Metabolic Behavior And Toxicity Mechanism Of Etoxazole In Citrus Fruits And Its Residual Risk

Citrus is one of the most important fruit in our daily life,however,it is always damaged by mites.Etoxazole is a new type of phenyl oxazoline chiral acaricide,and is widely used to control the eggs,larvae and adults of citrus mite.In agricultural processes,when pesticide is applied to fields,residues may remain on the surface of the fruit or other parts of the plants.Meanwhile,most of the pesticides that applied to the plants entered into environment such as soil and sediment etc.Due to the differences of physicochemical properties and environmental conditions,the behaviors of the pesticides were significantly different in the plants or ecological environment.Residues of pesticide in plant or fruit could enter to human body through eating which may raise certain risk to human health.Pesticide residues in the ecological environment may produce toxic effects on non-target organisms.Etoxazole as a new and widely used pesticides,studies on it at present still limited on the degradation behaviors and toxicity on target organism mites.Due to the lack of related information,this research evaluated other aspects of etoxazole such as the enantioselectve degradation etoxazole in citrus and soil,and enantioselective toxicity to cell activity and oxidative stress.Meanwhile,earthworms were used as model organisms to study the toxicological effect of etoxazole to non-targeted organisms and to understand the mode of action.After exposure to different concentration levels of etoxazole,earthworm samples were collected and analyzed by NMR.Changes of earthworm endogenous substances were monitored by ANVOA analysis.The metabolic pathway and related biomarkers were also detected.Besides,UPLC-QTOF/MS with full scan mode was used to identify etoxazole metabolites in citrus,soil and earthworms.Dissipation behaviors of five main metabolites in these three matrices were monitored.At last,the chronic risk of etoxazole in citrus were evaluated based on the final residues in citrus and toxicological datas.The main results of this research were as follows: 1.Enantioselective degradation of etoxazole in citrus and soilDegradation of R-and S-etoxazole in citrus and soil were studied at different sampling days.The results showed that the degradation of etoxaozle enantiomers in citrus and soil are selective.In citrus matrix,S-etoxazole always dissipated faster than its antipode which caused the enrichment R-etoxazole in citrus matrix.In the soil matrix,S-etoxazole also declined faster than R-etoxazole,causing R-etoxazole enrichment in the soil matrix.In two these matrices,the ratio of enantiomers(EF)value is always more than 0.5 which further confirmed the persistence of R-enantiomers in citrus and soil.The half-lives of R-and S-etoxazole in citrus were 19.2 and 10.8 d and in soil were 10.2 and 6.18 d.Compared with other reports in literatures,there were some differences in half-lives.The reason might be due to the differences of citrus fruit size,natural environment at different locations.1.Enantioselective of etoxazole on cell toxicity and oxidative damageR-etoxazole and S-etoxazole with pure monomer were parpared with cell culture solutions with the finial concentrations of 0.01,0.05,0.1,0.5 and 1.0 mM.After 24 h exposure,the effects of etoxazole enantiomers on cell activity and oxidative stress were evaluated.The results of MTT(cell activities)test and LDH release amount showed that with the increase of R-,Rac-and S-etoxaozle concentrations,cell activities declined and LDH release increased.At low concentrations,no significant differences of cell activities were found among three forms of etoxazole.However,when concentration increased,R-etoxazole showed the highest inhibitory rate to cell activity,followed by Rac-etoxazole and S-etoxazole.The indexes of ROS,CAT and SOD activities were used to detect the oxidative damage of etoxazole enantiomers to cells.Results showed that the oxidative damage induced by R-etoxazole was much higher than its enantiomer which indicated that R-etoxaole was more toxicity to cancer cells than S-etoxaole.3.Toxicity of etoxazole to earthworm based on NMR analysisThe earthworm individuals with the same growth period were selected and exposed in soil with etoxazole concentration of 10,50 and 100 mg/kg.After 2 d and 14 d's exposure,earthworm samples were collected and detected by NMR.Dates were analyed by PCA and OPLS-DA.The figures of PCA and OPLS-DA showed that good separations were observed between etoxazole treatment group and control group or different concentration groups.Clustering phenomena were found within the groups.Through ANOVA and VIP analysis,there are 14 kinds of earthworm endogenous including amino acids,sugars,and biological molecules with relative amount significant changes were detected after etoxazole treatment.Trimethylamine-N-oxide was considered to be the characteristic metabolite after etoxazole treatment.Through KEGG pathway analysis,related metabolic paths of these biomarkers were identified which was considered as the etoxazole affected earthworm metabolic pathways.4.Identification of etoxazole metabolites in different matricesCitrus,soil and earthworm samples were collected at different intervals after the treatment of etoxazole.Samples were detected by using UPLC-QTOF/MS with full scan mode to monitor etoxazole metabolites in three kinds of matrices.After 14 d's etoxazole exposure,4 etoxazole metabolites were detected in citrus matrix,11 etoxazole metabolites detected in soil matrix,and 8 etoxazole metabolites detected in earthworm matrix.The behaviors of five main metabolites and high toxicity metabolites were monitored.Results showed that most of the metabolites decreased gradually with the increase of exposure time.However,the amount of metabolites 1 and 3 were increased with the increase of exposure time,showing the persistence in soil and earthworm which should pay attention to.5.Potential risk evaluation of etoxazole residues in citrus to humanIn this experiment,etoxazole formulation was sprayed on the citrus fruits with different pesticide concentrations,different spraying intervals and different times.Blank group and treated groups were set with 3 replicates.Citrus sample were analyzed by UPLC-MS/MS with ESI+ mode.The highest etoxazole residues and average residues in citrus fruits under different application modes were detected.Acute and chronic human risks were evaluated through the related formulas.In this study,the acute dose reference value(ARfD)value of etoxazole is not available.Therefore,the acute risk of etoxazole to human can not be calculated.Chronic risk to human was very low despite the different application dosages,intervals and application times.Etoxazole residue in citrus peel provided high chronic risk to human,followed by the whole fruit and citrus pulp.The results of experiment also showed that the higher application dosage,shorter collection interval,higher application times,the higher of etoxazole risk to human.