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  • This metadata record contains the results from bioassays conducted to show the response of larval Antarctic Sterechinus neumayeri sea urchins to contamination from combinations if IFO 180 fuel and the fuel dispersant Slickgone NS. AAS project 4142. Experiments used an intermediate grade Fuel Oil (IFO 180) and an internationally approved fuel dispersant, Slickgone NS, produced by Dasic International LTD. Treatments included a physically dispersed treatment of IFO 180 only, a chemically dispersed treatment of IFO 180 treated with Slickgone NS and a Slickgone NS only treatment to determine the toxicity of the dispersant. Treatments were experimentally mixed using a magnetic stirrer to combine treatment substances and filtered seawater (FSW) in temperature-controlled cabinets at 0oC to create a Water Accommodated Fraction (WAF). WAFs were produced in 2 L and 5 L glass aspirator bottles following the methods of Singer, Aurand et al. (2000) with adaptations by Barron and Ka'aihue (2003) and Kostzakoulakis (chemistry section, project 4142) stirring for 42 h with a settling time of 6 h. WAF treatments used concentrations of 100%, 50%, 20% and 10%, CEWAF and dispersant only treatments used concentrations of 10%, 5%, 1% and 0.1%. Toxicity tests were conducted in temperature-controlled cabinets at 0 oC using uncapped, forty-millilitre glass headspace vials, each containing 15.5 ml of test solution and 0.5 ml of embryo suspension. Fertilisation methods followed standard procedures for Sterechinus neumayeri. Two tests were conducted to determine the effect of a single pollution event (test 1) compared with a recurring repeated pulse pollution event (test 2). Test 1 required no water changes, while test 2 required renewal of the test treatments on a 4-day basis. Three endpoints were used, un-hatched blastula (48 h to 48.5 h) to represent the embryonic phase, gastrula (10 d) and 4-armed pluteus (16 d to 18 d). At the termination of each endpoint, 1 ml of 10% buffered formalin was added to each relevant vial and recapped. At the conclusion of the experiments, preserved embryos were observed under a dissecting microscope to determine the number of normal, abnormal and unfertilised embryos relative to controls. Samples for analysis of total petroleum hydrocarbon content were taken throughout the 2 experiments to determine the actual concentrations to which embryos and larvae were exposed. The measured concentrations were integrated following the methods of Payne et al. (2014) to obtain a profile of hydrocarbon content over each test period. Two spreadsheets are included in this metadata record detailing survival data and results of hydrocarbon analysis. The survival data file includes test condition details on the first tab, with data for tests 1 and 2 on the second and third tabs. Test treatment and concentration are listed on the left of each data block and count categories are defined in the top left panel. Development stage, date preserved and age of organism is defined for each data block, representing the three endpoints included in the experiments: unhatched blastula, gastrula and 4-armed pluteus. The hydrocarbon analysis, TPH (total petroleum hydrocarbon) file details chemical analysis results produced by K. Kotzakoulais at Macquarie University as part of project 4142. Row terminology explanations are as follows: TPH metadata Test name- indicates the tested species Exp number-indicates whether the data belongs to test 1 or test 2 Water change- details the identification of the sample in relation to the 4-day water change regime. Start samples represent the beginning of the experiment. Pre samples are taken at the end of the corresponding 4-day period, before the water is changed. 'Post' samples are taken of newly made test solutions. The chronological order of sampling is therefore: Start, pre4d, post4d, pre8d, post 8d etc. Only 'pre' samples were taken for test 1, as there were no water changes. less than C9 - greater than C28- Hydrocarbon content of samples was broken down into four compound size classes detailed for each analysis. Contamination- contamination was detected in samples, the source of contamination remains unclear, however it was established that contamination occurred during the sampling process and therefore did not come into contact with organisms. Contamination was therefore excluded from calculations. The hydrocarbon content of 0.1% dilutions was unable to be reliably analysed due to accuracy of the equipment and interfering contamination. Control data indicates spot checks to confirm the presence or absence of fuel. Very small amounts of hydrocarbons were detected as lighter fuel components evaporated and dissolved into control water within the cabinet. These very small amounts are negligible. Abnormality metadata Tab 1 details test conditions Tab 2 'Test 1' includes the data for test 1 Tab 2 'Test 2' includes the data for test 2. All observational categories are defined within the spreadsheet.

