Toxicity of the insecticide imidacloprid and the fungicide propiconazole to the marine barnacle Amphibalanus amphitrite (Arthropoda/Crustacea) (NESP TWQ 3.1.5, AIMS)

This dataset shows the effects of of the insecticide imidacloprid and the fungicide propiconazole on larval development of the acorn barnacle Amphibalanus amphitrite experiments conducted in 2018 and 2019.

The aim of this project was to apply standard ecotoxicology protocols to determine the effects of the insecticide imidacloprid and the fungicide propiconazole on larval development rate of the acorn barnacle Amphibalanus Amphitrite. Larval development bioassays(4-d exposures) were conducted using a fungicide and insecticide that have been detected in the Great Barrier Reef catchment area (O'Brien et al., 2016). These toxicity data will enable improved assessment of the risks posed by pesticides to marine crustaceans for both regulatory purposes and for comparison with other taxa.

Methods:
Pesticide stock solutions were prepared using PESTANAL (Merck) analytical grade products (purity greater than or equal to 98%): imidacloprid (CAS 138261-41-3) and propiconazole (CAS 60207-90-1). This selection was based on application rates and detection in coastal waters of the GBR (O’Brien et al., 2016; Grant 2017). Pesticide stock solutions (100 – 1,000 mg L-1) were prepared by dissolving aliquots of the pure compounds in ultrapure water using clean, acid-washed (5% nitric acid) glass screw-top containers. Acetone was used to dissolve the imidacloprid and propiconazole (less than or equal to 0.01 % (v/v) in exposure solutions). Stock solutions were stored refrigerated and in the dark.

Broodstock barnacles had been grown for several generations in the AIMS-NT aquaria facility (originally sourced from Darwin Harbour – 12°26'57.48"S, 130°51'7.51"E). Broodstock were fed freshly hatched brine shrimp (Artemia salina) and live rotifers daily. Broodstock were spawned as previously described (van Dam et al., 2016) and nauplii collected. Tests were conducted as previously described (van Dam et al., 2016). Nauplii were exposed in a custom-designed experimental test system that allowed for constant movement of the exposure media. The system consisted of a series of silanized glass funnels in which nauplii were exposed to increasing concentrations of imidacloprid or propiconazole and tested against control nauplii. Generally, a total of 24 funnels were used for 7 treatment concentrations and a control group, thus allowing for 3 replicate funnels per treatment. Each treatment vessel contained 100 mL exposure media, 50 newly released stage II nauplii and 1 x 107 cells of rinsed Chaetoceros muelleri. Every 24 h, 1 x 107 cells of rinsed C. muelleri were added to each funnel. After 96 h exposure, funnel contents were drained over a 150 µm nitrile mesh. The mesh was examined under a stereomicroscope and the number of cyprids and settled larvae scored. Quality control criteria (> 70% survival in control group) for test acceptability were met for each test used to derive toxicity estimates. Treatment effects were quantified by the percentage successful transition to cyprid in treatment groups relative to controls.

Following prescribed statistical procedures (OECD 2006) the R package DRC (R-project 2015, Ritz & Streibig 2005), was used to model the test data and calculate toxicity estimates. Regression models evaluated included log-logistic and Weibull models of different levels of parametrisation. Model comparisons were conducted using the Akaike Information Criterion (AIC) and models that best described the data were applied to approximate pesticide concentrations eliciting 10 and 50% inhibition of successful transition relative to control animals (EC10 and EC50, respectively). The associated 95% confidence limits were estimated using the delta method.

Format:
The dataset is summarised in one file named ‘Amphibalanus amphitrite pesticide toxicity data_eAtlas.xlsx’

Data Dictionary:
The excel spreadsheet has one tab for each pesticide. The last tab of the dataset shows the measured (start and end of test) water quality (WQ) parameters (pH, salinity, dissolved oxygen (DO), and temperature) of each pesticide test.

For each ‘pesticide’_Development tab:

Nominal (µg/L) = nominal herbicide concentrations used in the bioassays
Measured (µg/L) = measured concentrations analysed by The University of Queensland
Rep = replicate notation is 1-3
No. of nauplii larvae at start = number of larvae per replicate at start of test
No. of cyprid larvae day 4 = number of cyprids observed per replicate at end of test

References:

O’Brien, D. et al. Spatial and temporal variability in pesticide exposure downstream of a heavily irrigated cropping area: application of different monitoring techniques. J. Agric. Food Chem. 64, 3975-3989 (2016).

Grant, S. et al. 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 (2017).

van Dam, J. W. et al. A novel bioassay using the barnacle Amphibalanus amphitrite to evaluate chronic effects of aluminium, gallium and molybdenum in tropical marine receiving environments. Mar Pollut Bull 112, 427-435, doi:http://dx.doi.org/10.1016/j.marpolbul.2016.07.015 (2016).

OECD. Current Approaches in the Statistical Analysis of Ecotoxicity Data., (OECD Publishing, 2006).

R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. (2015).

Ritz, C. & Streibig, J. C. Bioassay analysis using R. Journal of Statistical Software 12, 1-22 (2005).

