A novel, efficient, and ecologically relevant bioassay method using aquatic fungi and fungus-like organisms for fungicide ecological effect assessment.

Fungicides are used to control fungal plant pathogens, but they may also be highly toxic to aquatic fungi, which play an important role in natural aquatic ecosystems. However, a bioassay method using aquatic fungi has not been sufficiently developed. In the present study, a novel, efficient, and ecologically relevant bioassay method was developed for the ecological effect assessment of fungicides. Candidate test species were selected by considering the following 4 factors: 1) their ecological relevance (i.e., widely distributed and frequently observed) in freshwater habitats, 2) inclusion of a wide range of taxonomic groups, 3) availability from public culture collections, and 4) suitability for culture experiments using a microplate. The following 5 fungal species were selected: Rhizophydium brooksiaum (Chitridiomycota), Chytriomyces hyalinus (Chitridiomycota), Tetracladium setigerum (Ascomycota), Sporobolomyces roseus (Basidiomycota), and Aphanomyces stellatus (Oomycota, fungus-like organism). An efficient test method using the 5 species was developed based on a microplate assay using a 96-well white microplate and a test duration of 48 h. Fungal biomass was determined as adenosine 5'-triphosphate (ATP) luminescence, which is known to be proportional to live cell density and can be determined with a microplate reader. Test performance was evaluated by conducting bioassays of 3,5-dichlorophenol and malachite green as standard test substances. Fungal species were clearly more sensitive than other species to the fungicide malachite green. Environ Toxicol Chem 2018;37:1980-1989.