  • Two toxicity tests were conducted in the Davis station laboratories in December 2010. Tests used locally collected amphipods of the species Orchomenella pinguides. The tests were conducted by Bianca Sfiligoj, as part of her PhD research (Sfiligoj 2013), with results published in (Sfiligoj et al. 2015). Field and laboratory work was conducted under project AAS 2933, with analysis and write-up completed under AAS 4100 (both projects CI: King). Details are fully described in the published manuscript provided with this data record; file name: Sfiligoj et al 2015_Ecotoxicology.pdf. A subset of the data is also used in Candy et al. 2015 (Filename: Candy et al 2015_Ecotoxicology.pdf). Data files: Test data are provided in the .xlsx file: 'Orchomenella-Tests-Dec 2010.xlsx'. Each worksheet includes a "This worksheet provides…" description in cell A1. Laboratory notebook records are provided in the scanned file: Sfiligoj-LabBookScan-Davis10-11.pdf. In this notebook, tests are labelled LT1 and LT2 (referred to as: amphipod lentil test 1 and 2); with results recorded on pages: 1-19 and 26-28. Data associated with this record has also been presented at: - Candy SG, Sfiligoj BJ, King CK, Mondon JA (2013) Modelling interval-censored survival times in toxicological studies using generalized additive models, The International Biometric Society Australasian Region Conference 2013, Mandurah, Australia, 1-5 December 2013. - Sfiligoj BJ, King CK, Candy SG, Mondon JA (2012) Development of appropriate bioassay and statistical methods for determining survival sensitivities of Antarctic marine biota to metal exposure, 2nd Society for Environmental Toxicology and Chemistry (SETAC) Australasia Conference, Brisbane, Australia, 4-6 July 2012. - Sfiligoj BJ, King CK, Candy SG, Mondon JA (2012) Development of appropriate bioassay and statistical methods for determining survival sensitivities of Antarctic marine biota to metal exposure, Society for Environmental Toxicology and Chemistry (SETAC) World Congress, Berlin, Germany, 20-24 May 2012.

  • 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 metadata record contains the results of bioassays conducted to characterise the response of Antarctic nearshore marine invertebrates to hydrocarbon contaminants in fuels commonly used in Antarctica. AAS Project 3054. The results of Season 2 and Season 3 amphipod tests are in this dataset. Ecotoxicological bioassays were conducted at Davis and Casey Stations in 2009/10, 2010/11 and 2011/12 summer seasons to test the sensitivity of marine invertebrates to fuels in seawater. The three fuel types used in this project 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 control 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 5 L or 10 L 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. The mixture was settled for 6 h before the water portion was drawn from beneath the fuel. This dataset contains the results of ecotoxicological bioassays with near-shore marine amphipod species exposed to WAFs of SAB WAF, MGO WAF and IFO WAF (specified above). Experimental treatments consisted of undiluted 100% WAF and dilutions of 10% and 1% of WAFs in FSW, to test the toxicity of water accommodated fractions of these three fuels on Antarctic marine invertebrates. The majority of experiments tested WAFs of each of the three fuels, although one tested SAB only due to limited supply of test organisms. Bioassays were conducted in open vessels (glass jars or beakers) in temperature controlled cabinets. Mortality and/or sub-lethal effects were observed at endpoints of 24 h, 48 h, 96 h, 7 d, 8 d, 10 d, 12 d, 14 d, 16 d and 21 d. New WAF solutions were prepared at 4 d intervals to replenish the experimental treatments. Deionised water was added to test solutions as required to maintain test solution volume and salinity. Water quality data was collected at each water change. Hydrocarbon concentrations in WAFs were determined from replicate experiments to measure THC in WAFs over time (Dataset AAS_3054_THC_WAF). WAF exposure concentrations for each bioassay endpoint were derived from these hydrocarbon tests. An integrated concentration was calculated from measured hydrocarbon concentrations weighted to time. Calculations account for depletion of hydrocarbons from test treatments and any renewal of treatments. These integrated THC concentrations for endpoints from 24h to 21d are contained in dataset AAS_3054_THC_WAF_integ_conc_10_11_12. This dataset consists of Excel spreadsheets. The file name code for invertebrate bioassays is; Project number_Season_Taxa_Test name Eg AAS_3054_10_11_amphipod_2PWA1 Project number : AAS_3054 Season : 2010/11 season Taxa: amphipod Test name:2 for Season 2, PW for genus and species, A for adult, 1 for Test 1 Bioassay spreadsheets contain the results