This dataset contains the results of replicate experiments which measured the total hydrocarbon content (THC) in water accommodated fractions (WAFs) of three fuels; Special Antarctic Blend diesel, Marine Gas oil and intermediate fuel oil IFO 180.

This dataset contains results of toxicity tests with early life stages of the sea urchin Sterechinus neumayeri as part of 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.' Dataset consists of excel spreadsheets with separate spreadsheets for each test. Test details are outlined on worksheets 'Test conditions' and results of test in worksheet 'Counts'. This metadata record contains the results of toxicity tests conducted to characterise the response of Antarctic nearshore marine invertebrates to hydrocarbon contaminants in fuels commonly used in Antarctica as part of AAS Project 3054. This dataset contains results of toxicity tests conducted at Davis Station in 2010/11 summer season to test the sensitivity of fertilisation and early life stages of the sea urchin Sterechinus neumayeri to fuels in seawater. The three fuel types used were: Special Antarctic Blend diesel (SAB), Marine Gas Oil diesel (MGO) and an intermediate grade (180) of marine bunker Fuel Oil (IFO). Test treatments were obtained by experimentally mixing fuel and seawater in temperature controlled cabinets at -1 degrees 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 Pyrex glass bottles using a ratio of 1:25 fuel : FSW. This mixture was stirred at slow speed with minimal vortex for 18 h on a magnetic stirrer then settled for 6 h before the water portion was drawn from beneath the fuel. Mature S. neumayeri were collected from the outlet of Ellis Fjord, East Antarctica (68.62°S, 77.99°E) in December and early January 2010/11. Sea urchins were collected from shallow nearshore waters less than 1m deep, placed in 20 L buckets of seawater and transported to Davis station. They were held for 1–2 d in a flow-through aquarium at -1 plus or minus 1°C, with macroalgae from the collection site as a food source, before being used for testing. Seawater for experiments was collected ~20 m from the shoreline north of Davis station (68°34’ S, 77°57’ E). Collected seawater was filtered to 0.45 µm (FSW) and stored in 30 L polyethylene containers at 0°C. Fertilisation and early embryo toxicity tests. Effects of WAFs on fertilisation and on development to the 2 cell stage were determined in static tests in which both eggs and sperm were pre-exposed to SAB, MGO and IFO 180 WAFs, fertilised within treatments and developed to the 2 cell stage (G1, G2, G3). Gamete exposure and fertilisation was done in a temperature controlled room at 0°C. Test vessels were 22 mL borosilicate glass vials with foil lined lids holding 20 mL of test solution. There were 10 vials for each treatment; 5 replicates for fertilisation and 5 replicates for the 2 cell endpoint. To pre-expose eggs, 5 mL of prepared egg solution was added to vials that contained 5 mL of 2, 20 and 100% WAFs and FSW controls, to give final treatment concentrations of 1, 10 and 50% WAF dilutions and FSW controls. Vials were sealed, swirled gently to mix and left standing for 20 min. To pre-expose sperm, pooled sperm were activated by dilution in FSW to the density required for a sperm to egg ratio of 800:1. One µL of sperm solution was added to vials containing 5 mL of FSW and gently mixed. Five mL of this solution was then added to vials containing 5 mL of 2%, 20% and 100% WAFs (final treatments of 1, 10 and 50% WAF dilutions) and FSW controls. The vials were sealed, swirled gently to mix and left for 15 mins. After the gamete exposure period was complete, for each treatment the contents of the sperm vials were added to the egg vials with a final target concentration of ~10 eggs per mL. Vials were sealed and placed into temperature-controlled cabinets set at -1 plus or minus 1°C. Temperature was recorded at 10 min intervals using a data logger (Maxim ibutton) and averaged -1.3 plus or minus 0.5°C. Tests were terminated at 4 h for the fertilisation endpoint, and at 11 h for the 2 cell endpoint by the addition of 1 mL of 2.5% (v/v) buffered glutaraldehyde. Samples were viewed in a Sedgewick Rafter counting cell under a compound microscope at 10 times magnification. Fertilisation was assessed according to the presence or absence of a fertilisation membrane in the first 100 eggs counted, to obtain the percentage of eggs fertilised in each replicate. The 2 cell endpoint was assessed in the first 100 embryos counted, as the percentage of embryos in each replicate with normal first cleavage. Embryonic and larval toxicity tests. Effects of fuel WAFs on embryonic and larval development were tested with 1, 10, and 100% WAFs of SAB, MGO and IFO 180 and FSW control, with 5 replicates per treatment. Eggs and sperm were collected and density of solutions adjusted as described above to obtain the optimal sperm to egg ratio of 800:1. Two semi-static tests (EL1, EL2) were done to test effects of WAFs on embryos and larvae when first exposed as