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An ecotoxicological risk assessment of groundwater from two Macquarie Island fuel spill sites was conducted to assess the level of risk posed by the sites to the adjacent marine receiving environment. Experiments were conducted on Macquarie Island during the summer season of 2017/18. The two fuel spill sites (known as: Fuel Farm and Power House, see file: Map-macquarie_building_and_structures_14676.pdf) within the vicinity of the Macquarie Island research station had undergone intensive in situ remediation by the Australian Antarctic Division over the previous decade. Despite remediation efforts, groundwater leaching from the sites continued to contain some residual fuel contamination, with sheen observed at several shoreline seeps and chemical analysis of groundwater samples confirmed some hydrocarbon contamination remained. This study aimed to assess the level of residual risk posed by groundwater from these sites as it enters the adjacent marine environment. We ran a series of toxicity tests using composited samples of salinity-adjusted groundwater discharge, as an exposure medium to test the sensitivity of 11 locally collected marine invertebrate species to the groundwater. Groundwater sampling was conducted over two periods: 23-29/11/17 and 18-20/12/17, for use in two rounds of toxicity testing (referred to as test round 1 (A and B) and test round 2). Groundwater samples were collected from 22 groundwater monitoring points; 12 surface seeps and 7 previously installed piezometers. These monitoring points were located along the coastal margin of the of the fuel spill sites, at their boundary with the adjacent marine environment (see: Locations-Fuel Farm-groundwater monitoring.pdf and Locations-Powerhouse-groundwater monitoring.pdf). The 22 groundwater samples were used to prepare seven salinity-adjusted composite test solutions (TS), each composed of equal volumes of up to nine groundwater samples. Salinity adjustment was to approximately that of ambient seawater (34 ppt), using hypersaline brine (prepared from locally collected clean seawater, which was frozen, then partially defrosted to collect concentrated brine). A total of approximately 6 L of was prepared for each of the seven TSs. See file: MI Ecotox-2017-18_TestSolutions_v03.xlsx for TS details (including: collection, preparation and physicochemical analysis results). Eleven locally collected marine invertebrate species were used in the tests. Biota were collected from two sites on Macquarie Island, both within the vicinity of the research station but away from areas of known fuel contamination: 1). Garden Bay on the East Coast (54° 29' 56.9" S, 158° 56' 28.8" E) and 2). Hasselborough Bay on the West Coast (54° 29' 45.6" S, 158° 55' 55.8" E). See: Map-macquarie_building_and_structures_14676.pdf. Dates of collection of test biota were 1/12/2017 (for test round 1A), 6/12/2017 (for test round 1B) and 20 and 22/12/17 (for test round 2). The 11 test taxa were from six broad taxonomic groups: 2 amphipods (Paramoera sp., Parawaldeckia kidderi), 2 flatworms (Obrimoposthia wandeli, Obrimoposthia ohlini), 2 copepods (Tigriopus angulatus, Harpacticus sp.), 2 gastropods (Laevilitorina caliginosa, Macquariella hamiltoni), 2 bivalves (Gaimardia trapesina, Lasaea hinemoa) and 1 isopod (Exosphaeroma gigas). Test biota were observed for 14 or 21 days and survival observed periodically. Full details of toxicity test conditions are provided in the file: MI Ecotox-2017-18_RawTestObs v02.xlsx (worksheets: TestSummary, Species and Endpoints). This file also contains, on subsequent worksheets, the raw toxicity test observations for each text taxa. These raw result data are compiled in the file: MI Ecotox-2017-18_Test-DATA.xlsx, worksheet: Survival-ALL contains survival data for all tests and taxa. Subsequent worksheets provide data for each test taxa separately and also include any sublethal observations that were made. All data associated with test solution collection, composition and chemistry are provided in the file: MI Ecotox-2017-18_TestSolutions.xlsx. The following (A. – I.) provides a description for the files provided with this record: A. MI Ecotox-2017-18_A-Map-Groundwater monitoring sites.png Images of study sites. A.) Overall Macquarie Island station environment, with Fuel Farm (red) and Power House (blue) indicated and showing the close proximity of the two land based sites to the adjacent high energy marine receiving environment. B.) Line map indicating relative location sites; Power House (blue) and Fuel Farm (red) sites, within the Macquarie Island station area. C.) and D.) Aerial images of the two sites, showing groundwater monitoring point locations (piezometers and seeps) used to prepare the seven test solutions (TS) as per key; Power House (TS4 and TS5) and Fuel Farm (TS1, TS2, TS3, TS6 and TS7), respectively. Monitoring point labels correspond with those provided in the file: MI Ecotox-2017-18_D-TestSolutions.xlsx / TS-Collection. B. MI Ecotox-2017-18_B-Map-macquarie_building_and_structures_14676.pdf Map of overall Macquarie Island station area, showing locations referred to in this study relative to other station infrastructure; Fuel Farm and Power House (land based fuel contaminated sites) and Hasselborough Bay and Garden Bay (clean marine areas for collection of test biota). Produced by the Australian Antarctic Data Centre, July 2018. Map available at: https://data.aad.gov.au/aadc/mapcat/. Map Catalogue No. 14676. © Commonwealth of Australia 2018. C. MI Ecotox-2017-18_C-RawTestObs.xlsx Toxicity test condition details (in worksheets named: TestSummary, Species, Endpoints) and raw toxicity test observations for each text taxa (in subsequent worksheets). D. MI Ecotox-2017-18_D-TestSolutions.xlsx Details of test solutions, including collection, composition and chemistry. E. MI Ecotox-2017-18_E-Test-DATA.xlsx Compiled raw toxicity test results in long format. Worksheet: Survival-ALL contains survival data for all tests and taxa. Subsequent worksheets provide data for each test taxa separately and includes sublethal observations if made). F. MI Ecotox-2017-18_F-ScanLabBook.pdf Scanned copy of the laboratory notebook associated with these tests. Notes were recorded by Cath King and Jessica Holan during the 17/18 Macquarie Island field season. G. MI Ecotox-2017-18_G-ScanObservationSheets.pdf Scanned copy of the handwritten raw observation sheets used to record test observations (observations scored by: Cath King and Jessica Holan). H. MI Ecotox-2017-18_H-ChemicalAnalysis-ALS-COA.pdf Certificate of Analysis for chemistry results for samples analysed by Australian Laboratory Services (ALS) Environmental, Melbourne. Includes Total Recoverable Hydrocarbons (TRH; with and without silica gel clean up), nutrients (nitrogen) and a standard toxicity test (Microtox). Client sample ID with “Ecotox TS” prefix are those relevant to this study (other samples are associated with broader site remediation monitoring for the 17/18 season). I. MI Ecotox-2017-18_I-ChemicalAnalysis-ALS-QAQC.pdf Quality Assurance (QA) and Quality Control (QC) report provided by ALS, in association with the Certificate of Analysis. As previous, Client sample ID with “Ecotox TS” prefix are relevant to this study. J. MI Ecotox-2017-18_J-size measurements.zip Measures of specimen body lengths (mm). The .zip file contains a text file named: SizeMeasurements-README.txt, providing a description of the content associated with these data.
Study location and test species Subantarctic Macquarie Island lies in the Southern Ocean, just north of the Antarctic Convergence at 54 degrees 30' S, 158 degrees 57' E. Its climate is driven by oceanic processes, resulting in highly stable daily and inter-seasonal air and sea temperatures (Pendlebury and Barnes-Keoghan, 2007). Temperatures in intertidal rock pools (0.5 to 2 m deep) were logged with Thermochron ibuttons over two consecutive summers and averaged 6.5 (plus or minus 0.5) degrees C. The island is relatively pristine and in many areas there has been no past exposure to contamination. To confirm sites used for invertebrate collections were free from metal contamination, seawater samples were taken and analysed by inductively coupled plasma optical emission spectrometry (ICP-OES; Varian 720-ES; S1) The four invertebrate species used in this study were drawn from a range of taxa and ecological niches (Figure 1). The isopod Limnoria stephenseni was collected from floating fronds of the kelp Macrosystis pyrifera, which occurs several hundred meters offshore. The copepod Harpacticus sp. and bivalve Gaimardia trapesina were collected from algal species in the high energy shallow, subtidal zone. Finally, the flatworm Obrimoposthia ohlini was collected from the undersides of boulders throughout the intertidal zone. We hypothesised L. stephenseni would be particularly sensitive to changes in salinity and temperature due to its distribution in the deeper and relatively stable subtidal areas, while O. ohlini would be less sensitive due to its distribution high in the intertidal zone and exposure to naturally variable conditions. We reasoned that the remaining two species, G. trapesina, and Harpacticus sp. were intermediate in the conditions to which they are naturally exposed and hence would likely be intermediate in their response. Test procedure The combined effect of salinity, temperature and copper on biota was determined