Xi Li,Xiaoxin Chen,Jiye Hu*

College of Chemistry and Biological Engineering,University of Science and Technology Beijing,Beijing 100083,China

Key words:Chlorfenapyr Clothianidin Leek Dissipation behavior Residue distribution

ABSTRACT Commercial suspension emulsion(SE)of(8%chlorfenapyr+20%clothianidin),as a pre-registered product in China,was fi rstly investigated under Chinese open-fi eld conditions.A MWCNTs-based Qu ECh ERS method for simultaneous determination of chlorfenapyr and clothianidin in leek was established and validated through rapid resolution liquid chromatography triples quadrupole tandem mass spectrometry(RRLC-QqQ-MS/MS).Based on this method,the dissipation behaviors,residue distributions and dietary risk probability of these fungicides in leek from two representative locations in China in 2016,which were previously treated with these insecticides at the doses of 420–630 g a.i./ha twice applications,were further investigated for food safety.Dissipation behavior of chlorfenapyr and clothianidin in leek followed fi rst-order kinetics with the half-lives of 2.9–7.2 days,and the highest residues(HR)of these insecticides in leek were below 0.3811 mg/kg and 0.2989 mg/kg,respectively,at the pre-harvest interval(PHI,7 days).For dietary risk assessments,the risk quotients(RQs)of chlorfenapyr and clothianidin in leek were evaluated by comparing the value of national estimated daily intake(NEDI)with acceptable daily intake(ADI),based on the trials data under good agricultural practices(GAP)conditions.The results indicated that RQs of chlorfenapyr and clothianidin in leek were 21.5%and 0.29%,respectively,which exhibited an acceptably low health risk to leek consumption.The current study could not only guide reasonable usage of the formulation,but also facilitate the setting of maximum residue limits(MRLs)of chlorfenapyr and clothianidin in leek for Chinese authorities

Chlorfenapyr,as a new pyrrole insecticide,show sspecial effects for Bradysia odoriphaga,and it has been w idely used in vegetable and fruit fields for chemical control of various pests.Clothianidin,(E)-1-(2-chloro-1,3-thiazol-5-yl-methyl)-3-methyl-2-nitroguanidine,a new nicotinic insecticide,exhibits excellent performance in controlling aphids,thrips,leafhopper,planthopper,hemiptera,coleoptera,etc.Now adays,clothianidin has been considered as one of the most excellent pesticides in controlling Bradysia odoriphaga,and it has been w idely used in agricultural fi eld in many countries since its fi rst introduction,due to low-toxicity,high-ef fi ciency and broad-spectrum.The chemical structures of chlorfenapyr and clothianidin are show n in Fig.1.As a commercial formulation,suspension emulsion(SE)of(8%chlorfenapyr+20%clothianidin)exhibits more pronounced effect on vegetables and fruits,and it is fi rstly introduced to control Bradysia odoriphaga in leek by Qingdao Hansen Biologic Science Co.,Ltd.Presently,this new pesticide formulation is being registered by China,shortly afterw ards,it w ill be w idely popularized on a global scale.Ow ing to the fact that various pesticides are frequently used in or on plant products,food contamination issue induced by pesticide residues has received increasing attention in recent years[1–12].Hence,pesticides and toxicological metabolites residue in raw agricultural commodities(RACs)must be strictly monitored for environmental risk and food safety.

Up to now,no literatures are reported as to simultaneous determination of chlorfenapyr and clothianidin in leek.Therefore,to develop a sensitive method for multi-residue analysis of these insecticides in leek ecosystem is an important aspect of food analytical chemistry.Furthermore,scarcely any literatures are available about dissipation behavior and risk assessment of chlorfenapyr and clothianidin in leek under good agricultural practices(GAP)conditions.Therefore,to evaluate the environmental fate and dietary risk probability of these insecticidesin leek w ill be an important aspect of food safety and toxicology.

Fig.1.Chemical structures of chlorfenapyr(a)and clothianidin(b).

