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

This metadata record contains observed and predicted toxicity data from bioassays with two species of Antarctic marine microalgae: Phaeocystis antarctica (Prymnesiophyceae) and Cryothecomonas armigera (Cercoza). Bioassay exposures were of mixtures of 5 metals at two ratios, an Environmental (ENV) and Equitoxic (EC) mixture. The measured dissolved metal concentrations were used in two mixture reference models, Independent Action (IA) and Concentration Addition (CA), to predict toxicity as population growth rate inhibition. A Flow Cytometer (BD-FACSVerse) was used to measure the density of microalgae over time, which was then converted to a growth rate. An inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES), was used to measure metal concentrations. Data for each microalga is provided in individual excel spreadsheets, identified by the species tested. A word document is provided that contains the R code used to predict toxicity to the two microalgae by the reference models Independent Action and Concentration Addition. The R code also includes the steps required to extend the models to include a deviation parameter “a” that allows for departure from model additivity. A nested F-test then tests for significance between the fit of each test to observed toxicities. This R code has been adapted to use EC10 as parameter estimates, rather than EC50s. The code was adapted from the approach outlined in Hochmuth, J. D.; Asselman, J.; De, S. Are Interactive Effects of Harmful Algal Blooms and Copper Pollution a Concern for Water Quality Management? Water Res. 2014, 60, 41–53. DOI: 10.1016/j.watres.2014.03.041. Single-metal toxicity data and experimental protocols for P. antarctica from the following paper: and C. armigera used in this study can be found in the following papers: A robust bioassay to assess the toxicity of metals to the Antarctic marine microalga Phaeocyctis antarctica. Francesca Gissi, Merrin S. Adams, Catherine K. King, Dianne F. Jolley (2015). Environmental Toxicology and Chemistry. 2015 Feb 20. doi: 10.1002/etc.2949. Chronic toxicity of five metals to the polar marine microalga Cryothecomonas armigera – Application of a new bioassay. Darren J. Koppel, Francesca Gissi, Merrin S. Adams, Catherine K. King, and Dianne F. Jolley, (2017). Environmental Pollution, Volume 228, 2017, Pages 211-221, doi.org/10.1016/j.envpol.2017.05.034.

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).

Sediment cores (5cm diameter x 10cm deep), collected as part of the long-term monitoring of the Thala Valley waste disposal site clean-up (Casey station), were sectioned and a portion of each core analysed for a range of heavy metals. Metals were extracted from the sediment via a 4 hour 1M HCl acid extraction. Concentrations were gained from ICP-MS analysis of the resulting extracts (ICP-MS conducted at the School of Chemistry, University of Tasmania). Cores were collected from various control and potentially impacted sites in the Windmill Islands around Casey station. This work was conducted as part of ASAC 2201 (ASAC_2201).

This metadata record contains the results from bioassays conducted to show the response of the Antarctic gastropod, Skenella palludinoides to contamination from combinations of Special Antarctic Blend (SAB) diesel, chemically dispersed with fuel dispersant Ardrox 6120. 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. (2017). 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 approximately 20% 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 18 h with an additional settling time of 6 h at 0 plus or minus 1 oC. 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 approximately 10 S.palludinoides individuals per replicate. The healthiest and most active individuals were chosen. 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. Animals were not fed during experiments to prevent hydrocarbons being ingested, thereby introducing an additional exposure pathway. Experiments ran for a total of 35 d exposure duration for WAF and CEWAF experiments and 15 d for dispersant only experiments. Experiments were run in cold temperature-controlled cabinets set 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 test times of: 24 h, 48 h, 96 h, 7 d, 8 d, 10 d and 12 d, 14 d, 16 d, 20 d, 21 d, 28 d and 35 d for SAB + Ardrox 6120 experiments and 24 h, 48 h, 96 h, 7 d, 8 d, 10 d and 12 d, 14 d, 15 d for Ardrox 6120 only experiments. The health status of each individual was classified as per the criteria listed below: Attached to the vial with horns in or out Unattached (often upside down), horns out, will reattach if flipped over Not attached but if touched, will retract Closed but attached and out of water Operculum closed Dead, operculum open a little (muscles no longer working), if touched, operculum will not move and tissues might disintegrate Dead animals were removed and preserved in 80% ethanol at each observation period. 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

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/

This data describes the cellular metal concentrations of Phaeocystis antarctica and Cryothecomonas armigera following exposure to metals singly and in mixtures in laboratory studies. Microalgae were cultured in 80 mL of filtered (less than 0.45 um) seawater and low concentrations of nutrients supplemented with metal stocks to give a range of single and mixture exposures to the metals cadmium, copper, nickel, lead, and zinc. The cellular accumulation and partitioning are used to explain the metal's toxicity (cellular metal fractions are compared to the toxicity data provided in 10.4225/15/5ae93ff723ff8) and assess the risk bioaccumulation of metals to Antarctic marine microalgae may pose in the Southern Ocean food web.

