A rapid resazurin bioassay for assessing the toxicity of fungicides

Abstract

Fungicides are widely used in agriculture, and released in large amounts to the environment. Methods used for antifungal susceptibility testing are cumbersome and time-consuming. As a result, very little attention has been paid to including fungal tests in the routine screening of pesticides and there are no reports in the literature of fungicide focussed effects directed analysis (EDA). In addition very little is known on the toxicity of fungicides to environmentally significant fungi. Here we report a rapid microplate-based resorufin fluorescence inhibition bioassay and compare it with a 24 h microplate-based yeast growth inhibition bioassay using eight fungicides. The growth inhibition bioassay was sensitive, giving IC50 and IC90 values comparable to previously reported IC50 or MICs of these fungicides for Saccharomyces cerevisiae and other fungi. The resorufin fluorescence inhibition bioassay was both faster and more sensitive than the growth inhibition bioassay. Inhibitory concentrations obtained just after 30 min of incubation with amphotericin B (AMB) and captan were at least a hundred fold lower than IC50s in the literature for fungi. The fluorescence bioassay showed only a small response to pyrazophos and thiabendazole but these only inhibited growth at high concentrations so this may reflect low sensitivity of S. cerevisiae to these particular fungicides. This bioassay can detect toxic effects of a range of fungicides from different chemical classes with different modes of action. It will be valuable for screening chemical libraries for fungicides and as a biomarker for detecting the effects of fungicides to non-target fungi.