Validation of miniature bioassay for assessing herbicide toxicity to seagrass (NERP TE 4.2, AIMS and JCU)

This dataset shows the measured response of the photosystems of seagrasses to herbicides in experiments conducted in 2014. The purpose of the experiments were to develop and validate a miniature toxicity assay using isolated seagrass leaves in 12-well plate.

The aim of this study was to quantify the acute phytotoxicity of PSII herbicide, Diuron, on the seagrass Halophila ovalis while validating a 12-well plate fluorescence bioassay using the imaging pulse amplitude modulation fluorometry. Fluorescence-derived phytotoxicity endpoints in the isolated leaves were directly compared with potted and unpotted but intact (hydroponic) seagrasses and the influence of light on photosynthetic efficiency and damage to PSII were assessed. These data will enable improved assessment of the risks posed by PSII herbicides to tropical seagrass for both regulatory purposes and for comparison with other taxa.

Methods:

A miniature 12-well plate acute phytotoxicity assay was developed to assess the exposure of seagrass to PSII herbicides:-
1. All acute exposures (up to 24 h) were conducted in static conditions using measured concentrations of Diuron.
2. Pule amplitude modulation (PAM) fluorometry (see below) was applied as a sensitive indicator of PSII herbicide toxicity to isolated seagrass leaves and intact plants. Inhibition of photosynthesis was measured after 24 h exposure using (PAM) fluorometry. Two parameters were measured (effective quantum yield, deltaF/F’m and maximum quantum yield, Fv/Fm). The inhibition of photosynthetic yields relative to controls were plotted as dose-response curves by fitting inhibition data with measured concentrations using a 4-parameter logistic model (SigmaPlot 11). The herbicide inhibition concentrations (ICxx) that inhibited deltaF/Fm' and Fv/Fm by 10 and 50% (IC10 and IC50, respectively) were determined from each curve.
3. Leaves were screened for acceptable levels of photosynthetic efficiency before the start of each experiment.
4. Rapid light curves were used to assess the photosynthetic performance of the seagrass as a function of irradiance and to enable the selection of ambient illumination for the experiments.
5. Fluorescence images were taken using the I-PAM to spatially assess photosynthetic impact of Diuron in the isolated leaves.
6. The photosynthetic condition of plants were re-examined by I-PAM regularly over 24 h in the absence of herbicide to test for leaf deterioration over the exposure period.
7. Dose-response relationships were compared between I-PAM and Mini-PAM data to verify consistency with other studies.
8. Dose-response relationships were compared between isolated leaves in 12-well plates and intact plants (both potted and hydroponic) to validate the sensitivity of the well-plate method.
9. Dose-response relationships were compared using the well plate method at four light levels to (i) test consistency and repeatability under different irradiance conditions and (ii) examine the potential for Diuron to impact on seagrass under varying light conditions.
10. Potential interactions between irradiance and Diuron on effective and maximum quantum yields were explored using the Independent Action (IA) model.

Format:

Miniature bioassay dataset_Wilkinson_et_al_2014.xlsx:

This is the measured response of the photosystem of Halophila ovalis (a seagrass species) to Diuron over time.

- Plant rep: Plant replicate (3 plants used in each potted and hydroponic tank).
- Leaf rep: leaf replicate (21 leaves used for solvent control and 9 leaves used per treatment).
- deltaF/Fm': effective quantum (light adapted) yield measured by a Pulse Amplitude Modulated (PAM) fluorometer.
- Fv/Fm: maximum quantum (dark adapted) yield measured by a Pulse Amplitude Modulated (PAM) fluorometer.
- Solvent control: no herbicide but contains less than 0.03% v/v ethanol carrier as per the treatments.
- Time (hr): duration of exposure in hours (24 h was the duration of the herbicide exposure).
- PAR: Photosynthetically active radiation (light intensity) in µmol photons m-2s-1 (µE).

Leaves were plucked from plants and randomised in well plate assays so there should be no “plant rep” for those experiments. However plants rather than leaves were tracked in the potted and hydroponic system so here “plant rep” is valid.

Two measurements were made for each experiment: deltaF/Fm’ and Fv/Fm. These measurements are recorded in different columns as there were sometimes more solvent control measurements. As a result the matching Treatment, Plant Rep, Time columns were duplicated for each experiment.

For the Diuron-Light assay there were sometimes a different number of replicates if leaves were out of focus for the measurement or the fluorescence was too low.

For the RLC the replicate is a plant replicate.

