Effects of herbicide exposure on growth and photosynthetic efficiency of the aquatic fern Azolla pinnata (Pteridophyta) (NESP TWQ 3.1.5, AIMS and JCU)

This dataset shows the effects of herbicides (detected in the Great Barrier Reef catchments) on growth rates (from surface area and biomass) and photosynthesis (effective quantum yield) on the aquatic fern Azolla pinnata during laboratory experiments conducted in 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 aquatic fern Azolla pinnata. Growth bioassays were performed over 14-day exposures using herbicides that have been detected in the Great Barrier Reef catchment area (O’Brien et al. 2016). Chronic effects of herbicides on the photophysiology of A. pinnata, measured by chlorophyll fluorescence as the effective quantum yield (Delta F/Fm’) were investigated using PAM fluorometry after 14-day herbicide exposure. These toxicity data will enable improved assessment of the risks posed by PSII and alternative herbicides to aquatic macrophytes for both regulatory purposes and for comparison with other taxa.Methods:The aquatic fern Azolla pinnata was sourced from Watergarden Paradise Nursery, NSW. Cultures were established in IRRI2 medium (Pereira & Carrapiço 2009). Cultures were maintained in 10 L tubs containing 3–5 L IRRI2 as batch cultures with weekly transfers to fresh medium. Clean culture solutions were maintained at 26 ± 1 °C, under a 12:12 hr light:dark cycle (65-77µmol photons m–2 s–1).Herbicide stock solutions were prepared using PESTANAL (Sigma-Aldrich) analytical grade products (HPLC greater than or equal to 98%): diuron (CAS 330-54-1), fluometuron (CAS 2164-17-2), fluroxypyr (CAS 69377-81-7), haloxyfop-p-methyl (CAS 72619-32-0), imazapic (CAS 104098-48-8), isoxaflutole (CAS 141112-29-0) and triclopyr (CAS 5535-06-3). 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 100 mL glass volumetric flasks using milli-Q water. Diuron, haloxyfop-p-methyl and isoxaflutole were dissolved using analytical grade acetone (< 0.01% (v/v) in exposures). Imazapic was dissolved in methanol (less than 0.01% (v/v) in exposure). No solvent carrier was used for the preparation of the remaining herbicide stock solutions.Cultures of A. pinnata were exposed to a range of herbicide concentrations over a period of 14 days. Fronds were selected from actively growing cultures free of overt disease or deformity. Four triplicate fronds each comprising eight ramets were added to 100 mL of each herbicide solution concentration and control treatment. In each toxicity test, 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. Experiments were conducted in IRRI2 medium (Pereira & Carrapiço 2009) with solutions replaced at Day 7. Three replicates of each treatment solution and control were prepared and incubated at 26.6 ± 0.5 °C under a 12:12 h light:dark cycle (90 ± 6 µmol photons m–2 s–1). Each replicate treatment was photographed at a standard height to estimate surface area at Day 0 and Day 14. Biomass of a representative numbers of fronds were weighed to 4 significant figures using an analytical balance after blotting for 15 seconds to remove excess moisture. Fronds from each treatment replicate were weighed at Day 14 using the same technique. Specific growth rates (SGR) were expressed as the logarithmic increase in surface area or biomass 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 surface