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).

Study location and test species Subantarctic Macquarie Island lies in the Southern Ocean, just north of the Antarctic Convergence at 54 degrees 30' S, 158 degrees 57' E. Its climate is driven by oceanic processes, resulting in highly stable daily and inter-seasonal air and sea temperatures (Pendlebury and Barnes-Keoghan, 2007). Temperatures in intertidal rock pools (0.5 to 2 m deep) were logged with Thermochron ibuttons over two consecutive summers and averaged 6.5 (plus or minus 0.5) degrees C. The island is relatively pristine and in many areas there has been no past exposure to contamination. To confirm sites used for invertebrate collections were free from metal contamination, seawater samples were taken and analysed by inductively coupled plasma optical emission spectrometry (ICP-OES; Varian 720-ES; S1) The four invertebrate species used in this study were drawn from a range of taxa and ecological niches (Figure 1). The isopod Limnoria stephenseni was collected from floating fronds of the kelp Macrosystis pyrifera, which occurs several hundred meters offshore. The copepod Harpacticus sp. and bivalve Gaimardia trapesina were collected from algal species in the high energy shallow, subtidal zone. Finally, the flatworm Obrimoposthia ohlini was collected from the undersides of boulders throughout the intertidal zone. We hypothesised L. stephenseni would be particularly sensitive to changes in salinity and temperature due to its distribution in the deeper and relatively stable subtidal areas, while O. ohlini would be less sensitive due to its distribution high in the intertidal zone and exposure to naturally variable conditions. We reasoned that the remaining two species, G. trapesina, and Harpacticus sp. were intermediate in the conditions to which they are naturally exposed and hence would likely be intermediate in their response. Test procedure The combined effect of salinity, temperature and copper on biota was determined using a multi-factorial design. A range of copper concentrations were tested with each combination of temperatures and salinities, so that there were up to 9 copper toxicity tests simultaneously conducted per species (Table 1). Experiments on L. stephenseni and Harpacticus sp. were done on Macquarie Island within 2 to 3 days of collection, during which they were acclimated to laboratory conditions. While, G. trapesina and O. ohlini were transported by ship to Australia in a recirculating aquarium system and maintained in a recirculating aquarium at the Australian Antarctic Division in Hobart, both at 6 degreesC. These two taxa were used in experiments within 3 months of their collection. A limited number of G. trapesina and O. ohlini were available, resulting in fewer combinations of stressors tested. Controls for the temperature and salinity treatments were set at ambient levels of 35 plus or minus 0.1 ppt and 5.5 to 6 degreesC for all species. The lowered control temperature for the bivalve reflected the cooler seasonal temperatures at time of testing and lower position within the intertidal. Previous tests conducted under these ambient conditions provided information on the ranges of relevant copper concentrations, appropriate test durations, and water change regimes for each taxon (Holan et al., 2017, Holan et al., 2016b). From these previous studies, we determined that a test duration of 14 d was sometimes required with 7 d often being the best outcome for most species due to high control survival and sufficient response across concentrations. The bivalve G. trapesina was an exception to this due to unfavourable water quality after 3 days in previous work (Holan et al., 2016). For the other three species, this longer duration for acute tests, compared to tests with tropical and temperature species (24 to 96 h) was consistent with previous Antarctic studies that have required longer durations in order to elicit an acute response in biota (King and Riddle, 2001, Marcus Zamora et al., 2015, Sfiligoj et