Data Location:

This dataset is filed in the eAtlas enduring data repository at: data\nesp3\3.1.5_Pesticide-guidelines-GBR

本数据集记录了2018年与2019年开展的两项实验中，杀虫剂吡虫啉（imidacloprid）与杀菌剂丙环唑（propiconazole）对纹藤壶（Amphibalanus amphitrite）幼虫发育的影响。

本研究旨在采用标准生态毒理学（ecotoxicology）规程，明确吡虫啉与丙环唑对纹藤壶幼虫发育速率的影响。实验选取已在大堡礁流域（Great Barrier Reef catchment area）中检出的上述杀菌剂与杀虫剂，开展为期4天暴露的幼虫发育生物测定（bioassay）。本套毒性数据可为监管评估及与其他类群的对比研究提供支撑，以优化农药对海洋甲壳类生物的风险评估工作。

方法：
采用默克（Merck）公司的PESTANAL分析级试剂（纯度≥98%）配制农药母液：吡虫啉（CAS 138261-41-3）与丙环唑（CAS 60207-90-1）。试剂选择依据为大堡礁近岸水域的农药施用剂量与检出情况（O’Brien et al., 2016; Grant 2017）。将纯化合物分装后，使用经5%硝酸酸洗的洁净玻璃螺旋盖容器，溶于超纯水，配制浓度范围为100–1000 mg·L⁻¹的农药母液。吡虫啉与丙环唑需先用丙酮溶解，最终暴露液中丙酮体积占比≤0.01%。母液需避光冷藏保存。

亲代纹藤壶在澳大利亚海洋科学研究所北领地水族馆设施（AIMS-NT aquaria facility）中繁育多代，其最初采集自达尔文港（12°26'57.48"S, 130°51'7.51"E）。每日投喂刚孵化的卤虫（Artemia salina）与活体轮虫。按照既往研究方法（van Dam et al., 2016）对亲体进行产卵操作并收集无节幼体。实验操作参照既往方案（van Dam et al., 2016）开展。无节幼体在定制化实验系统中暴露，该系统可维持暴露介质持续流动。系统由一系列经硅烷化处理的玻璃漏斗组成，无节幼体在此中暴露于梯度浓度的吡虫啉或丙环唑溶液，并以对照组无节幼体作为参照。通常情况下，7个处理浓度组与1个对照组共设置24个漏斗，即每个处理设置3个重复漏斗。每个处理容器中加入100 mL暴露介质、50只刚孵化的II期无节幼体，以及1×10⁷个清洗后的角毛藻（Chaetoceros muelleri）细胞。每24小时向每个漏斗中添加1×10⁷个清洗后的角毛藻细胞。暴露96小时后，将漏斗内的液体通过150 µm丁腈滤网沥干。在体视显微镜下观察滤网，统计腺介幼体与定居幼虫的数量。每项用于推导毒性估算值的实验均满足质量控制标准：对照组存活率≥70%，即实验合格。处理效应以各处理组相对于对照组的成功变态为腺介幼体的百分比进行量化。

按照规定的统计流程（OECD 2006），使用R包DRC（R-project 2015, Ritz & Streibig 2005）对实验数据进行建模并计算毒性估算值。评估的回归模型包括不同参数化水平的对数逻辑斯蒂模型与威布尔（Weibull）模型。采用赤池信息准则（Akaike Information Criterion, AIC）进行模型比较，选取最适配数据的模型，以估算导致成功变态受到10%与50%抑制的农药浓度（分别记为EC10与EC50）。采用德尔塔法（delta method）估算对应的95%置信区间。

数据集格式：
本数据集汇总于名为‘Amphibalanus amphitrite pesticide toxicity data_eAtlas.xlsx’的单个文件中。

数据字典：
该Excel表格每个农药对应一个工作表。数据集的最后一个工作表展示了各农药实验的实测水质（water quality, WQ）参数（pH值、盐度、溶解氧（dissolved oxygen, DO）与水温），包含实验起始与结束时的检测结果。
针对每个「农药_Development」工作表：
Nominal (µg/L)：生物测定中使用的标称除草剂浓度（单位：µg/L）
Measured (µg/L)：由昆士兰大学分析测得的实际浓度（单位：µg/L）
Rep：重复编号，取值为1~3
No. of nauplii larvae at start：实验开始时每个重复的无节幼体数量
No. of cyprid larvae day 4：实验结束（第4天）时每个重复的腺介幼体数量

参考文献：
O’Brien, D. 等. 高灌溉度耕作区下游农药暴露的时空变异：不同监测技术的应用. 《农业与食品化学杂志》（J. Agric. Food Chem.）64, 3975-3989 (2016).
Grant, S. 等. 海洋监测计划：近岸农药监测2015-2016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 (2017).
van Dam, J. W. 等. 一种基于纹藤壶（Amphibalanus amphitrite）的新型生物测定方法，用于评估热带海洋受纳环境中铝、镓与钼的慢性效应. 《海洋污染公报》（Mar Pollut Bull）112, 427-435, doi:http://dx.doi.org/10.1016/j.marpolbul.2016.07.015 (2016).
经济合作与发展组织（OECD）. 《生态毒理学数据统计分析现行方法》. （经合组织出版, 2006）.
R语言与统计计算环境. R统计计算基金会, 奥地利维也纳. 网址https://www.R-project.org/. (2015).
Ritz, C. & Streibig, J. C. 基于R语言的生物测定分析. 《统计软件杂志》（Journal of Statistical Software）12, 1-22 (2005).

数据存储位置：
本数据集存档于eAtlas永久数据仓库：data\nesp3\3.1.5_Pesticide-guidelines-GBR