Development of a miniature seagrass bioassay for PSII herbicides and its application to mixtures and simultaneous stressors

Seagrass species are widely distributed along all major coasts of Australia and provide a number of crucial ecological functions in which they grow, but also to surrounding ecosystems. During the monsoon season along the Great Barrier Reef (GBR) increased rainfall causes significant freshwater runoff from adjacent agricultural land, transporting suspended sediment, nutrient and contaminants into nearby rivers and ultimately coastal inshore waters. Photosystem-II (PSII) herbicides are the most commonly applied herbicides along the Queensland coast, and diuron is among the most commonly detected PSII herbicides in this marine environment. Recent risk assessments have highlighted the need for new tools to assess: (i) the toxicity of alternative PSII herbicides to seagrass, (ii) the combined effects of mixtures of PSII herbicides on seagrass and (iii) the combined effects of PSII herbicides in the presence of other stressors. A miniature bioassay methodology was developed and validated in Chapter 2 to measure effects of photosystem-II herbicide exposure on the seagrass, Halophila ovalis. Three exposure types: (i) potted, intact plants, (ii) hydroponic and (iii) isolated leaves, were experimentally exposed to a range of concentrations of diuron for a 24 h experimental period. The effects of diuron on photosynthetic activity in PSII (the target activity of PSII herbicides) was measured, including the inhibition of effective quantum yield (ΔF/Fₘ') and maximum quantum yield (Fᵥ/Fₘ) using pulse amplitude modulation (PAM) fluorometry. Results from the Imaging-PAM were compared directly to the widely used mini-PAM to evaluate consistency between methods. Identical sensitivity was observed for all exposure types and with both instruments (p > 0.05). The diuron concentration which inhibited ΔF/Fₘ' by 50% (IC₅₀) ranged from 2.1 to 4.3 μg l⁻¹ in all cases, which was also similar to previously published values for other seagrass species (IC₅₀ = 2.41 – 2.47, Flores et al 2013). Validation of the miniature bioassay technique, which measured acute fluorescence responses in isolated leaves with an Imaging-PAM, enabled confident application of this technique to: 1. assess the comparative toxicity of alternative PSII herbicides and mixtures to H. ovalis and, 2. assess the combined effects of herbicides and simultaneous stressors. Fifteen PSII herbicides are registered for application on farms in the GBR catchment. The miniature bioassay was applied in Chapter 3 to measure the comparative acute toxicity (24-48 h exposure) of 10 of these herbicides to PSII activity in H. ovalis in isolation and in mixtures (binary and complex combinations). Results revealed a wide range of toxicities (IC₅₀), with diuron the most potent (4.3 μg l⁻¹) following 24 h exposure and fluometuron the least toxic (132 μg l⁻¹). This thesis includes the first toxicity data for seagrass for five of these emerging herbicides (metribuzin, fluometuron, ametryn, bromacil and prometryn). PSII herbicides are usually detected in mixtures with other PSII herbicides in the GBR and its catchments. Since all PSII herbicides share the identical mode of action (binding to the D1 protein of PSII, displacing plastoquinone, blocking electron transfer and suppression of ATP production), the combined effect in mixtures were expected to be additive. The model of joint action Concentration Addition (CA) was applied to demonstrate additivity. The effects of both mixture types were largely additive, demonstrating that additive effects models can be used for calculating the risk posed by multiple PSII herbicides to seagrasses. The exposure of seagrass to herbicides is greatest in summer months in conditions that may include elevated temperature and irradiance stress. In Chapter 2, the miniature seagrass assay was used to examine 28 combinations of diuron from 0 to 100 μg l⁻¹ (7 levels) and light from 40 to 400 μmol m⁻² s⁻¹ (4 levels). In Chapter 5, the miniature bioassay was applied to 36 combinations of temperature and PSII herbicides. Temperature ranged from 15 to 40°C (6 levels), and diuron concentrations from 0 to 30 μg L⁻¹ (6 levels). As temperature, light and herbicides affect PSII activity (but with different modes of action) the model of Independent Action (IA) was used to assess whether these combined effects were additive, antagonistic or synergistic. High light exposures (200 and 400 μmol photons m⁻²s⁻¹) caused greater PSII inhibition than low light (40 and 100 μmol photons m⁻²s⁻¹), particularly at lower diuron concentrations. However, when the inhibition of ΔF/Fₘ' was calculated relative to controls at the same light intensity there was no significant difference in IC₅₀ (IC₅₀ = 2.2 – 3.5 μg l⁻¹; p = >0.05) among light treatments. When inhibition was calculated relative to the control light intensity (100 μmol photons m⁻²s⁻¹), the impacts on ΔF/Fₘ' and Fᵥ/Fₘ were additive, with the blocking of electron transport by diuron likely compounding the photooxidative stress under high light conditions. Photosynthetic activity in H. ovalis leaves as measured in the miniature assay was greatest at ~28°C and declines in ΔF/Fₘ' and Fᵥ/Fₘ were observed for extreme temperatures of 15 and 40°C in the absence of diuron. There was a strong interaction between temperature and diuron on PSII activity with large differences among temperatures at low diuron concentration, and convergence of inhibition at all temperatures at high diuron concentrations (10 and 30 μg l⁻¹). Comparison of observed inhibition of ΔF/Fₘ' versus additive inhibition predicted from the IA model revealed that the majority of combinations of diuron and temperature stress led to sub-additive interactions. The diuron ΔF/Fₘ' IC₅₀ values were lowest at 30°C indicating greatest toxicity. However, when inhibition of ΔF/Fₘ' was calculated relative to 30°C extreme temperature treatments induced greater inhibition at low herbicide concentrations compared to all other treatments. The miniature bioassay methodology is a robust approach to test seagrass H. ovalis sensitivity to PSII herbicides, and combined stressors. It was applied to build upon current ecotoxicological data sets for existing and emerging PSII herbicides. PSII herbicides negatively impact numerous photosynthetic processes in seagrass species. Additive and interactive effects occur when PSII herbicides are encountered simultaneously with other stressors, such as in PSII herbicide mixtures and / or sub-optimal environmental conditions. This work can improve risk assessments of key photosynthetic habitats to PSII herbicides and has the potential to improve ecological guidelines for PSII herbicides, with the aim of marine species protection.