This metadata record contains the results from bioassays conducted to show the response of larval Antarctic Sterechinus neumayeri sea urchins to contamination from combinations if IFO 180 fuel and the fuel dispersant Slickgone NS. AAS project 4142. Experiments used an intermediate grade Fuel Oil (IFO 180) and an internationally approved fuel dispersant, Slickgone NS, produced by Dasic International LTD. Treatments included a physically dispersed treatment of IFO 180 only, a chemically dispersed treatment of IFO 180 treated with Slickgone NS and a Slickgone NS only treatment to determine the toxicity of the dispersant. Treatments were experimentally mixed using a magnetic stirrer to combine treatment substances and filtered seawater (FSW) in temperature-controlled cabinets at 0oC to create a Water Accommodated Fraction (WAF). WAFs were produced in 2 L and 5 L glass aspirator bottles following the methods of Singer, Aurand et al. (2000) with adaptations by Barron and Ka'aihue (2003) and Kostzakoulakis (chemistry section, project 4142) stirring for 42 h with a settling time of 6 h. WAF treatments used concentrations of 100%, 50%, 20% and 10%, CEWAF and dispersant only treatments used concentrations of 10%, 5%, 1% and 0.1%. Toxicity tests were conducted in temperature-controlled cabinets at 0 oC using uncapped, forty-millilitre glass headspace vials, each containing 15.5 ml of test solution and 0.5 ml of embryo suspension. Fertilisation methods followed standard procedures for Sterechinus neumayeri. Two tests were conducted to determine the effect of a single pollution event (test 1) compared with a recurring repeated pulse pollution event (test 2). Test 1 required no water changes, while test 2 required renewal of the test treatments on a 4-day basis. Three endpoints were used, un-hatched blastula (48 h to 48.5 h) to represent the embryonic phase, gastrula (10 d) and 4-armed pluteus (16 d to 18 d). At the termination of each endpoint, 1 ml of 10% buffered formalin was added to each relevant vial and recapped. At the conclusion of the experiments, preserved embryos were observed under a dissecting microscope to determine the number of normal, abnormal and unfertilised embryos relative to controls. Samples for analysis of total petroleum hydrocarbon content were taken throughout the 2 experiments to determine the actual concentrations to which embryos and larvae were exposed. The measured concentrations were integrated following the methods of Payne et al. (2014) to obtain a profile of hydrocarbon content over each test period. Two spreadsheets are included in this metadata record detailing survival data and results of hydrocarbon analysis. The survival data file includes test condition details on the first tab, with data for tests 1 and 2 on the second and third tabs. Test treatment and concentration are listed on the left of each data block and count categories are defined in the top left panel. Development stage, date preserved and age of organism is defined for each data block, representing the three endpoints included in the experiments: unhatched blastula, gastrula and 4-armed pluteus. The hydrocarbon analysis, TPH (total petroleum hydrocarbon) file details chemical analysis results produced by K. Kotzakoulais at Macquarie University as part of project 4142. Row terminology explanations are as follows: TPH metadata Test name- indicates the tested species Exp number-indicates whether the data belongs to test 1 or test 2 Water change- details the identification of the sample in relation to the 4-day water change regime. Start samples represent the beginning of the experiment. Pre samples are taken at the end of the corresponding 4-day period, before the water is changed. 'Post' samples are taken of newly made test solutions. The chronological order of sampling is therefore: Start, pre4d, post4d, pre8d, post 8d etc. Only 'pre' samples were taken for test 1, as there were no water changes. less than C9 - greater than C28- Hydrocarbon content of samples was broken down into four compound size classes detailed for each analysis. Contamination- contamination was detected in samples, the source of contamination remains unclear, however it was established that contamination occurred during the sampling process and therefore did not come into contact with organisms. Contamination was therefore excluded from calculations. The hydrocarbon content of 0.1% dilutions was unable to be reliably analysed due to accuracy of the equipment and interfering contamination. Control data indicates spot checks to confirm the presence or absence of fuel. Very small amounts of hydrocarbons were detected as lighter fuel components evaporated and dissolved into control water within the cabinet. These very small amounts are negligible. Abnormality metadata Tab 1 details test conditions Tab 2 'Test 1' includes the data for test 1 Tab 2 'Test 2' includes the data for test 2. All observational categories are defined within the spreadsheet.

