Untargeted Metabolomics Reveals Size-dependent Toxic Mechanisms Of Silver Nanoparticles In Chlorella Vulgaris

Silver nanoparticles(AgNPs)is widely used in lots of aspects of life due to its good properties.But it will migrate into natural water bodies along with rainfall,sewage discharge and landfill leachate in the process of production,transportation and application,posing a threat to aquatic organisms and aquatic ecological environment.Most current research focuses on the toxic effects of AgNPs on aquatic organisms,but the toxic mechanism of AgNPs with different particle sizes during exposure and recovery is still unclear.In this study,Chlorella vulgaris was used as the test organism.Silver nanoparticles with a particle size of 5 nm,20 nm and 70 nm(AgNPs5,AgNPs20 and AgNPs70)were selected as the exposed materials.Metabolomics combined with traditional toxicology methods will reveal the toxic effects and potential mechanisms of C.vulgaris during the exposure and recovery period.The main results obtained are as follows:(1)Traditional toxicology methods have revealed the size-dependent toxicity of AgNPs in C.vulgaris.Acute toxicity test results show stress-response hormesis of algae.The 50%growth inhibitory concentrations(EC₅₀)of AgNPs5,AgNPs20 and AgNPs70 after 72 h were 54.1?g/L,53.5?g/L and 111.3?g/L,respectively,showing a significant particle size dependence.The chlorophyll content exposed to the three particle sizes of AgNPs was significantly reduced.The light curve was not affected,and the size-dependent toxicity was not obvious.The enrichment process of AgNPs in algae showed that the mass of large-size AgNPs was more enriched in the unit of algae cell.Small-size AgNPs is easier to release more silver ions and can significantly inhibit the growth of algae at a lower concentration.That indicate the silver ions of AgNPs in the solution may be the main reason for inhibiting the growth of algae.(2)Metabolomics results showed that the metabolic toxicity of C.vulgaris exposed to EC₅₀ for 72 hours was mediated by particle size.AgNPs with different particle sizes caused different expressions of algae metabolites.AgNPs70 specifically regulated up to 49 different metabolites.Small-size silver nanoparticles(AgNPs5 and AgNPs20)mainly down-regulated small molecules of glycerol,glycosides and hypoxanthine.Large-size silver nanoparticles(AgNPs70)specifically down-regulate amino acids.The results of Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment pathways showed that small-size AgNPs via inhibited glycerophospholipid metabolism and purine metabolism to affect the synthesis of lipids and DNA in algae.Large-size AgNPs reduces the transport efficiency of amino acids by inhibiting aminoacyl t-RNA biosynthesis,affecting amino acid metabolism,and then inhibiting protein synthesis.(3)Part of cell functions of C.vulgaris returned to normal after the cell was transferred to fresh BG11 medium.After 72 hours of recovery,the photosynthetic capacity of algae was restored,and the silver in the algae cells was excreted.The number of differential expression metabolites induced by different sizes of AgNPs decreased.Compared with the exposed groups,the expression pattern of metabolites in the recovery groups was closer to that of the control group.The pathways and related metabolites inhibited by the small-size AgNPs are restored.However,the Aminoacyl-t RNA biosynthesis mediated by large-size AgNPs is still down-regulated,which indicated the persistence of the toxicity to the amino acid metabolism of algae.