using a multi-factorial design. A range of copper concentrations were tested with each combination of temperatures and salinities, so that there were up to 9 copper toxicity tests simultaneously conducted per species (Table 1). Experiments on L. stephenseni and Harpacticus sp. were done on Macquarie Island within 2 to 3 days of collection, during which they were acclimated to laboratory conditions. While, G. trapesina and O. ohlini were transported by ship to Australia in a recirculating aquarium system and maintained in a recirculating aquarium at the Australian Antarctic Division in Hobart, both at 6 degreesC. These two taxa were used in experiments within 3 months of their collection. A limited number of G. trapesina and O. ohlini were available, resulting in fewer combinations of stressors tested. Controls for the temperature and salinity treatments were set at ambient levels of 35 plus or minus 0.1 ppt and 5.5 to 6 degreesC for all species. The lowered control temperature for the bivalve reflected the cooler seasonal temperatures at time of testing and lower position within the intertidal. Previous tests conducted under these ambient conditions provided information on the ranges of relevant copper concentrations, appropriate test durations, and water change regimes for each taxon (Holan et al., 2017, Holan et al., 2016b). From these previous studies, we determined that a test duration of 14 d was sometimes required with 7 d often being the best outcome for most species due to high control survival and sufficient response across concentrations. The bivalve G. trapesina was an exception to this due to unfavourable water quality after 3 days in previous work (Holan et al., 2016). For the other three species, this longer duration for acute tests, compared to tests with tropical and temperature species (24 to 96 h) was consistent with previous Antarctic studies that have required longer durations in order to elicit an acute response in biota (King and Riddle, 2001, Marcus Zamora et al., 2015, Sfiligoj et al., 2015). Experimental variables (volume of water, density of test organisms, copper concentrations, temperatures and salinities) differed for each experiment due to differences between each species (Table 1). The temperature increases that were tested (2 to 4 degreesC) reflected the increased sea and air temperatures predicted for the region tested by 2100 (Collins et al., 2013). Treatments were prepared 24 h prior to the addition of animals. Seawater was filtered to 0.45 microns and water quality was measured using a TPS 90-FL multimeter at the start and end of tests. Dissolved oxygen was greater than 80% saturation and pH was 8.1 to 8.3 at the start of tests. All experimental vials and glassware were washed with 10% nitric acid and rinsed with MilliQ water three times before use. Salinity of test solutions was prepared by dilution through the addition of MilliQ water. Copper treatments using the filtered seawater at altered salinities were prepared using 500mg/L CuSO4 (Analytical grade, Univar) in MilliQ water stock solution. Samples of test solutions for metal analysis by ICP-OES were taken at the start and end of tests (on days 0 and 14). Details of ICP-OES procedures are described in the Supplemental material (S4). Samples were taken using a 0.45 µm syringe filter that had been acid and Milli-Q rinsed. Samples were then acidified with 1% diluted ultra-pure nitric acid (65% Merck Suprapur). Measured concentrations at the start of tests were within 96% of nominal concentrations. In order to determine approximate exposure concentrations for each treatment, we averaged the 0 d and 14 d measured concentrations (Table 1). Tests were conducted in temperature controlled cabinets at a light intensity of 2360 lux. At the Macquarie Island station a light-dark regime of 16:8 h was used to mimic summer conditions. In the laboratories in Kingston, Australia, a 12:12 h regime was used to simulate Autum light conditions (as appropriate for the time of testing). Test individuals were slowly acclimated to treatment temperatures over 1 to 2 h before being added to treatments. Temperatures were monitored using Thermochron ibutton data loggers within the cabinets for the duration of the tests. Determination of mortality of individuals differed for each taxon. Mortality was recorded for Gaimardia trapesina when shells were open due to dysfunctional adductor muscles; for Obrimoposthia ohlini when individuals were inactive and covered in mucous; for Limnoria stephenseni when individuals were inactive after gentle stimulation with a stream of water from a pipette; and for Harpacticus sp. when urosomes were perpendicular to prosomes (as used in other studies with copepods; see Kwok and Leung, 2005). All dead individuals were removed from test vials.