Researchers studied persistent organohalogen contaminants (POCs) in the eastern Antarctic sector. Samples were collected during January and February 2006 and originated from 12 sampling stations. They were analysed for greater than 100 organohalogen compounds including chlorinated pesticides, polychlorinated biphenyls (PCBs), polybrominated organic compounds and polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs). The suspected naturally occurring organohalogen, 2,4,6-tribromoanisole (TBA) as well as delta-HCH; o,p'- DDE; o,p'-DDD; p,p'-DDD; p,p'-DDT; penta-chlorobenzene (PeCB); HCB; heptachlor-exoepoxide; heptachlor; trans-nonachlor, mirex and toxaphene congeners Tox-26 (B8-1413), Tox-40+41 (B8-1414+ B8-1945) and Tox-50 (B8-2229) were quantified in all samples analysed whilst PCB-101, gamma-HCH, p,p'-DDE cis-nonachlor, Tox-42a (B8-806) and Tox-44 (B8-2229) were quantified in greater than or equal to 75% of samples analysed. Organochlorine pesticides dominated measured krill contaminant burdens with hexachlorobenzene (HCB) as the single most abundant compound quantified: 4.37 ng/glw (lipid weight) or 0.2 ng/gww (wet weight). HCB concentrations were comparable to those detected at this trophic level in both the Arctic and temperate northwest Atlantic, lending support to the hypothesis that HCB will approach global equilibrium at a faster rate than other POCs. Para, para'-dichlorodiphenylethene (p,p'- DDE) was detected at notable concentrations: 2.6 ng/glw 0.13 ng/gww. In contrast to the Arctic, PCBs did not feature prominently in contaminant burdens of Antarctic krill: 1.2 ng g- 1 lw and 0.05 ng/gww., dominant PCB congeners were PCB-18, PCB-28, PCB-31 and PCB- 153. The major commercial polybrominated diphenyl ether (PBDE) congeners -99 and -47 were quantified at low background levels (0.67 ng/glw , 0.03 ng/gww and 0.35 ng/glw, 0.007 ng/gww respectively) with clear concentration spikes observed at around 70 degrees E , in the vicinity of modern, active research stations. The suspected naturally occurring brominated organic compound, 2,4,6-tribromoanisole (TBA), was a ubiquitous contaminant in all samples 49 whereas the only PCDD/Fs quantifiable were trace levels of octachlorodibenzo-p-dioxin (OCDD) and 1,2,3,4,7,8/1,2,3,4,7,9-hexachlorodibenzofuran (HxCDF). This work has been incorporated in AAS project 3115 (ASAC_3115), Persistent Organic Pollutants and Emerging Contaminants of Concern; System Input From Local and Distant Contamination Sources.

