Toxicity of three insecticides and two fungicides to Acropora tenuis coral larvae (NESP TWQ 3.1.5, AIMS)

This dataset shows the effects of three insecticides (diazinon, fipronil, imidacloprid) and two fungicides (chlorothalonil, propiconazole) on larval metamorphosis in the coral Acropora tenuis. These five pesticides have been detected in the Great Barrier Reef lagoon and/or catchments. Settlement assays were conducted in Nov-Dec 2016 and Nov 2017.

The aim of this research is to add toxicity data for inclusion into water quality guidelines. In order to improve water quality guidelines and subsequent risk assessments for pesticides in tropical marine ecosystems, the current study investigated the effects of three insecticides (diazinon, fipronil, imidacloprid) and two fungicides (chlorothalonil and propiconazole) on larval settlement and metamorphosis of the common reef-building coral Acropora tenuis larvae following 48 h exposures. Concentration-response curves were plotted to estimate no effect concentration (NEC) and effect concentration (ECx) values that inhibited larval settlement by 10% and 50% (EC10 and EC50, respectively). NEC is the concentration below which the pesticides are not expected to cause a reduction in larval metamorphosis.

Methods:
Gravid colonies (25-40 cm diameter) of the coral Acropora tenuis (Dana, 1846) were collected from 4 – 8 m depth in November 2016 from Trunk Reef (18°18.2’ S, 146°52.2’ E) and in November 2017 from Falcon Island (18°46’ S, 146°32’ E), GBR under Great Barrier Reef Marine Park Authority Permit G12/35236.1. Colonies were transported to the National Sea Simulator at the Australian Institute of Marine Science (AIMS) in Townsville and maintained in 1700 l flow-through holding tanks until spawning. Temperatures were held at 26-27°C, which was equivalent to the water temperature at the collection site. Gametes were collected from 8 parental colonies on each occasion, fertilised and symbiont-free larvae were cultured at approximately 500 larvae L-1 in 500 L flow-through tanks (Negri and Heyward, 2001, Nordborg et al., 2018). Larvae were competent to settle after 5 d and we used 7-10-day old A. tenuis larvae, each 800-1000 µm in length for consistency in the pesticide exposure experiments.

The five pesticides in this study were > 98% pure and purchased from Sigma-Aldrich (NSW, Australia). Stock solutions (5 mg l-1) of all pesticides were dissolved in dimethyl sulfoxide (DMSO, final concentration < 0.01% (v/v) in exposures) and prepared in milli-Q water. A. tenuis larvae were exposed to diazinon (2.62 – 638 µg l-1), fipronil (1.57 – 1144 µg l-1), imidacloprid (3.88 – 947 µg l-1), chlorothalonil (0.69 – 507 µg l-1) and propiconazole (8.42 – 2053 µg l-1). Pesticide analyses were done by The University of Queensland, Queensland Alliance for Environmental Health Sciences (QAEHS), Woolloongabba, Australia.

Static exposures were conducted in 20 mL glass scintillation vials containing 12-14 larvae made up to 10 mL filtered seawater (0.5 µm) with 6-7 concentrations (per pesticide) and 6 replicate vials per concentration. All tests included solvent controls containing identical concentrations of DMSO carrier. Seawater and solvent carrier controls were run in 12-18 replicate vials. Copper (CuCl2) was used as a reference toxicant at 6 concentrations between 1.12 – 36 µg L-1 and 6 replicate vials per concentration. Glass vials were transferred in random positions within a refrigerated shaking incubator (TLM-530, Thermoline Scientific) at 70 RPM to maintain gentle water movement which prevents larvae from attaching and undergoing metamorphosis in the containers (Negri et al., 2016). Larvae were exposed under a light intensity of approximately 60 µmol photons m-2 s-1 (12:12 h L:D cycle) and at 26.7 ± 0.7 °C (range). Vials were re-randomised at 24 h. After 48 h exposure larvae and treatment water were transferred into 6-well polystyrene culture plates (Nunc, NY, USA) and returned to the incubator but without water movement. Metamorphosis was initiated by the addition of crustose coralline algae (CCA) extract (10 µL) prepared from 4 g CCA Porolithon onkodes (Heyward and Negri, 1999, Negri et al., 2005). Metamorphosis was assessed after a further 24 h and larvae were considered normal and functional if larvae had changed from free swimming or casually attached pear-shaped forms to squat, firmly attached, disc-shaped structures with pronounced flattening of the oral–aboral axis and with septal mesenteries radiating from the central mouth region (Heyward and Negri, 1999). Average settlement success greater than or equal to 70% in controls was considered indicative of a standard response to settlement inducers based on several previous studies using CCA or extracts of CCA to initiate settlement (Negri and Heyward, 2000, Negri et al., 2011b, Negri et al., 2016).

