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• Chlorfenapyr, as a new pyrrole insecticide, shows special effects for Bradysia odoriphaga, and it has been widely 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. Nowadays, clothianidin has been considered as one of the most excellent pesticides in controlling Bradysia odoriphaga, and it has been widely used in agricultural field in many countries since its first introduction, due to low-toxicity, high-efficiency and broad-spectrum. The chemical structures of chlorfenapyr and clothianidin are shown 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 firstly 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 afterwards, it will be widely popularized on a global scale. Owing 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.

  Figure 1

  

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

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  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 insecticides in leek will be an important aspect of food safety and toxicology.

  Firstly, a multi-walled carbon nanotubes (MWCNTs)-based QuEChERS 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 mm) was employed for liquid chromatography separation at 30 ℃. 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, five grams homogenised samples were weighed into 50 mL PTFE centrifuge tube, to which 10 mL 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 mL upper layer of soil was cleaned up with 150 mg anhydrous MgSO₄, 50 mg PSA, while 1.0 mL upper 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 filtered into an auto-sampler via with 0.22 mm syringe filter, and then it was analyzed using RRLC-QqQ-MS/MS analyzer.

  To validate the established method, the recovery experiments were carried out as follows. The mixed standard solutions were fortified 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 purified according to the mentioned procedure. Five parallel treatments for each fortified 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 quantification 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 shown in Table S1 (Supporting information). The LOQs were defined as the lowest spiked concentrations of target analytes in matrix with a signal-to-noise ratio of 10/1. In the condition 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) mg/L and 5.0 (4.0) mg/L, respectively (Table S1), which adequately embodied high-sensitivity of this method. The method's accuracy could be evaluated by recoveries with five replications n = 5) at three different fortifications. As shown 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 findings 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 two representative locations in China in 2016, were investigated. To study the dissipation kinetics of chlorfenapyr and clothianidin in leek ecosystem, a commercial SE of (8% chlorfenapyr + 20% clothianidin) was diluted with water 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 strawberry 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 pieces by food cutter, and then they were maintained at -20 ℃ 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 final residual experimental plots in the early stages of Bradysia odoriphaga by a portable sprayer for twice times with 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 ℃ 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 field-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 first-order kinetics, and the residues at the two 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 Anhui in 2016 with the half-lives of 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 significant 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 significant impact upon dissipation behavior of pesticides.

  Table 1

  

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

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  The terminal residues of chlorfenapyr and clothianidin in soil and leek from Anhui and 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 two representative locations in 2016 were lower than 0.3705 mg/kg and 0.2941 mg/kg, respectively, with an interval of 7 days postapplication twice 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 known, MRL is the highest concentration of a pesticide residue in food with the toxicological acceptability under GAP conditions, and setting an MRL needs to consider many factors, including residue data from mult-regions field 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.

  Table 2

  

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

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  Table 3

  

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

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  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 following formulas.

  where STMR_i (mg/kg) is the supervised trials median residue of chlorfenapyr or clothianidin in a certain kind of food registered by China; F_i (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^-1 bw) 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 STMR_i, the corresponding MRLs were used for NEDI calculation. The ADI of chlorfenapyr and clothianidin was 0.03 and 0.1 mg kg^-1 bw, 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 F_i was the 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 worth 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 NEDI in 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 NEDI was 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 twice times under the designed dosages. As is well known, the higher RQ value shows more pesticide residue, and RQ beyond 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 was not significant. The current study would be significant for providing guidance on the reasonable usage of these insecticides under the open-field leek production conditions. In this study, there is an additional contribution about the establishment of MRLs of chlorfenapyr and clothianidin in leek for Chinese authorities.

  In conclusion, the insecticides in leek under field-incurred conditions were rapidly degraded following first-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 twice 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 significant 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.

  Acknowledgments

  This work was financially supported by the Natural Science Foundation of Beijing (No. 8162029) and National Natural Science Foundation of China (No. 21677009). The authors acknowledged the Ministry of Agriculture of People's Republic of China for the project support.

  Appendix A. Supplementary data

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

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

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

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

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

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