area or biomass at day j and Xi is the surface area or biomass at day i (OECD 2006).SGR i-j = [(ln Xj - ln Xi )/(tj - ti )] (day-1)                              SGR relative to the control / solvent control treatment was used to derive chronic effect values for growth inhibition. A test was considered valid, if the SGR for frond number or surface area of control replicates was greater than or equal to 0.0.0495 day-1 (OECD 2014). Physical and chemical characteristics of each treatment were measured at 0, 7 and 14 days on new and old treatment solutions for pH, electrical conductivity and temperature. Temperature was also logged in 15-min intervals over the total test duration. Analytical samples were taken at 0 and 14 days.Chronic effects of herbicides on the photophysiology of A. pinnata, measured by chlorophyll fluorescence as the effective quantum yield (Delta F/Fm’), were investigated at 14 days using 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’                              Mini- PAM settings were set to ETR-F = 0.84, F-Offset = 46, measuring light frequency = 3, measuring intensity = 4, gain = 2; 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 2006), 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                              Format:Azolla pinnata herbicide toxicity data_eAtlas.xlsxData Dictionary:There are two or three tabs for each herbicide in the spreadsheet. The first tab corresponds to the specific growth rate – surface area (SGR-SA) data; the second tab is biomass (SGR-B) data; and 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.Diu – DiuronFluo - FluometuronFlur - FluroxypyrHalo – HaloxyfopImaz - ImazapicIsox - IsoxaflutoleTri – TriclopyrFor each ‘herbicide’_SGR tab:SGR = specific growth rate - the logarithmic increase from day 0 to day 14 as either surface area (SA) (mm2) or biomass (B) (g)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 treatmentsMeasured (µg/L) = measured concentrations analysed by The University of QueenslandRep = replicate notation is 1-3T14_Growth = surface area (mm2) or biomass (g) at day 14 ln(day14) = natural logarithm of surface area (mm2) or biomass (g) at day 14Average T0_Growth = surface area (mm2) or biomass (g) at day 0ln(day0) = natural logarithm of surface area (mm2) or biomass (g) at day 0For each ‘herbicide’_PAM tab:PAM = pulse amplitude modulation 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 Queenslandnotation 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 (2006) Current approaches in the statistical analysis of ecotoxicity data. OECD Publishing.OECD (2014) Test No. 238: Sediment-Free Myriophyllum Spicatum Toxicity Test, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris.Pereira, A.L., and Carrapiço, F. (2009) Culture of Azolla filiculoides in artificial conditions. Plant Biosystems, 143(3), 431-434 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2019年室内实验中，于大堡礁流域检出的除草剂对水生蕨类植物满江红（Azolla pinnata）生长速率（基于表面积和生物量）及光合作用（有效量子产率）的影响。本研究旨在开发并应用标准生态毒理学方案，以探究光系统II（Photosystem II, PSII）及其他类型除草剂对满江红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（O’Brien等，2016），开展为期14天的暴露生物测定。通过14天除草剂暴露后，采用脉冲振幅调制荧光法（PAM fluorometry）测定叶绿素荧光参数有效量子产率（ΔF/Fm’），以探究除草剂对满江红光生理特性的慢性影响。上述毒性数据可用于优化评估光系统II及其他除草剂对水生大型植物的风险，既可服务于监管目的，也可用于与其他类群的风险评估结果进行对比。

方法：