Study location and species The four species used in this study were collected from subantarctic Macquarie Island (54.6167 degrees S, 158.8500 degrees E), just north of the Antarctic Convergence in the Southern Ocean. Sea temperatures surrounding Macquarie Island are relatively stable throughout the year, with average temperatures ranging from ~4 to 7 degrees C [25]. Collection sites were free from any obvious signs of contamination and did not have elevated concentrations of metals as confirmed by analysis of seawater samples from the collection sites by inductively coupled plasma optical emission spectrometry (ICP-OES; Varian 720-ES). Toxicity tests were conducted at Macquarie Island over the 2013/14 austral summer, and at the Australian Antarctic Division (AAD) in Tasmania, Australia, from 2013 to 2015. The aquarium at the AAD used for culturing and for holding biota prior to their use in tests was maintained at a temperature of 5.8 degrees C under flow-through conditions (at 0.49L/sec). Individuals for toxicity tests on the island and individuals for return to Australia for culturing were collected from a range of habitats within the intertidal and subtidal zones. All species were highly abundant in each of their respective habitats. The gastropod Laevilittorina caliginosa was collected from pools high on the intertidal zone; the flatworm Obrimoposthia ohlini, from the undersides of boulders from the intertidal to shallow subtidal areas; the bivalve Gaimardia trapesina, from several macroalgae species in high energy locations in the shallow subtidal; and the isopod Limnoria stephenseni, from the floating fronds of the kelp Macrocystis pyrifera, which were located several hundred meters offshore. Test specimens were acclimated to laboratory conditions 24 h to 48 h prior to commencement of tests. Juvenile flatworms, isopods and gastropods were all products of reproduction in the laboratory at the AAD, and hence their approximate age at testing is known. The flatworms hatched from small (2 mm in diameter) brown eggs, laid on rocks or on the side of aquaria. The flatworms exhibited age based morphological differences; juvenile flatworms were light grey in colour, while the adults were black. The gastropods hatched from small (1 mm in diameter) translucent eggs laid on weed, often in a cluster. For flatworms and gastropods, juveniles were not all the same age at testing due to differing hatching times, with ages ranging from 2 weeks to 3 months. In contrast, juvenile isopods were all the same age. Although brooding isopods were not observed, juveniles were noticed during routine feeding, thus were likely within 2-3 days of being released, 6 months after adults were brought from the field to the aquarium. The tests with these juvenile isopods were done within 1 week of their being observed. Care was taken to collect adults from the field, for each species, within a narrow size range to minimise differences in ages between individuals tested (Table 1). However, ages of adults individuals used in tests are unknown. The smaller size class of bivalves tested (juveniles: 2.5 plus or minus 0.5 mm, Table 1) was also collected from the field along with the adults (8.0 plus or minus 1.0 mm, Table 1). Based on knowledge on the growth rate of this species (0.8 mm per year; Everson [26], the smaller size class likely represents a young adult of approximately 2.5 to 4 y old, as opposed to a juvenile stage, and adults collected were approximately 9 to 11 y old. Toxicity tests A static non-renewal test regime was used for all tests. Two replicate tests were done for each species at each life stage, with the exception of the juvenile isopods, where due to the limited number of individuals available, only one test was done. Longer tests durations of 14 days were done for acute responses due to the longer life span and response to contaminants compared to temperate and tropical species as determined in previous studies [7, 27]. All experimental

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

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

Metadata record for data from AAS (ASAC) Project 2933. While it is generally thought that Antarctic organisms are highly sensitive to pollution, there is little data to support or disprove this. Such data is essential if realistic environmental guidelines, which take into account unique physical, biological and chemical characteristics of the Antarctic environment, are to be developed. Factors that modify bioavailability, and the effects of common contaminants on a range of Antarctic organisms from micro-algae to macro-invertebrates will be examined. Risk assessment techniques developed will provide the scientific basis for prioritising contaminated site remediation activities in marine environments, and will contribute to the development of guidelines specific to Antarctica. Brown Ostracod toxicity tests, Kingston 2007 Test animals were collected from near shore environments at Casey Station, East Antarctica during Dec 2006 - Jan 2007, and transported to culturing facilities at the Australian Antarctic Division in Tasmania, where tests were conducted during 2007. The test animals were exposed to metals in non-renewable static tests in vials containing 50 mL of the test solution at ambient Antarctic coastal salinity of 34 ppt. Tests were held in temperature controlled cabinets (incubators) at a temperature of 0, 2 or 4 degrees C (+/- approximately 1 degrees C). Ten test animals were introduced into each of 3 to 5 replicate vials per treatment at test commencement, and were exposed for 10 weeks during which periodic observations were made. Test solutions were renewed in weekly water changes. Periodic observations (time since start of test) are given in hr (hours), d (days) or w (weeks). At each observation time, test animals were scored in one of the Endpoint categories described on each worksheet. Each worksheet provides data for a particular test taxa (Brown Ostracods - taxonomy to be verified) for a given test number (T01, T02, T03, T04, T05,) and a given metal contaminant (copper, zinc, cadmium and lead). Test information is provided in the first 14 rows of each worksheet, e.g. Site of collection, Test start date, Endpoint categories etc. ASU = artificial settlement units (plastic scourers used by Sarah Richards, which had been deployed in Newcomb Bay in approximately the year 2000). Concentration micro grams per litre are nominal concentrations. Measured concentrations are provided in the file: Brown Ostracod_chemistry.xls, as described below. Test temperature was 0 degrees C unless otherwise stated. Unit for all temperature data are degrees C. See the readme file in the download for more information.

