Effects of herbicide exposure on growth and photosynthetic efficiency of the microalgae Chlorella sp. (Chlorophyta) (NESP TWQ 3.1.5, AIMS and JCU)

This dataset shows the effects of herbicides (detected in the Great Barrier Reef catchments) on the growth rates (from cell density data) and photosynthesis (effective quantum yield) on the microalgae Chlorella sp. during laboratory experiments conducted from 2017-2019.The aims of this project were to develop and apply standard ecotoxicology protocols to determine the effects of Photosystem II (PSII) and alternative herbicides on the growth and photosynthetic efficiency of the microalgae Chlorella sp. Growth bioassays were performed over 3-day exposures using herbicides that have been detected in the Great Barrier Reef catchment area (O’Brien et al. 2016). Effects of herbicides on the photophysiology of Chlorella sp., measured by chlorophyll fluorescence as the effective quantum yield (Delta F/Fm’) were investigated using mini-PAM fluorometry after 72 h herbicide exposure. These toxicity data will enable improved assessment of the risks posed by PSII and alternative herbicides to microalgae for both regulatory purposes and for comparison with other taxa.Methods:The chlorophyte Chlorella sp. was sourced from the Supervising Scientist, Department Energy and Environment, Darwin. Cultures of Chlorella sp. were established in MBL medium (Riethmuller et al 2003, Pease et al 2016). Cultures were maintained in sterile 250 mL Erlenmeyer flasks as batch cultures in exponential growth phase with weekly transfers of 1 - 3 mL of a 7 day-old Chlorella sp. suspension to 100 mL MBL medium under sterile conditions. Clean culture solutions were maintained at 26 ± 2°C, and under a 12:12 h light:dark cycle (91 ± 12 µmol photons m–2 s–1).Herbicide stock solutions were prepared using PESTANAL (Sigma-Aldrich) analytical grade products (HPLC greater than or equal to 98%): bromacil (CAS 314-40-9), diuron (CAS 330-54-1), haloxyfop-p-methyl (CAS 72619-32-0), hexazinone (CAS 51235-04-2), imazapic (CAS 104098-48-8), isoxaflutole (CAS 141112-29-0), prometryn (CAS 7287-19-6) and propazine (CAS 139-40-2). The selection of herbicides was based on application rates and detection in coastal waters of the GBR (Grant et al. 2017, O’Brien et al. 2016). Stock solutions were prepared in sterile 1 L glass Schott bottles using milli-Q water. Diuron, haloxyfop-p-methyl, hexazinone, isoxaflutole and prometryn were dissolved using analytical grade acetone (< 0.01% (v/v) in exposures). Imazapic was dissolved in methanol (less than or equal to 0.01% (v/v) in exposure). No solvent carrier was used for the preparation of the remaining herbicide stock solutions.Cultures of Chlorella sp. were exposed to a range of herbicide concentrations over a period of 72 h. Inoculum was taken from cultures in exponential growth phase (4 – 7 day-old cultures). A Chlorella sp. working suspension was prepared in a 100 mL volumetric flask. A 1:10 and 1:100 dilution was prepared and counted using a haemocytometer under a compound microscope to determine appropriate dilution volumes. The pre-determined inoculum was added to 50 mL of each test and control treatment replicates to the required dilution (3 – 3.1 x 104 cells/ mL). In each toxicity test, a control (no herbicide) and solvent control (if used) treatments were added to support the validity of the test protocols and to monitor continued performance of the assays. All treatment solutions were prepared in 0.5x strength MBL medium. Replicates were incubated at 26.6 ± 0.5 °C under a 12:12 h light:dark cycle (190 ± 14 µmol photons m–2 s–1). Sub-samples were taken from each replicate to measure cell densities of algal populations at 72 h using a haemocytometer and photographed under phase contrast conditions. Cell counts were done either manually or using imageJ from microscope photographs (Rueden et al 2017). 