This metadata record contains the results from bioassays conducted to show the response of the common Antarctic amphipod, Paramoera walkeri to contamination from combinations of Special Antarctic Blend (SAB) diesel, Marine Gas Oil (MGO) and Intermediate Fuel Oil (IFO 180), chemically dispersed with fuel dispersants Ardrox 6120 and Slickgone NS. Fuel only water accommodated fractions (WAF), chemically enhanced water accommodated fractions (CEWAF) and dispersant only treatments were prepared following the methods in Singer et al. (2000) with adaptations from Barron and Ka’aihue (2003). WAF was made using the ratio of 1: 25 (v/v), fuel to filtered seawater (FSW) following the methods of Brown et al. (in prep). Ratios for chemically dispersed treatments were 1: 100 (v/v), fuel to FSW and 1: 20 (v/v) dispersant to fuel. Dispersant only treatments were made using ratios for CEWAF, substituting the fuel component with FSW. Mixes were made in 5 L or 10 L glass aspirator bottles using a magnetic stirrer to achieve a vortex of 20-25% in the FSW before the addition of test media. The same mixing energy was used to prepare all WAFs for enhanced reproducibility and comparability of results (Barron and Ka’aihue, 2003). Mixes were stirred in darkness to prevent bacterial growth for 42 h with an additional settling time of 6 h at 0 plus or minus 1 oC. Extended stirring times were used following the recommendations determined as part of the hydrocarbon chemistry component of this project (Kotzakoulakis, unpublished data). A dilution series of four concentrations were made from the full strength aqueous phase of each mix using serial dilution. WAF test concentrations were 100%, 50%, 20% and 10% while CEWAF concentrations were 10%, 5%, 1% and 0.1%. These concentrations were chosen in order to quantify the mortality curve and allow statistical calculation of LC50 values. To facilitate comparisons of dispersant toxicity in the presence and absence of fuel, dispersant only test concentrations reflected those of CEWAF treatments. WAF was sealed in airtight glass bottles stored at 0 plus or minus 1 oC for a maximum of 3 h before use. Fresh test solutions were prepared every four days to ensure consistent water quality and replace hydrocarbons that adsorbed or evaporated into the atmosphere. Each test concentration was represented by five replicates with five FSW control beakers, with 10 P. walkeri individuals per replicate. Only healthy and active individuals were chosen with a size range of 7.9 plus or minus 0.7 mm for adults and 2.5 plus or minus 0.2 for juveniles measured from the base of the antennae to the widest part of the dorsal curve. Larger individuals and brooding females were not used to avoid unrelated deaths related to age or reproductive state (Sagar, 1980). Beakers were filled to 200 ml and were left open to allow the natural evaporation of lighter monoaromatic hydrocarbon components that would occur during a real spill. A small square of plankton mesh was placed in each jar to provide a substratum to reduce the stress of laboratory conditions and to help to stem cannibalism. Animals were not fed during experiments to avoid hydrocarbons adsorbed onto food pellets being ingested by the amphipods, thereby introducing an additional exposure pathway. Experiments ran for a total of 12 d exposure duration. Experiments were run in cold temperature-controlled cabinets maintained at a temperature of 0 plus or minus 1 oC, fluorescent lights in the cabinets were set to a light regime of 18 h light, 6 h darkness, following the methods in Brown et al. (2017) to reflect Antarctic summer environmental conditions. Lethal and sublethal observations were made at standard ecotoxicology test times of 24 h, 48 h, 96 h, 7 d, 10 d and 12 d, with an additional observation at 8 d coinciding with one of the 4-day water changes. The health status of each individual was classified on a scale of one to four; one showing no effect up to four being mortality. Mortality was determined by a lack of movement and response to stimuli, particularly in the gills. Dead animals were removed and preserved in 80% ethanol at each observation period. Missing amphipods that may have been cannibalised were included in mortality counts as they were likely to have been moribund or already dead when eaten. In order to simulate a repeated pulse pollutant, 90 to 100% of the test solution volume of each beaker was renewed with freshly made test concentrations every four days to replenish hydrocarbons lost through evaporation and adsorption and ensure consistent water quality. Beakers were topped up to 200 ml between water changes with deionised water to maintain water quality parameters. Duplicate 25 ml aliquots of test concentrations were taken at the beginning and end of each experiment in addition to pre and post water change samples. Samples were immediately extracted with 0.7 μm of dichloromethane spiked with an internal standard of BrC20 (1-bromoeicosane) and cyclooctane. Samples were analysed using Gas Chromatography with Flame Ionisation Detection (GC-FID) and mass spectrometry (GC-MS). To determine actual exposure concentrations, four day measured TPH values were used to create a continuous exposure and evaporation profile over the 12 d test period following the methods outlined in Payne et al. (2014) and Brown et al. (2017).

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.

