Effects of herbicide exposure on growth of the stonewort Ceratophyllum demersum (Tracheophyta – Magnoliopsida) (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 stem length and biomass) on the stonewort Ceratophyllum demersum during laboratory experiments conducted in 2019.The aims of this project were to develop and apply standard ecotoxicology protocols to determine the effects of non-PSII herbicides on the growth of the stonewort Ceratophyllum demersum. Growth bioassays were performed over 7-day exposures using herbicides that have been detected in the Great Barrier Reef catchment area (O’Brien et al. 2016). This toxicity data will enable improved assessment of the risks posed by non-PSII herbicides to aquatic macrophytes for both regulatory purposes and for comparison with other taxa.Methods:The stonewort Ceratophyllum demersum was supplied by Watergarden Paradise Aquatic Nursery, Bass Hill, NSW. Cultures were maintained in 500 L outdoor plastic tanks in recirculating dechlorinated tap water, aerated and maintained at ambient outdoor temperature and lighting. Test replicates selected 48 h in advance and acclimated in dechlorinated tap water, 26 ± 2 °C, under a 12:12 hr light:dark cycle (102 ± 9 µmol photons m–2 s–1).Herbicide stock solutions were prepared using PESTANAL (Sigma-Aldrich) analytical grade products (HPLC less than or equal to 98%): haloxyfop-p-methyl (CAS 72619-32-0), imazapic (CAS 104098-48-8) 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 500 mL glass volumetric flasks using milli-Q water. Haloxyfop-p-methyl was 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 triclopyr.Cultures of Ceratophyllum demersum were exposed to a range of herbicide concentrations over a period of 7 days. Plants were sourced from actively growing cultures free of overt disease or deformity. Individual plants approximately 35 mm long with 5 whorls and an apical tip were added to 150 mL of each herbicide solution concentration and control treatment. 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. Experiments were conducted in autoclaved, recirculating dechlorinated tap water. Five 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 measure stem length at Day 0 and Day 7. Biomass of a representative numbers of fronds were weighed at Day 0 to 3 significant figures using an analytical balance after blotting for 15 seconds to remove excess moisture. Fronds from each treatment replicate were weighed at Day 7 using the same technique. Specific growth rates (SGR) were expressed as the logarithmic increase in stem length (mm) or biomass (g) 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 stem length or biomass at day j and Xi is the stem length or biomass at day i (OECD 2006).SGRi-j =[(ln Xj - ln X i) / (tj - ti)] (day-1)                              (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 0.0495 day-1 determined from (OECD 2006 and Riethmuller et al 2003). Physical and chemical characteristics of each treatment were measured at 0 and 7 days including 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 7 days.Format:Ceratophyllum demersum herbicide toxicity data_eAtlas.xlsxData Dictionary:There are one or two tabs for each herbicide in the spreadsheet. The first tab corresponds to the specific growth rate – biomass (SGR-B) data; the second tab is stem length (SGR-L) data. The last tab of the dataset shows the measured water quality (WQ) parameters (pH, electrical conductivity and temperature) of each herbicide test. Halo – HaloxyfopImaz – ImazapicTri – TriclopyrFor each ‘herbicide’_SGR tab:SGR = specific growth rate – the logarithmic increase from day 0 to day 7 as either biomass (B) (g/day) or stem length (L) (mm/day)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: for SGR, notation is 1-5 T7_Biomass or Length = Biomass or Stem Length at Day 7ln(day7) = natural logarithm of biomass (g) or stem length (mm) at Day 7T0_Surface Area or Frond = Biomass (g) or Stem Length (mm) at Day 0ln(day0) = natural logarithm of biomass (g) or stem length (mm) at Day 0References: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 Guideline 238: Sediment-free Myriophyllum spicatum toxicity test, OECD Publishing, Paris.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.Data 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金鱼藻（Ceratophyllum demersum）生长速率（基于茎长与生物量）的影响。本研究旨在开发并应用标准生态毒理学规程，以明确非光系统II（non-PSII）除草剂对金鱼藻生长的影响。实验采用于大堡礁流域检出的除草剂，开展为期7天的暴露生物测定（O’Brien等，2016）。本毒性数据可助力优化非PSII除草剂对水生大型植物的风险评估，既可用于监管目的，也可用于与其他类群的研究结果对比。

方法：金鱼藻由新南威尔士州巴斯山的Watergarden Paradise水族苗圃提供。培养物置于500 L室外塑料水箱中，采用循环脱氯自来水培养，持续曝气，维持在室外自然温度与光照条件下。试验重复组提前48小时选取，并于26±2 ℃、12:12小时光暗周期（102±9 μmol光子·m⁻²·s⁻¹）下在脱氯自来水中驯化。

