different from the non-irradiated sediments, i.e. differences in PCB or PAH profiles were not evident. Regions of the ECD chromatogram are characteristic in pattern to mixtures of Aroclors, especially beyond the region where tetrachlorinated PCB congeners generally elute. The early portion of some of the ECD chromatograms is obscured by a large sulphur peak, due to incomplete removal by the copper treatment. Three prominent groups of peaks were identified for the Winter Quarters Bay sediment in the regions of CI, C2 and C3 substituted naphthalenes, and beyond these peaks the larger PAHs contributed less than a few percent of the total PAH mass. Winter Quarters Bay sediment contained approximately 250ngg - l (dry weight) total PCBs and 20 ~tgg -1 total PAHs. The McMurdo Sound sediment contained 5 0 n g g - PCBs and less than 0.011xgg - l PAHs (approximate quantitation limit). Both PCBs and PAHs were below quantitation limits (20ngg -~ for PCBs) in the control sediments (Figs 3 and 4). Chromatographic profiles of the SPMD-sequestered PCBs and PAHs were similar to their respective sediment extracts. The chromatographic profiles for PCBs in the whole sediment extracts and the SPMD dialysates were similar; however, the SPMD dialysates contained additional lower molecular weight PCBs and other halogenated organic components. The SPMDs sequestered larger quantities of the smaller contaminants during a relatively short exposure interval (10days). Tentatively identified compounds sequestered by

TABLE 2

Toxicity Of Antarctic sediment extracts to Photobacterium phosphoreum (Microtox®).

Sample

Microtox ~ 5-min ECso

Sediment extracts Winter Quarters Bay (rag equivalent sediment) McMurdo Sound (nag equivalent sediment) Irradiated McMurdo Sound (nag equivalent sediment) Control 3 (mg equivalent sediment)

0.1 (o.05-0A1) 50 (49-51)

SPMD lipid extract Winter Quarters Bay (mg equivalent lipid) Irradiated Winter Quarters Bay (nag equivalent lipid) McMurdo Sound (rag equivalent lipid) Irradiated McMurdo Sound (nag equivalent lipid)

3.1 (2.9-3.3) 6.3 (5.0-7.7) 88.8 (28-275) 30.2 (13.9-65)

Quality control samples Procedural blank SPMD control Microtox Phenol reference toxicant (lag/mL) Mutatox Benzo(a)pyrene reference toxicant (1.0 lag/vial)

1.4 (1.2-1.6) 5.5 (2.9-10.7)

ND 4 ND 19.0 (17-21)

i Values are 5-min ECsos with +95% confidence intervals (in parentheses). 2 Minus signs indicate that sample was not mutagenic in the Mutatox test. 3 Control sediment extracts were obtained from a silt/clay-sized particle soil. 4 ND, Toxicity not detected.

198

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the SPMDs include alkylated benzenes, naphthalene and alkylated naphthalenes. Also, greater portions of the more volatile aromatics were lost during the drying of the Winter Quarters Bay sediments before extraction.

Discussion The overlying water quality was maintained well within the tolerance ranges of L. plumulosus during the 10-day test as indicated by high survivorship in the control and McMurdo Sound sediment treatments. Unionized ammonia concentrations were elevated and similar for the non-irradiated (1.6mgl -n) and irradiated ( l . 8 m g l - l ) McMurdo Sound sediment treatments; however, since marine amphipods have been shown to tolerate ammonia concentrations in excess of those measured in the present study (Kohn et al., 1994), it is unlikely that ammonia influenced amphipod responses to the McMurdo Sound sediments. However, further studies are needed to examine the potential interactive toxicity of ammonia and sediment-associated contaminants. Schlekat et al. (1992) demonstrated that L. plumulosus survives well under control conditions; is tolerant of salinities ranging from 1.0 to 33.0 ppt and sediment textures ranging from sand to mud; and is sensitive to environmental contaminants. Thus, L. plumulosus appear to be an ideal test species for assessing the toxicity of marine sediments in the laboratory.