zygotes (eggs fertilised in FSW then exposed to treatments before the first cleavage). To fertilise eggs, sperm were activated by their addition to 10 mL of FSW, and 1 µL of this sperm solution was added to beakers containing 700 mL of egg solution and gently mixed. After two hours, the mixture was stirred with a glass rod to maintain a homogeneous suspension while aliquots were transferred into 100 mL glass vials filled with 80 mL of test treatment, to a final density of ~10 zygotes per mL. Three tests (GL1, GL2, GLP) were done to test effects of WAFs on larval development with exposure commencing as gametes. One mL aliquots of egg mixture were added to vials containing 80 mL of test solution (to a density of ~10 eggs per mL) and left for 20 min. Sperm were activated in 10 mL of FSW and 0.1 mL aliquots added to the vials to fertilise eggs within treatments at a sperm to egg ratio of 800:1. Two exposure regimes were used; continuous semi-static WAF renewal (GL1 and GL2) and a single static pulse of WAF exposure up to the 4 d unhatched blastula stage, followed by post exposure recovery in FSW up to the 21 d pluteus stage (GLP). Vials were left uncovered and placed in a temperature controlled cabinet at -1 plus or minus 1°C with an 18 h light, 6 h dark photoperiod. Tests were under semi-static conditions, with test solutions renewed every 4 d. Water quality data was collected at each water change. Treatment renewals were done by removing and replacing approximately 90% of test solution. Disposable syringes with silicon tubing attached to the nozzle, and with the end of the tubing covered with plankton mesh, were used to withdraw test solution while preventing embryos/larvae from being removed. The vials were then refilled to the 80 mL mark with fresh test solutions. Treatment renewals for tests EL1, EL2 and GL1, GL2 were with freshly made WAFs every 4 d. For the single pulse WAF exposure test (GLP) on the first treatment renewal at 4 d, treatment solutions were removed as described above, and replaced with FSW. All subsequent 4 d renewals for test GLP were with FSW. To maintain the volume and salinity of test treatments a small volume of purified and deionised (Milli-Q) water at -1°C was stirred into the vials to the 80 mL mark every 2 d between water changes. Water quality measurements were made at the start of tests and pre and post treatment renewals. Mean water quality parameter measurements were pH 8.08 plus or minus 0.10, salinity 36.6 plus or minus 0.9‰ and dissolved oxygen 11.1 plus or minus 0.61 mg/L. Temperature was recorded at 10 min intervals using a data logger (Maxim ibutton) and averaged -1.0 plus or minus 1.0°C. In tests where exposure commenced as zygotes, endpoints were the embryonic 4-8 cell (20 h) and unhatched blastula (48 h) stages, and the larval blastula (6–7 d) and gastrula (14–15 d) stages. In tests with exposure commencing as gametes, endpoints were the larval blastula, gastrula and early 4-arm pluteus (21–24 d) stages. At each endpoint a sample was taken from each replicate by drawing an aliquot with a glass pipette and transferring it to a vial, to which 1 mL of 2.5% (v/v) buffered glutaraldehyde was added. Embryo and larvae were viewed in a Sedgewick Rafter counting cell under a compound microscope at 10 times magnification. The first 30 individuals in each sample at the 4-8 cell and unhatched blastula endpoints, and the first 100 individuals at the blastula, gastrula and pluteus endpoints, were assessed for normality. Test EL1 ended at the blastula stage and tests EL2 and GL2 at the gastrula stage as there were insufficient numbers of larvae remaining to continue the test beyond these stages. All remaining larvae were counted at the final endpoint. Chemical analysis of water accommodated fractions Total hydrocarbon content (THC) in WAFs were derived from replicate tests conducted under the same conditions but without test organisms. In these tests at 0°C, the concentrations of freshly made WAFs of each of the three fuels, and the depletion of hydrocarbons from 100%, 50%, 10% and 1% WAFs at multiple time points over 7 d were measured. Extracts were analysed for THC with GC-FID. Total hydrocarbon content was reported as the sum of hydrocarbons (µg/L) in the range less than n-C9 to C28 (Dataset AAS_3054_THC_WAF). For fertilisation, and 2 cell embryonic development assays that were done in sealed vials, measured values in freshly decanted 50% and 10% WAF dilutions were used as the exposure concentrations. For the embryonic and larval toxicity tests that were done in open vials, the exposure concentrations of THC in WAFs were modelled from the measured concentrations in WAF depletion tests. Exposure concentrations used to model sensitivity estimates were derived by calculating the time weighted mean THC between pairs of successive measurements in the 100% WAFs and dilutions to give overall exposure concentrations for each time point. These modelled concentrations integrated the loss of hydrocarbons over time, and renewal of test solutions at 4 d intervals.

