Combined toxic mechanism and risk assessment triggered by acetamiprid and tetraconazole in honey bees

The widespread use of pesticides driven by agricultural intensification has emerged as a major contributor to the global decline in pollinator populations. Among these, the neonicotinoid insecticide acetamiprid (ACT) and the triazole fungicide tetraconazole (TTC) are frequently co-detected in various bee-derived products, yet the potential synergistic toxicity and ecological risk of their combined exposure remains underexplored. In this research, we examined the co-exposure effects of ACT and TTC on honey bees (Apis mellifera). Biochemical analyses revealed significant alterations in the activities of caspase-3 (CASP-3), polyphenol oxidase (PPO), cytochrome P450 (CYP450), and carboxylesterase (CarE) following exposure to either compound alone or in combination. Notably, co-exposure induced a markedly greater increase in these enzymatic markers, implicating amplified mitochondrial dysfunction, immune perturbation, and metabolic disruption. At the transcriptional profile, both individual and mixture treatments triggered differential expression of key genes, including apidaecin, dorsal-2, relish, and nAChRα2. These molecular changes mirrored the biochemical responses, with co-exposure eliciting more pronounced immunotoxic and neurotoxic effects than single exposures. The observed synergistic toxicity underscored a concerning interactive effect between ACT and TTC, highlighting their potential to exacerbate neurological damage, mitochondrial impairment, and immune dysregulation in pollinators. The classical assessment approach, which relies on simple concentration addition, tends to underestimate the co-exposure risk due to the synergistic interaction. In contrast, the binary weight of evidence (BINWOE) method effectively addresses this issue, providing a more accurate estimate of the combined exposure risk in such cases. Collectively, these findings shed new light on the compounded ecological risks posed by concurrent pesticide residues and emphasize the urgent need for integrated strategies to mitigate their detrimental impact on pollination services.