Effects of herbicide exposure on growth and photosynthetic efficiency of the green algae Desmodesmus asymmetricus (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 green algae Desmodesmus asymmetricus during laboratory experiments conducted during 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 green algae Desmodesmus asymmetricus. 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 Desmodesmus asymmetricus, 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 Desmodesmus asymmetricus was purchased from the Australian National Algae Supply Service, Hobart (CSIRO). Cultures of Desmodesmus asymmetricus were established in MLA medium (Bolch and Blackburn 1996). 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 Desmodesmus asymmetricus suspension to 100 mL MLA 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), 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 100 - 500 mL glass volumetric flasks using milli-Q water. Diuron, haloxyfop-p-methyl, hexazinone and isoxaflutole 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 Desmodesmus asymmetricus 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 Desmodesmus asymmetricus working suspension was prepared in a 100 ml volumetric flask. A 1:10 and 1:100 dilutions were 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 concentrations were prepared in 0.5x strength MLA 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 Desmodesmus asymmetricus, 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 = [(Xcontrol - Xtreatment ) / Xcontrol] x 100                          (3)Format:Desmodesmus asymmetricus 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. Where value equals '-', measurement not taken. Brom - BromacilDiu – DiuronHalo – HaloxyfopHex - HexazinoneImaz – ImazapicIsox - IsoxaflutoleProp - 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:Bolch, C. J. S. and Blackburn S. I. (1996). Isolation and purification of Australian isolates of the toxic cyanobacterium Microcystis aeruginosa Kütz. Journal of Applied Phycology 8, 5-13Grant, 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).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

本数据集收录了2019年室内实验所得数据，展示了在大堡礁流域检出的除草剂对绿藻不对称栅藻（Desmodesmus asymmetricus）生长速率（基于细胞密度数据）与光合效能（有效量子产率）的影响。本研究的目标为开发并应用标准化生态毒理学方案，以探究光系统II（Photosystem II, PSII）及其他替代除草剂对不对称栅藻生长与光合效率的影响。

研究采用在大堡礁流域检出的除草剂开展了为期3天的暴露生长生物测定（O’Brien等，2016）。在除草剂暴露72小时后，采用mini-PAM荧光光度法，通过叶绿素荧光参数有效量子产率（ΔF/Fm'）对不对称栅藻的光生理响应受除草剂的影响开展了研究。本毒性数据集可为监管评估及与其他类群比对提供支撑，以优化光系统II类及替代除草剂对微藻的风险评估工作。

方法：
实验所用的绿藻不对称栅藻（Desmodesmus asymmetricus）购自澳大利亚霍巴特的澳大利亚国家藻类供应服务中心（CSIRO）。不对称栅藻的培养采用MLA培养基（Bolch和Blackburn，1996）。培养采用无菌250 mL锥形瓶进行批次培养，维持藻株处于指数生长阶段：每周将1~3 mL 7日龄的不对称栅藻悬浮液转接至100 mL新鲜MLA培养基中，全程保持无菌操作。无菌培养体系的培养条件为：温度26±2℃，光暗周期12:12小时，光照强度91±12 μmol光子·m⁻²·s⁻¹。

除草剂储备液采用PESTANAL（Sigma-Aldrich）分析纯级试剂（高效液相色谱纯度≥98%）配制，具体包括：溴谷隆（bromacil，CAS 314-40-9）、敌草隆（diuron，CAS 330-54-1）、精吡氟禾草灵甲酯（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）以及扑灭津（propazine，CAS 139-40-2）。除草剂的筛选基于其施用剂量及在大堡礁沿岸水域的检出情况（Grant等，2017；O’Brien等，2016）。储备液采用无菌100~500 mL玻璃容量瓶，以超纯水（milli-Q水）配制。敌草隆、精吡氟禾草灵甲酯、环嗪酮及异恶唑草酮采用分析纯丙酮溶解（暴露实验中溶剂占比<0.01%（体积比））；甲氧咪草烟采用甲醇溶解（暴露实验中溶剂占比≤0.01%（体积比））；其余除草剂储备液配制无需添加溶剂载体。

将不对称栅藻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。接种物取自处于指数生长阶段的培养物（培养4~7天）。配制不对称栅藻工作悬浮液：取100 mL容量瓶进行定容。分别制备1:10与1:100倍稀释液，采用血球计数板在复合显微镜下计数，以确定合适的接种稀释体积。将预先确定体积的接种物加入至50 mL各处理组与对照组的培养基中，使最终细胞密度达到3~3.1×10⁴ cells/mL。每一项毒性实验均设置空白对照组（无除草剂）与溶剂对照组（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运行状态。所有处理组的培养基均采用0.5倍浓度的MLA培养基配制。平行组培养条件为：温度26.6±0.5℃，光暗周期12:12小时，光照强度190±14 μmol光子·m⁻²·s⁻¹。

