Toxic Effect And Mechanism Of Two Pesticides (Pyraclostrobin And Pyroxsulam) Alone And Co-Exposed With ZnO Nanoparticles On Earthworms

Pyraclostrobin（PYRA）and pyroxsulam（PYRO）are the representative commercial formulation of strobilurin fungicide and triazolpyrimidine sulfonamide herbicides respectively,which are widely used in agricultural.With their extensive use,they produce residue in farmland soil environment and toxicity to non-target soil animals.Earthworms play an important role in soil ecosystems.Although PYRA has been proven to be toxic to earthworms in artificial soil,the toxicity of PYRA and PYRO to earthworms in actual farmland soils has not been studied.Zinc oxide nanoparticles（nZnO）is one of the most widely used nanomaterials at present.Extensive industrial and agricultural use will cause its release into the soil environment,resulting in joint exposure risk with PYRA and PYRO.However,the effect of nZnO on PYRA and PYRO toxicity remains unknown.The aim of this study was to clarify the toxicity differences of PYRA and PYRO in soils with different physicochemical properties and the effects of nZnO on PYRA and PYRO toxicity.Three representative farmland soils（red clay,black soil and fluvo-aquic soil）widely distributed in China were selected.Eisenia fetida was exposed to PYRA at different concentrations(0.1,1.0,and 2.5 mg kg^-1).The changes of 6biomarkers related to oxidative stress,including reactive oxygen species（ROS）content,superoxide dismutase（SOD）activity,catalase（CAT）activity,glutathione S-transferase（GST）activity,malondialdehyde（MDA）content and 8-hydroxy-deoxyguanosine（8-OHd G）content,were determined.Finally,the integrated biomarker response（IBR）index was calculated to compare the difference of oxidative stress of PYRA to E.fetida in different soils.In addition,the effects of PYRO(1 and 10 mg kg^-1)on oxidative stress of E.fetida were studied in black soil,artificial soil,artificial soil reduced organic matter,red clay,and red clay with increased organic matter or p H.In order to clarify the effect of nZnO on the toxicity of PYRA and PYRO and the molecular mechanism of the toxicity,we analyzed the toxicity mechanism of PYRA(0.5 mg kg^-1)single and co-exposure with nZnO(10.0 mg kg^-1)on E.fetida by using oxidative stress IBR index and transcriptomics.The mechanism of toxicity of PYRO(0.1 mg kg^-1)single and co-exposure with nZnO(10.0 mg kg^-1)on E.fetida was analyzed using oxidative stress IBR index,transcriptomics,metabolomics and lipidomics.The main research results are as follows:（1）Both PYRA and PYRO induced excessive ROS level in E.fetida in different soils,resulting in oxidative stress and oxidative damage.The oxidative stress IBR index showed that the oxidative stress toxicity of PYRA to E.fetida in artificial soil was smaller than that in farmland soils.The order of toxicity was artificial soil<fluvo-aquic soil<black soil<red clay.The comprehensive toxicity of PYRO to E.fetida in artificial soil was greater than that in farmland soils,and the toxicity order was red clay<black soil<artificial soil.Reducing organic matter content in artificial soil and increasing organic matter content in red clay can increase and decrease the oxidative stress toxicity of PYRO to E.fetida,respectively.In the same soil,the higher the organic matter content,the lower the toxicity of PYRO.（2）The comprehensive toxicity of PYRA to E.fetida was enhanced by adding nZnO,but the comprehensive toxicity of PYRO+nZnO treatment group is roughly equivalent to that of PYRO single treatment group.PYRA+nZnO group induced E.fetida to produce more excess ROS,and caused more severe changes in SOD and GST activities,resulting in more serious DNA oxidative damage.In the PYRO+nZnO group,excessive ROS was induced in E.fetida,activities of SOD and GST was inhibited,and eventually resulted in more serious DNA oxidative damage.（3）PYRA+nZnO group induced more differentially expressed genes than PYRA single pollution,and the number and significance of differentially expressed genes were greater.PYRA single contamination affected nuclear transport,cell morphology,feeding and metabolic behavior,and cell function of E.fetida.PYRA+nZnO caused E.fetida catalytic and detoxification enzyme abnormal,energy metabolism and utilization disorders,cell senescence apoptosis,the release of inflammatory factors.（4）In PYRO+nZnO group,E.fetida produced more differentially expressed genes,metabolites and lipid molecules than that in PYRO group.Transcriptomics GO enrichment analysis showed that both PYRO single and PYRO+nZnO combined contamination could affect the biological processes of E.fetida,including but not limited to protein degradation,DNA repair,endocytosis,autophagy,transcription,immunity,and inflammation,through the cellular signaling of ubiquitin-protein binding.In addition,PYRO single pollution can also affect biological processes such as cell fate,exoskeleton formation and cell components,and affect angiogenesis,catalytic activity,intracellular transport and carbohydrate metabolism of E.fetida through Notch receptor binding,chitin binding signal and scaveman receptor signal.PYRO+nZnO combined contamination may also induce inflammation and innate immunity through the activation of TNFαfactor by MAP kinase in E.fetida.Transcriptomics KEGG enrichment analysis showed that PYRO single contamination affected the lipid metabolism of E.fetida and the fluidity and flexibility of cell membrane,while there were no significant KEGG pathway enrichment in PYRO+nZnO group.Metabolomics KEGG enrichment analysis showed that both PYRO single and PYRO+nZnO combined pollution affected E.fetida amino acid metabolism,and affected energy metabolism and cell fate through ATP binding box transport and TCA cycling pathways.In addition,PYRO single pollution on butyric acid metabolism may lead to E.fetida intestinal epithelial barrier disorders.PYRO+nZnO combined pollution also affects more complex TCA cycles and upstream and downstream metabolic processes,including pyruvate metabolism,pantothenic acid and coenzyme A biosynthesis,and glycolysis/gluconogenesis.Lipidomics analysis showed that both PYRO single and PYRO+nZnO combined pollution had profound effects on E.fetida lipid metabolism.The main effects were that the contents of phosphatidyl ethanolamine（PE）and phosphatidyl choline（PC）increased,while the contents of lysate phosphatidyl choline（LPC）decreased.PYRO+nZnO combined pollution has more complex and far-reaching effects on E.fetida cell signal transduction and cell growth regulation than PYRO single pollution.This study enriched the toxicological data of single PYRA and PYRO pollution and combined with nZnO pollution on soil organisms,and provided a more comprehensive idea for ecological risk assessment of pesticide pollution:In addition to assessing the toxicity of a single exposure to a contaminant in model soil,there is also a need to assess the toxicity of a contaminant in farmland soils and the toxicity of co-exposure with contaminants that may coexist.