Toxicity of IFO 180 fuel and Slickgone NS dispersant to larval Sterechinus neumayeri Antarctic sea urchins

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.