Firstly,a multi-w alled carbon nanotubes(MWCNTs)-based Qu ECh ERS method for simultaneous determination of chlorfenapyr and clothianidin in leek was established and validated through rapid resolution liquid chromatography triples quadrupole tandem mass spectrometry(RRLC-QqQ-MS/MS).In this study,a rapid resolution liquid chromatography tandem triple quadrupled mass spectrometer(Agilent 6420,USA)equipped with a reversed phase C18 column(50 mm?3 mm I.D.,2.7 m m)was employed for liquid chromatography separation at 30?C.An electro spray ionization interface was operated in positive ion mode(ESI+).To extract and clean up the target analytes residue in soil and leek,fi ve grams homogenised samples were weighed into 50 m L PTFE centrifuge tube,to which 10 m L acetonitrile were added and shaked for 20 min.After then,NaCl(1.0 g)and anhydrous MgSO4(4.0 g)were sequentially added and vortexed again for 1.0 min.The extracts were centrifuged for 3.0 min at 4000 rpm.1.0 m Lupper layer of soil was cleaned up with 150 mg anhydrous MgSO4,50 mg PSA,while 1.0 m Lupper layer of leek was transferred to a centrifuge tube with 150 mg anhydrous MgSO4,50 mg PSA and 10 mg MWCNTs.Subsequently,the centrifugal procedure was performed at 10000 rpm for 3.0 min.The supernatant was fi ltered into an auto-sampler via with 0.22 m m syringe fi lter,and then it was analyzed using RRLC-QqQ-MS/MSanalyzer.

To validate the established method,the recovery experiments were carried out as follow s.The mixed standard solutions were forti fied to control leek(soil)to reach the concentrations of 0.1,1.0 and 10 mg/kg for chlorfenapyr,0.01,0.1 and 1.0 mg/kg for clothianidin.These samples were extracted and puri fied according to the mentioned procedure.Five parallel treatments for each forti fied level were carried out.Simultaneously,the dark control was also analyzed to calibrate the interference from leek matrix.In this study,the matrix-matched standard calibrations were used to calibrate possible interferences during quanti fi cation of analytes.The approving linearity and coefficient( R)over the reasonable concentration ranges were achieved,and all regression data for chlorfenapyr or clothianidin in soil and leek matrix were also obtained as show n in Table S1(Supporting information).The LOQs were de fined as the lowest spiked concentrationsof target analytes in matrix with a signal-to-noise ratio of 10/1.In the conditions mentioned previously,the LOQs of chlorfenapyr and clothianidin were 0.1 and 0.01 mg/kg,respectively.The LODs of this method could be calculated at a signal-to-noise ratio of 3/1 with acceptable precision and accuracy.Therefore,the LODs for chlorfenapyr and clothianidin in leek(soil)were corresponding to 0.2(4.5)m g/Land 5.0(4.0)m g/L,respectively(Table S1),which adequately embodied high-sensitivity of this method.The method’s accuracy could be evaluated by recoveries with fi ve replications(n=5)at three different forti fications.As show n in Table S2 (Supporting information),the mean recoveries of chlorfenapyr in leek and soil matrix were 90.1%–96.8%with the relative standard deviations(RSDs)of 3.34%–11.7%,while they ranged from 90.5%to 101%for clothianidin with the RSDs of 2.7%–8.55%.The above fi ndings demonstrated that the method was adequately for simultaneous multi-quantitation of chlorfenapyr or clothianidin residue in leek samples.