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)SGR relative to the control treatment 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 (OECD 2011). Physical and chemical characteristics of each treatment were measured at 0 h and 72 h including pH, electrical conductivity and temperature. Chamber temperature was also logged in 15-min intervals over the total test duration. Analytical samples were taken at 0 h and 72 h.Effects of herbicides on the photophysiology of Chlorella sp., measured by chlorophyll fluorescence as the effective quantum yield (Delta F/Fm’ ), were investigated at 72 h using mini-PAM fluorometry (mini-PAM, Walz, Germany). Light adapted minimum fluorescence (F) and maximum fluorescence (Fm’) were determined and effective quantum yield was calculated for each treatment as per equation (2)(Schreiber et al. 2002). Delta F/Fm’ = (Fm’-F)/Fm’                                              (2)Mini- PAM settings were set to ETR-F = 0.84, F-Offset = 92, measuring light frequency = 3, measuring intensity = 4, gain = 3; damp = 3. Saturation pulse settings: intensity = 6, width = 0.6.Mean percent inhibition in SGR and Delta F/Fm’ of each treatment relative to the control treatment was calculated as per equation (3)(OECD 2011), where Xcontrol is the average SGR or Delta F/Fm’ of control and Xtreatment is the average SGR or Delta F/Fm’ of single treatments.% Inhibition = [(X control - X treatment )/X control] x 100                             (3)Format:Chlorella sp.herbicide toxicity data_eAtlas.xlsxData Dictionary:There are two tabs for each herbicide in the spreadsheet. The first tab corresponds to the specific growth rate (SGR) data; the second tab is the pulse amplitude modulation (PAM) fluorometry data. The last tab of the dataset shows the measured water quality (WQ) parameters (pH, electrical conductivity and temperature) of each herbicide test. Brom - BromacilDiu – DiuronHalo – HaloxyfopHex - HexazinoneImaz – ImazapicIsox - IsoxaflutoleProm - PrometrynProp - PropazineFor each ‘herbicide’_SGR tab:SGR = specific growth rate – the logarithmic increase from day 0 to day 3Nominal (µg/L) = nominal herbicide concentrations used in the bioassays; SC denotes solvent control which is no herbicide and contains less than 0.01% v/v solvent carrier as per the treatmentsMeasured (µg/L) = measured concentrations analysed by The University of QueenslandRep = Replicate: for SGR, notation is 1-3; for PAM data, notation is 1-3T3_CellsPerMl = cell density at day 3 ln(day3) = natural logarithm of cell density at day 3Average T0_CellsPerMl = average cell density at day 0ln(Day0) = natural logarithm of cell density at day 0For each ‘herbicide’_PAM tab:PAM = pulse amplitude modulated fluorometry to calculate effective quantum yield (light adapted)Nominal (µg/L) = nominal herbicide concentrations used in the bioassays; SC denotes solvent control which is no herbicide and contains less than 0.01% v/v solvent carrier as per the treatments Measured (µg/L) = measured concentrations analysed by The University of QueenslandRep = Replicate: for SGR, notation is 1-3; for PAM data, notation is 1-3Delta F/Fm’ = effective quantum (light adapted) yield measured by a Pulse Amplitude Modulation (PAM) fluorometerReferences:Grant, S., Gallen, C., Thompson, K., Paxman, C., Tracey, D. and Mueller, J. (2017) 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 O’Brien, D., Lewis, S., Davis, A., Gallen, C., Smith, R., Turner, R., Warne, M., Turner, S., Caswell, S. and Mueller, J.F. (2016) Spatial and temporal variability in pesticide exposure downstream of a heavily irrigated cropping area: application of different monitoring techniques. Journal of Agricultural and Food Chemistry 64(20), 3975-3989.OECD (2011) OECD guidelines for the testing of chemicals: freshwater alga and cyanobacteria, growth inhibition test, Test No. 201. https://search.oecd.org/env/test-no-201-alga-growth-inhibition-test-9789264069923-en.htm (accessed 28 August 2019). Pease C, Mooney T, Trenfield M, Costello C & Harford A (2016). Updated procedure for the 72 hour algal growth inhibition toxicity test using Chlorella sp. Internal Report 645, September, Supervising Scientist, DarwinRiethmuller, N., Camilleri, C., Franklin, N., Hogan, A., King, A., Koch, A., Markich, S.J., Turley, C. and van Dam, R. (2003) Ecotoxicological testing protocols for Australian tropical freshwater ecosystems. Supervising Scientist Report 173, Supervising Scientist, Darwin NT.Rueden, C.T., Schindelin, J., Hiner, M.C. et al. (2017) ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18:529, PMID 29187165, doi:10.1186/s12859-017-1934-zData Location:This dataset is filed in the eAtlas enduring data repository at: dataesp3\3.1.5_Pesticide-guidelines-GBR

