Toxicity of eleven herbicides and one fungicide to the marine alga Tisochrysis lutea (Haptophyta) (NESP TWQ 3.1.5, AIMS)

This dataset shows the effects of herbicides and one fungicide (detected in Great Barrier Reef catchments) on the specific growth rates (from cell density data) of the microalgae Tisochrysis lutea during laboratory experiments conducted from 2018-2019.

The aim of this project was to apply standard ecotoxicology protocols to determine the effects of Photosystem II (PSII), alternative herbicides and one fungicide on the growth of the marine microalgae Tisochrysis lutea. Growth bioassays were performed over 3-day exposures using pesticides that have been detected in the Great Barrier Reef catchment area (O'Brien et al., 2016). These toxicity data will enable improved assessment of the risks posed by PSII and alternative herbicides as well as the fungicide propiconazole to microalgae for both regulatory purposes and for comparison with other taxa.

Methods:
The haptophyte Tisochrysis lutea (formerly known as Isochrysis galbana)(Grant etal. 2017) (strain CS-177) was purchased from the Australian National Algae Supply Service, Hobart (CSIRO). Cultures of T. lutea were established in EDTA-free Guillard’s f/2 marine medium (Trenfield et al. 2015) (1 ml L-1 of f/2 medium in autoclaved natural seawater). Cultures were maintained in sterile 500 ml Erlenmeyer flasks as batch cultures in exponential growth phase with weekly aseptically transfers of 10 ml T. lutea suspension to 300 ml f/2 medium. Culture were maintained at 28 ± 1°C, 33 ± 1.5 psu and under a 12:12 h light:dark cycle (80 – 100 µmol photons m–2 s–1).

Pesticide stock solutions were prepared using PESTANAL (Merck) analytical grade products (purity greater than or equal to 98%): diuron (CAS 330-54-1), metribuzin (CAS 21087-64-9), tebuthiuron (CAS 34014-18-1), bromacil (CAS 314-40-9), propazine (CAS 139-40-2), simazine (122-34-9), imazapic (CAS 104098-48-8), haloxyfop-p-methyl (CAS 72619-32-0), 2,4-D (CAS 94-75-7), MCPA (CAS 94-74-6), fluroxypyr (CAS 69377-81-7) and propiconazole (CAS 60207-90-1). The selection of pesticides was based on application rates and detection in coastal waters of the GBR (Grant et al. 2017, O’Brien et al. 2016). Pesticide stock solutions (100 – 1,000 mg L-1) were prepared by dissolving aliquots of the pure compounds in ultrapure water using clean, acid-washed (5% nitric acid) glass screw-top containers. Simazine, tebuthiuron and haloxyfop-p-methyl were dissolved using the carrier dimethyl sulfoxide (DMSO) (less than or equal to 0.02 % (v/v) in exposure solutions). Diuron, imazapic, metribuzin, bromacil, 2,4-D, propazine, MCPA, fluroxypyr and propiconazole were dissolved in acetone (less than or equal to 0.01 % (v/v) in exposure). Stock solutions were stored refrigerated and in the dark.

Tests were conducted as previously described (Trenfield et al. 2015). Cultures of T. lutea were exposed to increasing concentrations of individual pesticides over a period of 72 h. Inoculum was taken from cultures in exponential growth phase (4-d old culture) and starting cell density assessed using a haemocytometer. For each treatment, a total volume of 250 mL test media was prepared in a clean, acid-washed 500 mL Schott bottle. Test media consisted of filtered (0.45 µm) seawater spiked with the respective pesticide stock, quarter strength EDTA-free f/2 media as nutrient source and T. lutea at a starting density of 3x103 or 1x104 cells mL-1. In each toxicity test, the response (specific growth rate of the culture) of the treatments exposed to pesticide were assessed against a seawater control group (no herbicide).

For each test, 2 – 3 replicate 125 mL Erlenmeyer flasks (50 mL test volume) were assessed. Flasks were incubated at 27 – 29.0°C under a 12:12 h light:dark cycle (80 – 100 µmol photons m–2 s–1). After 72h, sub-samples (7 ml) were taken from each flask and cell densities measured using a flow cytometer (BD Accuri C6, BD Biosciences, CA, USA). Specific growth rates (SGR) were expressed as the logarithmic increase in cell density from day i (ti) to day j (tj) as per equation (1), where SGRi-j is the specific growth rate from time i to j; Xj is the cell density at day j and Xi is the cell density at day i (OECD 2011).

SGR i-j = [(ln Xj - ln Xi )/(tj - ti )] (day-1) (1)

Mean SGR for a pesticide treatment, relative to the mean control SGR was used to derive chronic effect values for growth inhibition. A test was considered valid, if the SGR of control replicates was greater than or equal to 0.92 day-1 4. Physical and chemical characteristics of each treatment were measured at 0 h and 72 h including pH, salinity, electrical conductivity and dissolved oxygen. Temperature was logged in 10-min intervals over the duration of the test. Sub-samples for chemical analysis were taken at 0 h and 72 h from each treatment.