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：精吡氟禾草灵甲酯（haloxyfop-p-methyl，CAS 72619-32-0）、咪唑烟酸（imazapic，CAS 104098-48-8）与三氯吡氧乙酸（triclopyr，CAS 5535-06-3）。除草剂的筛选基于其田间施用剂量与大堡礁近岸水域检出情况（Grant等，2017；O’Brien等，2016）。储备液于500 mL玻璃容量瓶中用超纯水（milli-Q水）配制。精吡氟禾草灵甲酯用分析级丙酮溶解（暴露体系中溶剂占比≤0.01%，v/v）；咪唑烟酸用甲醇溶解（暴露体系中溶剂占比≤0.01%，v/v）；三氯吡氧乙酸配制未使用溶剂载体。

金鱼藻培养物于7天周期内暴露于一系列除草剂浓度梯度。试验植株取自无明显病害或畸形的活跃生长培养物，选取约35 mm长、具5轮生体与顶芽的单株，接入各除草剂浓度溶液与对照组的150 mL体系中。每项毒性试验均设置空白对照（无除草剂）与溶剂对照（若使用），以验证试验规程有效性，并监测生物测定的持续性能。实验在经高压灭菌的循环脱氯自来水中开展。每个处理组与对照组均设置5个重复，于26.6±0.5 ℃、12:12小时光暗周期（90±6 μmol光子·m⁻²·s⁻¹）下培养。每个重复组均在标准高度拍摄照片，用于测量第0天与第7天的茎长。取代表性藻丛，用分析天平（精确至3位有效数字）称量第0天的生物量，称量前用吸水纸吸干15秒以去除多余水分。第7天的各处理重复组藻丛生物量采用相同方法称量。比生长速率（Specific Growth Rate, SGR）以第i天（ti）至第j天（tj）茎长（mm）或生物量（g）的对数增长表示，计算公式如式(1)，其中SGRi-j为时间i至j的比生长速率；Xj为第j天的茎长或生物量，Xi为第i天的茎长或生物量（OECD，2006）。
SGRi-j =[(ln Xj - ln Xi) / (tj - ti)] (day⁻¹) (1)
以空白对照/溶剂对照处理组为基准的相对比生长速率，用于推导生长抑制的慢性效应值。当对照组重复组的藻丛数量或表面积的比生长速率≥0.0495 day⁻¹时（依据OECD 2006与Riethmuller等2003），试验视为有效。于第0天与第7天测量各处理组的理化特性，包括pH、电导率与温度。试验全程以15分钟间隔记录温度。于第0天与第7天采集分析样品。

数据集格式：Ceratophyllum demersum除草剂毒性数据_eAtlas.xlsx
数据字典：电子表格中每种除草剂对应一个或两个工作表。第一个工作表对应比生长速率-生物量（SGR-B）数据；第二个工作表为茎长（SGR-L）数据。数据集的最后一个工作表为各除草剂试验的实测水质（WQ）参数（pH、电导率与温度）。
Halo：精吡氟禾草灵甲酯（haloxyfop-p-methyl）
Imaz：咪唑烟酸（imazapic）
Tri：三氯吡氧乙酸（triclopyr）
针对各‘除草剂’_SGR工作表：
SGR：比生长速率——第0天至第7天的对数增长，单位为生物量（B）（g/天）或茎长（L）（mm/天）
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，即无除草剂且溶剂载体占比≤0.01% v/v，与各处理组一致
Measured (µg/L)：由昆士兰大学（The University of Queensland）分析得到的实测浓度
Rep：重复组：SGR的标注为1-5
T7_Biomass或Length：第7天的生物量或茎长
ln(day7)：第7天生物量（g）或茎长（mm）的自然对数
T0_Surface Area或Frond：第0天的生物量（g）或茎长（mm）
ln(day0)：第0天生物量（g）或茎长（mm）的自然对数

参考文献：
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。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) 高度灌溉种植区下游农药暴露的时空变异性：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。
OECD (2014) 试验指南238：无沉积物穗状狐尾藻毒性试验，经合组织出版，巴黎。
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的生态毒理学试验规程。主管科学家报告173，主管科学家办公室，北领地达尔文。

数据存储位置：本数据集存档于eAtlas永久数据存储库，路径为：dataesp33.1.5_Pesticide-guidelines-GBR