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 al., 2015). Experimental variables (volume of water, density of test organisms, copper concentrations, temperatures and salinities) differed for each experiment due to differences between each species (Table 1). The temperature increases that were tested (2 to 4 degreesC) reflected the increased sea and air temperatures predicted for the region tested by 2100 (Collins et al., 2013). Treatments were prepared 24 h prior to the addition of animals. Seawater was filtered to 0.45 microns and water quality was measured using a TPS 90-FL multimeter at the start and end of tests. Dissolved oxygen was greater than 80% saturation and pH was 8.1 to 8.3 at the start of tests. All experimental vials and glassware were washed with 10% nitric acid and rinsed with MilliQ water three times before use. Salinity of test solutions was prepared by dilution through the addition of MilliQ water. Copper treatments using the filtered seawater at altered salinities were prepared using 500mg/L CuSO4 (Analytical grade, Univar) in MilliQ water stock solution. Samples of test solutions for metal analysis by ICP-OES were taken at the start and end of tests (on days 0 and 14). Details of ICP-OES procedures are described in the Supplemental material (S4). Samples were taken using a 0.45 µm syringe filter that had been acid and Milli-Q rinsed. Samples were then acidified with 1% diluted ultra-pure nitric acid (65% Merck Suprapur). Measured concentrations at the start of tests were within 96% of nominal concentrations. In order to determine approximate exposure concentrations for each treatment, we averaged the 0 d and 14 d measured concentrations (Table 1). Tests were conducted in temperature controlled cabinets at a light intensity of 2360 lux. At the Macquarie Island station a light-dark regime of 16:8 h was used to mimic summer conditions. In the laboratories in Kingston, Australia, a 12:12 h regime was used to simulate Autum light conditions (as appropriate for the time of testing). Test individuals were slowly acclimated to treatment temperatures over 1 to 2 h before being added to treatments. Temperatures were monitored using Thermochron ibutton data loggers within the cabinets for the duration of the tests. Determination of mortality of individuals differed for each taxon. Mortality was recorded for Gaimardia trapesina when shells were open due to dysfunctional adductor muscles; for Obrimoposthia ohlini when individuals were inactive and covered in mucous; for Limnoria stephenseni when individuals were inactive after gentle stimulation with a stream of water from a pipette; and for Harpacticus sp. when urosomes were perpendicular to prosomes (as used in other studies with copepods; see Kwok and Leung, 2005). All dead individuals were removed from test vials.

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.

A survey of macrobenthic assemblages in soft-sediments was done at Casey Station, East Antarctica. Samples were taken by divers using hand-held corers (core size - 10 cm diameter by 10 cm deep). The aims were: 1) To examine spatial variation at several scales in these assemblages; 2) To determine if there were differences between potentially impacted areas and control areas; 3) To determine the level of replication, taxonomic resolution and data transformation that are appropriate to studies of human impacts in Antarctic soft-sediment assemblages. Cores were collected by divers in a hierarchical, spatially nested design incorporating 4 scales: Locations (1000s of metres apart), Sites (100s of metres), Plots (10s of metres) and among replicates within plots (~1 metre). Four replicates for infaunal analysis were collected from each plot. Variation at the whole assemblage level was most significant at the largest scale: between Locations; but significant differences were also found between Plots within Sites, and between Sites within Locations. The impacted locations, near two waste tips, a sewage outfall and a wharf, as a group were significantly different to control locations. Impacted locations had less variable assemblages but more variable populations of dominant species than control locations. Control locations had greater richness and diversity than impacted locations. Patterns of assemblage structure were similar at fine (species and family) and medium (family to order) levels of taxonomic resolution but changed at coarse (phylum) levels of resolution. Assemblage patterns were similar between untransformed, square root and fourth root transformations but often different in presence/absence transformations. Concentrations of metals in sediments were also analysed and other environmental variables such as grain size and water depth were measured (two replicates from each plot). Multivariate correlations between the biological and environmental datasets were examined. Links to ASAC 1100. The fields in this dataset are: Location Site/Plot Replicate Antimony Arsenic Cadmium Chromium Copper Iron Lead Manganese Mercury Nickel Silver Tin Zinc Weight Toxicity

