Enantioselective Environmental Fate And Chiralspecific Molecular Toxicology Of Dinotefuran

Neonicotinoid chiral pesticides were increasingly used in the agricultural production,which posed a serious threat to our environment and biological safety because of its high toxic to honeybee and earthworm.It is helpful for the problem solving by conducting the environmental fate and different molecular toxicology form the perspective of enantiomer.In this thesis,a typical neonicotinoid chiral pesticide dinotefuran was selected.The enantioselective bioactivity on target pests Aphis gossypii and Apolygus lucorum and the enantioselective toxicology on non-target organism Eisenia fetida between the enantiomers was evaluated.The environmental fate of dinotefuran racemate and optical pure enantiomers were investigated in vegetable-soil ecosystem to disclose the main degradation products and metabolic pathways and to make sure the critical control points of the safe use of dinotefuran.Meanwhile,the enantioselective effects of chirality on the toxicology of dinotefuran enantiomers to Apis mellifera Linnaeus and Eisenia fetida was studied based on the molecular biology and chemistry methodology.It is helpful to provide a theoretical basis for environmental risk assessment,safe application,and development of efficient low-risk enantiomer of dinotefuran,and offer a new idea to solve the high-toxic to honeybee and earthworm.The main conclusions are as follows.First,enantioseparation of dinotefuran and its metabolites was developed using supercritical fluid chromatography/tandem mass spectrometry?SFC-MS/MS?.A statistical optimization strategy using central composite design was performed in the method optimization.The different chiral stationary phases?e.g.,column packing,size and inner diameter?,mobile phase?e.g.,organic phase variety,proportion and additives?,flow rate,automated backpressure regulator pressure?ABPR?,and column temperature were systematic investigated on the separation parameters of dinotefuran and metabolites enantiomers.The interactions among these independent variables and significant factors on the dependent variables were also elucidated in the study.S-?+?-dinotefuran and R-?–?-dinotefuran were further prepared individually with an enantiomeric excess of 99.9%.The proposed methodology could be a suitable alternative to trace dinotefuran enantiomers and provide the basis materials for the following studies of enantioselective biological activity,ecological toxicity and enanvironmental fate.Second,enantioselective bioactivity assays on the target pests Aphis gossypii and Apolygus lucorum were evaluated by contact and oral test modes,respectively.The bioactivitiy difference between S-?+?-dinotefuran and R-?–?-dinotefuran was not significant with P>0.01 and the fold change of 2.6–3.4 and 3.0–3.8 for Aphis gossypii and Apolygus lucorum,respectively.The enantioselective toxicity on non-target organism Eisenia fetida between the enantiomers was further evaluated in the study.S-?+?-dinotefuran was high toxic to Eisenia fetida and was of 71.5-fold greater toxicity than its antipode R-?–?-dinotefuran.This toxicology difference between the enantiomers was significant with P<0.01,indicating the S-?+?-dinotefuran was high-toxic enantiomer.Thrid,the enantioselective environmental fate of dinotefuran enantiomers were investigated in typical vegetables tomato?Lycopersicon esculentum Mill.?and collards green?Brassica oleracea var.acephala DC.?by foliar and root uptake pathways and isotope internal standard quantitation.The S-?+?-dinotefuran was more persistent in tomato than R-?–?-dinotefuran under greenhouse cultivation.Dinotefuran enantiomers were also metabolize to UF enantiomers,and in the foliar uptake pathway we found the?–?-UF was preferentially degraded in greenhouse tomato.However,there is no enantioselective degradation and metabolism in tomato during home canning.A great amount of dinotefuran enantiomers residues were retained by the tomato skin and therefore,it is essential to concern its commercial by product safety when tomato skin is used for lycopene extraction.The absorption rate,transformation rate and amount of S-?+?-dinotefuran was faster than R-?–?-dinotefuran in collards green.The metabolites UF and DN phosphate were both found in the collards cultivation,which suffered from the metabolism of parent and the degradation of themselves.The enantioselective metabolism was only found in the plant without generation in Hoagland solution.Forth,the enantioselective environmental fate of dinotefuran enantiomers were also studied in different soils under aerobic and anaerobic conditions.S-?+?-dinotefuran was more persistent in neutral sandy soil,however it was preferentially degraded in acidic and alkaline silty soils.S-?+?-dinotefuran also selectively metabolize to?+?-UF and R-?–?-dinotefuran generated to?–?-UF.The chiral configurations were not changed between the parent and metabolite enantiomers.The enantioselective metabolism trendency and yield of UF were different on different soils under different cultivation conditions.A higer metabolism yield was shown in the three soils under anaerobic condition than that of aerobic condition,illustrating soil anaerobic microorganisms had significant effects on the metabolism of dinotefuran enantiomers.The chiral configurations of dinotefuran enantiomers were stable because there is no transformation between S-?+?-dinotefuran and R-?–?-dinotefuran in the three soils under aerobic and unaerobic conditions.Fifth,the enantioselective effects of chirality on the toxicology of dinotefuran enantiomers to Apis mellifera Linnaeus was studied from the level of molecule.Electrophysiological studies on the pharmacological response of dinotefuran enantiomers to all the nAChR subunits highlight the S-?+?-dinotefuran as the high-toxic characteristic origin of dinotefuran,specifically acting on the AmeI?8 site in nAChR of Apis mellifera Linnaeus.Molecular docking further confirmed S-?+?-dinotefuran had a more stable,functional binding cavity towards AmeI?8 via a preferential configuration,which primarily resulted from multiple H-bond network.Sixth,the enantioselective effects of chirality on the toxicology of dinotefuran enantiomers to Eisenia fetida was also studied at protein levels.S-?+?-dinotefuran showed bigger effects?preferential active or inhibiting effects?on acetylcholinesterase?AChE?,antioxidase?SOD?CAT and GSH-Px?,detoxifying enzymes?GST and CarE?,and malondialdehyde?MDA?than its antipode.On the one hand,it promoted the neurotransmitter hydrolysis,interrupt acetylcholine transmission in synapsis and prevent the binding with nicotinic acetylcholine receptors?nAChRs?.On the other hand,it also induced a large number of reactive oxygen species?ROS?to damage the antioxidant defense system of Eisenia fetida.Furthermore,S-?+?-dinotefuran can more potency to inhibit the up-regulated expression of signal transduction gene?EW1_F1P07_H02?and Acyl carrier protein?EW1_F1P04_C04?and resulted in function obstacle on its signal transduction and GABA receptor transportation.It can significantly increase the aminocaproic glycosidase gene?EW1_F2P14_D06?to accelerate the hydrolysis of n-acetylgalactosamine,to produce GM3 gangliosides form GM2 gangliosides and to block the communication between cells in Eisenia fetida.S-?+?-dinotefuran also can inhibit the carrier protein?EW1_F1P10_E08?and Ca²⁺-ATPase Channel gene?Lr_PAHCF₆4C08_Skplus?which caused neurotoxicity by nerve dysfunction from inhibiting the active transport of calcium ions in earthworm.