Comprehensive analysis of zearalenone exposure and molecular effects in Rhodococcus pyridinivorans K404

Abstract Zearalenone (ZEA) is a Fusarium-derived mycotoxin that contaminates cereal crops and persists in soils and aquatic environments. Microbial biodegradation is increasingly proposed as a sustainable remediation strategy; however, a critical but often overlooked question is whether chemical disappearance of a toxin reflects actual detoxification. Here, we investigated the biotransformation of ZEA by Rhodococcus pyridinivorans K404 using an integrated analytical, toxicological, and molecular framework using time-resolved chemical profiling, bioluminescent yeast estrogen screening (BLYES/BLYR), zebrafish embryo toxicity assays, and transcriptome-wide RNA-sequencing. Although strain K404 reduced the ZEA concentration in the culture supernatant by up to 90.3% within 72 h, substantial amounts of ZEA and its metabolites were associated with the bacterial pellet, indicating that extracellular depletion reflected both biotransformation and redistribution into a cell-associated fraction. This decrease in supernatant ZEA concentration did not translate into a proportional decrease in biological hazard. In microinjection experiments, treated samples showed significantly higher developmental toxicity; embryo mortality peaked at 72% after 48-h treatments before falling to 53% at 120 h. This transient increase in toxicity aligned with accumulation of hydroxylated metabolites, mainly β-zearalenol and α-zearalenol, implicating biotransformation intermediates as key causes of acute toxicity. The bacterial strain showed a stress-associated transcriptional profile, dominated by genes of iron acquisition and oxidative stress defense. These results demonstrate that microbial ZEA degradation can transiently heighten toxicological risk, instead of reducing it, underscoring that microbial biocontrol strategies in agroecosystems must not rely solely on chemical depletion data but require comprehensive toxicological assessment, particularly through chronic exposure and long-term ecotoxicity studies. Key points • Rhodococcus pyridinivorans K404 efficiently removed ZEA (up to 90.3% in 72 h) • Transient toxicity increased via metabolites, while ZEA estrogenicity declined. • Transcriptomics at 48 h links peak cellular stress to metabolite driven toxicity.