This metadata record contains the results from 3 bioassays conducted with the Antarctic marine microalgae Cryothecomonas armigera (incertae sedis). These tests assessed the toxicity of copper, cadmium, lead, zinc and nickel. Test conditions for both algae are described in the excel spreadsheets. In summary, tests for P. antarctica and C.armigera, were carried out at 0 plus or minus 2 degrees C, 20:4 h light:dark (60-90 micromol/m2/s, cool white 36W/840 globes), in 80 mL natural filtered (0.22 microns) seawater (salinity - 35 ppt, pH - 8.1 plus or minus 0.2). Filtered seawater was supplemented with 1.5 mg/L NO3- and 0.15 mg/L of PO43-. All tests were carried out in silanised 250-mL glass flasks, with glass lids. Tests 1 and 2 consisted of metal treatments, with 3 replicates per treatment, alongside 3 replicate controls (natural filtered seawater). Test 3 consisted of metal treatments in an increasing series (no replicates) alongside 3 replicate controls. Seawater was spiked with metal solutions to achieve required concentration. Concentrations tested are recorded in excel datasheets as dissolved metal concentrations measured on day 0, and day 24. The average of the dissolved metal concentrations were used for further statistical analysis. The age of C.armigera at test commencement was 25-30 days. Algal cells were centrifuged and washed to remove nutrient rich media, and test flasks were inoculated with between 1-3 x10^3 cells/mL. Cell densities in all toxicity tests were determined by flow cytometry. Toxicity tests with C. armigera were carried out over 23-24 days, with cell densities determined twice a week. The growth rate (cell division; u) was calculated as the slope of the regression line from a plot of log10 (cell density) versus time (h). Growth rates for all treatments were expressed as a percentage of the control growth rates. The flow cytometer was also used to simultaneously measure changes in the following cellular parameters: chlorophyll a autofluorescence intensity (FL3), cell size (FSC) and cell complexity (SSC). The molecular stain BODIPY 493/503, was used to measure neutral lipid concentrations. Changes in cellular parameters were measured by applying a gate that captured greater than 95% of control cells in a region, R2. Changes in cellular parameters were observed in metal treatments as a shift of the cell population from the R2 region to R1 (for relative decreases) or to R3 (for relative increases). The proportion of cells in each region is expressed as a percentage of the total cell population. The pH was measured on the first and last day of the test. Sub-samples (5 mL) for analysis of dissolved metal concentrations were taken from each treatment on 24. Sub-samples were filtered through an acid washed (10% HNO3, Merck) 0.45-microns membrane filter and syringe, and acidified to 0.2% with Tracepur nitric acid (Merck). Metal concentrations were determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES) for Cu, Cd, Pb, Ni and Zn. Detection limits for Cu, Cd, Pb, Ni and Zn were 1.0, 0.3, 3.2, 1.4, and 1.0 micrograms per litre, respectively. Calculations of effect concentrations (EC 10 and 50) were made using the 'Dose Response Curve' package of R statistical analysis software. Concentration-response curves had several models applied to them, and were tested for best fit by comparing residual standard errors and Akaike's 'An Information Criterion' function . Generally, log-logistic models with 3 parameters provided the best fit. Data for each toxicity test is combined in a single excel spreadsheet, "Cryothecomonas armigera single metal toxicity". The first worksheet is titled "Test Conditions" which provides information on the toxicity test, e.g. species and metals tested, dates, test conditions, as well as explanation of abbreviations, definitions of toxicity values etc. The second worksheet includes the raw cell densities determined in each flask, the calculated growth rates, and measured metal concentrations. The third worksheet contains the measured physiological parameters: Neutral lipid concentrations (BODIPY 493/503), chlorophyll a autofluorescence (FL3), cell complexity (SSC), and cell size (FSC). The final worksheet contains the output of statistical analysis; dose-response curves for each metal with applied log-logistic model and 95% confidence interval, a table summarising the effect concentrations (EC10 and EC50), and No Effect Concentration (NEC) is also provided. The file "C. armigera combined.csv" contains rows representing individual exposures with columns for the metal treatment (Metal), averaged dissolved metal concentration for each exposure (Conc), growth rate (Growth), and growth rate as a percent of the control (Pcon). This data was used for data analysis in R statistics. Note that this contains data from all bioassays conducted with C. armigera, including those conducted by Francesca Gissi (doi:10.4225/15/551B2B65A73F3) The script used for data analysis is provided in the document "R statistics script for C. armigera single metal.docx"

