Freeze-thaw cycles intensified the synergistic inhibitory effects of enrofloxacin and UV-328 on rye seedling growth

Veterinary Enrofloxacin (ENR) and UV-328 residues from agricultural mulch are common co-occurring organic pollutants in agricultural fields. Freeze-thaw (FT) cycles represent a prevalent stressor for crop seedlings at high latitudes and altitudes. However, studies investigating the toxic effects of FT cycles combined with multi-pollutant stress (ENR and UV-328) on crop seedlings in alpine grasslands remain limited. In this study, we selected leaves as experimental material to investigate the coupled stress effects of FT, ENR and UV-328 on rye seedlings. The results demonstrated that compared with single pollution stress, the combined pollution of ENR and UV-328 significantly enhanced the toxicity to crops, synergistically exacerbating oxidative stress responses under FT conditions. This combination disrupted cell membrane integrity and increased the activities of antioxidant enzymes. Furthermore, ENR and UV-328 impaired rye photosynthesis under FT conditions, leading to reduced contents of photosynthetic pigments and significant decreases in photosynthetic parameters (net photosynthetic rate, transpiration rate, etc.). Molecular docking analysis revealed that ENR and UV-328 bind to antioxidant enzyme, thereby altering antioxidant enzyme activities and affecting crop growth and development. Integrated biomarker response index (IBRv2) analysis indicated that FT was the primary determinant of significant ecotoxicity. Collectively, these findings confirmed that FT cycles exacerbated the synergistic ecotoxicity of ENR and UV-328 to rye seedlings. Accordingly, it is recommended to reduce the application of antibiotics and UV-328 in agricultural practices of FT regions. Future research may focus on deciphering the underlying molecular mechanisms of this synergistic toxicity and developing remediation technologies based on soil substrates.