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"

A number of toxicity tests have been conducted using the marine microgastropod, Skenella paludionoides, between the years 2006 and 2010. Tests have determined sensitivity of this species to the a range of common metals contaminants; cadmium, copper, nickel, lead and zinc. Test biota were collected from Casey and Davis, with tests conducted either at Antarctic station laboratories or in AAD Kingston laboratories (after transport of animals back to Australia). See the child records for access to the data.

Metadata record for data from ASAC Project 2677 Data on the sensitivity of Antarctic marine organisms to contaminants is limited, and is essential to understanding the risks contaminants pose to the Antarctic environment. The use of traditional toxicity assessment approaches, to collect high quality sensitivity data for a range of species, is a time consuming and difficult process, especially in remote and hostile environments like Antarctica. In this project, we used a rapid toxicity test approach (described by Kefford et al. 2005) to determine the approximate sensitivity of a large and representative sample of Antarctic marine invertebrates to three common metals (cadmium, copper, zinc). Sensitivity estimates generated via this method are likely to be less precise than those derived from traditional toxicity test methods (due to lower replication and fewer exposure concentrations), but a much larger number of estimates for a wider and more representative range of taxa are able to be produced (under equivalent resourcing). This is advantageous for subsequent Species Sensitivity Distribution (SSD) models, which will include more species and will be more robust, producing protective concentration values that represent a greater proportion of the biodiversity of the region. In this study, a total of 88 different taxa were tested during the 2005/06 Austral summer at Casey station; specimens were collected from a wide range of intertidal and shallow sub-tidal marine sites, providing good representation of the nearshore marine invertebrate community as a whole for this region. Tests were of 10 day duration, with a water change at 4 days. Sensitivity estimates were modelled (LCx; concentrations lethal to x% of the test populations) at 4 and 10 days of exposure, calculated using measured metal concentrations. A series of SSDs were constructed using LC50 values, each one including sensitivity estimates for up to 87 taxa. SSDs were constructed using the Kaplan-Meier function (results provided here) and a log-likelihood based method (available via Kefford et al submitted 2018), both of which allowed inclusion of right- and interval-censored sensitivity data. The results of this work provides a basis for estimating the risk of exposure to three common metal contaminants to Antarctic marine invertebrates. Files: Four files are attached to this record: 1. ASAC_2677-1-Supplementary-Tables.xlsx Excel file containing: 1) LC50 values for all taxa tested, for 4 and 10 d exposure durations. Both modelled and non-modelled estimates are provided. 2) Taxonomic details for all taxa tested. 3) Hazardous concentrations (HCy) to 1%, 5%, 10%, 20% and 50% of the taxa tested (HC1, HC5, HC10, HC20 and HC50, respectively) in μg/L measured on various subgroups calculated from log-normal distributions. 2. AAS_2677-2-CaseyRapidTests_Modelled LCx.xlsx Excel file containing sensitivity estimate values. See ‘FileInfo’ worksheet for description of fields. 3. AAS_2677-3-CaseyRapidTests_Figs-Kaplan-Meier.docx Word document containing Species Sensitivity Distribution model plots, generated using the Kaplan-Meier function. Data are provided for cadmium, copper and zinc based on 4 day and 10 day LC50 values for Antarctic marine invertebrates (subgroup comparisons by phyla, Arthropoda order, abundance category), generating using a rapid testing approach. LC50 values used to generate these plots are provided in the Supplementary Information of Kefford et al (submitted 2018). 4. AAS_2677-4-CaseyRapidTests_Tables-Kaplan-Meier.xlsx Excel file containing results modelled using the Kaplan-Meier function. Includes two worksheets: - Table 1: Summary statistics of 4 and 10d LC50 values (µg/L measured) estimated from Kaplan-Meier functions for the taxa tested and various sub-groups. Values in brackets are 95% confidence intervals (CI). Values and CI omitted were not calculable with the data available. See Supplementary Figures S10-S22 for plots of the Kaplan-Meier functions. - Table 2: Hypothesis testing for differences in the Kaplan-Meier functions between SSD models (constructed using LC50 sensitivity estimates) for 3 metal and 2 exposure durations (4 and 10d) on various sub-groups using Log Rank (Mantel-Cox) test. NC = not calculable with the number of species tested.