This metadata record contains the results of two bioassays testing the response of Antarctic marine copepods to both individual and combined metals via 14 day toxicity tests. The tests were conducted during the 2012-2013 season at Davis Station, East Antarctica. Three metals (cadmium, copper and zinc) were tested singularly and in metal mixture combinations. The concentrations used in the two tests are outlined in the excel spreadsheet (AAS4100_12-13_MixedMetalTox.xlsx). Tests were carried out in 70 mL plastic vials (exposure vials) that contained 50 mL of test solutions. Test solutions were prepared by mixing stock solutions with filtered (0.45 microns) sea water and were stored in a constant temperature cabinet at 0 plus or minus 1 degree C for at least 2 hours prior to the start of tests in order to get to the required test temperature. Each treatment included four replicates and each test included eight controls. Within each replicate vial, 9-12 copepods were carefully added. No additional air, food or water was provided over the test period. At five days a water change was completed by removing the old test solution and replacing it with freshly prepared test solution at the same concentration. The tests were carried out in a constant temperature cabinet set at 0 plus or minus 1 degree C on a 16:8 light:dark photoperiod over 14 days. The number of surviving copepods were counted in each test container, at the same time each day, for 10 days and then a final count was completed on day 14. Mortality was determined by observing the copepods over 20 seconds and if there was no movement they were considered dead. Test solutions were sampled four times during the tests for measurement of metal concentrations. Samples were collected at day 0, day 5 pre-water change, day 5 post-water change and at day 10. Concentrations of the three test metals were determined in theses samples using Inductively-Coupled Plasma Optical Emission Spectrometer (ICP-OES) with appropriate matrix matched standards and blanks to ensure quality control. For all analyses, measured metal concentrations (as opposed to nominal concentrations) were used. Point estimates, including LC10 and LC50 values, were determined using the maximum likelihood-probit method using the software ToxCalc (version 5.0.26 Tidepool Scientific Software). Point estimates were calculated at 4, 7, 10 and 14 days of exposure. Whenever the assumptions for the maximum likelihood-probit method were not met then the Trimmed Spearman-Karber Analysis was used. Data are provided in an Excel workbook (filename: AAS4100_12-13_MixedMetalTox.xlsx). The first worksheet ("/Test Conditions") provides descriptive details for the tests and a key to abbreviations and units. Each worksheet includes a "This worksheet provides..." statement to assist interpretation of the data. A second data file is provided (filename: AAS4100_12-13_ToxCalc.xlsx) containing relevant test data from AAS4100_12-13_MixedMetalTox.xlsx, for input to ToxCalc software for analysis. This file also contains subsequent ToxCalc outputs, with key data (LC10 and LC50 values) provided in a summary worksheet. Other support files provided are seven images of the test species (images by Frances Alexander) and two figures showing copepod response to test solutions (% survival) over the exposure period of the two tests. Copepod samples were collected from the nearshore environment of Prydz Bay, offshore from Davis Station, on two days: 20 December 2012 and 9 January 2013. The 20 December collection was composed of Tisbe sp., collected from benthic habitats and the 9 January collection was composed of Paralabidocera Antarctica, collected from surface waters. Two 14-day laboratory-based toxicity tests were conducted in the Davis laboratories. The test dates were: 2 - 16 January 2013 (test 01; using Tisbe sp., collected 20 December 2012) and 10 - 24 January 2013 (test 02; using P. Antarctica, collected 9 January 2013).

