Antagonistic toxicity of co-exposure to DEHP and sulfadiazine in Eisenia fetida: Multi-level responses and molecular insights

s Phthalate esters (PAEs) are typically released from agricultural plastic films and veterinary antibiotics introduced through livestock manure. They often accumulate in agricultural soils, posing complex ecological risks and severe biological effects that are not yet fully understood. Therefore, this study investigated the ecotoxicity and risks of di(2-ethylhexyl) phthalate (DEHP), sulfadiazine (SDZ), and their co-exposure on earthworms. DEHP, SDZ, and their co-exposure was found to significantly impair earthworm growth and reproduction, induced oxidative stress, and altered the expression of functional genes ( tctp , ann , sod , cat , hsp70 , er ). Both DEHP and SDZ strongly bound to key earthworm proteins (SOD and TCTP), further supporting the evidence of oxidative stress and adverse effects on growth and development. Risk assessment revealed that DEHP exacerbated the reproductive and oxidative stress compared to SDZ and the co-exposure. Furthermore, histopathological and flow cytometric results suggested antagonistic interactions between DEHP and SDZ during co-exposure. Transcriptomics data demonstrated that SDZ activated pathways related to oxidative stress repair (peroxisome pathways) and detoxification (glutathione metabolism) in earthworms, which explains the relatively lower toxicity of co-exposure. Overall, these findings provide multi-level insights into the antagonistic effects of compound pollution in soil ecosystems and support the ecological risk assessment of PAEs and antibiotics.