本数据集呈现了2017-2019年室内控制实验中，于大堡礁流域检出的除草剂对微藻（microalgae）小球藻属（Chlorella sp.）生长速率（基于细胞密度数据）及光合作用（有效量子产率）的影响。本研究的核心目标为建立并应用标准化生态毒理学实验方案，以探究光系统II（Photosystem II, PSII）抑制剂类及其他替代除草剂对小球藻属生长与光合效率的影响。

实验采用已在大堡礁流域检出的除草剂开展为期3天的暴露生物测定（O’Brien等，2016）。在72小时除草剂暴露结束后，通过迷你脉冲振幅调制荧光仪（mini-PAM fluorometry）测定叶绿素荧光参数有效量子产率（ΔF/Fm’），以此分析除草剂对小球藻属光生理特性的作用效果。本毒性数据集可为优化评估光系统II抑制剂及替代除草剂对微藻的生态风险提供支撑，同时可用于监管决策制定及与其他生物类群的毒性数据对比。

### 实验方法
实验所用绿藻小球藻属（Chlorella sp.）菌株源自澳大利亚达尔文能源与环境部监管科学家项目组。采用MBL培养基（MBL medium，Riethmuller等，2003；Pease等，2016）建立小球藻属培养体系。将培养物置于250 mL无菌锥形瓶中作为分批培养物，维持其处于指数生长阶段：每周将1~3 mL 7日龄的小球藻属悬浮液转接至100 mL新鲜MBL培养基中，操作全程保持无菌环境。洁净培养体系维持在26±2 ℃，光暗周期为12:12 h，光照强度为91±12 μmol光子·m⁻²·s⁻¹。

除草剂储备液采用西格玛奥德里奇（Sigma-Aldrich）PESTANAL分析级产品（高效液相色谱纯度≥98%）配制，包括：溴谷隆（bromacil，CAS 314-40-9）、敌草隆（diuron，CAS 330-54-1）、精氟吡甲禾灵（haloxyfop-p-methyl，CAS 72619-32-0）、环嗪酮（hexazinone，CAS 51235-04-2）、甲氧咪草烟（imazapic，CAS 104098-48-8）、异恶唑草酮（isoxaflutole，CAS 141112-29-0）、扑草净（prometryn，CAS 7287-19-6）以及丙津（propazine，CAS 139-40-2）。除草剂的筛选基于其田间施用剂量及大堡礁近岸水域检出情况（Grant等，2017；O’Brien等，2016）。储备液采用超纯水在1 L无菌肖特玻璃瓶中配制。其中，敌草隆、精氟吡甲禾灵、环嗪酮、异恶唑草酮及扑草净采用分析级丙酮溶解（暴露体系中溶剂体积占比≤0.01%）；甲氧咪草烟采用甲醇溶解（暴露体系中溶剂体积占比≤0.01%）；其余除草剂储备液无需添加溶剂载体。

将小球藻属培养物暴露于一系列梯度浓度的除草剂中，暴露时长为72小时。接种物取自指数生长阶段的培养物（培养4~7日龄）。将100 mL容量瓶制备为小球藻属工作悬浮液，分别进行1:10和1:100倍稀释后，采用血细胞计数板（haemocytometer）在正置显微镜下计数，以确定合适的接种体积。将预定量的接种物加入至50 mL各处理组及对照组体系中，使最终细胞密度达到3~3.1×10⁴ 细胞·mL⁻¹。每组毒性实验均设置空白对照组（无除草剂）及溶剂对照组（若使用溶剂），以验证实验方案的有效性并监测生物测定的持续性能。所有处理体系均采用0.5倍浓度的MBL培养基配制。培养体系置于26.6±0.5 ℃环境中，光暗周期为12:12 h，光照强度为190±14 μmol光子·m⁻²·s⁻¹。

从每个重复体系中取样，于72小时后采用血细胞计数板测定藻类细胞密度，并通过相差显微镜拍摄图像。细胞计数可采用人工计数或通过ImageJ软件分析显微镜图像（Rueden等，2017）。比生长速率（specific growth rate, SGR）以第i天（ti）至第j天（tj）的细胞密度对数增长值计算，计算公式如式(1)所示，其中SGRi-j为第i天至第j天的比生长速率；Xj为第j天的细胞密度，Xi为第i天的细胞密度（OECD，2011）：
$$ ext{SGR}_{i-j} = frac{ln X_j - ln X_i}{t_j - t_i} quad ( ext{day}^{-1}) ag{1}$$
采用相对于对照组的相对比生长速率，推导生长抑制的慢性效应值。若对照组重复体系的比生长速率≥0.92 day⁻¹，则认为该实验有效（OECD，2011）。在实验0小时及72小时时测定各处理体系的理化参数，包括pH、电导率及温度。实验全程以15分钟为间隔记录培养箱温度。分别于0小时及72小时采集分析样品。

于72小时后，采用迷你脉冲振幅调制荧光仪（mini-PAM, Walz, 德国）测定叶绿素荧光参数有效量子产率（ΔF/Fm’），以此分析除草剂对小球藻属光生理特性的影响。测定光适应下的最小荧光（F）及最大荧光（Fm’），并根据式(2)计算各处理组的有效量子产率（Schreiber等，2002）：
$$Delta F/Fm' = frac{Fm' - F}{Fm'} ag{2}$$
mini-PAM的参数设置为：ETR-F=0.84，F-Offset=92，测量光频率=3，测量强度=4，增益=3，阻尼=3；饱和脉冲参数：强度=6，宽度=0.6。