Based on this method,the dissipation behaviors and residue distributions of these fungicides in leeks from tw o representative locations in China in 2016,were investigated.To study the dissipation kinetics of chlorfenapyr and clothianidin in leek ecosystem,a commercial SEof(8%chlorfenapyr+20%clothianidin)was diluted with w ater and sprayed on blank leek at a dosage of 630 g a.i./ha(1.5 times of recommended high dosage).A separate plot with the same size and no-pesticide application was compared simultaneously.The representative straw berry samples(2.0 kg)were collected randomly from each plot at different time intervals(0,1,3,5,7,14,21 and 30 days).These samples were cut into 0.5 cm piecesby food cutter,and then they were maintained at?20?C until analyzed.To investigate the terminal residues of chlorfenapyr and clothianidin,a low-dose level of 420 g a.i./ha(recommended high dosage)and a high-dose level of 630 g a.i./ha(1.5 times of the recommended high dosage)were applied to the fi nal residual experimental plots in the early stages of Bradysia odoriphaga by a portable sprayer for tw ice timeswith an interval of 7 days.Samples(2.0 kg)were collected from each plot at 7 and 14 days after the last application according to FAO/WHO(1986)recommendations.Sampling was performed by randomly collecting from untreated plots.All samples were stored in a cooled chamber at?20?C before further analysis.These actual samples were pre-treated in triplicate using the above-mentioned method.The dissipation dynamics and parameters of chlorfenapyr or clothianidin in fi eld-incurred leek and soil were investigated.As presented in Table 1,the loss in amplitude of these insecticides in leek and soil from Anhui and Shandong followed fi rst-order kinetics,and the residuesat the tw o evaluated fields were declined quickly as time passes.The validity of these models was demonstrated by the coefficient( R)as summarized in Table 1.The initial deposits of chlorfenapyr in leeks were easily observed as a result of 1.669–2.882 mg/kg,with the dissipation half-lives of 2.9–5.1 days.The initial concentrations and half-lives of clothianidin in leek were in the ranges of 4.289–8.011 mg/kg and 3.2–7.2 days,respectively.Obviously,the fastest dissipation rate was chlorfenapyr residue in leek from Anhuiin 2016 with the half-livesof 2.9 days.The initial deposits of chlorfenapyr and clothianidin in soil ranged from 0.2279 mg/kg to 2.501 mg/kg with the half-lives of 9.2–24.5 days.Moreover,no signi fi cant positive or negative correlations were observed between dissipation rates and initial concentrations.From the mentioned above,these distinctions for both chlorfenapyr and clothianidin in leek and soil matrix mainly depended on chemical properties of pesiticides.Secondly,some environmental factors such as temperature,light intensity and dilution factor have a signi fi cant impact upon dissipation behavior of pesticides.

Table 1 Dissipation dynamics parameters of chlorfenapyr and clothianidin residue in soil and leek.

Table 2 Mean terminal residues(in triplicate for each plot)of chlorfenapyr and clothianidin in soil and leek.

The terminal residues of chlorfenapyr and clothianidin in soil and leek from Anhuiand Shandong in 2016 were listed in Table 2.It was easily observed that the residue of chlorfenapyr and clothianidin under different treatments followed the trend that residues were lower with a longer harvest interval.The ultimate concentrations of chlorfenapyr and clothianidin in soil at the evaluated locations were below 1.558 mg/kg and 3.173 mg/kg,respectively,at the pre-harvest interval(PHI,7 days).The terminal residues of chlorfenapyr and clothianidin in leeks from tw o representative locations in 2016 were lower than 0.3705 mg/kg and 0.2941 mg/kg,respectively,with an interval of 7 days postapplication tw ice applications under the designed dosages.Although no corresponding maximum residue limits(MRLs)of chlorfenapyr and clothianidin in leek was recommended by China,1.0 mg/kg might be considered as a referenced MRL of these insecticides in leek,based on the 2–4 times HR(0.3811 mg/kg for chlorfenapyr and 0.2989 mg/kg for clothianidin)(Table 3).As is well know n,MRLis the highest concentration of a pesticide residue in food with the toxicological acceptability under GAPconditions,and setting an MRL needs to consider many factors,including residue data from mult-regions fi eld trials,the dietary intake data and the food processing factors[13].Therefore,the results in this study can provide some reference data in setting a reasonable MRL for chlorfenapyr and clothianidin in leek.