本数据集记录了2014年开展的海草光系统（photosystem）响应除草剂的实测结果。本实验的目的为开发并验证一套基于12孔板（12-well plate）分离海草叶片的微型毒性试验方法。

本研究的核心目标为定量评估光系统II（PSII）除草剂敌草隆（Diuron）对卵形喜盐草（Halophila ovalis，一种海草物种）的急性植物毒性，同时验证采用成像脉冲振幅调制荧光仪（I-PAM）的12孔板荧光生物检测方法。研究中将分离叶片的荧光衍生毒性终点与盆栽及非盆栽完整（水培）海草进行直接比对，并评估光照对光合效率及光系统II损伤的影响。本数据集可为监管需求及与其他生物类群比对提供更精准的PSII除草剂对热带海草的风险评估依据。

方法：
本研究开发了微型12孔板急性植物毒性试验方法，用于评估海草暴露于PSII除草剂的情况：
1. 所有急性暴露实验（最长时长24h）均采用静态体系，使用定量添加的敌草隆进行处理。
2. 脉冲振幅调制（Pulse Amplitude Modulation，PAM）荧光法（详见下文）作为检测分离海草叶片及完整植株受PSII除草剂毒性影响的灵敏指标。暴露24h后采用PAM荧光法测定光合抑制水平，测定两项参数：有效量子产率（deltaF/Fm'）与最大量子产率（Fv/Fm）。以对照组为基准，将光合产率抑制数据与实测浓度拟合，采用四参数Logistic模型（SigmaPlot 11）绘制剂量-反应曲线，并通过每条曲线计算得到使deltaF/Fm'与Fv/Fm分别降低10%与50%的除草剂抑制浓度（IC10与IC50，对应标注）。
3. 每项实验开始前，均对叶片的光合效率水平进行筛选，确保其处于合格范围。
4. 采用快速光曲线（RLC）评估海草在不同辐照度下的光合性能，以此确定实验所需的环境光照条件。
5. 使用I-PAM拍摄荧光图像，以空间可视化方式评估敌草隆对分离叶片的光合影响。
6. 在无除草剂的条件下，于24h暴露期间定期通过I-PAM重新检测植株的光合状态，以验证暴露周期内叶片是否出现退化。
7. 比对I-PAM与Mini-PAM的剂量-反应关系数据，以验证本研究与其他相关研究的一致性。
8. 比对12孔板中分离叶片与完整植株（包括盆栽与水培植株）的剂量-反应关系，以验证孔板试验方法的灵敏度。
9. 在4种光照梯度下采用孔板试验方法测定剂量-反应关系，以：(i) 验证不同辐照度条件下试验的一致性与可重复性；(ii) 探究不同光照条件下敌草隆对海草产生影响的可能性。
10. 采用独立作用（Independent Action，IA）模型，探究辐照度与敌草隆对有效量子产率及最大量子产率的潜在交互作用。

数据集格式：
微型生物测定数据集_Wilkinson等（2014）.xlsx：
本数据集记录了卵形喜盐草（Halophila ovalis）的光系统随时间变化对敌草隆的响应结果。
- 植株重复（Plant rep）：植株重复样本（每个盆栽与水培培养槽各使用3株植株）。
- 叶片重复（Leaf rep）：叶片重复样本（溶剂对照组使用21片叶片，每个处理组使用9片叶片）。
- deltaF/Fm'：有效量子（光适应态）产率，通过脉冲振幅调制（PAM）荧光仪测定。
- Fv/Fm：最大量子（暗适应态）产率，通过脉冲振幅调制（PAM）荧光仪测定。
- 溶剂对照组：不添加除草剂，但按照处理组的标准添加体积分数低于0.03%的乙醇载体。
- 暴露时长（Time, hr）：以小时为单位的暴露时长（除草剂标准暴露时长为24h）。
- 光合有效辐射（PAR）：以µmol photons m-2s-1（即µE）为单位的光强。

叶片重复试验中，叶片采自植株并随机分配至孔板，因此该类试验无需设置“植株重复”。但在盆栽与水培体系中，研究对象为植株而非叶片，因此此处“植株重复”的标注有效。

每项实验均测定两项参数：deltaF/Fm'与Fv/Fm。由于溶剂对照组的测定次数通常更多，因此两项参数被分至不同列中记录。由此导致每组实验对应的处理、植株重复、暴露时长列均存在重复。

在敌草隆-光照试验中，若叶片成像失焦或荧光信号过弱，则该样本的重复数量可能存在差异。

快速光曲线（RLC）试验的重复样本为植株重复。