Full title: Diatom and associated data for a manipulative field experiment which translocated control and contaminated sediments between locations within the Windmill Islands, Antarctica. A manipulative field experiment was performed to assess the effects of heavy metals and petroleum hydrocarbons on benthic diatom communities in the Windmill Islands. Three treatments were used (control, metal contaminated, and petroleum hydrocarbon contaminated), with replicates of each treatment deployed at three locations (Sparkes Bay, Brown Bay and O'Brien Bay). The datasets associated with this experiment include the concentrations of metals within the sediments as well as diatom data (raw counts, and the relative abundance of benthic species). This work was completed as part of ASAC project 1130 (ASAC_1130) and project 2201 (ASAC_2201). Public summary from project 1130: Algal mats grow on sea floor in most shallow marine environments. They are thought to contribute more than half of the total primary production in many of these areas, making them a critical food source for invertebrates and some fish. We will establish how important they are in Antarctic marine environments and determine the effects of local sewerage and tip-site pollution. We will also investigate the impact on the algal mats of the additional UV radiation which results from the ozone hole. Public summary from project 2201: As a signatory to the Protocol on Environmental Protection to the Antarctic Treaty Australia is committed to comprehensive protection of the Antarctic environment. This protocol requires that activities in the Antarctic shall be planned and conducted on the basis of information sufficient to make prior assessments of, and informed judgements about, their possible impacts on the Antarctic environment. Most of our activities in the Antarctic occur along the narrow fringe of ice-free rock adjacent to the sea and many of our activities have the potential to cause environmental harm to marine life. The Antarctic seas support the most complex and biologically diverse plant and animal communities of the region. However, very little is known about them and there is certainly not sufficient known to make informed judgements about possible environmental impacts. The animals and plants of the sea-bed are widely accepted as being the most appropriate part of the marine ecosystem for indicating disturbance caused by local sources. Attached sea-bed organisms have a fixed spatial relationship with a given place so they must either endure conditions or die. Once lost from a site recolonisation takes some time, as a consequence the structure of sea-bed communities reflect not only present conditions but they can also integrate conditions in the past. In contrast, fish and planktonic organisms can move freely so their site of capture does not indicate a long residence time at that location. Because sea-bed communities are particularly diverse they contain species with widely differing life strategies, as a result different species can have very different levels of tolerance to stress; this leads to a range of subtle changes in community structure as a response to gradually increasing disturbance, rather than an all or nothing response. This project will examine sea-bed communities near our stations to determine how seriously they are affected by human activities. This information will be used to set priorities for improving operational procedures to reduce the risk of further environmental damage. The fields in this dataset are: Species Arsenic Cadmium Copper Lead Silver Zinc Concentration Location Treatment Abundance Benthic Site

These results are for the 4 hour extraction of HCl. See also the metadata records for the 0.5 hour extraction of HCl, and the time trial data for 1 M HCl extractions. A regional survey of potential contaminants in marine or estuarine sediments is often one of the first steps in a post-disturbance environmental impact assessment. Of the many different chemical extraction or digestion procedures that have been proposed to quantify metal contamination, partial acid extractions are probably the best overall compromise between selectivity, sensitivity, precision, cost and expediency. The extent to which measured metal concentrations relate to the anthropogenic fraction that is bioavailable is contentious, but is one of the desired outcomes of an assessment or prediction of biological impact. As part of a regional survey of metal contamination associated with Australia's past waste management activities in Antarctica, we wanted to identify an acid type and extraction protocol that would allow a reasonable definition of the anthropogenic bioavailable fraction for a large number of samples. From a kinetic study of the 1 M HCl extraction of two certified Certified Reference Materials (MESS-2 and PACS-2) and two Antarctic marine sediments, we concluded that a 4 hour extraction time allows the equilibrium dissolution of relatively labile metal contaminants, but does not favour the extraction of natural geogenic metals. In a regional survey of 88 marine samples from the Casey Station area of East Antarctica, the 4 h extraction procedure correlated best with biological data, and most clearly identified those sediments thought to be contaminated by runoff from abandoned waste disposal sites. Most importantly the 4 hour extraction provided better definition of the low to moderately contaminated locations by picking up small differences in anthropogenic metal concentrations. For the purposes of inter-regional comparison, we recommend a 4 hour 1 M HCl acid extraction as a standard method for assessing metal contamination in Antarctica. The fields in this dataset are Location Site Replicate Antimony Arsenic Cadmium Chromium Copper Iron Lead Manganese Nickel Silver Tin Zinc

These results are for the 0.5 hour extraction of HCl. See also the metadata records for the 4 hour extraction of HCl, and the time trial data for 1 M HCl extractions. A regional survey of potential contaminants in marine or estuarine sediments is often one of the first steps in a post-disturbance environmental impact assessment. Of the many different chemical extraction or digestion procedures that have been proposed to quantify metal contamination, partial acid extractions are probably the best overall compromise between selectivity, sensitivity, precision, cost and expediency. The extent to which measured metal concentrations relate to the anthropogenic fraction that is bioavailable is contentious, but is one of the desired outcomes of an assessment or prediction of biological impact. As part of a regional survey of metal contamination associated with Australia's past waste management activities in Antarctica, we wanted to identify an acid type and extraction protocol that would allow a reasonable definition of the anthropogenic bioavailable fraction for a large number of samples. From a kinetic study of the 1 M HCl extraction of two certified Certified Reference Materials (MESS-2 and PACS-2) and two Antarctic marine sediments, we concluded that a 4 hour extraction time allows the equilibrium dissolution of relatively labile metal contaminants, but does not favour the extraction of natural geogenic metals. In a regional survey of 88 marine samples from the Casey Station area of East Antarctica, the 4 h extraction procedure correlated best with biological data, and most clearly identified those sediments thought to be contaminated by runoff from abandoned waste disposal sites. Most importantly the 4 hour extraction provided better definition of the low to moderately contaminated locations by picking up small differences in anthropogenic metal concentrations. For the purposes of inter-regional comparison, we recommend a 4 hour 1 M HCl acid extraction as a standard method for assessing metal contamination in Antarctica. The fields in this dataset are Location Site Replicate Antimony Arsenic Cadmium Chromium Copper Iron Lead Manganese Nickel Silver Tin Zinc