This metadata record contains the results from 11 bioassays conducted with 2 species of Antarctic marine microalgae. Seven tests were conducted with Phaeocystis antarctica (Prymnesiophyceae), assessing the toxicity of copper, cadmium, lead, zinc and nickel. Four tests were conducted with Cryothecomonas armigera (Incertae sedis), assessing the toxicity of copper only. Test conditions for both algae are described in the excel spreadsheets. In summary, tests for P. antarctica and C.armigera, were carried out at 0 plus or minus 2 degrees C, 20:4 h light:dark (150-200 micro mol/m2/s, cool white 36W/840 globes), in natural filtered (0.45 microns for P.antarctica and 0.22 microns filtered for C. armigera) seawater (salinity - 35 ppt, pH - 8.1 plus or minus 0.2). For both species, filtered seawater was supplemented with 1.5 mg/L NO3- and 0.15 mg/L of PO43-. All tests were carried out in silanised 250-mL glass flasks, with glass lids. Test volumes for P.antartica and C.armigera were 50 mL and 80 mL, respectively. All tests consisted of 3-5 metal treatments, with 3 replicates per treatment, alongside 3 replicate controls (natural filtered seawater). Seawater was spiked with metal solutions to achieve required concentration. Concentrations tested are recorded in excel datasheets. The following replicate toxicity tests were completed for P. antarctica: - 5 tests with copper (1-20 micro g/L) - 4 tests with lead (10-500 micro g/L) - 3 tests with cadmium (100-2000 micro g/L) - 3 tests with zinc (100-2000 micro g/L) - 3 tests with nickel (200-1000 micro g/L) For C. armigera, 1 rangefinder test was carried out testing 6 concentrations (1-100 micro g/L), and 3 definitive test, with 5 concentrations (15-100 micro g/L). The age of P. antarctica and C.armigera at test commencement was 8-12 days, and 25-30 days, respectively. Algal cells were centrifuged and washed to remove nutrient rich media, and test flasks were inoculated with between 1-3 x103 cells/mL. Cell densities in all toxicity tests were determined by flow cytometry. The flow cytometer was also used to simultaneously measure change sin chlorophyll a fluorescence intensity, cell size and internal cell granularity. Toxicity tests were continued until cell densities in the control treatments had increased 16-fold. Toxicity tests with P. antarctica were carried out over 10 days, with cell densities in each replicate flask measured every 2 days. Toxicity tests with C. armigera were carried out over 23-24 days, with cell densities determined twice a week. The growth rate (cell division; u) was calculated as the slope of the regression line from a plot of log10 (cell density) versus time (h). Growth rates for all treatments were expressed as a percentage of the control growth rates. The pH in all treatments was measured on the first and last day of the test, as well as on day 6 for P. antarctica tests and an additional two times per week for C. armigera tests. Sub-samples (5 mL) for analysis of dissolved metal concentrations were taken from each treatment on days 0, 6 and 10 for P. antarctica tests, and on days 0, 7, 14, 21, and 24 for C. armigera tests. Sub-samples were filtered through an acid washed (10% HNO3, Merck) 0.45-micron membrane filter and syringe, and acidified to 0.2% with Tracepur nitric acid (Merck). All toxicity test results were calculated using measured dissolved metal concentrations, which were determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES) for Cu, Cd, Pb, Ni and Zn and using inductively coupled plasma-mass spectrometry (ICP-MS; Agilent 7500CE) for lowest concentration Cu samples (nominal concentration 1 micro g/L). Detection limits for Cu, Cd, Pb, Ni and Zn were 1, 0.12, 1.7, 1.2 and 0.1 micro g/L, respectively (ICP-AES) and 0.05 micro g/L (ICP-MS) for low concentration Cu samples. The specific growth rates (u) and corresponding measured metal concentrations were used to calculate toxicity test values using Toxcalc (Version 5.0.23, TidePool Scientific Software, San Francisco, CA, USA). Data were tested for normal distribution using Shapiro-Wilk's test (p greater than 0.01); and equal variances using Bartlett's test (p = 0.09). The inhibitory concentration which reduced population growth rate by x% (ICx) compared to controls was calculated using linear interpolation. The Dunnett's multiple comparison test was used to determine which treatments were significantly different to the control (2 tailed, p less than or equal to 0.05), and to calculate the no observable effect concentration (NOEC) and the lowest observable effect concentration (LOEC). Data for each toxicity test are provided in individual excel spreadsheets, identified by the species tested, the test number for that species and the date the test started. A summary table of details for the 11 tests is provided in the file: Summary table.xlsx. The first worksheet for each test file is titled "Test Conditions". This