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).

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.

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.