水生蕨类满江红（Azolla pinnata）购自新南威尔士州Watergarden Paradise苗圃。采用IRRI2培养基建立培养体系（Pereira & Carrapiço, 2009）。培养体系置于10 L容器中，装入3–5 L IRRI2培养基作为分批培养体系，每周转接至新鲜培养基中。无菌培养体系维持于26 ± 1 ℃，光照周期为12:12小时光暗循环（光照强度65–77 µmol photons m–2 s–1）。
除草剂储备液采用PESTANAL（Sigma-Aldrich）分析级产品（高效液相色谱纯度≥98%）配制，包括敌草隆（diuron，CAS 330-54-1）、伏草隆（fluometuron，CAS 2164-17-2）、氟草烟（fluroxypyr，CAS 69377-81-7）、精氟吡甲禾灵（haloxyfop-p-methyl，CAS 72619-32-0）、甲氧咪草烟（imazapic，CAS 104098-48-8）、异恶唑草酮（isoxaflutole，CAS 141112-29-0）以及三氯吡氧乙酸（triclopyr，CAS 5535-06-3）。除草剂的选择基于其田间施用剂量及在大堡礁沿海水域的检出情况（Grant等，2017；O’Brien等，2016）。储备液采用超纯水在100 mL玻璃容量瓶中配制。敌草隆、精氟吡甲禾灵与异恶唑草酮采用分析级丙酮溶解（暴露体系中丙酮体积占比<0.01%）；甲氧咪草烟采用甲醇溶解（暴露体系中甲醇体积占比<0.01%）；其余除草剂储备液无需使用溶剂载体。
将满江红培养体系暴露于一系列梯度浓度的除草剂溶液中，暴露周期为14天。选取生长旺盛、无明显病害或畸形的藻体，每株包含8个分株，每份处理设置3个重复，每重复取4株藻体加入100 mL相应浓度的除草剂溶液或对照溶液。每项毒性实验均设置空白对照（无除草剂）与溶剂对照（若使用溶剂），以验证实验方案有效性并监测生物测定的持续性能。实验采用IRRI2培养基（Pereira & Carrapiço, 2009），于第7天更换培养基。每份处理及对照设置3个重复，置于26.6 ± 0.5 ℃、12:12小时光暗循环（光照强度90 ± 6 µmol photons m–2 s–1）的环境中培养。在第0天与第14天，以标准高度拍摄每个重复处理的藻体，用于估算表面积。称取代表性藻体的生物量：先用吸水纸吸干表面多余水分15秒，再采用分析天平称量至4位有效数字。所有处理重复的藻体均于第14天采用相同方法称量。比生长速率（SGR）以第i天（ti）至第j天（tj）的表面积或生物量对数增量表示，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天的表面积或生物量，Xi为第i天的表面积或生物量（OECD, 2006）：
SGR_i-j = [(ln X_j - ln X_i)/(t_j - t_i)] (day^-1)
采用相对于空白对照/溶剂对照处理的比生长速率，推导生长抑制的慢性效应值。若空白重复的藻体数量或表面积的比生长速率≥0.0495 day^-1，则认为实验有效（OECD, 2014）。于第0、7、14天分别测定各处理溶液的pH、电导率与温度，同时采集新旧处理溶液的样品。实验全程以15分钟为间隔记录温度。于第0天与第14天采集分析样品。
于暴露14天后，采用脉冲振幅调制荧光法（PAM fluorometry，mini-PAM，Walz，德国）测定叶绿素荧光参数有效量子产率（ΔF/Fm’），以探究除草剂对满江红光生理特性的慢性影响。测定光适应下的最小荧光（F）与最大荧光（Fm’），并按照式(2)计算各处理的有效量子产率（Schreiber等，2002）：
ΔF/Fm’ = (Fm’ - F)/Fm’
Mini-PAM参数设置为：ETR-F = 0.84，F-Offset = 46，测量光频率=3，测量强度=4，增益=2，阻尼=3。饱和脉冲参数设置为：强度=6，宽度=0.6。
按照式(3)计算各处理相对于空白对照的比生长速率与有效量子产率的平均抑制百分比（OECD, 2006），其中X_control为空白对照的平均比生长速率或有效量子产率，X_treatment为单处理组的平均比生长速率或有效量子产率：
% Inhibition = [(X_control - X_treatment)/X_control] × 100

数据格式：Azolla pinnata herbicide toxicity data_eAtlas.xlsx

数据字典：
电子表格中每种除草剂对应2或3个工作表。第一个工作表为比生长速率-表面积（SGR-SA）数据；第二个工作表为生物量（SGR-B）数据；第三个工作表为脉冲振幅调制（PAM）荧光法数据。数据集的最后一个工作表展示了各除草剂实验的水质（WQ）参数（pH、电导率与温度）。值为‘-’的条目表示未进行测量。
缩写说明：
Diu – 敌草隆（Diuron）
Fluo – 伏草隆（Fluometuron）
Flur – 氟草烟（Fluroxypyr）
Halo – 精氟吡甲禾灵（Haloxyfop-p-methyl）
Imaz – 甲氧咪草烟（Imazapic）
Isox – 异恶唑草酮（Isoxaflutole）
Tri – 三氯吡氧乙酸（Triclopyr）
针对各‘除草剂’_SGR工作表：
SGR = 比生长速率——以表面积（SA，单位mm²）或生物量（B，单位g）计的第0天至第14天的对数增量
Nominal (µg/L) = 生物测定中使用的标称除草剂浓度；SC表示溶剂对照，即无除草剂且溶剂载体体积占比<0.01%的处理组，与实验处理一致
Measured (µg/L) = 昆士兰大学分析测定的实际除草剂浓度
Rep = 重复编号，取值为1-3
T14_Growth = 第14天的表面积（mm²）或生物量（g）
ln(day14) = 第14天表面积（mm²）或生物量（g）的自然对数
Average T0_Growth = 第0天的平均表面积（mm²）或生物量（g）
ln(day0) = 第0天表面积（mm²）或生物量（g）的自然对数
针对各‘除草剂’_PAM工作表：
PAM = 用于计算有效量子产率（光适应下）的脉冲振幅调制荧光法数据
Nominal (µg/L) = 生物测定中使用的标称除草剂浓度；SC表示溶剂对照，即无除草剂且溶剂载体体积占比<0.01%的处理组，与实验处理一致；Measured (µg/L) = 昆士兰大学分析测定的实际除草剂浓度
重复编号为1-3；PAM数据的重复编号为1-3
Delta F/Fm' = 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：
Grant, S., Gallen, C., Thompson, K., Paxman, C., Tracey, D. and Mueller, J. (2017) 海洋监测项目：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
O’Brien, D., Lewis, S., Davis, A., Gallen, C., Smith, R., Turner, R., Warne, M., Turner, S., Caswell, S. and Mueller, J.F. (2016) 强灌溉耕作区下游农药暴露的时空变异：不同监测技术的应用。《农业与食品化学期刊》64(20), 3975-3989.
OECD (2006) 生态毒性数据统计分析现行方法。经合组织出版社。
OECD (2014) 测试方法238：无沉积物狐尾藻毒性测试，《OECD化学品测试准则》第2部分，经合组织出版社，巴黎。
Pereira, A.L., and Carrapiço, F. (2009) 人工条件下细叶满江红（Azolla filiculoides）的培养。《植物生物系统》143(3), 431-434
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永久数据仓库中，路径为：dataesp33.1.5_Pesticide-guidelines-GBR