Study On The Steroselective Degradation And Toxicity Difference Of Cyflumetofen In Soil-Earthworm Microcosms

Pesticides play an important role in ensuring crop health,increasing production and income.However,due to the large-scale use of pesticides,a series of problems such as pesticide residues,environmental pollution,ecological damage and agricultural product safety have also been caused.During the 13th Five-Year Plan period,the national key research and development plan"Research and Development of Comprehensive Technology for Chemical Fertilizers and Pesticide Application Reduction and Efficiency Improvement"was successfully implemented,and the amount of pesticides achieved zero growth,while the utilization rate of pesticides increased significantly.In 2020,the pesticide utilization rate of the three major food crops is 40.6%,an increase of 4percentage points over 2015.At the same time,the state will further strengthen scientific and technological support,increase policy support,and strive to increase the pesticide utilization rate by another 3%in 2025.Even so,most pesticides will remain in the environment,causing a series of chain problems such as environmental pollution,ecological damage and resource shortage.Therefore,it is the main development trend of pesticides in the future to eliminate pesticides with high toxicity to agriculture,forestry,non-target organisms and ecological environment,and to encourage the development and use of safe,efficient and economical pesticides.About 40%of commonly used pesticides are chiral pesticides,and with the development and marketization of new pesticides,the proportion will continue to increase.However,only 7%of chiral pesticides have been developed as a commercial single isomer or highly potent isomer.The enantiomers of chiral pesticides have almost identical physicochemical properties,but often have huge differences in biological activity,residue behavior,and toxic effects.Therefore,the selective biological effects and toxicological mechanisms of chiral pesticide enantiomers have received extensive attention and have become an important research hotspot.Studying the differences in biological activity,residue behavior,environmental fate,and toxic effects of chiral pesticide enantiomers,and elucidating the relevant selectivity mechanisms will provide the basis for accurate environmental risk assessment of chiral pesticides and the development of efficient single isomers.CYF has been widely used in about 15 countries around the world due to its unique target,good acaricidal effect,and not easy to produce cross-resistance.It has a good market prospect.CYF contains two enantiomers（+）-CYF and（-）-CYF.Studies have shown that the degradation of CYF enantiomers in soil is significantly different,and the toxicity to human hepatoma cells（Hep G2）and zebrafish embryonic cells is significantly different.However,as soil is an important environmental carrier of pesticide residues,researches on the degradation behavior of buflufenafen enantiomers in soils with different properties,the differences in soil microbial structure,soil nitrogen cycle function,and toxic effects on earthworms have not been reported yet.Based on this,we carried out studies on the effects of CYF enantiomer degradation differences on soil microbial structure and function in different types of soil,as well as on the differences in earthworm toxicity,and obtained the following main research results:1.Enantiomer detection of cyflumetofen and its degradation trend in soilA method for the analysis of CYF enantiomers in soil was established by high performance liquid chromatography,and its degradation behavior in soil was studied accordingly.The half-lives of CYF enantiomers at a concentration of 10 mg/kg in three soils with different properties were:T1/2 Heilongjiang-clay loam>T1/2 Chongqing-clay>T1/2 Inner _{Mongolia-silty sandy soil}.Microorganisms promoted the degradation of CYF,and the half-life of enantiomers in sterilized soil and natural soil was:T_{1/2 sterilized soil}>T_{1/2 natural soil}.In addition,the selective degradation behavior of high concentrations of CYF enantiomers was analyzed.The enantiomeric half-life of CYF is different in soils with different concentrations,p H values and properties:In Heilongjiang-clay loam soil（p H=5.51）,low concentration T_{1/2（+）-CYF}=2.19 d,T_{1/2（-）-CYF}=2.82 d;high concentration T_{1/2（+）-CYF}=7.73 d,T_{1/2（-）-CYF}=10.28 d;In Inner Mongolia-silty sandy soil（p H=8.82）,low concentration T_{1/2（+）-CYF}=1.00 d,T_{1/2（-）-CYF}=2.03 d;high concentration T_{1/2（+）-CYF}=1.45d,T_{1/2（-）-CYF}=1.83 d.In both types of soils,（+）-CYF degraded faster than（-）-CYF.At the same time,in both soils,the enantiomer transformation was confirmed at high concentrations,and the transformation trends were different.In Heilongjiang-clay loam soil,（-）-CYF was converted to（+）-CYF;in Inner Mongolia-silty sandy soil,（+）-CYF was converted to（-）-CYF.2.The effect of the enantiomers of cyflumetofen on soil microbial population structureThe effects of different concentrations（0.5 mg/kg and 100 mg/kg）of CYF enantiomers on soil microbial population structure and bacterial abundance were detected by high-throughput sequencing and q PCR techniques.The results showed that different concentrations of CYF enantiomers inhibited the abundance of bacteria in the soil.In Heilongjiang-clay loam soil,（+）-CYF-treated bacterial abundances were lower;while in Inner Mongolia-silty sandy soil,（-）-CYF-treated bacterial abundances were lower.The effects of different concentrations of CYF enantiomers on soil microbial Alpha diversity and Beta diversity were as follows:both enantiomers inhibited Alpha diversity.In Heilongjiang-clay loam soil,（-）-CYF inhibited more