Format:
The dataset is summarised in one file named ‘Tisochrysis lutea pesticide toxicity data_eAtlas.xlsx’

Data Dictionary:
The excel spreadsheet has one tab for each pesticide which incl specific growth rate (SGR) data. The last tab of the dataset shows the measured (start and end of test) water quality (WQ) parameters (pH, salinity, dissolved oxygen (DO), and temperature) of each pesticide test.

For each ‘pesticide’_SGR tab:

SGR = specific growth rate - the logarithmic increase from day 0 to day 3
Nominal (µg/L) = nominal herbicide concentrations used in the bioassays
Measured (µg/L) = measured concentrations analysed by The University of Queensland
Rep = replicate notation is 1-3
Average T3_CellsPer_ml = cell density at day 3
Average ln(day3) = natural logarithm of cell density at day 3
T0_CellsPer_ml = average cell density at day 0
ln(day0) = natural logarithm of cell density at day 0

References:
O’Brien, D. et al. Spatial and temporal variability in pesticide exposure downstream of a heavily irrigated cropping area: application of different monitoring techniques. J. Agric. Food Chem. 64, 3975-3989 (2016).

Grant, S. et al. Marine Monitoring Program: Annual Report for inshore pesticide monitoring 2015-2016. Report for the Great Barrier Reef Marine Park Authority, Great Barrier Reef Marine Park Authority, Townsville, Australia. 128 pp, http://dspace-prod.gbrmpa.gov.au/jspui/handle/11017/13325 (2017).

Trenfield, M. A. et al. Aluminium, gallium, and molybdenum toxicity to the tropical marine microalga Isochrysis galbana. Environ. Toxicol. Chem. 34, 1833-1840, doi:10.1002/etc.2996 (2015).

OECD. Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test. (OECD Publishing, 2011).

Data Location:

This dataset is filed in the eAtlas enduring data repository at: data\nesp3\3.1.5_Pesticide-guidelines-GBR

本数据集呈现了2018-2019年室内实验中，除草剂及一种在大堡礁（Great Barrier Reef）流域中检出的杀菌剂对微藻（microalgae）鲁氏四鞭藻（Tisochrysis lutea）比生长速率（基于细胞密度数据计算）的影响。

本研究的目标为采用标准生态毒理学（ecotoxicology）规程，探究光系统II（Photosystem II, PSII）抑制剂类除草剂、替代除草剂及一种杀菌剂对海洋微藻鲁氏四鞭藻（Tisochrysis lutea）生长的影响。实验采用大堡礁流域已检出的农药（O'Brien等，2016）开展为期3天的暴露生长生物测定（bioassays）。本毒性数据可优化评估光系统II抑制剂类除草剂、替代除草剂以及杀菌剂丙环唑（propiconazole）对微藻的生态风险，可用于监管决策及与其他分类单元（taxa）的毒性数据对比。

### 实验方法
本实验所用定鞭藻门（haptophyte）鲁氏四鞭藻（Tisochrysis lutea，原名为Isochrysis galbana，Grant等，2017）菌株CS-177购自澳大利亚霍巴特市澳大利亚国家藻类供应中心（CSIRO）。采用无乙二胺四乙酸（EDTA）的Guillard f/2海水培养基（Guillard’s f/2 marine medium，Trenfield等，2015，即每升高压灭菌天然海水中添加1mL f/2培养基）构建鲁氏四鞭藻培养体系。将菌株置于无菌500mL锥形瓶（Erlenmeyer flasks）中以分批培养方式维持于指数生长相（exponential growth phase），每周以无菌操作转接10mL鲁氏四鞭藻悬浮液至300mL新鲜f/2培养基中。培养条件设置为：温度28±1℃，盐度33±1.5实用盐度单位（psu），光暗周期（light:dark cycle）12:12小时，光照强度80~100 μmol光子·m⁻²·s⁻¹。

采用默克（Merck）PESTANAL品牌分析纯（analytical grade）试剂（纯度≥98%）配制农药储备液（stock solutions）：敌草隆（diuron，CAS 330-54-1）、嗪草酮（metribuzin，CAS 21087-64-9）、丁噻隆（tebuthiuron，CAS 34014-18-1）、溴谷隆（bromacil，CAS 314-40-9）、扑灭津（propazine，CAS 139-40-2）、西玛津（simazine，CAS 122-34-9）、甲氧咪草烟（imazapic，CAS 104098-48-8）、精吡氟禾草灵（haloxyfop-p-methyl，CAS 72619-32-0）、2,4-二氯苯氧乙酸（2,4-D，CAS 94-75-7）、4-氯-2-甲基苯氧乙酸（MCPA，CAS 94-74-6）、氟草烟（fluroxypyr，CAS 69377-81-7）及丙环唑（propiconazole，CAS 60207-90-1）。农药筛选依据为大堡礁近岸水域的检出情况及田间施用剂量（Grant等，2017；O’Brien等，2016）。将对应纯品称样后，使用经5%硝酸酸洗的洁净玻璃螺口容器，溶于超纯水（ultrapure water）配制浓度为100~1000 mg·L⁻¹的农药储备液。其中西玛津、丁噻隆及精吡氟禾草灵以二甲基亚砜（dimethyl sulfoxide, DMSO）为助溶剂，最终暴露体系中助溶剂体积占比≤0.02%；敌草隆、甲氧咪草烟、嗪草酮、溴谷隆、2,4-D、扑灭津、MCPA、氟草烟及丙环唑以丙酮（acetone）为助溶剂，最终暴露体系中助溶剂体积占比≤0.01%。储备液需于避光冷藏条件下保存。