Format:
Acropora tenuis pesticide settlement data_eAtlas.xlsx

Data Dictionary:

The dataset comprises of three tabs:

‘Settlement_2016’ denotes the settlement assay conducted in 2016 and consists of seawater and solvent controls, blanks, copper (reference toxicant), diazinon, fipronil, imidacloprid and chlorothalonil settlement data.
‘Propiconazole_2017’ denotes the settlement assay conducted in 2017 and consists of seawater and solvent controls, copper (reference toxicant) and propiconazole settlement data.
‘WQ’ denotes the measured water quality parameters at the initiation and finalisation of the test exposure.

SW control - seawater control; no herbicide and no solvent carrier; 12-18 scintillation vials (2-3 x 6-well plates: A/B/C); in well plates, crustose coralline algae (CCA) was added to initiate settlement
Solvent control = no herbicide and contains less than 0.01% v/v DMSO (dimethyl sulfoxide) solvent carrier as per the treatments; 12-18 scintillation vials (2-3 x 6-well plates: A/B/C); in well plates, CCA was added to initiate settlement
Blank = no herbicide and no solvent carrier and no CCA added; 12 scintillation vials (2 x 6-well plates: A, B)
Nominal conc (µg/L) = nominal pesticide concentrations used in the settlement assays
Measured conc (µg/L) = measured pesticide concentrations analysed by The University of Queensland
Rep = replicate vials 1-6
# Settled = number of coral larvae settled in the 6-well plates
# Total = number of total coral larvae counted in each well of a 6-well plate
# Failed = number of coral larvae that did not metamorphose: (# Total - # Settled) = # Failed
% Settled = percent of coral larvae that metamorphosed and settled: (# Settled/# Total) x 100

WQ = water quality parameters measured at start and end of exposure
DO = dissolved oxygen

References:
Dana, J.D. 1846. United States Exploring Expedition during the years 1838-1842. Zoophytes 7: 1-740. Lea and Blanchard, Philadelphia., available online at http://www.sil.si.edu/digitalcollections/usexex/navigation/ScientificText/USExEx19_08select.cfm

Negri, A.P. and Heyward, A.J. 2001. Inhibition of coral fertilisation and larval metamorphosis by tributyltin and copper. Marine Environmental Research 51(1): 17-27.

Nordborg, M., Flores, F., Brinkman, D.L., Agusti, S., Negri, A.P. 2018. Phototoxic effects of two common marine fuels on the settlement success of the coral Acropora tenuis. Scientific Reports 8(1). https://doi.org/10.1038/s41598-018-26972-7

Negri, A.P., Brinkman, D.L., Flores, F., Botte, E.S., Jones, R.J., Webster, N.S. 2016. Acute ecotoxicology of natural oil and gas condensate to coral reef larvae. Scientific Reports 6. https://doi.org/10.1038/srep21153
Heyward, A.J. and Negri, A.P. 1999. Natural inducers for coral larval metamorphosis. Coral Reefs 18(3): 273 – 279.

Negri, A.P., Vollhardt, C., Humphrey, C., Heyward, A.J., Jones, R., Eaglesham, G. and Fabricius, K. 2005. Effects of the herbicide diuron on the early life history stages of coral. Marine Pollution Bulletin 51(1-4); 370-383.

Negri, A.P. and Heyward, A.J. 2000. Inhibition of fertilization and larval metamorphosis of the coral Acropora millepora (Ehrenberg, 1834) by petroleum products. Marine Pollution Bulletin 41(712): 420-427.

Negri, A.P., Harford, A.J., Parry, D.L., van Dam, R.A. 2011. Effects of alumina refinery wastewater and signature metal constituents at the upper thermal tolerance of: 2. The early life stages of the coral Acropora tenuis. Marine Pollution Bulletin 62(3): 474-82.

