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.
An technical failure occurred at the Australian Antarctic Division Marine Research Facility occurred in July 2013 in which organisms were exposed to temperatures up to 10oC for an exposed period of several days. Experiments using chemically and physically dispersed SAB were conducted in August and September 2013 using organisms that survived the increased temperatures. Organisms used in experiments may therefore be hardier than those occurring in the environment and results may suggest a higher tolerance to hydrocarbon exposure.
Some contamination of hydrocarbon samples occurred during sampling, which test organisms were not exposed to. Contamination levels were therefore excluded from calculations of exposure concentrations. The hydrocarbon content of 0.1% dilutions was unable to be reliably analysed due to accuracy of the equipment and the interfering contamination. These values were extrapolated from analysis of 1% dilutions. It was determined that the extraction method was insufficient for the accurate quantitative recovery of hydrocarbons for the dispersant due to the presence of strongly polar molecules. As such, nominal concentrations were used for all dispersant only treatments and THC data represent the hydrocarbons from the fuel only for chemically dispersed treatments.