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.

Tags: marine
Organization: Australian Ocean Data Network