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.xlsx

Data 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 - Bromacil
Diu – Diuron
Halo – Haloxyfop
Hex - Hexazinone
Imaz – Imazapic
Isox - Isoxaflutole
Prom - Prometryn
Prop - Propazine

For each ‘herbicide’_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; 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 Queensland
Rep = Replicate: for SGR, notation is 1-3; for PAM data, notation is 1-3
T3_CellsPerMl = cell density at day 3
ln(day3) = natural logarithm of cell density at day 3
Average T0_CellsPerMl = average cell density at day 0
ln(Day0) = natural logarithm of cell density at day 0

For 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 Queensland
Rep = Replicate: for SGR, notation is 1-3; for PAM data, notation is 1-3
Delta F/Fm’ = effective quantum (light adapted) yield measured by a Pulse Amplitude Modulation (PAM) fluorometer

References:

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, Darwin

Riethmuller, 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-z

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2017-2019年室内实验中，除草剂（于大堡礁流域检出）对微藻小球藻属（Chlorella sp.）生长速率（基于细胞密度数据）与光合作用（有效量子产率）的影响。

本研究旨在开发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）类及其他类除草剂对微藻小球藻属（Chlorella sp.）生长及光合效率的影响。实验采用2016年于大堡礁流域检出的除草剂（O’Brien等，2016），开展了为期3天的暴露生长生物测定。在除草剂暴露72小时后，通过微型脉冲振幅调制（mini-PAM）荧光计测量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（Chlorella sp.）光生理特性的影响。本毒性数据集可用于优化评估光系统II类及其他类除草剂对微藻的风险，既可为监管工作提供支撑，也可用于与其他类群的毒性数据开展对比。

### 实验方法
#### 实验材料与培养
实验所用绿藻小球藻属（Chlorella sp.）菌株由澳大利亚达尔文能源与环境部监管科学家项目组提供。采用MBL培养基（MBL medium）构建小球藻属（Chlorella sp.）培养体系（Riethmuller等，2003；Pease等，2016）。将培养物置于无菌250 mL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（Chlorella sp.）悬浮液转接至100 mL新鲜MBL培养基中，操作全程保持无菌。洁净培养体系维持在26±2℃，光照周期为12:12 h光暗循环，光照强度为91±12 µmol photons m–2 s–1。

#### 除草剂储备液制备
本实验所用除草剂均采用PESTANAL（Sigma-Aldrich）分析纯级产品（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）、精氟禾草灵（haloxyfop-p-methyl, CAS 72619-32-0）、嗪草酮（hexazinone, CAS 51235-04-2）、咪唑乙烟酸（imazapic, CAS 104098-48-8）、异恶唑草酮（isoxaflutole, CAS 141112-29-0）、扑草净（prometryn, CAS 7287-19-6）及扑灭津（propazine, CAS 139-40-2）。除草剂的筛选基于其田间施用量及大堡礁近岸水域检出情况（Grant等，2017；O’Brien等，2016）。储备液采用无菌1 L肖特玻璃瓶配制，溶剂为超纯水（milli-Q水）。其中，敌草隆、精氟禾草灵、嗪草酮、异恶唑草酮及扑草净采用分析纯丙酮溶解（暴露体系中溶剂体积占比<0.01% (v/v)）；咪唑乙烟酸采用甲醇溶解（暴露体系中溶剂体积占比≤0.01% (v/v)）；其余除草剂储备液无需添加溶剂载体。

