Dissipation, Residue, and Dietary Risk Assessment of Methoxyfenozide, Chlorantraniliprole, Indoxacarb, Lufenuron, and Chlorfenapyr in Spinach Using a Modified QuEChERS Method Combined with a Tandem Mass Spectrometry Technique

Abstract

Spinach is a frequently consumed vegetable worldwide. Chemical pesticides are widely used to produce spinach with high yield and quality. However, the unregulated use of pesticides negatively impacts human and environmental health. A simple and efficient method using dispersive solid-phase extraction (d-SPE) combined with field experiments was conducted to study the residue dissipation of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr in spinach under different planting conditions. The results showed that the half-lives of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr in spinach were 1.1–3.0 d, 2.6–4.0 d, 1.7–4.2 d, 3.4–4.4 d, and 2.8–4.3 d, respectively. The five pesticides rapidly degraded without significant differences between the open and greenhouse fields. The final residue of indoxacarb in spinach was not higher than the maximum residue limit (MRL) in China (3 mg kg⁻¹); the highest residual value was 1.0 mg kg⁻¹. Although the MRLs of methoxyfenozide, chlorfenapyr, and lufenuron for spinach are yet to be formulated in China, the long-term dietary risk for the four pesticides was acceptable, with an RQ < 100%, according to the international and national assessments. These results are necessary to provide guidance for the proper and safe use of these pesticides.

1. Introduction

Spinach (Spinacia oleracea L.) is widely consumed in various countries as a fresh vegetable or processed product [1]. China is one of the major spinach producers and consumers due to its strong adaptability, diverse cultivation methods, and year-round supply [2]. Chemical pesticides are widely used for controlling pests and diseases during planting to meet the demand for high-yielding and quality spinach. Therefore, the registration of safe, efficient, and economic pesticides by the appropriate regulatory bodies should be encouraged to ensure sufficient certified pesticides for spinach [3]. We identified five highly effective and low-toxic pesticides registered for cabbage, including methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr. Since these pesticides have not been registered for spinach, it is essential to investigate the residual levels of the above five pesticides applied to spinach and assess their dietary intake risks.

Currently, a few studies on the assessment of residue dissipation and dietary risk of the five pesticides have been reported in various crops. Xu et al. [4,5] showed that chlorfenapyr displays great half-life differences among the different crops, with the fastest degradation rate of 1.2–1.5 d in asparagus and the slowest rate of 6.9–12.4 d in tea and chive. However, its long-term dietary risk in the five crops was within the acceptable range. Furthermore, Sun et al. [6] showed that the half-life of chlorfenapyr on mustard is 4.2–5.9 d, and its short-term dietary intake risk is unacceptable for children aged 1–6 years. The study suggested that the short-term dietary intake risk level of chlorfenapyr should be reduced by extending the harvest interval. Huang et al. [7] and Feng et al. [8] showed that the degradation of chlorfenapyr in these crops was rapid, and its dietary risk was controllable. A study conducted by Wang et al. [9] suggested that the residue dissipation of chlorfenapyr in cabbage varied greatly among different regions, but it would not cause unacceptable risk levels to the health of the general population. Tang et al. [10] combined the QuEChERS method with UPLC-MS/MS to study the residue status and dietary intake risk of lufenuron and methoxyfenozide in pak choi under two different planting conditions (open and protected fields). The results showed that the half-lives of lufenuron and methoxyfenozide were 5.7 d and 3.9 d in the open field, respectively, which were significantly prolonged to 11 d and 8.6 d, respectively, in the greenhouse. The results of dietary risk indicated that the risk quotients (RQ) of the two pesticides were acceptable for different gender and age groups in China. Furthermore, methoxyfenozide degradation was found to be rapid in cauliflower, tea, [11] and rice, [12] and its dietary risk was controllable. Paramasivam [13] found that the half-life of chlorantraniliprole in tomato was 1.26 d, which is an acceptable level for dietary risk, but caused moderate harm to soil earthworms and arthropods. Kansara et al. [14] and Mandal et al. [15] studied the residue dissipation and dietary risks of chlorantraniliprole on pigeon peas and berseem. The results showed that the half-life of chlorantraniliprole in the two crops was 4.95–5.78 d and 0.93–1.33 d, respectively. Thus, the study suggests rapid degradation and controllable dietary risk of chlorantraniliprole when used according to the recommended dosage.