We investigated the toxicity of copper, zinc and cadmium to the following taxa: copepods Tigriopus angulatus (Lang) and Harpacticus sp. (Order Harpacticoida, Family Harpacticidae); flatworm Obrimoposthia ohlini (Bergendal) (Order Seriata, Family Procerodidae); bivalve Gaimardia trapesina (Lamarck) (Order Veneroida, Family Gaimardiidae); sea cucumber Pseudopsolus macquariensis (Dendy) (Order Dendrochirotida, Family Cucumriidae); sea star Anasterias directa (Koeler) (Order Forcipulatida, Family Asteriidae). Sites chosen for the collection of invertebrates for this study were free of obvious signs of metal contamination, as verified by the analysis of seawater samples from collection sites by inductively coupled plasma optical emission spectrometry (ICP-OES). Six invertebrate species were selected for toxicity tests to represent a range of taxa and ecological niches. Individuals of the copepod Tigriopus angulatus were collected using fine mesh dip nets from rock pools high on the intertidal zone. Individuals of the flatworm Obrimoposthia ohlini were collected from the undersides of boulders, high in the intertidal zone. The copepod Harpacticus sp. and bivalve Gaimardia trapesina were collected from several macroalgae species at high energy locations in the intertidal zone. Individuals of the sea cucumber Pseudopsolus macquariensis were collected from rocks from high energy locations from the intertidal to subtidal zones. Juveniles of the sea star Anasterias directa were collected from rocks in deep pools, low in the intertidal zone. All experimental tests using O. ohlini, T. angulatus, P. macquariensis and A. directa were conducted at the AAD Kingston laboratories, while some tests with Harpacticus sp. and all tests with G. trapesina were conducted in the laboratory facilities on Macquarie Island. Adult life-stages were tested for all species except for P. macquairensis and A. directa in which juvenile stages were tested. Psedopsolus macquariensis released eggs in the aquarium which developed into juveniles prior to being used in tests, and juvenile A. directa were collected from the field. Each test involved exposure to copper, zinc or cadmium solution under a static non-renewal test regime over 14 days. Five metal concentrations plus a control were used for each test, with 3-5 replicates of each concentration. Where possible, tests were replicated. Concentrations used in replicate tests sometimes varied, as species sensitivity information accrued in tests was used to optimise subsequent tests. Metal test solutions in seawater were prepared 24 hours prior to the addition of animals, using 500 micrograms/L CuSO4, 500 micrograms/L ZnCl2 and 500 micrograms/L Cd SO4 MilliQ stock solutions. Seawater was filtered to 0.45 microns and water quality parameters were measured using a TPS 90-FL multimeter at the start and end of tests. Dissolved oxygen (DO) was greater than 80% saturation, salinity 35 ppt plus or minus 0.5, and pH was ~8.1-8.3 at the start of tests. All experimental vials and glassware were acid washed with 10% nitric acid and rinsed with MilliQ three times before use. Metal concentrations were determined using ICP-OES; samples of test solutions were taken at the start (day 0) and end of tests (day 14), filtered through a 0.45 microns syringe filter and acidified with 1% ultra-pure nitric acid. Measured concentrations at the start of tests were within 96% of nominal concentrations. In order to estimate exposure concentrations, the measured concentrations at days 0 and 14 were averaged. Tests were conducted in lidded plastic vials of varying sizes, depending on the size and number of individuals in the test. For both copepod species, there were 10 individuals per 50 mL in 70 mL vials; for P. macquariensis there were 8 individuals per 50 mL in 70 mL vials; and for O. ohlini, A. directa and G. trapesina, 10 individuals per 100 mL in 120 mL vials. Tests were conducted under a light-dark regime (at 2360 lux) of 18:6h light:dark in summer, 12:12 for tests for the rest of the year. Tests were kept in controlled temperature cabinets set at 6 degrees C, and temperatures within cabinets were monitored throughout the test using data loggers. Vials were checked daily and survival recorded on days 1, 2, 4, 7, 10 and 14. Individuals were considered dead, and removed from test vials, when for G. trapesina adductor muscles no longer closed shell; O. ohlini were inactive and covered in mucous; P. macquariensis and A. directa tube feet were no longer moving; T. angulatus and Harpacticus sp. urosomes were perpendicular to prosomes. Data are provided in a series of excel workbooks; one workbook per test species.