Toxicity of physically and chemically dispersed fuels to the common Antarctic amphipod, Paramoera walkeri

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following the recommendations determined as part of the hydrocarbon chemistry component of this project (Kotzakoulakis, unpublished data).A dilution series of four concentrations were made from the full strength aqueous phase of each mix using serial dilution.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).

本元数据记录包含了针对南极常见端足类沃氏副端足虫（Paramoera walkeri）的生物测定结果，旨在揭示其对南极专用混合柴油（Special Antarctic Blend, SAB）、船用轻柴油（Marine Gas Oil, MGO）以及180号中间燃料油（Intermediate Fuel Oil, IFO 180）与燃油分散剂Ardrox 6120、Slickgone NS复配污染的响应。本研究参照Singer等（2000）的方法，并结合Barron与Ka’aihue（2003）的改良方案，制备了纯燃油水容组分（water accommodated fractions, WAF）、化学强化水容组分（chemically enhanced water accommodated fractions, CEWAF）以及仅含分散剂的处理组。其中WAF按照燃油与过滤海水（filtered seawater, FSW）1:25（体积比）的比例配制，方法源自Brown等（待刊）。化学分散处理组的比例为燃油与FSW 1:100（体积比），分散剂与燃油1:20（体积比）。仅含分散剂的处理组以FSW替代CEWAF中的燃油组分，其余配制流程一致。所有混合体系均在5 L或10 L玻璃抽滤瓶中制备，使用磁力搅拌器在加入受试介质前于FSW中形成20%~25%体积的涡流。为保证实验结果的可重复性与可比性，所有WAF的搅拌能量均保持一致（Barron和Ka’aihue, 2003）。混合体系在避光条件下搅拌42 h，随后在0±1 ℃下静置6 h。本项目烃类化学组分研究建议采用延长搅拌时长的方案（Kotzakoulakis, 未发表数据）。通过对各混合体系的全浓度水相进行梯度稀释，制备4个浓度梯度的受试液。WAF的测试浓度分别为100%、50%、20%和10%，CEWAF的测试浓度则为10%、5%、1%和0.1%。设置上述浓度旨在量化死亡率曲线，实现半致死浓度（LC50）的统计学计算。为便于比较有无燃油存在时分散剂的毒性，仅含分散剂的测试浓度与CEWAF处理组保持一致。WAF需密封于气密玻璃瓶中，于0±1 ℃下储存，最长不超过3 h后方可使用。每4天重新配制新鲜受试液，以保证水质稳定，并补充因吸附或挥发进入大气的烃类物质。每个测试浓度设置5个重复，同时设置5个FSW对照组；每个重复中放置10只P. walkeri个体。仅选取健康活跃的个体，成年个体体长范围为7.9±0.7 mm，幼体为2.5±0.2 mm（测量起点为触角基部，终点为背侧曲线最宽处）。避免使用体型较大的个体及抱卵雌性，以排除年龄或繁殖状态相关的非目标死亡（Sagar, 1980）。每个烧杯中加入200 mL受试液，保持敞口以模拟真实溢油场景下轻质单环芳烃组分的自然挥发。在每个容器中放置一小块浮游生物网布，为端足类提供附着基质，以缓解实验室环境胁迫并减少同类相食现象。实验期间不投喂饵料，避免端足类摄食吸附了烃类的食物颗粒，引入额外的暴露途径。实验总暴露时长为12 d。实验在温度控制为0±1 ℃的低温培养箱中进行，培养箱内的荧光灯设置为18 h光照、6 h黑暗的光周期，参照Brown等（2017）的方法以模拟南极夏季的环境条件。在生态毒理学测试的标准时间点——24 h、48 h、96 h、7 d、10 d和12 d，以及与每4天换水周期重合的第8 d，分别进行致死与亚致死效应观测。每个个体的健康状态按1~4级评分：1级表示无效应，4级代表死亡。死亡判定标准为无运动反应且对刺激无响应，尤其是鳃部无活动。每次观测后需移除死亡个体，并将其保存于80%乙醇中。对于可能被同类捕食的缺失个体，计入死亡计数，因为其在被吞食前大概率已处于濒死或死亡状态。为模拟重复脉冲式污染物暴露，每4天更换90%~100%的烧杯内受试液，使用新鲜配制的同浓度受试液，以补充因挥发和吸附损失的烃类物质，维持水质稳定。换水间隙使用去离子水将烧杯补液至200 mL，以稳定水质参数。在每个实验的开始与结束，以及换水前后，分别采集两份25 mL的受试液平行样。样品立即用加有BrC20（1-溴二十烷）和环辛烷内标的二氯甲烷进行萃取，萃取滤膜孔径为0.7 μm。采用气相色谱-火焰离子化检测法（Gas Chromatography with Flame Ionisation Detection, GC-FID）与气相色谱-质谱联用法（mass spectrometry, GC-MS）对样品进行分析。为确定实际暴露浓度，参照Payne等（2014）与Brown等（2017）的方法，使用4天实测的总石油烃（TPH）值，构建12 d实验周期内的连续暴露与挥发轮廓。