Fungicide effects on diclofenac uptake, translocation and tolerance in zucchini and its endophytes

Pharmaceutical pollutants like diclofenac, a widely used non-steroidal anti-inflammatory drug (NSAID), are frequently found in agricultural soils, yet their effects on plant-associated microbiota remain poorly understood. Diclofenac is classified as hazardous under regulations (e.g., EU REACH, US EPA), monitored as a contaminant of emerging concern, and listed for priority monitoring due to its persistence and ecotoxicity. Cucurbit species, including zucchini (Cucurbita pepo cv. Atena Polka), can bioaccumulate such pollutants, affecting crop performance and human exposure. We examined the effects of diclofenac (2.5 mg/kg), alone and with the fungicide benomyl, on physiological, biochemical, and microbiological traits in zucchini over 28 days. We quantified diclofenac uptake and translocation, measured biomass, chlorophyll, and phenolic compounds, and assessed endophytic bacterial diversity using Biolog EcoPlates™ and 16S rRNA gene sequencing. Diclofenac primarily accumulated in roots, but benomyl co-application significantly increased shoot accumulation (17-fold) and translocation (49-fold). Diclofenac exposure reduced plant biomass, phenylpropanoids, and endophytic microbial activity and diversity. Benomyl partially mitigated these effects, enhancing microbial function and plant resilience. Our findings reveal that co-occurring agrochemicals can modulate pharmaceutical behavior in plants and their microbiomes, with important implications for environmental risk assessment and food safety in contaminated agroecosystems.