Study On Toxicity Of Degradation Products Of Nicosulfuon To Environmental Organisms

A strain of Bacillus subtilis,which degrades nicosulfuron efficiently,was isolated from the sewage of pesticide factory previously in our laboratory.Three enzymes,the manganese ABC transporter,vegetative catalase 1 and acetoin dehydrogenase E1,were separated from the extracellular enzymes of YB1 by DEAE-Sepharose Fast Flow chromatography,Native PAGE and SDS-PAGE.Based on this,soluble enzyme solutions of those three extracellular enzymes were successfully obtained through a prokaryotic expression method.Hydrolysis cycle test and liquid chromatography were used to verify the activity of extracellular enzyme solutions for nicosulfuron degradation.The degradation products of nicosulfuron were further determined by liquid chromatography-mass spectrometry between three enzymes and the YB1 strain.The degradation pathway of the three enzymes to nicosulfuron were initially determined.Nicosulfuron and two major degradation products(4,6-dihydroxypyrimidine and 2-amino-4,6-dimethoxypyrimidine)were tested for environmental bioassay.By measuring the toxicity of the two major degradation products against three species of aquatic organisms,earthworms and six non-target plants,it was clarified that the two degradation products had low toxicity levels for the three aquatic organisms,earthworms and six non-target plants.The specific results are as follows:1.This study successfully expressed three genes encoding nicosulfuron degrading enzymes in E.coli.Three enzymes,the manganese ABC transporter,vegetative catalase 1 and acetoin dehydrogenase E1 were used for prokaryotic expression and protein purification.All of them can degrade nicosulfuron in different degrees.Among them,the vegetative catalase 1 had the best degradation effect,and the nicosulfuron degradation rate was 76.70% at the initial concentration of 1000 mg/L at 3 h,followed by acetoin dehydrogenase E1,and the degradation rate was 57.11%.Finally,the degradation rate of the manganese ABC transporter was 40.25%.2.This study determined the nicosulfuron degradation products of the YB1 strain and the manganese-binding ABC transporter,plant catalase 1 and acetoin dehydrogenase E1 proteins.It was found that the three enzymes and the YB1 strain on nicosulfuron have the same degradation products.The three fragments with a mass-to-charge ratio of 156 m/z,230 m/z,and 274 m/z in the positive ion mode were determined as 2-ylamine-4,6-dimethoxy pyrimidine,N,N-dimethyl-2-sulfamoyl-isonicotinamide,((4-(dimethycarbamoyl)pyridine-2-yl)sulfonyl)carbamic acid,the production of these three degradation products were from the cleavage of the C-N bond.The fragment of 304 m/z is from the condensation reaction and the cleavage of the sulfonylurea bridge,which is 1-(4,6-dimethoxy-pyrimidin-2-yl)-3-(2-methyliminomethanesulfonyl-acetyl)-urea,while the mass/charge ratio of 111 m/z is 4,6-dihydroxypyrimidine,coming from the degrading of the 2-amino-4,6-dimethoxypyrimidine.3.The toxicity of nicosulfuron and the two main degradation products on three aquatic organisms was determined.The results of toxicity test showed that the LC50 of nicosulfuron was 16.949 mg ai/L for Brachydaniorerio 96 h,the LC50 of 2-amino-4,6-dimethoxypyrimidine and 4,6-dihydroxypyrimidine for Brachydaniorerio 96 h.>100.00 mg ai/L.According to the environmental safety assessment guidelines for chemical pesticides,the acute toxicity levels of nicosulfuron,2-amino-4,6-dimethoxypyrimidine and 4,6-dihydroxypyrimidine to Brachydaniorerio are all "low toxicity";The ErC50 of nicosulfuron,2-amino-4,6-dimethoxypyrimidine,and 4,6-dihydroxypyrimidine for 72 h of ScenedesmusobliquusTurp.Kuetzing was 8.070 mg ai/L,142.657 mg ai/L and 14.068 mg ai/L,respectively.According to the environmental safety evaluation criteria for chemical pesticides,the toxicity grades of nicosulfuron,2-amino-4,6-dimethoxypyrimidine and 4,6-dihydroxypyrimidine to ScenedesmusobliquusTurp.Kuetzing are "low toxicity";the EC50 values of nicosulfuron and 2-amino-4,6-dimethoxypyrimidine at 48 h for Daphnia magna are 9.190 mg ai/L and 51.954 mg ai/L.The EC50 of 4,6-dihydroxypyrimidine against Daphnia magna for 48 h was >100.00 mg ai/L.According to the environmental safety evaluation guidelines for chemical pesticides,the acute activity inhibition toxicity of nicosulfuron against Daphnia magna was “poisoning”,the acute activity-inhibitory toxicity ratings of 2-amino-4,6-dimethoxypyrimidine and 4,6-dihydroxypyrimidines against Daphnia magna were all "low toxicity".The comprehensive evaluation shows the acute toxicity of the two main degradation products to three aquatic organisms was lower than the acute toxicity of nicosulfuron to three aquatic organisms.4.By analyzing the acute activity inhibition toxicity of nicosulfuron and two major degradation products on Eisenia foetida,the results showed that nicosulfuron and the two main degradation products all had a LC50 > 100.00 mg ai/L 14 d on Eisenia foetida.According to the test guidelines,nicosulfuron and the two main degradation products on the acute activity inhibition toxicity level of earthworms are "low toxicity."5.Through analyzing the efeect of nicosulfuron and two major degradation products had effect on six non-target plants in biomass,nicosulfuron was most sensitive to six non-target plants,of which the semi-effect concentration at 14 d was 0.086 mg ai/kg dry soil of nicosulfuron to Triticum aestivum L.,and the sensitivity to Lactuca sativaL.was the worst,with the semi-effect concentration at 14 d of 0.644 mg ai/kg dry soil.The sensitivity of nicosulfuron to these six non-target plants were respectively Triticum aestivum L.,Brassica pekinensisR.,Cucumis sativusL.,Brassica napusL.,Raphanus sativusL.,Lactuca sativaL..2-amino-4,6-dimethoxypyrimidine was the second sensitive to six non-target plants,and the most sensitive was Brassica pekinensisR.The semi-effect concentration at the 14 d was 45.111 mg ai/kg dry soil.The sensitivity to Triticum aestivum L.was the worst.The semi-effect concentration at 14 d was 171.516 mg ai/kg dry soil.The sensitivity of 2-amino-4,6-dimethoxypyrimidine to six non-target plants was Brassica pekinensisR.,Brassica napusL.,Cucumis sativusL.,Lactuca sativaL.,Raphanus sativusL.and Triticum aestivum L..The sensitivity of 4,6-dihydroxypyrimidine to six non-target plants was the worest,the semi-effect concentrations at 14 d were all greater than 100.00 mg ai/kg dry soil.In summary,according to the evaluation criteria of environmental assessment criteria for chemical pesticides,the results showed that nicosulfuron has the toxicity level of "poisoning" to Cucumis sativusL.,Brassica pekinensisR.,Lactuca sativaL.and Raphanus sativusL.,and the toxicity level of " high toxicity " to Triticum aestivum L..The toxicity levels of 2-amino-4,6-dimethoxypyrimidine and 4,6-dihydroxypyrimidine to six non-target plants were all "low toxicity".Therefore,the sensitivity of nicosulfuron and two degradation products to six non-target plants was nicosulfuron,2-amino-4,6-dimethoxypyrimidine,and 4,6-dihydroxypyrimidine,respectively.