Multiple environmental factors mediated-toxicity and mechanisms of environmentally relevant silver nanoparticles in Chlorella vulgaris

The extensive application of silver nanoparticles (Ag NPs) has raised growing concerns regarding their potential ecological risks in aquatic environments. However, current studies remain largely on high dose exposure upon single factor, overlooking the complexity and heterogeneity of natural aquatic systems. This study investigated seven environmental factors on the response of Chlorella vulgaris ( C. vulgaris ) upon exposure of Ag NPs at environmentally relevant concentrations. The results showed that chloride ions (Cl - ), nitrogen and phosphorus nutrients, and light intensity are the dominant regulatory factors. Cl - exhibited the strongest mitigation, reducing C. vulgaris growth inhibition rate from 10.5% to −13.6% by promoting Ag NPs aggregation and reducing Ag bioavailability. Significant interactions were observed between dissolved organic maters (DOM) and pH, as well as phosphorus and temperature, which jointly regulated Ag NPs toxicity. Given the key role of DOM, the extracellular polymeric substances (EPS) secreted by C. vulgaris were further examined. EPS mitigates Ag NPs toxicity, by forming a protective layer, reducing direct contact between NPs and cells, and enhancing the Cl - buffering. Transmission electron microscopy showed that Cl - maintained cellular structural integrity, with EPS further enhancing organelle integrity. Metabolomic analysis indicated that Cl - significantly upregulated glutathione and α-linolenic acid metabolism, elevating reduced glutathione to 21.8-fold of the control (only 2.61-fold with EPS). Levels of 12-OPDA and 9-HpODE decreased significantly, while the milder reduction in 13-HOTrE with EPS indicated a buffering effect on membrane metabolic disorders. This study provides insights for elucidating the biological responses of Ag NPs in complex aquatic environments.