significantly,while in Inner Mongolia-silty sandy soil,（+）-CYF inhibition was more pronounced.According to PCo A analysis,different concentrations of CYF enantiomers changed the original soil microbial population structure,and some beneficial microorganisms were significantly enriched in（-）-CYF treatment.3.Effects of cyflumetofen on soil nitrogen content and abundance of nitrogen cycling genesBy detecting nitrogen content in soil,it was found that the effect of CYF enantiomer on soil NH₄⁺-N and NO₃^--N content was significantly different.The effect of CYF enantiomer on NH₄⁺-N content is as follows:in Heilongjiang-clay loam soil,（+）-CYF treatment decreased by 11.88%-52.05%,（-）-CYF treatment decreased by 12.31%-35.64%;in Inner Mongolia-silty sandy soil On the 30th day,the content of NH₄⁺-N in（+）-CYF treatment was significantly lower than that in（-）-CYF treatment.Effects of CYF enantiomers on NO₃^--N content:in Heilongjiang-clay loam soil,（+）-CYF treatment increased by 11.51%-36.56%,（-）-CYF treatment inhibited by 6.60%-29.81%;while in Inner Mongolia-silty sandy soil Both showed significant inhibition,and the contents were decreased by 22.47%-82.10%and 8.82%-94.85%after（+）-CYF and（-）-CYF treatment,respectively.Long-term exposure of two soils to（+）-CYF resulted in a significant decrease in soil NH₄⁺-N content.The effects of CYF enantiomers on nitrogen cycle-related nitrogen-fixing bacteria（nif H）,nitrifying bacteria（AOB）,and nitrifying archaea（AOA）abundances were examined by q PCR.The effect of CYF enantiomers on nif H abundance was that in Heilongjiang-clay loam soil,（+）-CYF inhibited 25.36%-73.11%,（-）-CYF inhibited2.35%-72.94%;in Inner Mongolia-silty sandy soil,（+）-CYF inhibited 2.35%-72.94%,（-）-CYF inhibited 24.20%-75.02%,while（-）-CYF had no obvious regularity.Effects of CYF enantiomers on AOB and AOA gene abundance:In Heilongjiang-clay loam soil,（+）-CYF increased by 3.90%-105.31%and 5.08%-71.12%,while（-）-CYF inhibited by18.78%-62.30%and 0.54%-27.45%;in Inner Mongolia-silty sandy soil,（+）-CYF and（-）-CYF inhibited by 21.48%-63.93%and 5.35%-84.81%,respectively.In two typical soils,（+）-CYF suppressed nif H abundance more severely,while（-）-CYF suppressed AOB and AOA abundance more severely.Microorganisms related to environmental factors were obtained by combined analysis of LEf Se,RDA and q PCR.Among them,g-Massilia and g-Arthrobacter were highly correlated with nitrogen fixation and nitrification,and Arthrobacter was closely related to nitrogen fixation and nitrification.The degradation of CYF is closely related.4.Study on the selectivity and acute toxicity differences of the enantiomers of cyflumetofen in earthwormsA method for the analysis of CYF enantiomers in earthworms was established,and the results showed that the degradation of CYF enantiomers in artificial soils containing earthworms had significant differences.Compared with（-）-CYF,earthworms had a stronger ability to enrich（+）-CYF,and（+）-CYF remained in earthworms for a longer time.The acute toxicity of Rac-CYF,（+）-CYF and（-）-CYF to earthworms was detected by the filter paper method in order:Rac-CYF（162.5μg/cm²）>（+）-CYF（309.4μg/cm²）≈（-）-CYF（323.1μg/cm²）.The combined toxic effect analysis showed that the toxic effects of the two enantiomers were additive.5.Differences in the chronic toxicity of cyflumetofen enantiomers to earthwormsThe chronic toxicity of CYF enantiomers to earthworms was investigated using five indicators including body weight change,oxidative stress response,detoxification metabolic enzyme activity,cellular DNA damage and transcriptomics.The results showed that long-term exposure of earthworms to artificial soil containing（-）-CYF significantly reduced their body weight and had a dose-effect;the oxidative stress marker superoxide dismutase（SOD）of earthworms under（+）-CYF treatment,catalase（CAT）and malondialdehyde（MDA）specific activities were significantly higher than（-）-CYF treatment,while（-）-CYF treatment of detoxification metabolic enzymes,such as multifunctional oxidase（MFO）and Glutathione-S-transferase（GST）activity was higher;DNA comet patterns in（+）-CYF-treated earthworm cells had longer trailing distances,indicating greater genotoxicity to earthworms.The results of transcriptome sequencing showed that the number of differentially expressed genes（DEGs）in earthworms treated with（+）-CYF was higher than that in（-）-CYF treatment,and the higher the concentration,the more DEGs.Functional annotation of DEGs mainly focuses on binding,catalysis and biological metabolism.Further focusing on the functional annotation of DEGs related to human disease pathways and detoxification metabolic pathways,it was found that（+）-CYF-treated earthworms significantly increased the number of human disease-related genes in the up-regulated DEGs of（+）-CYF-treated earthworms.The number of genes related to drug metabolism and P450 and glutathione metabolism were higher in CYF-treated earthworms.In conclusion,the two enantiomers of CYF showed significant differences in degradation and ecotoxicological properties:both enantiomers degraded faster in alkaline and silty sandy soils at low concentrations,But（-）-CYF has a longer half-life and longer residual time;in addition to both enantiomers can significantly inhibit nitrification in Heilongjiang-clay loam soil and Inner Mongolia-silty sandy soil,（+）-CYF also significantly inhibits nitrogen fixation in Inner Mongolia-silty sandy soils,（-）-CYF is more likely to activate the detoxification metabolism of earthworms,while（+）-CYF is more likely to enrich and activate its oxidative stress response in earthworms,causing more obvious cytogenetic toxicity and activating more disease-related gene expression.Overall,（+）-CYF showed a higher ecotoxicological risk than（-）-CYF.