The Residue Distribution And Metabolism Of ¹⁴C-Dufulin In Soybeans

Dufulin is a plant immune induction antiviral agent,which has the characteristics of high efficiency and low toxicity.It is widely used in the prevention and control of various plant diseases,such as tobacco,tomato,cucumber,rice,etc.,and has a good market prospect.In recent years,studies on the environmental behavior of Dufulin have been carried out gradually.Information about their metabolites must be provided in the registration and safety evaluation of pesticides.However,up to now,there are a few related reports on the metabolism of Dufulin in plants.Therefore,it is of great practical significance to study the metabolism of Dufulin in plants for its safety evaluation and residue definition.According to the requirements of the Guideline for the testing of pesticide metabolism in crops,the residual distribution and metabolism of[thiazyl-2-¹⁴C]-Dufulin(¹⁴C-Dufulin A)and[benzyl-¹⁴C]-Dufulin(¹⁴C-Dufulin B)in soybean were explored using the ¹⁴C tracer method and modern instrumental analysis.Four metabolites of Dufulin in soybeans were identified.The possible metabolic pathways of Dufulin were also inferred.The results provided data support for the objective evaluation of the safety of Dufulin and technical guidance for the scientific and rational use of Dufulin.The main results were as follows:（1）The residue of Dufulin was mainly detected in soybean leaves,and the amount transferred to other parts was limited after treatment.During the whole experiment,the distribution ratio and total residual concentration of ¹⁴C-Dufulin A and ¹⁴C-Dufulin B generally decreased in the following order:leaves＞stems＞shells＞roots＞beans.And the residue content in edible beans was less than 0.3%of the total radioactive residue at harvest time.（2）The extractable residues of Dufulin in the leaves gradually declined over time.At harvest,the extractable residues of ¹⁴C-Dufulin A and ¹⁴C-Dufulin B in leaves accounted for86.58%and 83.06%of the total radioactive residues,while the bound residues gradually increased over time.And the bound residues of ¹⁴C-Dufulin A and ¹⁴C-Dufulin B in leaves accounted for less than 20%of the total radioactive residues.（3）The radioisotope tracing technology,liquid scintillation measuring instrument,and high performance liquid chromatography-mass spectrometry technology were applied,and four metabolites of Dufulin in leaves and beans were identified.Among them,DFL-M1 and DFL-M2 were the hydroxylated metabolites of Dufulin,which were[（2-Fluoro-phenyl）-（4-methanol-benzothiazol-2-ylamino）-methyl]-phosphonic acid diethyl ester and[（2-Fluoro-phenyl）-（4-methyl-6-hydroxy-benzothiazol-2-ylamino）-methyl]-phosphonic acid diethyl ester.DFL-M3（[（2-Fluoro-phenyl）-（4-methyl-6-glucosyl-benzothiazol-2-ylamino）-methyl]-phosphonic acid diethyl ester）was formed by conjugated with glucose after hydroxylation of Dufulin;DFL-M4（6-（2-{[Diethoxy-phosphoryl）-（2-fluoro-phenyl）-methyl]-amino}-4-methyl-benzothiazol-6-yloxy）-3,4,5-trihydroxy-tetrahydro-pyran-2-ylmethoxy-3-oxopropanoic acid)was formed by conjugated with glucose malonate after the hydroxylation of Dufulin.（4）The residual concentration of metabolites in leaves gradually increased over time.At harvest,the residual concentrations of the metabolites DFL-M4,DFL-M3 and（DFL-M1+DFL-M2）from ¹⁴C-Dufulin A were 4.03 mg·kg^-1,2.93 mg·kg^-1,and 1.71 mg·kg^-1,while the same metabolites from ¹⁴C-Dufulin B were 3.84 mg·kg^-1,2.43 mg·kg^-1 and 1.46 mg·kg^-1.The residual concentration of ¹⁴C-Dufulin A and ¹⁴C-Dufulin B in leaves were 9.86 mg·kg^-1and 6.96 mg·kg^-1,accounting for 62.23%and 53.44%of the total radioactivity at harvest,respectively.The degradation process of Dufulin in leaves conformed to the first-order kinetic equation,and their half-lives were 16.9 d and 15.4 d,respectively.（5）At harvest,the relative content of Dufulin and DFL-M4 in edible beans were49.30%～51.93%and 20.30%～23.13%of the extractable residues,and the residual concentration of both were more than 0.01 mg·kg^-1.According to the"uniform standard,"the residues of Dufulin in edible beans were defined as the parent compound and DFL-M4.（6）The possible metabolic pathways of Dufulin in leaves and beans were divided into two processes.In the phaseⅠ,DFL-M1 and DFL-M2 were produced by hydroxylation reaction of Dufulin.In the phase II,DFL-M3 and DFL-M4 were formed by the conjugation reaction between the-OH of the phaseⅠmetabolites with endogenous substances such as glucose and malonate.Under the catalysis of UGTs and MTs,DFL-M2 may be conjugated with glucose to form DFL-M3,and then combined with malonate to form DFL-M4.DFL-M2 may directly combine with glucose malonate to form DFL-M4 under the catalysis of UGTs.