Toxicity of cadmium, copper, nickel, lead and zinc, as single metals and in metal mixtures, to the Antarctic limno-terrestrial rotifer Adineta editae.

This dataset contains results of toxicity testing with the Antarctic limno-terrestrial bdelloid rotifer Adineta editae. This study assessed the toxicity of copper, zinc, cadmium, lead and nickel, singly and in metal mixtures, to Adineta editae in aqueous exposures. Toxicity tests were done at Casey in the austral summer of 2017/18 using rotifers collected from the field and isolated in the laboratory.Rotifer collectionRotifers were sourced from Prassiola crispa algal mat collected from ephemeral melt-streams on Shirley Island in the Windmill Islands, East Antarctica Adineta editae were isolated into a single species aqueous culture prior to use in toxicity tests. Culture maintenance and toxicity testing was within temperature-controlled cabinet set at 5°C with 18/6 h light dark cycle. Rotifer species identification was confirmed by DNA sequencing (BOLD sequence OZBOL4598-21) with a 99.52% match to haplotype Bd8_02.Toxicity testsToxicity testing with A. editae rotifers was done in the Casey station laboratory during January and February of 2018. Exposures were with single metals cadmium, copper, nickel, lead, and zinc, and in metal mixtures of the five metals at two ratios, an Environmental mixture (EvM, Mixture 1) and Equitoxic mixture (EqM, Mixture 2) in aqueous exposures. Each mixture had a different fixed ratio of the five metals, which was multiplied by different factors to produce a concentration series. Exposures were with single metals cadmium, copper, nickel, lead, and zinc, and in metal mixtures of the five metals at two ratios, an Environmental mixture (EvM, Mixture 1) and Equitoxic mixture (EqM, Mixture 2) in aqueous exposures. Each mixture had a different fixed ratio of the five metals, which was multiplied by different factors to produce a concentration series. For EvM Mixture 1, the ratio was broadly based on metal concentrations reported in Brown Bay, a marine site adjacent to a legacy waste site near Casey station in East Antarctica. The metal ratio was 1 Cu: 0.1 Cd: 0.3 Ni: 0.3 Pb: 4.5 Zn, and this mixture was tested at multiples from 5 to 70 times. Mixture 2 was closer to an equimolar ratio, with lower concentrations of lead. This metal ratio was 1.1 Cu: 0.7 Cd: 0.8 Ni: 0.3 Pb: 1.0 Zn, and this mixture was tested at multiples from 0.2 to 2 times. Measured concentrations of each metal in each treatment were used in mixture toxicity modelling to give predicted toxicities, which are compared with observed toxicity using toxic units.Metal stock solutions were prepared using analytical grade reagents in ultrapure water acidified to 0.2% with HCl (Suprapur, MilliporeSigma). Exposures were in plastic microwell plates with 5 metal treatments plus controls, 3 replicate wells per treatment and 8 rotifers in each replicate. To add rotifers to test wells, collection dishes were viewed under the microscope and small, non-ovigerous individuals were selected. Using a micropipette, each organism was transferred from the collection dishes in an 8 µL aliquot of the culture water, with eight organisms added to each well, representing a single replicate. Following transfer of the organisms to a well, 236 µL of test solution was added, giving a final volume of 300 µL. Rotifers were not fed, although there was some suspended algal detritus carried over in the transfer water that provided a limited quantity of food during the test. Well plates were sealed with Parafilm and placed in trays in cabinets at 5°C with 18/6 h light dark cycle.Rotifers were observed under a stereomicroscope at day 4 and day 7 of the test and assessed with survival and behavioural endpoints. Survival (Alive/Dead) was defined by body morphology and mobility. Dead rotifers had a distinct relaxed and flattened body morphology with no muscular contraction or movement evident. The individuals scored as Alive were also scored with a sub-lethal endpoint based on their movement behaviour (Active/Inactive). Active individuals showed normal movement behaviours (creeping, feeding) and Inactive individuals had their bodies fully contracted to form a tun, with no external body movement.Chemical analysis To measure the metal exposure concentrations in test treatments, two sets of sacrificial well plates containing a total of 6 mL of each exposure solution were prepared at the beginning of each toxicity test. These well plates were identical to well plates used for toxicity testing, but did not contain rotifers. One sacrificial well plate was sampled by syringe at the beginning (day 0) and end (day 7) of each toxicity test into clean polypropylene tubes. Subsamples were acidified to 0.2% HNO3 (Suprapur, MilliporeSigma) and stored at 4 °C until analysis by inductively coupled plasma-atomic emission spectrophotometry (ICP-AES; Varian 730-ES). The measured dissolved concentrations (µg/L) of each metal in tests was calculated as the geometric mean of initial and final concentrations for each treatment.Statistical analysesSingle metal response data from day 4 and day 7 counts were modelled to estimate the effect concentrations (EC) and lethal concentrations (LC) for 10 and 50 percent response relative to controls (EC10 and EC50, LC10 and LC50) for each metal.Response data and measured metal concentrations from toxicity tests are shown in spreadsheet 'Rotifer_toxicity test responses and metal conc.xlsx'The R code used to model dose response and estimate the effect concentrations (EC) and lethal concentrations (LC) for 10 and 50 percent response relative to controls (EC10 and EC50, LC10 and LC50) are shown in file'2023_Rotifer dose response modelling.R'This R file also contains the code used to model mixture toxicity to give predicted toxicities, which are compared with observed toxicity using toxic units. The dose response data file used as input to the R analysis is shown in file '2023 Antarctic Rotifer.csv'The results of this study are described in the manuscript 'High sensitivity to metals of the Antarctic rotifer Adineta editae and their ecological relevance in contaminated site risk assessments'