Metadata record for data from AAS (ASAC) Project 2933. While it is generally thought that Antarctic organisms are highly sensitive to pollution, there is little data to support or disprove this. Such data is essential if realistic environmental guidelines, which take into account unique physical, biological and chemical characteristics of the Antarctic environment, are to be developed. Factors that modify bioavailability, and the effects of common contaminants on a range of Antarctic organisms from micro-algae to macro-invertebrates will be examined. Risk assessment techniques developed will provide the scientific basis for prioritising contaminated site remediation activities in marine environments, and will contribute to the development of guidelines specific to Antarctica. Amphipod and Isopod toxicity tests, Kingston 2007 Filename: Amphipod and Isopod test results.xls Test animals were collected from near shore environments at Casey Station, East Antarctica during Dec 2006 - Jan 2007, and transported to culturing facilities at the Australian Antarctic Division in Tasmania, where tests were conducted during 2007. The test animals were exposed to metals in non-renewable static tests in vials containing 50 mL of the test solution at ambient Antarctic coastal salinity of 34 ppt. Tests were held in temperature controlled cabinets (incubators) at a temperature of 0 degrees C (plus or minus approximately 1 degrees C). Five to eight test animals were introduced into each of 3 replicate vials per treatment at test commencement, and were exposed for 10 to 12 weeks during which periodic observations were made. Test solutions were renewed in weekly water changes. Periodic observations (time since start of test) are given in hr (hours), d (days) or w (weeks). At each observation time, test animals were scored in one of the Endpoint categories described on each worksheet. Each worksheet provides data for a particular test taxa (slater isopods, small red isopods, spider amphipods and Orange Long Antennae Amphipods - taxonomy to be verified) for a given test number (T01, T02) and a given metal contaminant (copper, zinc, cadmium). Test information is provided in the first 14 rows of each worksheet, e.g. Site of collection, Test start date, Endpoint categories etc. ASU = artificial settlement units (plastic scourers used by Sarah Richards, which had been deployed in Newcomb Bay in approximately the year 2000). Conc micrograms/L are nominal concentrations. Measured concentrations are provided in the worksheet: /Amph and Isop T01-02 CHEMISTRY SUMM Test temperature was 0 degrees C unless otherwise stated. Unit for all temperature data is degrees C. The file contains the following worksheets: Worksheet: /Amph and Isop T01-02 CHEMISTRY SUMM Chemistry data as provided also in Kingston 07 Chemistry_Amph and Iso.xls described below. Worksheet: /Slater isopods T01 Cu Test taxa: Slater isopod; Test ID: T01, Kingston 2007; Metal contaminant: copper Worksheet: /Slater isopods T01 Zn Test taxa: Slater isopod; Test ID: T01, Kingston 2007; Metal contaminant: zinc Worksheet: /Slater isopods T01 Cd Test taxa: Slater isopod; Test ID: T01, Kingston 2007; Metal contaminant: cadmium Worksheet: /Small red isopods T02 Cu Test taxa: Small red isopods; Test ID: T02, Kingston 2007; Metal contaminant: copper Worksheet: /Small red isopods T02 Zn Test taxa: Small red isopods; Test ID: T02, Kingston 2007; Metal contaminant: zinc Worksheet: /Spider Amphipods T01 Cu Test taxa: Spider Amphipods; Test ID: T01, Kingston 2007; Metal contaminant: copper Worksheet: /Orange LongAnt Amph T01 Cu Test taxa: Orange Long Antenae Amphipods; Test ID: T01, Kingston 2007; Metal contaminant: copper Filename:Kingston 07 Chemistry_Amph and Iso.xls Metal concentrations in test solutions were analysed using an ICP-AES, by Ashley Townsend at the Central Science Laboratory, University of Tasmania, Hobart. Worksheet: /Amph and Isop T01-02 Summary Summary of chemistry data for Amphipod and Isopod

This data set describes the concentrations of copper, lead and iron in the calcareous tests of heart urchins that were exposed to spiked sediments for 60 days. Porewater is the water extracted from between sand grains. Filtered porewater has been filtered. DGT stands for Diffuse Gel Transfer. The HCl extraction listed in one of the excel spreadsheets is an extraction from the actual sediment. The fields in this dataset are: Isotope Concentration Porewater Filtered Porewater DGT Porewater

Metadata record for data from AAS (ASAC) Project 2933. While it is generally thought that Antarctic organisms are highly sensitive to pollution, there is little data to support or disprove this. Such data is essential if realistic environmental guidelines, which take into account unique physical, biological and chemical characteristics of the Antarctic environment, are to be developed. Factors that modify bioavailability, and the effects of common contaminants on a range of Antarctic organisms from micro-algae to macro-invertebrates will be examined. Risk assessment techniques developed will provide the scientific basis for prioritising contaminated site remediation activities in marine environments, and will contribute to the development of guidelines specific to Antarctica. Juvenile Gastropod toxicity tests, Kingston 2007 During 2007 a series of toxicity tests, using Antarctic marine invertebrates, were conducted at Australian Antarctic Division laboratories in Kingston, Tasmania, to test the sensitivity of Antarctic nearshore biota to a range of common metal contaminants. This data record describes two such tests, using juveniles of the microgastropod Skenella paludionoides. The first test (T01) was a 14 day test (start date: 20/08/2007) using juveniles less than 7 days old. The second test (T02) was a 12 week test (start date: 10/09/2007) using juveniles of the same cohort, that were less than 28 days at the commencement of the test. A range of concentrations of three single metals (cadmium, copper and zinc) were applied as test treatments to determine this species sensitivity to these common metal contaminants. T01 included all three metals, T02 used copper and zinc only. Data are provided in the excel file: CaseyKingston0607_Microgastropod.xlsx This file includes descriptive test details, test data and measured metal concentrations of test solutions. Scanned copies of laboratory notebook and test scoresheets are provided in PDF files: - CKing_ Ecotox Kingston 0607.pdf - Kingston07-microgastropod-juv-T01.pdf