This metadata record contains the results from bioassays conducted to show the response of the common Antarctic amphipod, Paramoera walkeri to contamination from combinations of Special Antarctic Blend (SAB) diesel, Marine Gas Oil (MGO) and Intermediate Fuel Oil (IFO 180), chemically dispersed with fuel dispersants Ardrox 6120 and Slickgone NS. Fuel only water accommodated fractions (WAF), chemically enhanced water accommodated fractions (CEWAF) and dispersant only treatments were prepared following the methods in Singer et al. (2000) with adaptations from Barron and Ka’aihue (2003). WAF was made using the ratio of 1: 25 (v/v), fuel to filtered seawater (FSW) following the methods of Brown et al. (in prep). Ratios for chemically dispersed treatments were 1: 100 (v/v), fuel to FSW and 1: 20 (v/v) dispersant to fuel. Dispersant only treatments were made using ratios for CEWAF, substituting the fuel component with FSW. Mixes were made in 5 L or 10 L glass aspirator bottles using a magnetic stirrer to achieve a vortex of 20-25% in the FSW before the addition of test media. The same mixing energy was used to prepare all WAFs for enhanced reproducibility and comparability of results (Barron and Ka’aihue, 2003). Mixes were stirred in darkness to prevent bacterial growth for 42 h with an additional settling time of 6 h at 0 plus or minus 1 oC. Extended stirring times were used following the recommendations determined as part of the hydrocarbon chemistry component of this project (Kotzakoulakis, unpublished data). A dilution series of four concentrations were made from the full strength aqueous phase of each mix using serial dilution. WAF test concentrations were 100%, 50%, 20% and 10% while CEWAF concentrations were 10%, 5%, 1% and 0.1%. These concentrations were chosen in order to quantify the mortality curve and allow statistical calculation of LC50 values. To facilitate comparisons of dispersant toxicity in the presence and absence of fuel, dispersant only test concentrations reflected those of CEWAF treatments. WAF was sealed in airtight glass bottles stored at 0 plus or minus 1 oC for a maximum of 3 h before use. Fresh test solutions were prepared every four days to ensure consistent water quality and replace hydrocarbons that adsorbed or evaporated into the atmosphere. Each test concentration was represented by five replicates with five FSW control beakers, with 10 P. walkeri individuals per replicate. Only healthy and active individuals were chosen with a size range of 7.9 plus or minus 0.7 mm for adults and 2.5 plus or minus 0.2 for juveniles measured from the base of the antennae to the widest part of the dorsal curve. Larger individuals and brooding females were not used to avoid unrelated deaths related to age or reproductive state (Sagar, 1980). Beakers were filled to 200 ml and were left open to allow the natural evaporation of lighter monoaromatic hydrocarbon components that would occur during a real spill. A small square of plankton mesh was placed in each jar to provide a substratum to reduce the stress of laboratory conditions and to help to stem cannibalism. Animals were not fed during experiments to avoid hydrocarbons adsorbed onto food pellets being ingested by the amphipods, thereby introducing an additional exposure pathway. Experiments ran for a total of 12 d exposure duration. Experiments were run in cold temperature-controlled cabinets maintained at a temperature of 0 plus or minus 1 oC, fluorescent lights in the cabinets were set to a light regime of 18 h light, 6 h darkness, following the methods in Brown et al. (2017) to reflect Antarctic summer environmental conditions. Lethal and sublethal observations were made at standard ecotoxicology test times of 24 h, 48 h, 96 h, 7 d, 10 d and 12 d, with an additional observation at 8 d coinciding with one of the 4-day water changes. The health status of each individual was classified on a scale of one to four; one showing no effect up to four being mortality. Mortality was determined by a lack of movement and response to stimuli, particularly in the gills. Dead animals were removed and preserved in 80% ethanol at each observation period. Missing amphipods that may have been cannibalised were included in mortality counts as they were likely to have been moribund or already dead when eaten. In order to simulate a repeated pulse pollutant, 90 to 100% of the test solution volume of each beaker was renewed with freshly made test concentrations every four days to replenish hydrocarbons lost through evaporation and adsorption and ensure consistent water quality. Beakers were topped up to 200 ml between water changes with deionised water to maintain water quality parameters. Duplicate 25 ml aliquots of test concentrations were taken at the beginning and end of each experiment in addition to pre and post water change samples. Samples were immediately extracted with 0.7 μm of dichloromethane spiked with an internal standard of BrC20 (1-bromoeicosane) and cyclooctane. Samples were analysed using Gas Chromatography with Flame Ionisation Detection (GC-FID) and mass spectrometry (GC-MS). To determine actual exposure concentrations, four day measured TPH values were used to create a continuous exposure and evaporation profile over the 12 d test period following the methods outlined in Payne et al. (2014) and Brown et al. (2017).

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.