To assess the intake risk probability of chlorfenapyr and clothianidin for leek consumption,the risk quotients(RQ)were calculated by comparing the national estimated daily intake(NEDI)with acceptable daily intake(ADI).The NEDI(mg)of chlorfenapyr and clothianidin and the RQ were calculated using the follow ing formulas.

NEDI=P(STMRi?Fi)

RQ=NEDI/(ADI?bw) w here STMRi(mg/kg)is the supervised trials median residue of chlorfenapyr or clothianidin in a certain kind of food registered by China;Fi(kg)is the dietary reference intake for a certain kind of food used to plan and assess nutrient intakes of healthy Chinese people;ADI(mg kg?1bw)is the acceptable daily intake of chlorfenapyr or clothianidin;bw is the average body weight of a Chinese adult(63 kg).If there is no suitable STMRi,the corresponding MRLs were used for NEDI calculation.The ADI of chlorfenapyr and clothianidin was 0.03 and 0.1 mg kg?1bw,respectively.In order to accurately assess the NEDI,Chinese dietary pattern,the registered crops of these fungicides in China and their corresponding MRLs registered by various countries should be considered simultaneously.As listed in Tables S3 and S4(Supporting information),the Fiwasthe dietary reference intake of a certain kind of food used to plan and assess nutrient intakes of healthy Chinese people.The registered crops of chlorfenapyr and clothianidin in China and their corresponding MRLs recommended by various countries were summarized in Table S5(Supporting information).It is w orth noting that the selection of reference residue limits followed the priority order of China>CAC>USA>European Union>Australia>Korea>Japan.Secondly,only the reference residue limit that can ensure the highest NEDIin a kind of food was selected to calculate the corresponding NEDI values.Emphatically,the reference residue limit of the evaluated food derived from its supervised trials median residue(STMR).As displayed in Table 3,the STMRs of chlorfenapyr and clothianidin in leek at the PHI 7 day were 0.1810 mg/kg and 0.1334 mg/kg,respectively,thus their total NEDIwas calculated as the results of 0.4068 mg(Table S3)and 0.01884 mg(Table S4).Therefore,the RQs of chlorfenapyr and clothianidin in the evaluated leek to Chinese consumers were 21.5%and 0.29%,respectively,with an interval of 7 day after post-application tw ice times under the designed dosages.As is well know n,the higher RQ value show s more pesticide residue,and RQbeyond 100%indicates that the evaluated food exhibits an unacceptably high health risk to consumers[14].Hence,the above results in current study indicated that potential health risk induced by chlorfenapyr and clothianidin in leek wasnot signi fi cant.The current study w ould be signi fi cant for providing guidance on the reasonable usage of these insecticides under the open-fi eld leek production conditions.In this study,there is an additional contribution about theestablishment of MRLs of chlorfenapyr and clothianidin in leek for Chinese authorities.

Table 3 Terminal residues summary of chlorfenapyr and clothianidin in leek from Anhui and Shandong in 2016.

In conclusion,the insecticides in leek under fi eld-incurred conditions were rapidly degraded follow ing fi rst-order kinetic models.The terminal residues of these insecticides in leeks were lower than 0.3705 mg/kg with an interval of 7 days post-application tw ice times under the designed dosages.The results of dietary risk assessment indicated that the RQs of chlorfenapyr and clothianidin were far below 100%,which demonstrated the potential health risk induced by these insecticides in leek was not signi fi cant for Chinese consumers.In addition,1.0 mg/kg might be recommended as a reasonable MRL of chlorfenapyr and clothianidin in leek in China,based on the results of residue distribution and dietary risk assessment.

Acknow ledgments

This work was fi nancially supported by the Natural Science Foundation of Beijing(No.8162029)and National Natural Science Foundation of China(No.21677009).The authors acknow ledged the Ministry of Agriculture of People’s Republic of China for the project support.

Appendix A.Supplem entary data

Supplementary data associated with thisarticle can be found,in the online version,at https://doi.org/10.1016/j.cclet.2018.02.001.