The above studies suggested that pesticide residue dissipation and dietary risks, such as with chlorfenapyr, vary significantly in different crops and planting patterns. However, to date, the residue dissipation and dietary risk assessment of the five pesticides have not been reported for spinach. In order to evaluate the safety of the five pesticides in spinach, we explored and validated a sensitive and effective method for six compounds in spinach based on the QuEChERS method. We conducted the experiments in eight main spinach-producing areas to determine the residue dissipation of the five pesticides in spinach under two different planting conditions: a greenhouse and open fields. Based on the residue dissipation data, the dietary risks of the five pesticides in spinach were assessed, providing supporting data for their possible registration for future application on spinach.

2. Materials and Methods

2.1. Reagents and Materials

Methoxyfenozide (98.98%), chlorantraniliprole (97.28%), indoxacarb (97.37%), lufenuron (98.97%), and chlorfenapyr (99.0%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Tralopyril (99.0%) was purchased from Honeywell Specialty Chemicals, while sodium chloride (analytical purity), formic acid (chromatographic purity), and acetonitrile (chromatographic purity) were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd (Tianjin, China). The C18 packing (40–60 μm), primary secondary amine (PSA) (40–60 μm), and graphitized carbon black (GCB) absorbents were purchased from Agela Technologies (Tianjin, China), whereas the organic filter (0.22 μm) was purchased from Ameritech. The methoxyfenozide (240 g/L suspension concentrate), chlorantraniliprole (5% suspension concentrate), indoxacarb (150 g/L suspension concentrate), lufenuron (50 g/L suspension concentrate), and chlorfenapyr (10% suspension concentrate) formulations were provided by the Shandong Province Institute for the Control of Agrochemicals.

2.2. Instrumental Conditions

A liquid chromatography-tandem mass spectrometer (LC-MS/MS) equipped with an electrospray ionization (ESI) source was used to detect methoxyfenozide, chlorantraniliprole, indoxacarb, tralopyril, and lufenuron. Conversely, chlorfenapyr was detected using gas chromatography (Agilent 8890, Santa Clara, CA, USA) and mass spectrometry (Agilent 7000d, Santa Clara, CA, USA) equipped with an EI source. The details of all chemical compounds and the working instrument conditions are listed in Tables S1 and S2. Other instruments used for the analysis were a MTV-100 multi-tube vortex mixer (Allsheng Instrument Co., Ltd., Hangzhou, China), a Sorvall ST16 desktop ventilated centrifuge (Thermo Fisher, Shanghai, China), and a AL204 electronic balance [Mettler Toled (Shanghai, China)]. The laboratory ultrapure water system (Exceed-Cd-08) was purchased from Chengdu Tangshi Kangning Science Development Co., Ltd (Chengdu, China).

2.3. Field Experiment Design

Field experiments were designed according to the Guidelines for Test Area for the Registration of Pesticide Residue and the Guideline for Testing Pesticide Residues in Crops. [16,17] One treatment plot and one control plot (without treatment) were set for each of the five test pesticides. Each plot measured at least 50 m², and the plots were delineated with protection zones. The dosages of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr used to control Spodoptera exigua infestation on spinach were 50.4, 22.5, 40.5, 22.5, and 49.5 g a.i. ha⁻¹, respectively. The pesticides were sprayed once during the larvae stage of S. exigua, and a spray volume of 450 L per hectare was applied by an electric knapsack sprayer. The details of each test site, harvest interval, and planting pattern are listed in

Table 1. Information on field experiments in eight representative areas.