实验参照已发表方法开展（Trenfield等，2015）。将鲁氏四鞭藻暴露于系列梯度浓度的单一农药中，暴露时长为72小时。接种藻种取自指数生长相的4天龄培养物，初始细胞密度通过血球计数板（haemocytometer）测定。每组处理均在经5%硝酸酸洗的洁净500mL肖特瓶中配制250mL试验培养基：试验培养基由经0.45μm过滤的天然海水、1/4浓度无EDTA的f/2培养基（作为营养源）及鲁氏四鞭藻藻液组成，藻液初始密度设置为3×10³或1×10⁴ cells·mL⁻¹。每个毒性实验均设置海水对照组（不添加除草剂），以农药暴露组的培养物比生长速率作为响应指标，与对照组进行对比。

每个处理设置2~3个重复，取125mL锥形瓶装入50mL试验培养基。将锥形瓶置于27~29℃、光暗周期12:12小时、光照强度80~100 μmol光子·m⁻²·s⁻¹的条件下培养。暴露72小时后，从每个锥形瓶中采集7mL子样本，使用流式细胞仪（flow cytometer，BD Accuri C6，美国加利福尼亚州BD生物科学公司）测定细胞密度。比生长速率（SGR）按照公式(1)计算，以细胞密度从时间点i（ti）到时间点j（tj）的对数增长表示，其中SGRi-j为时间i至j的比生长速率，Xj为j时刻的细胞密度，Xi为i时刻的细胞密度（OECD，2011）：
$$ ext{SGR}_{i-j} = frac{ln X_j - ln X_i}{t_j - t_i} quad ( ext{day}^{-1}) ag{1}$$
以农药处理组的平均比生长速率与对照组平均比生长速率的比值，推导生长抑制的慢性效应值（chronic effect values）。若对照组重复的比生长速率≥0.92 day⁻¹，则判定该实验有效。分别于暴露0小时和72小时测定每组处理的理化指标，包括pH、盐度、电导率（electrical conductivity）及溶解氧（dissolved oxygen, DO）。实验全程以10分钟为间隔记录温度数据。分别于0小时和72小时采集每组处理的子样本用于化学分析。

### 数据集格式
本数据集汇总于名为《鲁氏四鞭藻农药毒性数据_eAtlas.xlsx》的单一文件中。

### 数据字典
本Excel文件的每个工作表（tab）对应一种农药，包含比生长速率（SGR）数据；最后一个工作表展示各农药毒性实验的实测水质参数（WQ），包括实验初始与结束时的pH、盐度、溶解氧（DO）及温度。
每个“农药名_SGR”工作表的字段说明如下：
- SGR：比生长速率——第0天至第3天的细胞密度对数增长值
- Nominal (µg/L)：生物测定中所用除草剂的名义浓度
- Measured (µg/L)：昆士兰大学测定的实测浓度
- Rep：重复编号，取值为1~3
- Average T3_CellsPer_ml：第3天的平均细胞密度
- Average ln(day3)：第3天细胞密度的自然对数
- T0_CellsPer_ml：第0天的平均细胞密度
- ln(day0)：第0天细胞密度的自然对数

### 参考文献
1. O’Brien, D. 等. 高灌溉量农区下游农药暴露的时空变异：不同监测技术的应用. 农业与食品化学杂志, 64, 3975-3989 (2016).
2. Grant, S. 等. 海洋监测计划：2015-2016近岸农药监测年度报告. 大堡礁海洋公园管理局报告, 澳大利亚汤斯维尔, 大堡礁海洋公园管理局. 128页, http://dspace-prod.gbrmpa.gov.au/jspui/handle/11017/13325 (2017).
3. Trenfield, M. A. 等. 铝、镓及钼对热带海洋微藻Isochrysis galbana的毒性. 环境毒理与化学, 34, 1833-1840, doi:10.1002/etc.2996 (2015).
4. OECD. 试验方法No.201：淡水藻类及蓝细菌生长抑制试验. 经合组织出版社, 2011.

### 数据集存储位置
本数据集存储于eAtlas永久数据仓库中，路径为：data\nesp3\3.1.5_Pesticide-guidelines-GBR