Absorption,Translocation,Directional Accumulation And Metabolism Of ¹⁴C-Cycloxaprid Enantiomers In Oilseed Brassica Spp.

With frequent use of neonicotinoid insecticides worldwide,the ecological and environmental problems caused by this kind of insecticides such as imidacloprid,thiamethoxam and clothianidin have become increasingly prominent.Therefore,research on the environmental behavior and fate of neonicotinoid insecticides and searching for efficient and environmentally friendly alternatives to such pesticides have become one of the most important and urgent scientific and technical problems in the world.Cycloxaprid is a new variety of neonicotinoid pesticides with independent intellectual property rights in China,and has a good prospect.Cycloxaprid,with two stereogenic centers,has an oxabridged ring and consists of two stereoisomers（5R,8S-CYC and 5S,8R-CYC）.However,compared with imidacloprid,which has been commercially used for many years,there is still limited scientific knowledge about the absorption,translocation,directional accumulation and metabolism of cycloxaprid.This research took the enantiomers of cycloxaprid as the research object and comprehensively used the isotope tracer technology and advanced instrument analysis technology.The isotope tracer technology used ¹⁴C-labeled compound that can reflect the molecular characteristics of cycloxaprid as the tracer.The study focused on the absorption,translocation,directional accumulation and metabolism of the enantiomers of cycloxaprid in oilseed Brassica spp..The aim is to provide a theoretical basis for more objective assessment and scientific understanding of the environmental safety of cycloxaprid.This work guides the scientific and rational use of this neonicotinoid substitute,and creates the same kind of other chiral new pesticides in China research,provides research technology systems and methods which can be used for reference.The main results obtained were as follows:1)The absorption,translocation and directional accumulation of the cycloxaprid enantiomers in Brassica campestris L.seedlings.The results showed that most enantiomers of cycloxaprid remained in the treated leaves（TL）.The cycloxaprid enantiomers can be transferred in both acropetal and basipetal directions,but it was easier to move from the TL to LATL.There was no significant difference in the total amount of absorption of CYC enantiomers.93.4%of 5R,8S-CYC and 93.7%of 5S,8R-CYC of the total radioactive residue（TRR）remained in the TL.0.12%of the TRR of CYC enantiomers translocated in the roots,0.45%and 0.47%in the stalks,2.39%and 2.36%in the leaves above treated leaves（LATL）,1.52%and 1.51%in the leaves below treated leaves（LBTL）,and there was no significant difference between the enantiomers in each plant part.The results revealed that there was no significant difference in the directional accumulation of CYC enantiomers in seedlings.The activity-based concentration of 5R,8S-CYC generally decreased in the following order:TL>LATL>stalks>LBTL>roots.The concentration of 5S,8R-CYC followed a similar pattern,but the range was slightly different.At the end of the experiment,the concentrations of two enantiomers in LBTL and stalks were significant different（p<0.05）,while the concentrations in TL,LATL and roots showed no significant difference.The translocation concentration factors of 5R,8S-CYC generally decreased in the following order:LATL>stalks>LBTL>roots.The TCF of 5 S,8R-CYC followed a similar pattern.Compared to the former,the TCF values of 5S,8R-CYC in LBTL and stalks were significantly different（p<0.05）,while the TCF values in LATL and roots were not significantly different.On the whole,¹⁴C-CYC underwent stereoselective translocation in seedlings.2)The absorption,translocation and directional accumulation of the cycloxaprid enantiomers in Brassica napus L.during the bud period.The majority of the enantiomers of cycloxaprid remained in the treated leaves,accounting for about 84.0% and 84.8%.There was no significant difference between CYC enantiomers in the TL,which were similar to the experiment of Brassica campestris seedlings.Comprehensively considering all organs at the upper and lower parts of TL at 87 d of maturity,the total residues transported to the upper parts of two enantiomers accounted for 6.31% and 6.15% of the TRR respectively,and the total residues transported to the base accounted for 1.89% and 3.09%of the TRR.This result indicated that the two enantiomers were more easily translocated to the top in Brassica napus at the bud stage,which was in accordance with the translocation of Brassica campestris at the seedling stage.The concentration of 5R,8S-CYC and 5S,8R-CYC in the vegetative organs of Brassica napus during the bud period were in order of:TL>LBTL>LATL>BS（Stalks below treated leaf）and AS（stalks above treated leaf）>root.The total amount of CYC enantiomers in the reproductive organs of Brassica napus at bud stage was less than that of vegetative organs.There was no significant difference between the concentration of 5R,8S-CYC and 5S,8R-CYC in the bud（0.56-0.87 mg/kg and 0.40-1.00 mg/kg）,whole flower（0.85-0.52 mg/kg and 0.78-0.48 mg/kg）and silique（1.33-2.59 mg/kg and 0.93-2.51 mg/kg）.However,it was worth noting that there was a significant difference