Development and validation of MRM analysis of pesticide metabolites for the environmental safety assessment of biobeds disposal

Biobeds are low-cost bioreactors used for the remediation of contaminated wastewater generated during agricultural practices. Despite their effectiveness in degrading parent compounds, limited attention has been paid to the potential accumulation of toxic metabolites in the biomixture before its disposal in the soil. In this study, the development and validation of a multiresidue analytical method for determining 30 pesticide metabolites, corresponding to 23 active substances, was carried out in biomixture samples from biobed systems. The method was based on acetonitrile extraction and cleanup steps compatible with both gas chromatography and liquid chromatography coupled to triple quadrupole mass spectrometry (GC-MS/MS and LC-MS/MS) operated in multiple reaction monitoring (MRM) mode. Two extraction methods were compared, and the one offering better overall recovery and precision was selected for full validation. Method performance parameters-including linearity, matrix effect, limits of quantification (LOQ), trueness, and precision-were assessed according to SANTE guidelines. A cross-validation was successfully conducted between two laboratories using different instrumental platforms. The validated method was then applied to real biomixture samples from biobed experiments. Among the metabolites monitored, boscalid 5-hydroxy (M510F01), included in the MRL definition but not in environmental databases, was quantified in nine samples, with concentrations ranging from 241.8 to 1053.4 µg kg^-1. This study highlights the need to assess not only parent compound degradation but also the formation and persistence of metabolites when evaluating the environmental safety of biobed disposal. The proposed method is a valuable tool for supporting risk assessment of used bio-mixtures before their release into the environment.