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the recommendations determined as part of the hydrocarbon chemistry component of this project (Kotzakoulakis, unpublished data).A dilution series of four concentrations were made from the full strength aqueous phase of each mix using serial dilution.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

本元数据记录包含一系列生物测定（bioassay）实验的结果，旨在探究南极常见端足类动物帕氏异脚钩虾（Paramoera walkeri）对南极专用混合柴油（Special Antarctic Blend, SAB）、船用瓦斯油（Marine Gas Oil, MGO）与中间燃料油180（Intermediate Fuel Oil 180, IFO 180）组合污染的响应，上述燃油经燃油分散剂Ardrox 6120与Slickgone NS进行化学分散处理。实验设置了纯燃油水可溶组分（water accommodated fractions, WAF）、化学强化水可溶组分（chemically enhanced water accommodated fractions, CEWAF）以及仅分散剂处理组，实验方法参照Singer等（2000）的方案，并结合Barron与Ka’aihue（2003）的调整方法。WAF按照燃油与过滤海水（filtered seawater, FSW）体积比1:25的比例配制，具体方法参照Brown等（待刊）的流程。化学分散处理组的配制比例为燃油与FSW体积比1:100、分散剂与燃油体积比1:20。仅分散剂处理组参照CEWAF的配制方法，仅将燃油组分替换为FSW。所有混合液均在5L或10L玻璃抽气瓶中配制，使用磁力搅拌器在加入受试介质前于FSW中形成20%~25%的涡流。为提升实验重现性与结果可比性，所有WAF的搅拌参数保持一致（Barron和Ka’aihue，2003）。混合液在黑暗条件下搅拌42小时，随后在0±1℃环境中静置6小时。本次实验采用了延长搅拌时长的方案，该方案基于本项目烃类化学组分研究的推荐方法（Kotzakoulakis，未发表数据）。从各混合液的全浓度水相出发，通过梯度稀释（serial dilution）制备4个浓度梯度的受试液。WAF的测试浓度为100%、50%、20%与10%，CEWAF的测试浓度则为10%、5%、1%与0.1%。设置上述浓度梯度旨在量化死亡率曲线并统计计算半致死浓度（LC50）。为便于对比有/无燃油条件下分散剂的毒性，仅分散剂处理组的浓度与CEWAF处理组保持一致。WAF需密封于气密玻璃瓶中，在0±1℃环境下储存，且需在3小时内使用。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空白对照烧杯，每个重复中放入10只帕氏异脚钩虾个体。仅选择健康活跃的个体，成年个体体长范围为7.9±0.7mm，幼体为2.5±0.2mm（测量起点为触角基部，终点为背部曲线最宽处）。排除体型较大的个体与抱卵雌性个体，以避免因年龄或生殖状态导致的非目标死亡（Sagar，1980）。每个烧杯加入200ml受试液，保持开口状态以模拟真实溢油场景下轻质单环芳烃（monoaromatic hydrocarbon）组分的自然挥发。在每个烧杯中放置一小块浮游生物网布（plankton mesh），为端足类提供附着基质以缓解实验室环境压力，并减少同类相食行为。实验期间不投喂动物，以避免受试端足类摄食吸附了烃类的食物颗粒，从而引入额外的暴露途径。实验总暴露时长为12天。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小时光照、6小时黑暗的光周期，参照Brown等（2017）的方法以模拟南极夏季的环境条件。在标准生态毒理学测试时间点24h、48h、96h、7d、10d与12d进行致死与亚致死效应观测，同时在第8天额外开展一次观测，以匹配每4天一次的换液周期。个体健康状况按1~4级进行分级：1级表示无效应，4级代表死亡。死亡判定标准为无运动能力且对刺激无响应，尤其是鳃部无反应。每次观测后需移除死亡个体，并将其保存于80%乙醇溶液中。因同类相食而失踪的端足类个体被计入死亡数，因为它们在被吞食时很可能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%的受试液，使用新鲜配制的同浓度受试液，以补充因蒸发与吸附损失的烃类物质，并维持水质稳定。换液间隙使用去离子水将烧杯补至200ml以维持水质参数。在每个实验的开始与结束阶段，以及换液前后，分别采集2份25ml的受试液平行样。样品立即用0.7μm二氯甲烷萃取，萃取液中加入内标物BrC20（1-溴二十烷，1-bromoeicosane）与环辛烷（cyclooctane）。采用气相色谱-火焰离子化检测器（Gas Chromatography with Flame Ionisation Detection, GC-FID）与气相色谱-质谱联用（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）概述的方法，使用4天实测总石油烃（total petroleum hydrocarbon, TPH）值构建12天测试周期内的连续暴露与蒸发变化曲线。