Full title: Diatom and associated data for a manipulative field experiment examining the effects of heavy metal and petroleum hydrocarbon contamination on benthic diatom communities in the Windmill Islands, Antarctica. A manipulative field experiment was performed to assess the effects of heavy metals and petroleum hydrocarbons on benthic diatom communities in the Windmill Islands. Three treatments were used (control, metal contaminated, and petroleum hydrocarbon contaminated), with replicates of each treatment deployed at three locations (Sparkes Bay, Brown Bay and O'Brien Bay). The datasets associated with this experiment include the concentrations of metals and hydrocarbons within samples, as well as diatom data (raw counts, and the relative abundance of benthic species). This work was completed as part of ASAC project 1130 (ASAC_1130) and project 2201 (ASAC_2201). Public summary from project 1130: Algal mats grow on sea floor in most shallow marine environments. They are thought to contribute more than half of the total primary production in many of these areas, making them a critical food source for invertebrates and some fish. We will establish how important they are in Antarctic marine environments and determine the effects of local sewerage and tip site pollution. We will also investigate the impact on the algal mats of the additional UV radiation which results from the ozone hole. Public summary from project 2201: As a signatory to the Protocol on Environmental Protection to the Antarctic Treaty Australia is committed to comprehensive protection of the Antarctic environment. This protocol requires that activities in the Antarctic shall be planned and conducted on the basis of information sufficient to make prior assessments of, and informed judgements about, their possible impacts on the Antarctic environment. Most of our activities in the Antarctic occur along the narrow fringe of ice-free rock adjacent to the sea and many of our activities have the potential to cause environmental harm to marine life. The Antarctic seas support the most complex and biologically diverse plant and animal communities of the region. However, very little is known about them and there is certainly not sufficient known to make informed judgements about possible environmental impacts. The animals and plants of the sea-bed are widely accepted as being the most appropriate part of the marine ecosystem for indicating disturbance caused by local sources. Attached sea-bed organisms have a fixed spatial relationship with a given place so they must either endure conditions or die. Once lost from a site recolonisation takes some time, as a consequence the structure of sea-bed communities reflect not only present conditions but they can also integrate conditions in the past. In contrast, fish and planktonic organisms can move freely so their site of capture does not indicate a long residence time at that location. Because sea-bed communities are particularly diverse they contain species with widely differing life strategies, as a result different species can have very different levels of tolerance to stress; this leads to a range of subtle changes in community structure as a response to gradually increasing disturbance, rather than an all or nothing response. This project will examine sea-bed communities near our stations to determine how seriously they are affected by human activities. This information will be used to set priorities for improving operational procedures to reduce the risk of further environmental damage. The fields in this dataset are: Species Site Abundance Treatment Type Antimony Arsenic Cadmium Chromium Copper Iron Lead Manganese Mercury Nickel Silver Tin Zinc Special Antarctic Blend Lube

These results are for the time trial extraction of HCl. See also the metadata records for the 0.5 and 4 hour extractions of HCl. A regional survey of potential contaminants in marine or estuarine sediments is often one of the first steps in a post-disturbance environmental impact assessment. Of the many different chemical extraction or digestion procedures that have been proposed to quantify metal contamination, partial acid extractions are probably the best overall compromise between selectivity, sensitivity, precision, cost and expediency. The extent to which measured metal concentrations relate to the anthropogenic fraction that is bioavailable is contentious, but is one of the desired outcomes of an assessment or prediction of biological impact. As part of a regional survey of metal contamination associated with Australia's past waste management activities in Antarctica, we wanted to identify an acid type and extraction protocol that would allow a reasonable definition of the anthropogenic bioavailable fraction for a large number of samples. From a kinetic study of the 1 M HCl extraction of two certified Certified Reference Materials (MESS-2 and PACS-2) and two Antarctic marine sediments, we concluded that a 4 hour extraction time allows the equilibrium dissolution of relatively labile metal contaminants, but does not favour the extraction of natural geogenic metals. In a regional survey of 88 marine samples from the Casey Station area of East Antarctica, the 4 h extraction procedure correlated best with biological data, and most clearly identified those sediments thought to be contaminated by runoff from abandoned waste disposal sites. Most importantly the 4 hour extraction provided better definition of the low to moderately contaminated locations by picking up small differences in anthropogenic metal concentrations. For the purposes of inter-regional comparison, we recommend a 4 hour 1 M HCl acid extraction as a standard method for assessing metal contamination in Antarctica. The fields in this dataset are Concentration Extraction Time Antimony Arsenic Cadmium Chromium Copper Iron Lead Manganese Nickel Silver Tin 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, 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