of bioassays for a species. Where replicate tests were conducted, each experiment is on a separate spreadsheet. The worksheet labelled "Test conditions" shows details of Test name, dates, animal collection details, laboratory holding conditions, details of water accommodated fractions (WAF), bioassay conditions, scoring criteria and water quality data. The worksheet labelled "Counts" has columns for Replicate number and columns with the Score for all the animals in that replicate at every time endpoint. A full description of the scoring criteria is on the "Test conditions" worksheet. Totals, means and standard deviations are calculated for each treatment. The worksheet labelled "Totals, means, percent, StDev" has calculations of Survival, Unaffected, including mean and standard deviation, Percent Survival and Unaffected including means and standard deviation. Also included is column for the Total number of moults in each treatment. During the research to obtain early life stages of invertebrates for experiments, the number of Paramoera walkeri amphipod neonates per female, the timing of their release from the brood pouch and their early growth rate were recorded. These data are also included in AAS_3054_10_11_PW_neonates Samples were collected from: Ellis Narrows, Vestfold Hills Airport Beach, Davis, Vestfold Hills Prydz Bay, Davis (Between Anchorage Island and Bluff Island) Bailey Peninsula, Windmill Islands

  • This data record has been compiled for a statistical methods study, conducted by Abigael Proctor as part of her PhD research in 2018. The data in this record have been used to showcase a new statistical method for determining no effect concentration (NEC). The study uses the data in this record to compare NEC and LCx estimates for copper in four Antarctic marine invertebrate species. The data associated with this record are a subset of four existing larger datasets: 1. amphipod: AAS_2933_Orchomenella_pinguides_Sensitivity_metals_Davis_2010-11 2. copepod: AAS_4100_Toxicity_Copepods 3. gastropod: AAS_2933_MetaToxicityMarine_JuvenileGastropods_Kingston2007 4. ostracod: AAS_2933_MetalToxicityMarine_BrownOstracods_Kingston2007 Subset details are described in the excel file provided.

  • This metadata record contains the results from bioassays conducted to show the response of the Antarctic gastropod, Skenella palludinoides to contamination from combinations of Special Antarctic Blend (SAB) diesel, chemically dispersed with fuel dispersant Ardrox 6120. 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. (2017). 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 approximately 20% 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 18 h with an additional settling time of 6 h at 0 plus or minus 1 oC. 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 approximately 10 S.palludinoides individuals per replicate. The healthiest and most active individuals were chosen. 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. Animals were not fed during experiments to prevent hydrocarbons being ingested, thereby introducing an additional exposure pathway. Experiments ran for a total of 35 d exposure duration for WAF and CEWAF experiments and 15 d for dispersant only experiments. Experiments were run in cold temperature-controlled cabinets set 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 test times of: 24 h, 48 h, 96 h, 7 d, 8 d, 10 d and 12 d, 14 d, 16 d, 20 d, 21 d, 28 d and 35 d for SAB + Ardrox 6120 experiments and 24 h, 48 h, 96 h, 7 d, 8 d, 10 d and 12 d, 14 d, 15 d for Ardrox 6120 only experiments. The health status of each individual was classified as per the criteria listed below: - Attached to the vial with horns in or out - Unattached (often upside down), horns out, will reattach if flipped over - Not attached but if touched, will retract - Closed but attached and out of water - Operculum closed - Dead, operculum open a little (muscles no longer working), if touched, operculum will not move and tissues might disintegrate Dead animals were removed and preserved in 80% ethanol at each observation period. 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). Brown, K.E., King, C.K., Harrison, P.L., 2017. Lethal and behavioural impacts of diesel and fuel oil on the Antarctic amphipod Paramoera walkeri. Environmental Toxicology and Chemistry. Animal collection, 2013 experiments: animals sourced from AAD aquarium, collected in previous seasons. Animal collection, 2014 experiments: January and February 2014 Experiments were conducted at the Marine Research Facility at the Australian Antarctic Division in Kingston, Tasmania. Experiments using SAB fuel and the fuel dispersant Ardrox 6120 were conducted in August and September 2013, with additional experiments conducted in May 2014 using Ardrox 6120 only.

