The Residue Determination And Biomarker Study Of Clothianidin In Brassica Chinensisl

ObjectiveClothianidin is a new second filial generation broad-spectrum systemic insecticide.It kills insects by affecting acetylcholine receptor,makes the never keeping activated and the bodies keeping spasmodic.Comparing with the first generation,the main distinctions of the molecular structure are:chlor-thiazole takes the place of pyridyl and nitro imine takes the place of nitro methane.Few clothianidin residues will not create immediate and direct poisoning to the human body,but it can cause cancer and internal secretion turbulence if we eat these polluted vegetables for a long time because the molecular structure of clothianidin is quite stable.This research intends to establish one kind examination technology of clothianidin residue which is simple,fast and highly effective on one hand.On the other hand this research intends to find one kind or several kinds of quite sensitive biomarkers which can use as early diagnosis guideline in clothianidin polluted vegetables.Methods1.Field experimentField experiment is taken to spray high-dose(200g/hm²) and low-dose(50g/hm²) clothianidin water-solubility granule.The clothianidin water-solubility granule was sprayed every other day and 3 times altogether.At the same time,make a blank without clothianidin.The brassica chinensisl samples were gathered by the method of Z form in 0d,1d,3d,5d,7d,10d,12d,15d after the last time clothianidin water-solubility granule was sprayed.When taking the samples,make three replications to get the average value.2.The determination of ciothianidin residueBrassica chinensisl samples were withdrawn by acetone,purified by silica gel chromatographic column concentrated by rotary evaporator and detected by HPLC.3.The detection of biomarkerThe activities of peroxidase,polyphenoloxidase,the content of glutathione,malondialdehyde,superoxide anion and protein were measured by guaiacol method,spectrophotography,Ellman method,thiobarhituric assay,hydroxylamine oxidation method and Coomassie brilliant blue G-250 separately.Results1.The dynamic degradation of the clothianidin residuesThe recovery rate of clothianidin in brassica chinensisl is 93.02%～94.4%and the relative standard deviation is 3.3%～5.4%(n=6).The result showed:the half-value period of clothianidin residues in low-dose group is 1.61 days and its degeneration equation is C_T=28.633e^-0.4301T,r=0.9455(n=6).The half-value period of clothianidin residues in high-dose group is 2.05 days and its degeneration equation is C_T=85.816e^-0.3374T,r=0.9578(n=6),after 12 days the dose of clothianidin that residues in brassica chinensisl are all bellow 0.2mg/kg.2.Effect of clothianidin on protein in brassica chinensislThe content of protein in both groups are obviously lower than control group,The content of protein in low-dose group minimized in the 5th day,increase gradually after that and restored to basically approach with the control group in the 12th day.The content of protein in high-dose group begin to reduce in the 1st day,maximized in the 7th day,and still have significant difference with control group in the 12th day.3.Effect of clothianidin on PPO activity in brassica chinensislThe activities of PPO in low-dose group maximized in the 5th day were equal to 1.17 times of the control group,and then gradually reduced.The activities of PPO in high-dose group maximized in the 7th day were equal to 1.4 times of the control group,gradually reduced after that.4.Effect of ciothianidin on GSH in brassica chinensislThe content of GSH in low-dose group minimized in the 5th day.The content of GSH in high-dose group minimized in 7 days,and restored to basically approach with the control group in the 12th day.5.Effect of clothianidin on MDA in brassica chinensislThe content of MDA was highly improved in both high-dose group and low-dose group,and the content of MDA of in high-dose group is higher than low-dose group. On the day of the last time clothianidin was sprayed,the content of MDA in high-dose group equalled to 3.3 times of the comparison group and the content of MDA in low-dose group equaled to 1.8 times of the comparison group.The content of MDA in two processing group had the same change tendency and both gradually reduced.6.Effect of clothianidin on POD activity in brassica chinensislThe activities of POD in low-dose group maximized in the 5th day while the activities of POD in high-dose group maximized in the 7th day and both restored to basically approach with the comparison group in the 12th day.7.Effect of clothianidin on O₂^-·in brassica chinensislThe content of O₂^-·in cell has positive correlation with the pesticide dose,the content of O₂^-·in high-dose group was higher than the low-dose group during the whole experiment,gradually reduced follow time-lapse.The content of O₂^-·in low-dose group was higher than control group in the first 5 days,then lower than that.Conclusion1.The sample pretreatment and detection which this experiment used are suitable for fast examination under the clothianidin pesticide residue in vegetable.2.The clothianidin residues in brassica chinensisl degraded rapidly.3.POD,PPO,GSH,MDA,protein and O₂^-·all can be served as the biomarker under the clothianidin pollution,among these,protein and GSH are more sensitive than others.