between the peak concentration in the anther and BEA（Blossom except anther）.The peak concentration of 5R,8S-CYC and 5 S,8R-CYC in the anther were 1.15 mg/kg and 0.96 mg/kg,respectively.The peak concentration of 5R,8S-CYC and 5S,8R-CYC in the BEA were 1.59 mg/kg and 1.25 mg/kg,respectively.In view of the residual concentration of the CYC enantiomers in whole flowers and anthers was greater than 0.5 mg/kg（the maximum residual limit of the neonicotinoid insecticide imidacloprid）,it was necessary to consider possible ecological risks of nontarget organisms such as bees.The concentration of 5R,8S in seeds was 1.03 mg/kg,and 5S,8R was 0.75 mg/kg,the difference was not significant.In order to scientifically evaluate the possible influences of CYC residues in rapeseed on the human body,the formula was used to calculate the estimated daily intake（EDI）of the human.The range was 0.003-0.004 mg/kg bw,much lower than the ADI of neonicotinoid insecticide imidacloprid in GB 2763-2019,which was 0.06 mg/kg bw.Therefore,under the conditions of this experiment,a lower dietary risk was brought by the application of CYC enantiomers in rapeseed,but the concentration of the CYC enantiomers detected in rapeseed was relatively high.Thus,the residues of CYC in rapeseed were still worth concerning.3)The residual morphology,product composition and metabolic pathways of ¹⁴C-CYC in the leaves of Brassica napus.The bound residues of cycloxaprid in the leaves gradually increased with time,and at 87 d,the bound residues of 5S,8R was 33.7%,significantly higher than that of 5R,8S（21.1%）.The extractable residues gradually decreased with time.The extractable residues of 5R,8S and 5S,8R at 87d were reduced by 16.7%and 31.8%,respectively,compared to the 2d.Based on LSC and HPLC-QTOF-MS technology,we identified seven metabolites of cycloxaprid.CYC-M1 was 2-chloro-5-（（4,5-dihydro-lH-imidazolidin-1-yl）methyl）pyridine.CYC-M2 was（formamido（（3,4,5-trihydroxybenzoyl）oxy）methyl）carbamic acid combined with three molecules of glucose to form a glycoconjugate.CYC-M3 was（formamido（（3,4,5-trihydroxybenzoyl）oxy）methyl）carbamic acid combined with four molecules of glucose to form a glycoconjugate.CYC-M4 was 2-chloro-5-（（nitromethylene）imidazolidin-1-yl）methyl)pyridine.CYC-M5 was（（（3,4,5-trihydroxybenzoyl）oxy）methylene）dicarbamic acid combining two molecules of glucose formed a glycoconjugate.CYC-M6 was（formamido（（3,4,5-trihydroxybenzoyl）oxy）methyl）carbamic acid combined with two molecules of glucose to form a glycoconjugate.CYC-M7 was（formamido（（3,4,5-trihydroxybenzoyl）oxy）methyl）carbamic acid combined with one molecules of glucose to form a glycoconjugate.After treated with 5R,8S-CYC in Brassica,CYC-M1 accounted for 39.6-18.3%of TRR.The content of CYC-M2 was 9.6-13.7%of TRR.CYC-M3 was 2.9-28.5%.CYC-M4 was 32.3-13.5%.CYC-M5 was 8.2-4.8%.CYC-M6 was 2.4-1.2%.CYC-M7 was only detected in 2 d,which accounted for 0.8%.As for 5S,8R-CYC,CYC-M1 was 9.4-30.3%.CYC-M2 was 8.7-15.2%.CYC-M3 was 5.7-26.0%.CYC-M4 was 38.5-16.9%.CYC-M5 was 6.8-3.5%.CYC-M6 was 5.9-1.0%.CYC-M7 accounted for 1.4%,only detected in 2 d.The content of CYC-M4 was the highest among the seven metabolites of cycloxaprid in Brassica at the early trials.The product has biological toxicity.Therefore,it should be included in the CYC residue definition and given special attention when formulating the residue limits of CYC,especially when CYC residues remained in the plant with short growth cycles.The proportion of phase Ⅰ metabolites in CYC enantiomers in Brassica decreased over time.Phase Ⅱ metabolites gradually changed from monosaccharide and disaccharide conjugates to trisaccharide and tetrasaccharide conjugates.Based on the above metabolites,we inferred two possible metabolic pathways of cycloxaprid in Brassica.CYC had one-phase metabolism in Brassica.CYC-M4 was produced by breaking the seven-membered heterocycle where the CYC oxygen bridge is located to.With denitration and C=C bond fracture,the metabolite CYC-M4 was further converted into CYC-M1.CYC also had two-phase metabolism,suggesting two metabolic pathways.One was（formamido（hydroxy）methyl）carbamic acid which related to the imidazole ring,combined with gallic acid,a product of the shikimic acid pathway in plant secondary metabolism,to form（formamide（（3,4,5-trihydroxybenzoyl）oxy）methyl）carbamic acid.This compound combined with one molecule,two molecules,three molecules,and four molecules of glucose to form glycoconjugate CYC-M7,CYC-M6,CYC-M2,CYC-M3,respectively.The other one was（hydroxymethylene）dicarbamic acid combined with gallic acid to form（（（3,4,5-trihydroxybenzoyl）oxy）methylene）dicarbamic acid.Then this compound combined with two molecules of glucose to form gly coconjugate CYC-M5.4)No stereoselectivity was observed for the absorption and residues in edible parts of CYC in Brassica,but the translocation and the directional accumulation of residual in some organs showed the significant difference.Based on this phenomenon,this paper proposes that the studies on the absorption,translocation,directional accumulation and metabolism of CYC in crops should be carried out from enantiomers aspect,in order to gain a deeper understanding of the behavioral differences and metabolic characteristics of different enantiomers in different plants.