  • This study assessed the performance of diffusive gradients in thin-films (DGT) with a binding resin that used Chelex-100 (iminodiacetic acid functional groups) to measure cadmium, copper, nickel, lead, and zinc contaminants in Antarctic marine conditions. To do this, three sets of experiments were done: (I) the uptake of metals to DGT samplers was assessed over time when deployed to three metal mixtures of known concentrations (DGT performance page). This allowed for the determination of metal diffusion coefficients in Antarctic marine conditions and demonstrated when metal competition for binding sites were likely to occur. (II) the DGT were deployed in the presence of the microalga Phaeocystis antarctica at a concentration of 1000-3000 cells/mL to investigate how environmentally realistic concentrations of an Antarctic marine microalgae affect the uptake of metals (DGT uptake with algae page). Finally, the DGT-labile concentrations from part (II) were used in reference toxicity mixture models to predict toxicity to the microalgae so they could be compared to a previous study that investigated the toxicity of metal mixtures to Phaeocystis antarctica and Cryothecomonas armigera (DGT toxicity modelling page).

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

  • This metadata record contains observed and predicted toxicity data from bioassays with two species of Antarctic marine microalgae: Phaeocystis antarctica (Prymnesiophyceae) and Cryothecomonas armigera (Cercoza). Bioassay exposures were of mixtures of 5 metals at two ratios, an Environmental (ENV) and Equitoxic (EC) mixture. The measured dissolved metal concentrations were used in two mixture reference models, Independent Action (IA) and Concentration Addition (CA), to predict toxicity as population growth rate inhibition. A Flow Cytometer (BD-FACSVerse) was used to measure the density of microalgae over time, which was then converted to a growth rate. An inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES), was used to measure metal concentrations. Data for each microalga is provided in individual excel spreadsheets, identified by the species tested. A word document is provided that contains the R code used to predict toxicity to the two microalgae by the reference models Independent Action and Concentration Addition. The R code also includes the steps required to extend the models to include a deviation parameter “a” that allows for departure from model additivity. A nested F-test then tests for significance between the fit of each test to observed toxicities. This R code has been adapted to use EC10 as parameter estimates, rather than EC50s. The code was adapted from the approach outlined in Hochmuth, J. D.; Asselman, J.; De, S. Are Interactive Effects of Harmful Algal Blooms and Copper Pollution a Concern for Water Quality Management? Water Res. 2014, 60, 41–53. DOI: 10.1016/j.watres.2014.03.041. Single-metal toxicity data and experimental protocols for P. antarctica from the following paper: and C. armigera used in this study can be found in the following papers: A robust bioassay to assess the toxicity of metals to the Antarctic marine microalga Phaeocyctis antarctica. Francesca Gissi, Merrin S. Adams, Catherine K. King, Dianne F. Jolley (2015). Environmental Toxicology and Chemistry. 2015 Feb 20. doi: 10.1002/etc.2949. Chronic toxicity of five metals to the polar marine microalga Cryothecomonas armigera – Application of a new bioassay. Darren J. Koppel, Francesca Gissi, Merrin S. Adams, Catherine K. King, and Dianne F. Jolley, (2017). Environmental Pollution, Volume 228, 2017, Pages 211-221, doi.org/10.1016/j.envpol.2017.05.034.