sheet provides information on the toxicity test e.g. species and metals tested, dates, test conditions, as well as explanation of abbreviations, definitions of toxicity values etc. The second worksheet includes the raw cell densities determined in each flask, the calculated growth rates, and the measured pH and metal concentrations. For C. armigera data sheets, there is an additional worksheet, "Measured Cu and pH" which includes all measured pH values and metal concentrations across the 24-day period. Following the growth rate sheets are the statistical outputs for each metal, which were all generated using Toxcalc. Finally, if additional cellular parameters were measured (Chlorophyll a fluorescence, cell size and internal cell granularity), the raw data for each parameter is include in a worksheet, "Metal cellular parameters". Data were collected in an Australian laboratory (CSIRO Land and Water, Centre for Environmental Contaminants Research, Lucas Heights, 2234, NSW) during May 2013 - April 2014. The tests used microalgal strains that had been previously collected from the Southern Ocean and are cultured within the microalgal collection at the Australian Antarctic Division (AAD). Daughter daughter cultures were transferred to CSIRO, where they were cultured for this work.

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.

Ecotoxicological tests were done at Davis and Casey Stations in 2009/10, 2010/11 and 2011/12 summer seasons under AAS Project 3054 to test the sensitivity of near-shore marine invertebrates to fuels in seawater. The three fuel types used in this project were: Special Antarctic Blend diesel (SAB), Marine Gas Oil diesel (MGO) and an intermediate grade (180) of marine bunker fuel oil (IFO). This dataset contains the results of tests with the near-shore amphipod species Paramoera walkeri exposed to WAFs of SAB, MGO and IFO 180 (specified below) conducted at Davis Station in 2009/10 summer (Season 1). Test treatments were obtained by experimentally mixing fuel and seawater in temperature control cabinets at -1°C to prepare a mixture of fuel hydrocarbons in filtered seawater (FSW) termed the water accommodated fraction (WAF). WAF was produced by adding fuel to seawater in 5 L or 10 L Pyrex glass bottles using a ratio of 1:40 fuel : FSW. This mixture was stirred at slow speed with minimal vortex on a magnetic stirrer. The water portion was then drawn from beneath the fuel. Test treatments consisted of undiluted 100% WAF and dilutions of 10% and 1% of WAFs in FSW. Toxicity tests were conducted in open glass vessels in temperature controlled cabinets. Mortality and/or sub-lethal effects were observed at endpoints of 24 h, 48 h, 96 h, 7 d, 14 d, and 21 d. Treatments were renewed at 7 d intervals. Water quality data was collected at each water change. Hydrocarbon concentrations in WAFs were determined from replicate experiments to measure THC in WAFs over time (Dataset AAS_3054_THC_WAF). WAF exposure concentrations for each test endpoint were derived from these hydrocarbon tests to account for depletion of hydrocarbons from test treatments and any renewal of treatments. An integrated concentration was calculated from measured hydrocarbon concentrations weighted to time. These integrated THC concentrations for endpoints from 24h to 21d are contained in dataset AAS_3054_THC_WAF_integrated_conc_09_10 and are the exposure concentrations used for analysis of sensitivity. Species tested; Paramoera walkeri amphipod; adults This dataset consists of Excel spreadsheets. The file name code for invertebrate tests is; Project number_Season_Taxa_Test name Eg AAS_3054_09_10_amphipod_1PWA1 Project number : AAS_3054 Season : 2009/10 season Taxa: amphipod Test name: 1 for Season 1, PW for genus and species, A for adult, 1 for Test 1 Spreadsheets contain the results of tests with this species. Where replicate tests were conducted, each experiment is on a separate spreadsheet. The worksheet labelled 'Test conditions' shows details of Test name, dates, animal collection details, laboratory holding conditions, details of water accommodated fractions (WAF), test conditions, scoring criteria and water quality data. The worksheet labelled 'Counts' has columns for Replicate number and columns with the Score for all the animals in that replicate at every time endpoint. A full description of the scoring criteria is on the 'Test conditions' worksheet. Totals, means and standard deviations are calculated for each treatment. The worksheet labelled 'Totals, means, percent, StDev' has calculations of Survival, Unaffected, including mean and standard deviation, Percent Survival and Unaffected including means and standard deviation. Amphipod tests also show the Total number of moults in each treatment. Samples were collected at the following locations: - Airport Beach, Davis, Vestfold Hills

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.