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3 min Retention time 75 rain Fig. 4 Gas chromatographic(GC-PID) analysis of sediment extracts and SPMDs exposed to sedimentsunder laboratory conditions. The PAH standard peaks are identifiedwith letters above the peaks as follows: a=naphthalene; b=internal standard; c = acenapthylene; d = acenaphthene; e = fluorene; f= phenanthrene and anthracene, g=fluoranthene; h=pyrene; i=benzo [a]anthracene and chrysene;j = benzo[b] and [k] fluoranthene; k=benzo[a]pyrene; l=indeno[123cd]pyreneand dibenz[ah]anthracene; m = benzo[ghilperylene. Both the acute toxicity test with L. plumulosus and the Microtox test indicated that the sediments from Winter Quarters Bay are extremely toxic compared to the McMurdo Sound and control sediments. The amphipods avoided the Winter Quarters Bay sediment during the first 3 days and none survived to the end of the 10day exposure. Microtox 5-min ECso estimates for extracts of the sediment samples revealed that the Winter Quarters Bay sediment was about 6-fold more toxic than the McMurdo Sound sediment and about 50fold more toxic than the control sediment. The results of the present study were consistent with the chemical screening, which identified a complex mixture of approximately 250 ng g - 1 and 50 lag g - ~ of PCBs and PAHs, respectively, in the Winter Quarters Bay sediment. Other organic and inorganic contaminants were probably also present, but were not identified. These unidentified constituents may be of natural or anthropogenic sources and probably contributed to the toxicological responses observed in the present study. The responses of benthic organisms to anthhropogenic sources of organic enrichment are similar to those from other sources (Spies et al., 1988). Further, sedimentassociated PCBs, PAHs, pesticides and metals have 199

Marine Pollution Bulletin been shown to be toxic to freshwater (Ingersoll & Nelson, 1990; Nelson et al., 1993) and marine invertebrates (Swartz et al., 1991; Plesha et al., 1988; DeWitt et al., 1989). Caution should be exercised in extrapolation of the results of the present study to define potential effects in the Antarctic environment. This study was conducted with surrogate species under temperate conditions and results obtained under true Antarctic conditions (i.e. lower temperatures, marine geochemical conditions, and use of endemic species) could be different. The present study does identify a potential risk that should be studied further under ambient Antarctic conditions to fully delineate the ecological impacts of contaminated sediments in Antarctica. For example, the results of our amphipod and Microtox tests are supported by those of Lenihan (1995) who showed that benthic invertebrate communities changed dramatically along contamination gradients in McMurdo Sound. Also, in field and laboratory tests conducted by Lenihan et al. (1995), the response of Antarctic crustaceans to Winter Quarters Bay sediments were similar to those of the amphipods tested in our study, i.e. the crustaceans incurred high mortality (80%) and also avoided the sediments. None of the samples tested in the present study was genotoxic in the Mutatox test, although the Mutatox test has been shown to be highly effective in identifying genotoxins. Contaminants such as polycyclic aromatic hydrocarbons, complex chemical mixtures, and organic sediment extracts have been identified as genotoxins with Mutatox (Johnson, 1992a,b; Ho & Quinn, 1993). In the present study, the irradiated and nonirradiated Winter Quarters Bay sediments elicited the same toxicity in the amphipod test (i.e. no amphipods survived the 10-day exposure), and SPMD lipid extracts of irradiated and nonirradiated Winter Quarters Bay sediment elicited similar responses in the Microtox test. To characterize toxicity induced through the photomodification of a compound, e.g. a PAH, the exposure concentration of the parent compound must not exceed the effective concentration for endpoints measured in the test organisms. Thus, the lack of differences between toxic responses elicited with the irradiated and nonirradiated Winter Quarters Bay sediments suggest that the sediments may have been contaminated to the extent that the toxicity of any potentially photomodified compounds could not be distinguished from that of the parent compounds. In contrast, the irradiated McMurdo Sound sediment elicited significantly higher mortality of the amphipods and was more than 50-fold more toxic in the Microtox tests compared to the nonirradiated McMurdo Sound sediments. Apparently, some substance(s) associated with the McMurdo Sound sediment was photolytically modified to a more toxic form or became more bioavailable. Prior research indicates that enhanced toxicity of PAHs occurs during exposure to UV radiation (e.g. Ren et al., 1994). The photoactivated toxic agent, at least for PAHs, appears to be short-lived. In the present study, toxicity was enhanced by UV irradiation of sediments before the amphipods were exposed. Also, there was no 200