Experiments were done to quantify the Total Hydrocarbon Content (THC) in water accommodated fractions (WAF) of three fuels; Special Antarctic Blend diesel (SAB), Marine Gas Oil diesel (MGO) and an intermediate grade of marine bunker Fuel Oil (IFO 180).These tests measured the hydrocarbon content in freshly decanted WAFs and the resulting loss of hydrocarbons over time when WAFs were exposed in temperature controlled cabinets at 0°C. These tests are detailed in Dataset AAS_3054_THC_WAF. The results of hydrocarbon WAF tests were used to calculate integrated concentration from measured hydrocarbon concentrations weighted to time to be used as the exposure concentrations for toxicity tests with Antarctic invertebrates. 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 Exposure concentrations of THC in µg/L are shown for endpoints from 24 h to 21 d

This metadata record contains the results from bioassays conducted to show the response of an Antarctic nemertean Antarctonemertes unilineata to contamination from combinations of Special Antarctic Blend (SAB) diesel, Marine Gas Oil (MGO) and Intermediate Fuel Oil (IFO 180), chemically dispersed with fuel dispersants Ardrox 6120, Slickgone LTSW and Slickgone NS. Note that the corresponding PhD thesis chapter refers to the species as Antarctonemertes sp., prior to being named Antarctonemertes unilineata in 2018. Experiments using SAB, MGO and IFO 180 with the dispersant Ardrox 6120, including fuel only and dispersant only treatments were conducted at Casey station. Experiments involving IFO 180 and the fuel dispersants Slickgone LTSW and Slickgone NS were conducted at the Antarctic Division’s Marine Research Facility quarantine labs. All experimental procedures, including test mix preparation and bioassays were conducted at 0 plus or minus 1 degree C. Water accommodated fractions (WAF; fuel mixed in water) and chemically enhanced water accommodated fractions (CEWAF) were made according to the specifications of Singer, Aurand et al. (2000), Barron and Ka’aihue (2003) and Kotzakoulakis (unpublished at time of writing). Dispersant only mixes were also made using filtered seawater (FSW) and dispersant volumes proportional to those used for CEWAF production. WAF was made using a loading ratio of 1: 25 (v/v) fuel to FSW, CEWAF was prepared using 1:100 (v/v) fuel to FSW ratio, and 1: 20 (v/v) dispersant to fuel ratio. Following the 48 h preparation time, the seawater WAF components of the mix were drained from the bottom of aspirator bottles and serially diluted. WAF treatment concentrations were 100%, 50%, 20% and 10%, CEWAF and dispersant only concentrations were 10%, 5%, 1% and 0.1%. Treatment solutions were replenished every four days to simulate a repeated pulse exposure to contaminants and to replace hydrocarbons lost through evaporation and adsorption and to maintain water quality parameters. WAF, CEWAF and dispersant only test solutions were remade every four days using identical methods. Tests were done in temperature-controlled cabinets set to 0 plus or minus 1 degree C following a 6 h light to 18 h dark photoperiod. Beakers were left uncovered to allow for the natural evaporation of lighter hydrocarbon components to reflect real fuel spill conditions. Experiments ran for 24 d except for the Ardrox 6120 only experiment, which ran for 16 d due to high mortality in this treatment. Sublethal and lethal endpoints were assessed at 1, 2, 4, 7, 8, 12, 14, 16, 20 and 24 d observations. Aliquot water samples for analysis of total hydrocarbon content (THC) were taken for initial and final test concentrations, and before and after each four-day water change, to obtain accurate profiles of hydrocarbon loss over the test period. Duplicate samples were taken for every treatment concentration and extracted with dichloromethane, spiked with an internal standard of 1-bromoeicosane and cyclooctane. Samples were analysed using gas chromatography with flame ionization detection (GC-FID) and gas chromatography mass spectrometry (GC-MS). Average THC concentrations for the duration of the experiment were obtained by integrating the measured concentrations to which animals were exposed following the methods of Brown et al. (2016) and Payne et al. (2014). This data submission includes one file detailing the TPH experiment analyses and one detailing the bioassay tests and results. The thesis that relates to this work is available from: https://epubs.scu.edu.au/theses/533/

This data set contains chemical parameters determined for marine sediment samples collected in the 2014-15 summer field season as part of the Thala Valley Long term Monitoring (TV-LTM) project. The aim of this project is to examine changes in the marine benthic ecosystem in the vicinity of Casey station following clean-up of the abandoned Thala Valley waste disposal ('tip') site in 2003-04. The chemical parameters are: (1) 1 M hydrochloric acid-extractable elements (mainly metals) by ICP-AES (inductively coupled plasma - atomic emission spectrometry) (2) water-extractable nutrients by FIA (flow injection analysis) (3) petroleum hydrocarbon fractions (TPH: total petroleum hydrocarbons) and persistent organic pollutants (POPs) - polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) - by GC-FID, GC-ECD and GC-MS (gas chromatography - flame ionization detector / electron capture detector / mass spectrometry), respectively (4) Loss on Ignition at 550 degrees Celsius (LOI; as a proxy for Total Organic Matter) and Dry Matter Fraction (DMF) by gravimetric analysis. Data sets 1, 3 and 4 were obtained for composite samples prepared from the 0-5 cm section of 51 marine sediment cores collected by SCUBA divers from impacted (contaminated) and control (pristine) locations around Casey. Data set 2 was obtained for a subsample of the surface section (0-1 cm) of each of 74 marine sediment cores collected during the same sampling campaign. Sample locations: * Brown Bay (BB) - inner, mid and outer sites * Casey Wharf * McGrady Cove * O'Brien Bay (OB) - OB1, OB2, OB3 sites * Shannon Bay * Wilkes (adjacent to abandoned station) Analytical labs involved: * Wild Lab, AAD, Kingston, Tasmania (data sets 1 and 4; sample preparation for data set 2) * Analytical Services Tasmania (AST), New Town, Tasmania (data set 2) * Analytical Services Unit (ASU), Queen's University, Kingston, Ontario, Canada (data set 3) Information concerning analytical data quality (method reporting limits, accuracy and precision), are included with each data set. Complete analytical method details are available in a separate summary document.