This metadata record contains the results from bioassays conducted to show the response of an Antarctic nemertean Antarctonemertes unilineata 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, Slickgone LTSW and Slickgone NS. Note that the corresponding PhD thesis chapter refers to the species as Antarctonemertes sp., prior to being named Antarctonemertes unilineata in 2018. Experiments using SAB, MGO and IFO 180 with the dispersant Ardrox 6120, including fuel only and dispersant only treatments were conducted at Casey station. Experiments involving IFO 180 and the fuel dispersants Slickgone LTSW and Slickgone NS were conducted at the Antarctic Division’s Marine Research Facility quarantine labs. All experimental procedures, including test mix preparation and bioassays were conducted at 0 plus or minus 1 degree C. Water accommodated fractions (WAF; fuel mixed in water) and chemically enhanced water accommodated fractions (CEWAF) were made according to the specifications of Singer, Aurand et al. (2000), Barron and Ka’aihue (2003) and Kotzakoulakis (unpublished at time of writing). Dispersant only mixes were also made using filtered seawater (FSW) and dispersant volumes proportional to those used for CEWAF production. WAF was made using a loading ratio of 1: 25 (v/v) fuel to FSW, CEWAF was prepared using 1:100 (v/v) fuel to FSW ratio, and 1: 20 (v/v) dispersant to fuel ratio. Following the 48 h preparation time, the seawater WAF components of the mix were drained from the bottom of aspirator bottles and serially diluted. WAF treatment concentrations were 100%, 50%, 20% and 10%, CEWAF and dispersant only concentrations were 10%, 5%, 1% and 0.1%. Treatment solutions were replenished every four days to simulate a repeated pulse exposure to contaminants and to replace hydrocarbons lost through evaporation and adsorption and to maintain water quality parameters. WAF, CEWAF and dispersant only test solutions were remade every four days using identical methods. Tests were done in temperature-controlled cabinets set to 0 plus or minus 1 degree C following a 6 h light to 18 h dark photoperiod. Beakers were left uncovered to allow for the natural evaporation of lighter hydrocarbon components to reflect real fuel spill conditions. Experiments ran for 24 d except for the Ardrox 6120 only experiment, which ran for 16 d due to high mortality in this treatment. Sublethal and lethal endpoints were assessed at 1, 2, 4, 7, 8, 12, 14, 16, 20 and 24 d observations. Aliquot water samples for analysis of total hydrocarbon content (THC) were taken for initial and final test concentrations, and before and after each four-day water change, to obtain accurate profiles of hydrocarbon loss over the test period. Duplicate samples were taken for every treatment concentration and extracted with dichloromethane, spiked with an internal standard of 1-bromoeicosane and cyclooctane. Samples were analysed using gas chromatography with flame ionization detection (GC-FID) and gas chromatography mass spectrometry (GC-MS). Average THC concentrations for the duration of the experiment were obtained by integrating the measured concentrations to which animals were exposed following the methods of Brown et al. (2016) and Payne et al. (2014). This data submission includes one file detailing the TPH experiment analyses and one detailing the bioassay tests and results. The thesis that relates to this work is available from: https://epubs.scu.edu.au/theses/533/