  • This metadata record contains the results from 3 bioassays conducted with the Antarctic marine microalgae Cryothecomonas armigera (incertae sedis). These tests assessed the toxicity of copper, cadmium, lead, zinc and nickel. Test conditions for both algae are described in the excel spreadsheets. In summary, tests for P. antarctica and C.armigera, were carried out at 0 plus or minus 2 degrees C, 20:4 h light:dark (60-90 micromol/m2/s, cool white 36W/840 globes), in 80 mL natural filtered (0.22 microns) seawater (salinity - 35 ppt, pH - 8.1 plus or minus 0.2). Filtered seawater was supplemented with 1.5 mg/L NO3- and 0.15 mg/L of PO43-. All tests were carried out in silanised 250-mL glass flasks, with glass lids. Tests 1 and 2 consisted of metal treatments, with 3 replicates per treatment, alongside 3 replicate controls (natural filtered seawater). Test 3 consisted of metal treatments in an increasing series (no replicates) alongside 3 replicate controls. Seawater was spiked with metal solutions to achieve required concentration. Concentrations tested are recorded in excel datasheets as dissolved metal concentrations measured on day 0, and day 24. The average of the dissolved metal concentrations were used for further statistical analysis. The age of C.armigera at test commencement was 25-30 days. Algal cells were centrifuged and washed to remove nutrient rich media, and test flasks were inoculated with between 1-3 x10^3 cells/mL. Cell densities in all toxicity tests were determined by flow cytometry. Toxicity tests with C. armigera were carried out over 23-24 days, with cell densities determined twice a week. The growth rate (cell division; u) was calculated as the slope of the regression line from a plot of log10 (cell density) versus time (h). Growth rates for all treatments were expressed as a percentage of the control growth rates. The flow cytometer was also used to simultaneously measure changes in the following cellular parameters: chlorophyll a autofluorescence intensity (FL3), cell size (FSC) and cell complexity (SSC). The molecular stain BODIPY 493/503, was used to measure neutral lipid concentrations. Changes in cellular parameters were measured by applying a gate that captured greater than 95% of control cells in a region, R2. Changes in cellular parameters were observed in metal treatments as a shift of the cell population from the R2 region to R1 (for relative decreases) or to R3 (for relative increases). The proportion of cells in each region is expressed as a percentage of the total cell population. The pH was measured on the first and last day of the test. Sub-samples (5 mL) for analysis of dissolved metal concentrations were taken from each treatment on 24. Sub-samples were filtered through an acid washed (10% HNO3, Merck) 0.45-microns membrane filter and syringe, and acidified to 0.2% with Tracepur nitric acid (Merck). Metal concentrations were determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES) for Cu, Cd, Pb, Ni and Zn. Detection limits for Cu, Cd, Pb, Ni and Zn were 1.0, 0.3, 3.2, 1.4, and 1.0 micrograms per litre, respectively. Calculations of effect concentrations (EC 10 and 50) were made using the 'Dose Response Curve' package of R statistical analysis software. Concentration-response curves had several models applied to them, and were tested for best fit by comparing residual standard errors and Akaike's 'An Information Criterion' function . Generally, log-logistic models with 3 parameters provided the best fit. Data for each toxicity test is combined in a single excel spreadsheet, "Cryothecomonas armigera single metal toxicity". The first worksheet is titled "Test Conditions" which provides information on the toxicity test, e.g. species and metals tested, dates, test conditions, as well as explanation of abbreviations, definitions of toxicity values etc. The second worksheet includes the raw cell densities determined in each flask, the calculated growth rates, and measured metal concentrations. The third worksheet contains the measured physiological parameters: Neutral lipid concentrations (BODIPY 493/503), chlorophyll a autofluorescence (FL3), cell complexity (SSC), and cell size (FSC). The final worksheet contains the output of statistical analysis; dose-response curves for each metal with applied log-logistic model and 95% confidence interval, a table summarising the effect concentrations (EC10 and EC50), and No Effect Concentration (NEC) is also provided. The file "C. armigera combined.csv" contains rows representing individual exposures with columns for the metal treatment (Metal), averaged dissolved metal concentration for each exposure (Conc), growth rate (Growth), and growth rate as a percent of the control (Pcon). This data was used for data analysis in R statistics. Note that this contains data from all bioassays conducted with C. armigera, including those conducted by Francesca Gissi (doi:10.4225/15/551B2B65A73F3) The script used for data analysis is provided in the document "R statistics script for C. armigera single metal.docx"