Live O. orensanzi were found in the AAD's Marine Research Facility emerging from sediments during feeding on 3 July 2014. It is likely that live specimens were included in samples collected for another species, Antarctonemertes sp. from intertidal rocky areas at Beall Island near Casey station (66 30.4265 degree S, 110 45.851 degrees E), East Antarctica in January and February 2014. It is also possible that the O. orensanzi were collected from southeast Newcomb Bay, adjacent to Casey station on 2 and 3 of February 2012 (Figure 4), and survived in the Marine Research Facility's aquarium, but this is considered less likely.
Experiments were conducted at the AAD's quarantine facility in Kingston, Tasmania, between 19 July and 2 September 2014.
This metadata record contains the results from bioassays conducted to show the response of Antarctic Polychaetes Ophryotrocha orensanzi to contamination from combinations if IFO 180 fuel and the fuel dispersants Ardrox 6129, Slickgone LTSW and Slickgone NS.
Test solutions were prepared following the methods of Singer et al. (2000) with modifications by Barron and Ka'aihue (2003) and others. Water accommodated fractions of fuel in water (WAF) were produced using a 1:25 (v/v) fuel to FSW ratio in accordance with studies by Payne et al. (2014) and Brown et al., (2016) to facilitate comparability of results. Chemically enhanced water accommodated fractions (CEWAF) were made following a lower 1:100 (v/v) fuel to FSW ratio. A 1:20 (v/v) dispersant to fuel ratio was used for all three dispersants, an application rate of 1:20 dispersant to fuel rate was used both because this is the standard default application rate used in the field and to increase comparability to previous studies. Dispersant only mixes were made according to CEWAF specifications, substituting FSW for fuel.
Test mixes were prepared in dark temperature-controlled cabinets at 0 plus or minus 1 degree C. Mixes were made in two L or five L glass aspirator bottles using a magnetic stirrer. Mix preparation followed the pre-vortex method in which a 20 - 25 % vortex was achieved in 0 plus or minus 1 degree C FSW before addition of the test materials. Once added, fuel was allowed to cool for a further 10 minutes before subsequent addition of dispersants during CEWAF preparation. Mixes were stirred for a total of 42 h with an additional settling time of 6 h following the recommendations determined as part of the hydrocarbon chemistry component of this project (Kotzakoulakis, unpublished data). The mixture was subsequently serially diluted to achieve the desired concentrations. Test concentrations were 100%, 50%, 20% and 10% for WAF and 10%, 5%, 1% and 0.1% for CEWAF. Concentrations for dispersant only treatments mimicked CEWAF in order to be directly comparable. Test solutions were kept in sealed glass bottles with minimal headspace at 0 plus or minus 1 degree C for a maximum of 3 h before use.
Test dilutions were remade each four day period to replenish hydrocarbons lost through evaporation and absorption to simulate a repeated pulse exposure to the contaminant. Ninety percent of the test solution volume was replaced for each beaker during each water change by gently tipping out the solution with minimal disturbance to the test organisms. Replacement solutions were chilled to the correct temperature and replenished immediately to avoid any temperature shock to test animals. Beakers were topped up with deionized water between water changes to maintain water quality and solution volume.
Bioassays were conducted in cold temperature cabinets at 0 plus or minus 1 degree C and light regimes were set to 18 h light and 6 h dark to mimic Antarctic conditions used by Brown et al. (2017). Exposure vessels were 100 ml glass beakers containing 80 ml of test solution. Beakers were left open to allow for the evaporation of lighter fuel components. Each experiment consisted of four replicates per treatment concentration, with eight to 10 individuals per replicate (8 each for Slickgone NS, 10 each for Ardrox and LTSW). Experiments ran for 12 days with observations at 24 h, 48 h, 96 h, 7 d, 8 d, 10 d and 12 d. Mortality was assessed at each observation using a Leica MZ7.5 dissecting microscope. Mortality was determined by the absence of response to stimuli, specifically lack of movement in the maxillae or mandibles. No food was added during experiments to avoid inclusion of an additional exposure pathway.
Aliquots of each test concentration were taken at the beginning and end of each experiment, as well as before and after each water change to analyse the total petroleum hydrocarbon (TPH) content. Duplicate 25 ml samples were taken for each test dilution and immediately extracted with a mixture of Dichloromethane spiked with an internal standard of BrC20 (1-bromoeicosane) and cyclooctane. Extractions were analysed using Gas Chromatography with Flame Ionisation Detection (GC-FID) and Gas Chromatography mass spectrometry (GC-MS). The measured concentrations were integrated following the methods of Payne et al. (2014) to obtain a profile of hydrocarbon content over each 12 d test period.
