Single metal toxicity to the Antarctic marine microalga Cryothecomonas armigera

This metadata record contains the results from 3 bioassays conducted with the Antarctic marine microalgae Cryothecomonas armigera (incertae sedis). These tests assessed the toxicity of copper, cadmium, lead, zinc and nickel. Test conditions for both algae are described in the excel spreadsheets. In summary, tests for P. antarctica and C.armigera, were carried out at 0 plus or minus 2 degrees C, 20:4 h light:dark (60-90 micromol/m2/s, cool white 36W/840 globes), in 80 mL natural filtered (0.22 microns) seawater (salinity - 35 ppt, pH - 8.1 plus or minus 0.2). Filtered seawater was supplemented with 1.5 mg/L NO3- and 0.15 mg/L of PO43-. All tests were carried out in silanised 250-mL glass flasks, with glass lids. Tests 1 and 2 consisted of metal treatments, with 3 replicates per treatment, alongside 3 replicate controls (natural filtered seawater). Test 3 consisted of metal treatments in an increasing series (no replicates) alongside 3 replicate controls. Seawater was spiked with metal solutions to achieve required concentration. Concentrations tested are recorded in excel datasheets as dissolved metal concentrations measured on day 0, and day 24. The average of the dissolved metal concentrations were used for further statistical analysis. The age of C.armigera at test commencement was 25-30 days. Algal cells were centrifuged and washed to remove nutrient rich media, and test flasks were inoculated with between 1-3 x10^3 cells/mL. Cell densities in all toxicity tests were determined by flow cytometry. Toxicity tests with C. armigera were carried out over 23-24 days, with cell densities determined twice a week. The growth rate (cell division; u) was calculated as the slope of the regression line from a plot of log10 (cell density) versus time (h). Growth rates for all treatments were expressed as a percentage of the control growth rates. The flow cytometer was also used to simultaneously measure changes in the following cellular parameters: chlorophyll a autofluorescence intensity (FL3), cell size (FSC) and cell complexity (SSC). The molecular stain BODIPY 493/503, was used to measure neutral lipid concentrations. Changes in cellular parameters were measured by applying a gate that captured greater than 95% of control cells in a region, R2. Changes in cellular parameters were observed in metal treatments as a shift of the cell population from the R2 region to R1 (for relative decreases) or to R3 (for relative increases). The proportion of cells in each region is expressed as a percentage of the total cell population. The pH was measured on the first and last day of the test. Sub-samples (5 mL) for analysis of dissolved metal concentrations were taken from each treatment on 24. Sub-samples were filtered through an acid washed (10% HNO3, Merck) 0.45-microns membrane filter and syringe, and acidified to 0.2% with Tracepur nitric acid (Merck). Metal concentrations were determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES) for Cu, Cd, Pb, Ni and Zn. Detection limits for Cu, Cd, Pb, Ni and Zn were 1.0, 0.3, 3.2, 1.4, and 1.0 micrograms per litre, respectively. Calculations of effect concentrations (EC 10 and 50) were made using the 'Dose Response Curve' package of R statistical analysis software. Concentration-response curves had several models applied to them, and were tested for best fit by comparing residual standard errors and Akaike's 'An Information Criterion' function . Generally, log-logistic models with 