The effect of location, depth and sediment contamination on recruitment of soft-sediment assemblages were examined in a pilot experiment at Casey Station, East Antarctica. Two locations were used, a polluted bay adjacent to an old disused tip site (Brown Bay) and an undisturbed control (O'Brien Bay). At each location two types of defaunated sediment (polluted and control) were placed at 2 depths, 15 m and 25 m. Sediments were left in place over the Austral winter, from March - November. There were large differences in recruitment between the two locations and depths and some differences between the two sediment types. Brown Bay had greater recruitment than O'Brien Bay. Shallow sites had generally greater recruitment than deep, but deep sites had greater diversity (H'), richness (d) and evenness (J'). Control sediment recruited greater numbers of arthropod, gammarid and isopod taxa. There were not only differences in abundance of taxa and assemblage structure but also in spatial variability and variability of populations of certain taxa, with recruitment to the control and deep locations more variable, and recruitment in the control sediment more variable than the polluted sediment. Recruitment was influenced by a combination of location, depth and sediment type. There is some evidence of an environmental impact at the polluted site. The majority of fauna recruiting to the experiment were highly motile colonizing species with non-pelagic lecithotrophic larvae, usually brooded and released as dispersing juveniles, such as gammarids, tanaids, isopods and gastropods. A total of 56 recruitment samples were collected. Samples were sieved at 500 micro metres and sorted mainly to species. Metal concentrations and total organic carbon concentrations are also included. Also links to ASAC 1100. The fields in this dataset are: Species Location Site Treatment (tmt) Site and replicate Toxicity Arsenic Cadmium Copper Lead Silver Zinc

The effects of hyrdocarbon and heavy metal contamination of marine sediments on recruitment of soft-sediment assemblages were examined in a field experiment at Casey Station, East Antarctica. Three locations were used, a polluted bay adjacent to an old disused tip site (Brown Bay) and two control locations (O'Brien Bay and Sparkes Bay). At each location three types of defaunated sediment (hydrocarbon treated, heavy metal treated and control) were placed at approximately 15 m depth and left in place for 3 months, from December to February. Sediments were artificially contaminated with hydrocarbons and metals at concentrations which were representative of levels found in sediments at contaminated sites around Casey Station. There were large differences in recruitment between the three locations and significant differences between the control and contaminated sediment. Sediments in the experiment were also examined for evidence of degradation and attenuation of hydrocarbons and heavy metals. A total of 104 recruitment samples were collected. Samples were sieved at 500 micro m and sorted mainly to species. Other work to arise from this experiment includes examination of the effects on diatom communities and microbial communities. Data includes fauna, metals and hydrocarbon concentrations in experiment. Pre-deployment concentrations (before experiment was deployed in water) are indicated as 'pre-deployment'. Concentrations of contaminants in sediments surrounding the experiment (within several metres) are indicated as 'surrounding'. This project also links to ASAC 1100. The fields in this dataset are: Location Site Treatment (tmt) Site and replicate Species Toxicity Arsenic Cadmium Copper Lead Silver Zinc Special Antarctic Blend Fuel (SAB) Lube TPH

The effect of location and sediment contamination on recruitment of soft-sediment assemblages were examined in field experiment at Casey Station, East Antarctica. Four locations were used, a polluted bay adjacent to an old disused tip site (Brown Bay), a bay adjacent to the Casey Station sewage outfall, and two undisturbed control locations in O'Brien Bay. At each location two types of defaunated sediment (polluted and control) were placed 12 - 18 m, in experimental trays. Half of the experimental sediments were left in place over the Austral winter, from March - November, and the remaining sediments were collected after a total of one year, in February 1999. There were large differences in recruitment between the two locations and significant differences between the polluted and control sediment. There were not only differences in abundance of taxa and assemblage structure but also in spatial variability and variability of populations of certain taxa, with recruitment to the control locations more variable than polluted locations, and recruitment in the control sediment more variable than the polluted sediment. The majority of fauna recruiting to the experiment were highly motile colonizing species with non-pelagic lecithotrophic larvae, usually brooded and released as dispersing juveniles, such as gammarids, tanaids, isopods and gastropods. A total of 64 recruitment samples were collected after 9 months and 52 samples after one year. Samples were sieved at 500 micro m and sorted mainly to species. Samples are rows in data sheet. Site codes include place name (e.g. BB2) and experimental treatment (e.g. C1 - control 1). See accompanying sheet for full details of codes, including species names. Sediment chemistry data are means (and standard errors) for each treatment (averaged over 2 trays). Also links to ASAC 1100. The fields in this dataset are: Species Site Sample Abundance Toxicity Arsenic Cadmium Copper Lead Silver Zinc