从每个平行组中采集子样本，在72小时时采用血球计数板测定藻类细胞密度，并在相差显微镜下拍摄样本。细胞计数可采用人工计数或通过ImageJ软件分析显微镜照片完成（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量表示，计算公式如式(1)所示，其中SGRi-j为时间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（经济合作与发展组织，2011）。
式(1)：SGR_i-j = [(ln X_j - ln X_i )/(t_j - t_i )] (d⁻¹)

以对照组为参照的相对比生长速率可用于推导生长抑制的慢性效应值。若对照组平行组的比生长速率≥0.92 d⁻¹，则认为该实验有效（经济合作与发展组织，2011）。在实验开始时（0小时）与结束时（72小时）测定各处理组的理化参数，包括pH值、电导率与温度。在整个实验周期内，以15分钟为间隔记录培养箱温度。分别在0小时与72小时采集分析样本。

在除草剂暴露72小时后，采用德国Walz公司生产的mini-PAM荧光光度仪，通过叶绿素荧光参数有效量子产率（ΔF/Fm'）对不对称栅藻的光生理响应受除草剂的影响开展了研究。测定光适应下的最小荧光（F）与最大荧光（Fm'），并根据式(2)计算各处理组的有效量子产率（Schreiber等，2002）。
式(2)：ΔF/Fm' = (Fm' - F)/Fm'

mini-PAM仪器参数设置如下：ETR-F=0.84，F-Offset=92，测量光频率=3，测量光强度=4，增益=3，阻尼系数=3；饱和脉冲参数设置：强度=6，脉冲宽度=0.6。

以对照组为参照，各处理组的比生长速率与ΔF/Fm'的平均抑制百分率按式(3)计算（经济合作与发展组织，2011），其中X_control为对照组的平均比生长速率或ΔF/Fm'值，X_treatment为单一组处理的平均比生长速率或ΔF/Fm'值。
式(3)：抑制率（%）= [(X_control - X_treatment) / X_control] × 100

数据集格式：不对称栅藻除草剂毒性数据_eAtlas.xlsx

数据字典：
本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）荧光光度法数据。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（pH值、电导率与温度）。若单元格值为‘-’，则表示未获取该测量数据。

缩写说明：Brom—溴谷隆（Bromacil）；Diu—敌草隆（Diuron）；Halo—精吡氟禾草灵甲酯（Haloxyfop-p-methyl）；Hex—环嗪酮（Hexazinone）；Imaz—甲氧咪草烟（Imazapic）；Isox—异恶唑草酮（Isoxaflutole）；Prop—扑灭津（Propazine）

各‘除草剂名_SGR’工作表字段说明：
SGR：比生长速率（Specific Growth Rate）——第0天至第3天的细胞密度对数增长量
Nominal (μg/L)：标称除草剂浓度——生物测定中使用的除草剂标称浓度；SC代表溶剂对照组，即无除草剂且溶剂载体占比<0.01%（体积比）的处理组
Measured (μg/L)：实测除草剂浓度——由昆士兰大学分析得到的实测除草剂浓度
Rep：平行组编号——SGR数据的平行组编号为1~3；PAM数据的平行组编号亦为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

各‘除草剂名_PAM’工作表字段说明：
PAM：脉冲振幅调制荧光光度法——用于计算光适应下的有效量子产率
Nominal (μg/L)：标称除草剂浓度——生物测定中使用的除草剂标称浓度；SC代表溶剂对照组，即无除草剂且溶剂载体占比<0.01%（体积比）的处理组
Measured (μg/L)：实测除草剂浓度——由昆士兰大学分析得到的实测除草剂浓度
Rep：平行组编号——SGR数据的平行组编号为1~3；PAM数据的平行组编号亦为1~3
ΔF/Fm'：有效量子产率——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. Bolch, C. J. S. 与Blackburn S. I. (1996)。有毒蓝藻铜绿微囊藻（Microcystis aeruginosa Kütz）澳大利亚分离株的分离与纯化。《应用藻类学杂志》（Journal of Applied Phycology），8卷，5-13页。
2. Grant, S.等(2017)。海洋监测计划：2015-2016年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，共128页。链接：http://dspace-prod.gbrmpa.gov.au/jspui/handle/11017/13325
3. O’Brien, D.等(2016)。强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》（Journal of Agricultural and Food Chemistry），64卷(20期)，3975-3989页。
4. 经济合作与发展组织（Organization for Economic Co-operation and Development, OECD）(2011)。化学品测试指南：淡水藻类与蓝藻生长抑制试验，测试编号201。链接：https://search.oecd.org/env/test-no-201-alga-growth-inhibition-test-9789264069923-en.htm（2019年8月28日访问）。
5. Rueden, C.T.等(2017)。ImageJ2：面向下一代科学图像数据的ImageJ软件。《BMC生物信息学》（BMC Bioinformatics），18卷:529，PMID 29187165，doi:10.1186/s12859-017-1934-z。

数据集存储位置：
本数据集存储于eAtlas永久数据仓储中，路径为：dataesp33.1.5_Pesticide-guidelines-GBR