本数据集包含南极淡水陆生蛭形轮虫Adineta editae的毒性测试结果。本研究评估了铜、锌、镉、铅和镍这五种金属单独及混合暴露时对Adineta editae的水生毒性。毒性测试于2017/18年南半球夏季在凯西站进行，所用轮虫采自野外并在实验室中分离纯化。

轮虫收集
轮虫来源于南极东部风车群岛雪莉岛短暂融水溪流中的Prassiola crispa藻垫。Adineta editae被分离为单物种水培体系，以备毒性测试使用。培养维持和毒性测试均在温度控制箱中进行，条件为5°C、18/6小时光暗周期。轮虫物种鉴定通过DNA测序（BOLD序列OZBOL4598-21）确认，与单倍型Bd8_02的匹配度为99.52%。

毒性测试
Adineta editae的毒性测试于2018年1月至2月在凯西站实验室开展。暴露实验包括五种金属的单独处理及两种比例的混合处理：环境混合组（EvM，混合组1）和等毒性混合组（EqM，混合组2）。每种混合组采用不同的固定金属比例，通过乘以不同因子生成浓度系列。环境混合组1的比例大致基于南极东部凯西站附近遗留废物点相邻海域布朗湾的金属浓度报告，比例为1 Cu: 0.1 Cd: 0.3 Ni: 0.3 Pb: 4.5 Zn，测试倍数为5至70倍。混合组2接近等摩尔比例，铅浓度较低，比例为1.1 Cu: 0.7 Cd: 0.8 Ni: 0.3 Pb: 1.0 Zn，测试倍数为0.2至2倍。各处理组中每种金属的实测浓度用于混合毒性模型以预测毒性，并通过毒性单位将预测毒性与观察到的毒性进行比较。

金属储备液使用分析纯试剂在超纯水中制备，并用HCl（Suprapur，MilliporeSigma）酸化至0.2%。暴露实验在塑料微孔板中进行，每个处理包括5个金属浓度组及对照组，每组3个重复孔，每孔8只轮虫。向测试孔添加轮虫时，在显微镜下观察收集皿，选择小型非怀卵个体。使用微量移液器将每只轮虫从收集皿中转移至测试孔，每孔转移8只（代表一个重复），转移体积为8 µL培养水。转移完成后，每孔加入236 µL测试溶液，最终体积为300 µL。测试期间轮虫不喂食，但转移水中携带的少量悬浮藻类碎屑可提供有限食物。微孔板用Parafilm密封，置于托盘内放入5°C、18/6小时光暗周期的培养箱中。

在测试第4天和第7天，通过体视显微镜观察轮虫，评估存活和行为终点。存活状态（存活/死亡）通过体型和活动性定义：死亡轮虫表现为明显松弛扁平的体型，无肌肉收缩或活动迹象；存活个体进一步通过运动行为评分（活跃/不活跃）：活跃个体表现出正常运动行为（爬行、摄食），不活跃个体身体完全收缩形成囊状，无外部活动。

化学分析
为测定测试处理中的金属暴露浓度，在每个毒性测试开始时制备两组不含轮虫的牺牲微孔板，每组含各暴露溶液共6 mL。这些微孔板与毒性测试用板相同，但不含轮虫。每组牺牲板在测试开始（第0天）和结束（第7天）时通过注射器取样至干净聚丙烯管中。子样品用HNO3（Suprapur，MilliporeSigma）酸化至0.2%，并在4°C下储存，直至通过电感耦合等离子体原子发射光谱法（ICP-AES；Varian 730-ES）分析。各处理中每种金属的实测溶解浓度（µg/L）为初始和最终浓度的几何平均值。

统计分析
基于第4天和第7天的单金属响应数据建模，以估算每种金属相对于对照组的10%和50%效应浓度（EC）及致死浓度（LC）（即EC10、EC50、LC10和LC50）。

毒性测试的响应数据和实测金属浓度见电子表格“Rotifer_toxicity test responses and metal conc.xlsx”。用于剂量响应建模及估算效应浓度（EC）和致死浓度（LC）的R代码见文件“2023_Rotifer dose response modelling.R”，该文件还包含混合毒性建模代码，用于预测毒性并通过毒性单位与观察毒性比较。R分析的输入数据文件为“2023 Antarctic Rotifer.csv”。本研究结果详见手稿《南极轮虫Adineta editae对金属的高敏感性及其在污染场地风险评估中的生态相关性》