根据式(3)计算各处理组相对于对照组的比生长速率及有效量子产率的平均抑制百分比（OECD，2011），其中Xcontrol为对照组的比生长速率或有效量子产率平均值，Xtreatment为单一组处理的比生长速率或有效量子产率平均值：
$$\% ext{ Inhibition} = frac{X_{ ext{control}} - X_{ ext{treatment}}}{X_{ ext{control}}} imes 100 ag{3}$$

### 数据集格式
本数据集存储为Excel文件："Chlorella sp. herbicide toxicity data_eAtlas.xlsx"
#### 数据字典
该电子表格中每种除草剂对应两个工作表：第一个工作表为比生长速率（SGR）数据，第二个工作表为脉冲振幅调制（pulse amplitude modulation, PAM）荧光测定数据。数据集的最后一个工作表为各除草剂毒性实验的实测水质（water quality, WQ）参数，包括pH、电导率及温度。
缩写对应如下：
Brom：溴谷隆（Bromacil）
Diu：敌草隆（Diuron）
Halo：精氟吡甲禾灵（Haloxyfop-p-methyl）
Hex：环嗪酮（Hexazinone）
Imaz：甲氧咪草烟（Imazapic）
Isox：异恶唑草酮（Isoxaflutole）
Prom：扑草净（Prometryn）
Prop：丙津（Propazine）

针对每个`*herbicide*_SGR`工作表：
- SGR：比生长速率——第0天至第3天的对数增长值
- 标称浓度（Nominal, µg/L）：生物测定中使用的标称除草剂浓度；SC代表溶剂对照组，即无除草剂且溶剂体积占比≤0.01%的处理组，与实验组溶剂条件一致
- 实测浓度（Measured, µg/L）：由昆士兰大学分析测定的实际除草剂浓度
- Rep：重复样本编号：比生长速率数据中为1~3；PAM荧光数据中为1~3
- T3_CellsPerMl：第3天的细胞密度
- ln(day3)：第3天细胞密度的自然对数
- Average T0_CellsPerMl：第0天的平均细胞密度
- ln(Day0)：第0天细胞密度的自然对数

针对每个`*herbicide*_PAM`工作表：
- PAM：脉冲振幅调制荧光仪，用于计算光适应下的有效量子产率
- 标称浓度（Nominal, µg/L）：生物测定中使用的标称除草剂浓度；SC代表溶剂对照组，即无除草剂且溶剂体积占比≤0.01%的处理组，与实验组溶剂条件一致
- 实测浓度（Measured, µg/L）：由昆士兰大学分析测定的实际除草剂浓度
- Rep：重复样本编号：比生长速率数据中为1~3；PAM荧光数据中为1~3
- Delta F/Fm’：通过脉冲振幅调制（PAM）荧光仪测定的光适应下有效量子产率

#### 参考文献
1. Grant, S., Gallen, C., Thompson, K., Paxman, C., Tracey, D. and Mueller, J. (2017) 海洋监测计划：2015-2016年近岸农药监测年度报告。提交给大堡礁海洋公园管理局，大堡礁海洋公园管理局，澳大利亚汤斯维尔。共128页，http://dspace-prod.gbrmpa.gov.au/jspui/handle/11017/13325
2. O’Brien, D., Lewis, S., Davis, A., Gallen, C., Smith, R., Turner, R., Warne, M., Turner, S., Caswell, S. and Mueller, J.F. (2016) 重度灌溉种植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(20)，3975-3989。
3. OECD (2011) 化学品测试指南：淡水藻类及蓝细菌生长抑制试验，试验编号201。https://search.oecd.org/env/test-no-201-alga-growth-inhibition-test-9789264069923-en.htm（2019年8月28日访问）。
4. Pease C, Mooney T, Trenfield M, Costello C & Harford A (2016). 小球藻属72小时藻类生长抑制毒性试验更新规程。内部报告645，2016年9月，达尔文监管科学家项目组。
5. Riethmuller, N., Camilleri, C., Franklin, N., Hogan, A., King, A., Koch, A., Markich, S.J., Turley, C. and van Dam, R. (2003) 澳大利亚热带淡水生态系统生态毒理学测试规程。监管科学家报告173，澳大利亚北领地达尔文监管科学家项目组。
6. Rueden, C.T., Schindelin, J., Hiner, M.C. et al. (2017) ImageJ2：面向下一代科学图像数据的ImageJ。BMC生物信息学，18:529，PMID 29187165，doi:10.1186/s12859-017-1934-z

### 数据存储位置
本数据集存储于eAtlas持久化数据存储库中，路径为：dataesp3\3.1.5_Pesticide-guidelines-GBR