Systematic evaluation of fluoride quantification methods identifies an appropriate measurement for studying fungal defluorination of per- and Polyfluoroalkyl substances.

The large-scale manufacturing and disposal of Per- and Polyfluoroalkyl substances (PFASs) are causing global concerns, and investigations are needed to understand their ecological impacts. Saprotrophic fungi are dominant nutrient recyclers in ecosystems, while their roles in transforming PFAS remain largely untapped. Particularly, there is a lack of appropriate means to rapidly measure fluoride anions released by fungal cultures to understand their defluorination (deF) capacities and mechanisms. In this research, we systematically evaluated three prevalent means for quantifying free fluoride anions under cultural conditions of wood decay fungal species. Two spectrophotometric measurings, including one based on the fluoride-catalyzed kinetochromic reaction of Xylenol Orange-Zirconium (XO-Zr) and another one based on Alizarin-Lanthanum-Fluoride (Al-La-F) ternary complexone reaction, were successfully adapted to a high-throughput micro-well setup for detecting micromolar-level fluoride, but essential fungal media components pose significant interference to both assays. The third method, relying on the potentiometer and fluoride ion-selective electrode (F-ISE), demonstrated a strong capacity for anti-interferences of fungal nutrients, metabolites, and PFAS, leaving it a most appropriate method to study fungal deF. With F-ISE, we further demonstrated a deF test using a model PFAS, 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid, and a wood rot fungus Trametes versicolor. Together, our research identifies a reliable fluoride quantification method that can allow assessing fungal cultures for their PFAS-defluorinating phenotypes.