These data sets describe the toxicity of lead, coppyer, zinc and cadmium spiked seawater to the juveniles of Abatus ingens and Abatus nimrodi. Metals were tested separately over exposure periods of 96 and 240 hours. The experimental endpoint was mortality as defined by cessation of observable movement. The coding system for the data files is J(juvenile)An (Abatus nimrodi) or Ai (Abatus ingens) - Metal name_Dates of the experiment the period of the observation (96 hour or 240 hour). This work falls under the umbrella project ASAC_2201. The fields in this dataset are: Species Toxicant Date Replicate Concentration Moving pH Salinity Dissolved Oxygen

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

This metadata record will contain the results of bioassays conducted to characterise the response of Antarctic near-shore marine zooplankton to hydrocarbon contaminants in Special Antarctic Blend (SAB) diesel fuels commonly used in Antarctica. The results from one summer season (2010-11) are in this record. This was conducted under the AAS Project 3054: Ecological risks from oil products used in Antarctica: characterising hydrocarbon behaviour and assessing toxicity on sensitive early life stages of Antarctic marine invertebrates. Exposure solutions of fuel were experimentally mixed by slow stir of fuel and seawater in temperature control cabinets at -1 degree 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 1:24 Fuel : FSW. This mixture was stirred at slow speed with minimal vortex for 18 h on a magnetic stirrer. The mixture was settled for 6 h before the water portion was drawn from beneath the fuel. Ecotoxicological bioassays were conducted at Davis Stations in the 2010/11 summer season using SAB WAF to prepare experimental treatments consisting of WAF dilution series. For each bioassay, treatments consisted of undiluted 100% WAF and dilutions of 10%, 17%, 25% and 50% of WAFs in FSW, to test the toxicity of water accommodated fractions of these three fuels on Antarctic both the zooplankton community and single copepod species. Bioassays were conducted in open vessels (glass jars or beakers) in temperature controlled cabinets. Mortality was observed at endpoints of 24 hrs, 48 hrs, 96 hrs, 7 days, 8 days, 9 days, 10 days, 11days, 12 days, 14 days, 15 days, and 16 days. New WAF solutions were prepared at the 7 day interval to replenish the experimental treatments. Deionised water was added to test solutions as required to maintain test solution volume and salinity. Water quality data was collected at each water change. Samples of test treatments for chemical analysis of hydrocarbon concentration were taken at each water change. Results of these analyses are not included as delayed progress with HC analyses impacted on quality of samples and these data were not used. This dataset consists of Excel spreadsheets. The file name code for zooplankton bioassays is; Project number_Season_Taxa_Test name Eg AAS_3054_10-11_zooplankton_m1 Project number : AAS_3054 Season : 2010/11 season Taxa: Zooplankton Community Test name: M1 =Multi-species test 1 Bioassay spreadsheets contain the results of bioassays for a species or the zooplankton community. Where replicate tests were conducted, each experiment is on a separate worksheet. The worksheet labelled "Test conditions" shows details of Test name, dates, animal collection details, laboratory holding conditions, details of water accommodated fractions (WAF), and bioassay conditions. The worksheet labelled "Counts" has a table for each of the replicates, arranged into a column for each treatment type. These tables show the number or dead individuals which were found and removed at each of the observation days. The worksheet labelled "Totals" has calculations of total number of individuals (of all species) which were found dead at each observation day in each replicate. It also gives the mean and standard deviation for each of the treatments. Further information on the zooplankton community structure in the 6 samples taken across the summer, based on the community in the toxicity tests and trials, is also included in the spreadsheet "AAS_3054_10-11_zooplankton_CommStructure". Sampling locations were near-shore from Davis Station, Vestfold Hills and from O'Gorman Rocks, southwest of Anchorage Island and northwest of Plough Island.