3 parameters provided the best fit. Data for each toxicity test is combined in a single excel spreadsheet, "Cryothecomonas armigera single metal toxicity". The first worksheet is titled "Test Conditions" which provides information on the toxicity test, e.g. species and metals tested, dates, test conditions, as well as explanation of abbreviations, definitions of toxicity values etc. The second worksheet includes the raw cell densities determined in each flask, the calculated growth rates, and measured metal concentrations. The third worksheet contains the measured physiological parameters: Neutral lipid concentrations (BODIPY 493/503), chlorophyll a autofluorescence (FL3), cell complexity (SSC), and cell size (FSC). The final worksheet contains the output of statistical analysis; dose-response curves for each metal with applied log-logistic model and 95% confidence interval, a table summarising the effect concentrations (EC10 and EC50), and No Effect Concentration (NEC) is also provided. The file "C. armigera combined.csv" contains rows representing individual exposures with columns for the metal treatment (Metal), averaged dissolved metal concentration for each exposure (Conc), growth rate (Growth), and growth rate as a percent of the control (Pcon). This data was used for data analysis in R statistics. Note that this contains data from all bioassays conducted with C. armigera, including those conducted by Francesca Gissi (doi:10.4225/15/551B2B65A73F3)The script used for data analysis is provided in the document "R statistics script for C. armigera single metal.docx"

本元数据记录包含针对南极海洋微藻刺胞隐藻（Cryothecomonas armigera，分类地位未定）开展的3项生物测定结果。上述试验评估了铜、镉、铅、锌及镍的毒性。两种藻类的试验条件详见各电子表格。总体而言，南极某藻（P. antarctica）与刺胞隐藻（C.armigera）的试验均在温度为0±2℃、光暗周期20:4 h（光照强度60~90 μmol/(m²·s)，采用冷白光36W/840灯泡）、体积80 mL经0.22 μm过滤的天然海水（盐度35 ppt，pH 8.1±0.2）的体系中开展。过滤海水额外添加1.5 mg/L NO₃⁻与0.15 mg/L PO₄³⁻。所有试验均在硅烷化处理的250 mL玻璃烧瓶及配套玻璃瓶盖中进行。试验1与试验2设置金属处理组，每组设3个生物学重复，同时设置3个重复对照组（天然过滤海水）。试验3采用梯度递增的金属处理组（无重复），并设置3个重复对照组。通过向海水中加入金属标准溶液以达到目标浓度。试验所采用的浓度以第0天与第24天测得的溶解金属浓度形式记录于电子数据表中。溶解金属浓度的平均值用于后续统计分析。试验开始时，刺胞隐藻的培养时长为25~30天。将藻细胞离心并洗涤以去除富含营养物的培养基，随后以1~3×10³ cells/mL的密度接种至试验烧瓶中。所有毒性试验的细胞密度均通过流式细胞术（flow cytometry）测定。刺胞隐藻的毒性试验时长为23~24天，每周测定两次细胞密度。生长速率（细胞分裂速率μ）通过以log₁₀(细胞密度)为纵坐标、时间（小时）为横坐标绘制的散点图的回归直线斜率计算得到。所有处理组的生长速率均以对照组生长速率的百分比表示。流式细胞仪还可同时测定以下细胞参数：叶绿素a自发荧光强度（FL3）、细胞大小（前向散射光，FSC）与细胞复杂度（侧向散射光，SSC）。采用分子染色剂BODIPY 493/503测定中性脂浓度。通过设置门控圈取95%以上的对照组细胞至区域R2，以此测定细胞参数的变化。在金属处理组中，细胞群体从R2区域向R1区域（相对减少）或R3区域（相对增加）的偏移体现了细胞参数的变化。各区域内的细胞占总细胞群体的百分比予以记录。试验的第1天与最后1天测定pH值。于第24天从各处理组中采集5 mL子样本用于溶解金属浓度分析。子样本经10%硝酸（默克公司）酸洗的0.45 μm滤膜与注射器过滤后，使用Tracepur硝酸（默克公司）酸化至0.2%。采用电感耦合等离子体原子发射光谱法（inductively coupled plasma-atomic emission spectrometry, ICP-AES；型号Varian 730-ES）测定Cu、Cd、Pb、Ni及Zn的浓度。上述5种金属的检出限分别为1.0、0.3、3.2、1.4与1.0 μg/L。效应浓度（EC10与EC50）的计算采用R统计分析软件的"Dose Response Curve"包完成。浓度-反应曲线拟合采用多种模型，并通过比较残差标准误与赤池信息准则（Akaike's An Information Criterion, AIC）函数筛选最优拟合模型。通常而言，三参数对数逻辑斯蒂模型可提供最优拟合效果。每项毒性试验的数据均整合至单个电子表格"Cryothecomonas armigera single metal toxicity"中。首个工作表标题为"Test Conditions"，包含毒性试验的相关信息，如受试物种与金属、试验日期、试验条件，以及缩写说明、毒性值定义等。第二个工作表包含各烧瓶中测得的原始细胞密度、计算得到的生长速率与实测金属浓度。第三个工作表包含实测生理参数：中性脂浓度（BODIPY 493/503）、叶绿素a自发荧光（FL3）、细胞复杂度（SSC）与细胞大小（FSC）。最后一个工作表包含统计分析结果：各金属经对数逻辑斯蒂模型拟合的剂量反应曲线及95%置信区间、汇总效应浓度（EC10与EC50）的表格，同时提供无效应浓度（NEC）。文件"C. armigera combined.csv"包含代表单次暴露的行数据，列字段包括金属处理组（Metal）、单次暴露的平均溶解金属浓度（Conc）、生长速率（Growth）以及生长速率占对照组的百分比（Pcon）。该数据用于R语言统计分析。需注意，该数据集包含所有针对刺胞隐藻开展的生物测定数据，包括Francesca Gissi完成的试验（doi:10.4225/15/551B2B65A73F3）。数据分析所用脚本已收录于文档"R statistics script for C. armigera single metal.docx"中。