Data Location:

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

本数据集记录了三种杀虫剂（二嗪农（diazinon）、氟虫腈（fipronil）、吡虫啉（imidacloprid））与两种杀菌剂（百菌清（chlorothalonil）、丙环唑（propiconazole））对纤细鹿角珊瑚（Acropora tenuis）幼虫变态的影响。上述五种农药已在大堡礁潟湖及/或集水区中被检出。附着实验分别于2016年11-12月及2017年11月开展。

本研究旨在补充毒性数据，以纳入水质指南。为完善热带海洋生态系统中农药的水质指南及后续风险评估，本研究探究了经48小时暴露后，上述三种杀虫剂与两种杀菌剂对常见造礁珊瑚纤细鹿角珊瑚幼虫附着与变态的影响。通过绘制浓度-反应曲线，估算无观察效应浓度（NEC）以及使幼虫附着率分别降低10%、50%的效应浓度（ECx，即EC10与EC50）。其中，NEC指不会导致幼虫变态率下降的农药最高浓度。

### 方法
2016年11月，研究人员于大堡礁的特伦克礁（18°18.2’ S, 146°52.2’ E）海域，2017年11月于猎鹰岛（18°46’ S, 146°32’ E）海域，从4-8米水深采集怀卵的纤细鹿角珊瑚（Acropora tenuis (Dana, 1846)）群体（直径25-40 cm），采集许可为大堡礁海洋公园管理局G12/35236.1号许可。将采集的珊瑚群体转运至位于汤斯维尔的澳大利亚海洋科学研究所（AIMS）国家海洋模拟器，置于1700升流水储水槽中暂养至产卵，水温维持在26-27℃，与采集海域的水温一致。每次实验从8个亲本群体中收集配子，进行受精，无共生藻的幼虫以约500枚/升的密度在500升流水水槽中培养（Negri和Heyward, 2001; Nordborg等, 2018）。幼虫在培养5天后具备附着能力，本实验使用培养7-10天的纤细鹿角珊瑚幼虫，幼虫体长为800-1000 μm，以保证农药暴露实验的一致性。

本实验使用的五种农药纯度均高于98%，购自澳大利亚新南威尔士州的西格玛奥德里奇（Sigma-Aldrich）公司。所有农药的母液（5 mg·l⁻¹）均以二甲基亚砜（DMSO，暴露实验中溶剂终浓度<0.01% (v/v)）溶解，并用超纯水配制。纤细鹿角珊瑚幼虫分别暴露于以下浓度梯度的农药中：二嗪农（2.62–638 μg·l⁻¹）、氟虫腈（1.57–1144 μg·l⁻¹）、吡虫啉（3.88–947 μg·l⁻¹）、百菌清（0.69–507 μg·l⁻¹）以及丙环唑（8.42–2053 μg·l⁻¹）。农药浓度分析由澳大利亚伍伦加拉巴的昆士兰大学昆士兰环境卫生科学联盟（QAEHS）完成。

静态暴露实验在20 mL玻璃闪烁瓶中开展，每个瓶中放入12-14只幼虫，加入10 mL经过0.5 μm过滤的海水，每个农药设置6-7个浓度梯度，每个浓度设置6个重复瓶。所有实验组均设置溶剂对照组，加入与处理组相同浓度的DMSO载体。海水对照组与溶剂对照组各设置12-18个重复瓶。以氯化铜（CuCl₂）作为参考毒物，设置6个浓度梯度（1.12–36 μg·l⁻¹），每个浓度设置6个重复瓶。将玻璃瓶随机放置于制冷振荡培养箱（TLM-530，Thermoline Scientific）中，以70 RPM转速振荡以维持温和的水流，防止幼虫在容器中附着并发生变态（Negri等, 2016）。暴露实验的光照强度约为60 μmol photons·m⁻²·s⁻¹，光暗周期为12:12 h，温度维持在26.7 ± 0.7 ℃。实验进行24小时后，重新随机放置瓶体位置。暴露48小时后，将幼虫与处理液转移至6孔聚苯乙烯培养板（Nunc，美国纽约州），放回培养箱但停止水流。通过加入由4克多孔石孔藻（Porolithon onkodes）制备的壳状珊瑚藻（crustose coralline algae, CCA）提取物（10 μL）来诱导幼虫变态（Heyward和Negri, 1999; Negri等, 2005）。继续培养24小时后评估幼虫变态情况：若幼虫从自由游泳或偶然附着的梨形形态转变为匍匐、牢固附着的盘状结构，且口-反口轴明显扁平、中隔膜从中央口部区域呈放射状分布，则视为正常且具有功能的变态个体（Heyward和Negri, 1999）。根据此前多项利用壳状珊瑚藻或其提取物诱导附着的研究，当对照组的平均附着成功率≥70%时，可认为附着诱导剂的响应符合标准（Negri和Heyward, 2000; Negri等, 2011b; Negri等, 2016）。