#### 暴露实验与生长速率测定
将小球藻属（Chlorella sp.）培养物暴露于一系列梯度浓度的除草剂溶液中，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）。先将小球藻属（Chlorella sp.）工作悬浮液配制于100 mL容量瓶中，随后按1:10和1:100比例稀释，通过血球计数板（haemocytometer）在复合显微镜下计数，以确定合适的接种体积。将预定量的接种物加入至50 mL的各处理组及对照组中，使最终细胞密度达到3~3.1×104 cells/mL。
每一组毒性实验均设置空白对照组（无除草剂）及溶剂对照组（若使用溶剂载体），以验证实验方案的有效性并监测生物测定的持续稳定性。所有处理溶液均采用0.5倍浓度的MBL培养基配制。重复组置于26.6±0.5℃、12:12 h光暗循环（光照强度190±14 µmol photons m–2 s–1）的培养箱中培养。
在培养72小时后，从每个重复组中取样，采用血球计数板（haemocytometer）计数藻类细胞密度，并通过相差显微镜（phase contrast）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）定义为从时间ti（第i天）至时间tj（第j天）细胞密度的对数增长，计算公式如式(1)所示，其中SGRi-j为从时间i至j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）：
$$ ext{SGR}_{i-j} = frac{ln X_j - ln X_i}{t_j - t_i} quad ( ext{day}^{-1}) ag{1}$$
以对照组的比生长速率为基准，计算得到生长抑制的慢性效应值。当对照组重复组的比生长速率≥0.92 day-1时，实验判定为有效（OECD，2011）。
在实验开始（0 h）与结束（72 h）时，测量各处理组的理化参数，包括pH、电导率及温度。培养箱温度在整个实验周期内以15分钟为间隔进行记录。分别在0 h和72 h采集分析样品。

#### 光生理特性测定
在除草剂暴露72小时后，采用微型脉冲振幅调制荧光计（mini-PAM, Walz, 德国）测量叶绿素荧光参数，以此分析除草剂对小球藻属（Chlorella sp.）光生理特性的影响，其中有效量子产率（ΔF/Fm’）为核心指标。测定前先获取光适应下的最小荧光（F）与最大荧光（Fm’），随后根据式(2)计算各处理组的有效量子产率（Schreiber等，2002）：
$$Delta F/F_m' = frac{F_m' - F}{F_m'} ag{2}$$
微型PAM的参数设置如下：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)计算各处理组相较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为对照组的平均比生长速率或有效量子产率，Xtreatment为单个处理组的平均比生长速率或有效量子产率：
$$\% ext{Inhibition} = frac{X_{ ext{control}} - X_{ ext{treatment}}}{X_{ ext{control}}} imes 100 ag{3}$$

### 数据集格式
本数据集存储为Excel文件：Chlorella sp.除草剂毒性数据_eAtlas.xlsx

### 数据字典
该电子表格中每个除草剂对应两个工作表：第一个工作表存储比生长速率（SGR）数据，第二个工作表存储脉冲振幅调制（PAM）荧光计测定数据。数据集的最后一个工作表为各除草剂实验的实测水质（water quality, WQ）参数，包括pH、电导率及温度。
各除草剂缩写对应如下：
- Brom：溴谷隆（bromacil）
- Diu：敌草隆（diuron）
- Halo：精氟禾草灵（haloxyfop-p-methyl）
- Hex：嗪草酮（hexazinone）
- Imaz：咪唑乙烟酸（imazapic）
- Isox：异恶唑草酮（isoxaflutole）
- Prom：扑草净（prometryn）
- Prop：扑灭津（propazine）

#### 各‘除草剂缩写_SGR’工作表字段说明
- SGR：比生长速率——第0天至第3天的对数增长值
- 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% (v/v)的处理组
- Measured (µg/L)：由昆士兰大学分析测定的实测除草剂浓度
- Rep：重复组编号：比生长速率数据组的重复编号为1-3，PAM数据组的重复编号为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值
- 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值

#### 各‘除草剂缩写_PAM’工作表字段说明
- PAM：脉冲振幅调制荧光计数据，用于计算有效量子产率（光适应下）
- 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% (v/v)的处理组
- Measured (µg/L)：由昆士兰大学分析测定的实测除草剂浓度
- Rep：重复组编号：比生长速率数据组的重复编号为1-3，PAM数据组的重复编号为1-3
- Delta F/Fm’：通过脉冲振幅调制（PAM）荧光计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的报告，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) 高灌溉强度种植区下游农药暴露的时空差异：不同监测技术的应用。《农业与食品化学期刊》，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（Chlorella sp.）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，达尔文北领地监管科学家项目组
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软件。《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永久数据仓库中，路径为：data
esp33.1.5_Pesticide-guidelines-GBR