evidence of in vivo photoenhancement of toxicity of accumulated contaminants during 48-h UV exposures following the 10-day sediment exposure. Since the McMurdo Sound sediments contained no PAHs above the detection limit, another class of chemicals with a different mechanism of photoactivation may be responsible for the observed effects. Antarctic marine biota may be significantly impacted due to interactions of ultraviolet radiation and contaminants. The photolytic modification of sediment-associated contaminants observed in the present study, coupled with the ozone hole that occurs in Antarctica with its concomitant increase in ultraviolet radiation (Smith et al., 1992) may significantly increase hazards to Antarctic marine life, particularly in nearshore areas. Although marine sediments in Antarctica are shielded from solar radiation by ice cover for long periods during the year, results of the present study indicate that photolytic modification of sedimentassociated contaminants can occur in a relatively brief period (< 24h). This interaction between contaminants and solar radiation can occur at the water surface, within the water column, and at the surface of sediments. Ultraviolet radiation alone can induce injury to marine organisms (Damkaer et al., 1980) that compound the stresses posed by environmental contaminants. Further, ultraviolet radiation can generate secondary impacts to aquatic life through the generation of toxic peroxides and free radicals in seawater (Zepp & Baughman, 1978). Also, the photomodification of sediment-associated contaminants to more toxic forms would pose a threat to aquatic biota in temperate climates where contaminated sediments in shallow coastal and inland waters may be exposed to solar radiation for extended periods annually. The analytical results of the present study corroborate, in part, the findings of Kennicutt et al. (1995) who analysed samples of sediment and biota from Arthur Harbor and McMurdo Sound, Antarctica. Their study revealed that sediment samples from Winter Quarters Bay contained total PAH concentrations that ranged from 0.36 to 13ggg - l (dry weight), which predominantly consisted of naphthalenes and phenanthrenes with lower concentrations of pyrogenic PAHs (e.g. pyrene, fluoranthene, chrysene). Sediment samples from remote stations in McMurdo Sound contained trace concentrations of PAHs near their method's detection limit of about 150-200 ng g - ~. Concentrations of PCBs in sediment samples from Winter Quarters Bay ranged from 0.68 to 1.2 gg g-1 and PCBs in sediment samples from remote station in McMurdo Sound were below 0.001 g g g - i (Kennicutt et al., 1995).

Conclusions The sediment samples collected from Winter Quarters Bay elicited toxic responses in the amphipod and Microtox tests. These sediments elicited avoidance and inhibited burrowing among L. plumulosus, which implies that recolonization along existing contaminant gradients in Winter Quarters Bay would be unlikely. The Winter Quarters Bay sediments contained PAHs,

Volume 34/Number 3/March 1997 P C B s a n d p r o b a b l y o t h e r c o n t a m i n a n t s s u c h as m e t a l s (e.g. see K e n n i c u t t e t al., 1995), thus, t h e t o x i c r e s p o n s e s o b s e r v e d c a n n o t b e a t t r i b u t e d to specific s e d i m e n t associated contaminants. The present study was not d e s i g n e d to assess i n t e r a c t i v e t o x i c i t y (i.e. s y n e r g i s m , a n t a g o n i s m a n d a d d i t i v i t y ) t h a t m a y b e elicited by complex contaminant mixtures. The McMurdo Sound sediments did not contain measurable quantities of PAHs and only trace amounts of PCBs; however, the McMurdo Sound sediments apparently contained some unidentified substance that w a s p h o t o l y t i c a l l y m o d i f i e d to a m o r e t o x i c f o r m . T h e photolytic modification of sediment-associated cont a m i n a n t s o b s e r v e d in t h e p r e s e n t s t u d y , c o u p l e d w i t h the o z o n e h o l e t h a t o c c u r s in A n t a r c t i c a w i t h its concomitant i n c r e a s e in u l t r a v i o l e t r a d i a t i o n m a y i n c r e a s e h a z a r d s to m a r i n e life, p a r t i c u l a r l y in n e a r s h o r e areas. A d d i t i o n a l r e s e a r c h is r e q u i r e d to fully e x p l o r e this p o s s i b i l i t y . We thank Aaron DeLonay and Ginger Gibson at the MSC for assistance in conducting the tests and Janet Lamberson, US EPA, Pacific Ecosystems Branch, Newport, OR for providing the control sediment.

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