数据集文件为：Acropora tenuis pesticide settlement data_eAtlas.xlsx

#### 数据字典
本数据集包含三个工作表：
1. `Settlement_2016`：对应2016年开展的附着实验，包含海水对照组、溶剂对照组、空白组、参考毒物铜、二嗪农、氟虫腈、吡虫啉及百菌清的附着数据。
2. `Propiconazole_2017`：对应2017年开展的附着实验，包含海水对照组、溶剂对照组、参考毒物铜及丙环唑的附着数据。
3. `WQ`：记录实验暴露起始与结束时的水质参数。

各字段说明：
- SW control（海水对照组）：无农药与溶剂载体，设置12-18个闪烁瓶（对应2-3块6孔板：A/B/C）；培养板中加入壳状珊瑚藻（CCA）以诱导附着。
- Solvent control（溶剂对照组）：无农药，含有低于0.01% (v/v)的DMSO溶剂载体，与处理组一致，设置12-18个闪烁瓶（对应2-3块6孔板：A/B/C）；培养板中加入CCA以诱导附着。
- Blank（空白组）：无农药与溶剂载体，且未添加CCA，设置12个闪烁瓶（对应2块6孔板：A、B）。
- Nominal conc (µg/L)（标称浓度）：附着实验中使用的标称农药浓度。
- Measured conc (µg/L)（实测浓度）：由昆士兰大学分析得到的实测农药浓度。
- Rep（重复）：重复瓶编号1-6。
- # Settled（已附着数量）：6孔培养板中已附着的珊瑚幼虫数量。
- # Total（总数量）：6孔培养板每孔中计数的总珊瑚幼虫数量。
- # Failed（未变态数量）：未发生变态的珊瑚幼虫数量，计算公式为`# Total - # Settled = # Failed`。
- % Settled（附着率）：发生变态并附着的珊瑚幼虫百分比，计算公式为`(# Settled/# Total) × 100`。
- WQ（水质参数）：实验暴露起始与结束时测量的水质参数。
- DO（溶解氧）：溶解氧含量。

#### 参考文献
> Dana, J.D. 1846. United States Exploring Expedition during the years 1838-1842. Zoophytes 7: 1-740. Lea and Blanchard, Philadelphia. 可在线获取：http://www.sil.si.edu/digitalcollections/usexex/navigation/ScientificText/USExEx19_08select.cfm
> Negri, A.P. 和 Heyward, A.J. 2001. 三丁基锡与铜对珊瑚受精及幼虫变态的抑制作用. 《Marine Environmental Research》51(1): 17-27.
> Nordborg, M., Flores, F., Brinkman, D.L., Agusti, S., Negri, A.P. 2018. 两种常见船用燃料对纤细鹿角珊瑚附着成功率的光毒性影响. 《Scientific Reports》8(1). https://doi.org/10.1038/s41598-018-26972-7
> Negri, A.P., Brinkman, D.L., Flores, F., Botte, E.S., Jones, R.J., Webster, N.S. 2016. 天然石油与凝析油对珊瑚礁幼虫的急性生态毒性. 《Scientific Reports》6. https://doi.org/10.1038/srep21153
> Heyward, A.J. 和 Negri, A.P. 1999. 珊瑚幼虫变态的天然诱导剂. 《Coral Reefs》18(3): 273–279.
> Negri, A.P., Vollhardt, C., Humphrey, C., Heyward, A.J., Jones, R., Eaglesham, G. 和 Fabricius, K. 2005. 除草剂敌草隆对珊瑚早期生活史阶段的影响. 《Marine Pollution Bulletin》51(1-4): 370-383.
> Negri, A.P. 和 Heyward, A.J. 2000. 石油产品对珊瑚 millepora（Ehrenberg, 1834）的受精与幼虫变态的抑制作用. 《Marine Pollution Bulletin》41(712): 420-427.
> Negri, A.P., Harford, A.J., Parry, D.L., van Dam, R.A. 2011. 铝土矿精炼废水及特征金属成分在高温耐受上限下对纤细鹿角珊瑚早期生活史阶段的影响：2. 《Marine Pollution Bulletin》62(3): 474-82.

#### 数据位置
本数据集存储于eAtlas持久数据存储库中，路径为：data
esp33.1.5_Pesticide-guidelines-GBR