The Study Of Detection Methods And Toxicity Analysis On Chloroacetamide Herbicides And Their Metabolites

In this paper,chloroacetamide herbicides and their metabolites were studied.Firstly,a rapid and sensitive detection method was established to detect three herbicides and four metabolites simultaneously,which provided technical support for toxicological analysis.Secondly,the interaction mechanism of the metabolites of chloroacetamide herbicides and bovine serum protein was studied to explore the binding mode of chloroacetamide herbicides metabolites to macromolecules.Thirdly,the application of human hepatoblastoma cells(HepG2)was used to establish a model to investigate the toxic effects and apoptotic mechanisms of chloroacetamide herbicides and their metabolites in vitro.Finally,toxicity on embryonic development and possible apoptotic mechanism of chloroacetamide herbicides and their metabolites were studied using zebrafish embryos as models,with a view to extracting relevant forensic toxicological analysis work and environmental ecotoxicological research on chloroacetamide herbicides poisoning cases as a certain theoretical basis.The main contents were as follows:Part 1 Study on the simultaneous detection of chloroacetamide herbicides and their metabolites by heart-cutting two dimensional liquid chromatographyObjective:To establish a new method for simultaneous determination of chloroacetamide herbicides and their metabolites by heart-cutting two-dimensional liquid chromatography.Method:The on-line solid phase extraction and separation conditions were optimized,and the simultaneous determination of Acetochlor,Metolachlor,Butachlor and their metabolites CDEPA,CMEPA,DEA and MEA was realized.Results:The linearity of the seven substances was good in the range of 0.018-3.400mg/L.The correlation coefficient R2 was greater than 0.99,and the detection limit was 0.009-0.026mg/L.The blank water samples were used as matrix and analyzed according to the optimum conditions.The recoveries of blank water samples were 78.1%-98.6%and RSD was 2.85%-8.62%.Conclusion:The method was rapid and sensitive for simultaneous determination of various substances,and could be used for simultaneous determination of chloroacetamide herbicides and metabolites in environmental water.Part 2 Study on interaction between chloroacetamide herbicides metabolites and bovine serum albuminObjective:To study the interaction of three metabolites of chloroacetamide herbicides with bovine serum albumin(BSA).Method:The rate constants(Kq),quenching constants(Ksv),number of binding sites(n),binding constants(Kc)and Hill coefficient(nH)of DCA,DEA and MEA with BSA at different temperatures were studied by fluorescence spectroscopy.Thermodynamic parameters,enthalpy change(?H),entropy change(?S)and Gibbs free energy(?G)were determined by Van't Hoff equation.Results:All three metabolites could bind to bovine serum albumin.The fluorescence quenching mode between DEA,MEA and BSA was dynamic quenching,while static quenching occured between DCA and BSA.The binding ratio of three metabolites to BSA was 1:1,and high temperature was not conducive to the interaction between metabolites and BSA.The combinations of DEA,MEA and BSA were spontaneous exothermic reaction,in which hydrophobic force played a major role.Conclusion:Three main metabolites of chloroacetamide herbicides could bind to bovine serum albumin under simulated conditions in vitro.Part 3 Cytotoxicity and apoptotic effects of chloroacetamide herbicides and their metabolites on human hepatoblastoma cells(HepG2)Objective:To study the cytotoxicity and apoptotic mechanism of two herbicides(acetochlor and metolachlor)and their common metabolites CMEPA and ME A in vitro.Method:After the cells were treated,the toxic effects of herbicide and its metabolites on cells were studied by CCK-8 method.The oxidative damage of cells after exposure was detected.The DNA damage was analyzed by single cell gel electrophoresis(Comet assay),and the apoptosis rate and expression of apoptosis related proteins were analyzed by flow cytometry and western blot.Results:Both herbicides(acetochlor and metolachlor)and their common metabolites had different degrees of toxicity to cells,among which the metabolite CMEPA was more toxic;the level of ROS in cells increased significantly;and the SOD activity,GSH-Px level and LDH level changed;DNA damage was higher than control group.With the increasing of concentrations of chloroacetamide herbicides and their metabolites,the apoptosis rate increased.Endoplasmic reticulum stress markers BIP,CHOP and Caspase-3 expressed higher than control group.Conclusion:The two herbicides(acetochlor,metolachlor)and their common metabolites CMEPA and MEA had toxic effects on human hepatoblastoma cells(HepG2),which were related to apoptosis and endoplasmic reticulum stress pathway.Part 4 Apoptotic and Toxic effects of acetochlor,metolachlor and theirmetabolites on early embryonic development of zebrafishObjective:To study the toxicity and mechanism of acetochlor,metolachlor and their metabolites on zebrafish embryos in early development stage.Method:Morphological changes of zebrafish embryos after exposure were observed to explore developmental toxicity;apoptosis was observed by AO staining;oxidative damage factors of zebrafish tissues were detected;expression of apoptotic genes and endoplasmic reticulum stress markers was determined by RT-PCR and western blot,and toxicity mechanism was studied.Results:Acetochlor,metolachlor and their metabolites could produce developmental toxicity and oxidative damage to zebrafish embryos.Zebrafish cells showed apoptosis,and the expression of apoptotic marker genes and the proteins mentioned endoplasmic reticulum stress were changed.Conclusion:Acetochlor,metolachlor and their metabolites could induce embryonic developmental toxicity,oxidative damage and apoptosis in zebrafish,which might be related to endoplasmic reticulum-related stress mechanism,and basically consistent with the results of the research.