Sites	Longitude and Latitude	Trial Date	Average Temperature during the Test (°C)	Precipitation during the Test (mm)	Varieties	Planting Type	Crop Status	Sample Intervals (d)
							BBCH
Inner Mongolia Autonomous Region	40.42 N, 110.48 E	2021.06.17–6.24	15–30	22	Spinach-no.001	Green house	41	5,7
Shaanxi	37.33 N, 112.40 E	2021.05.06–5.13	13–28	30	Qiulu-spinach	Open field	47	5,7
Beijing	40.17 N, 116.36 E	2021.05.18–5.25	12–28	25	Halimu	Green house	45	5,7
Shandong	36.07 N, 116.94 E	2021.05.07–5.17	25–37	15	Royal Dutch F1	Green house	45	0,3,5,7,10
Anhui	31.85 N, 117.01 E	2021.05.23–6.2	18–32	36	Huabo no.1	Green house	47	0,3,5,7,10
Hunan	28.53 N, 113.13 E	2021.05.31–6.7	18–30	45	Big leaf spinach	Open field	43	5,7
Guizhou	26.41 N, 106.40 E	2021.04.23–5.3	15–23	37	Hybrid spinach no.168	Open field	45	0,3,5,7,10
Guangdong	23.25 N, 113.06 E	2021.10.21–10.31	22–32	65	Transcend no.3	Open field	47	0,3,5,7,10

. Leaf samples (at least 1 kg) were collected randomly from at least 24 mature and disease-free spinach plants, showing normal growth at each sampling site. Thereafter, the spinach plants were cut into smaller sections (less than 1 cm), mixed well, and separated into groups of 150 g using the quartering method. The samples were placed into sealable sample bags and stored at −18 °C until further analysis.

2.4. Sample Preparation by Modified QuEChERS

For the analysis of the six compounds, the proposed extraction and clean-up procedures were based on a modified QuEChERS method [18]. The spinach samples were mashed with a food processor, after which 5.0 g of the samples were accurately weighed and put into 50 mL plastic centrifuge tubes with stoppers. Thereafter, 10 mL of acetonitrile was added, and the mixture was vortexed on an automatic vortex analyzer for 5 min at 2500 r/min. Sodium chloride (4 g) was then added, and the mixture was vigorously shaken for 1 min, followed by centrifugation at 4000 r/min for 5 min. Aliquots (1 mL) of the supernatants were transferred into purification tubes containing 20 mg of GCB and vortexed for 30 s. The mixture was then left to stand for 1 min and passed through the 0.22 µm organic filter membrane for further analysis.

2.5. Calculation Equations

2.5.1. Definition of Chlorfenapyr Assessment

The dietary assessment of chlorfenapyr was defined as chlorfenapyr + 10 × tralopyril, [19] and its defined residue was calculated according to the following equation:

X = C1 + 10 × C2

where X denotes the defined chlorfenapyr residue (mg kg⁻¹) in the sample, and C1 denotes the actual chlorfenapyr residue in the sample (mg kg⁻¹). If the chlorfenapyr residue is less than the limit of quantitation (LOQ), then the LOQ value is directly substituted into the equation. C2 denotes the tralopyril residue in the sample (mg kg⁻¹); if C2 is less than the LOQ, only the chlorfenapyr residue is calculated.

2.5.2. Long-Term Dietary Risk Assessment

The RQ was used to assess the long-term dietary exposure risk of the pesticides. Higher RQ values indicated higher risk levels, while RQ values higher than 100% indicated unacceptable risk levels to human health [20,21]. The calculation equation is as follows:

$$\mathrm{NEDI} (\mathrm{mg} {\mathrm{kg}}^{-1})=\frac{{{\displaystyle \sum }}^{ }\mathrm{FI}\left(\mathrm{kg}\right)\times \mathrm{STMR}\left(\mathrm{mg} {\mathrm{kg}}^{-1}\right)}{\mathrm{bw}\left(\mathrm{kg}\right)}$$

(1)

$$\mathrm{RQ}=\frac{\mathrm{NEDI}\left({\mathrm{mg} \mathrm{kg}}^{-1} \mathrm{bw}\right)}{\mathrm{ADI}\left({\mathrm{mg} \mathrm{kg}}^{-1} \mathrm{bw}\right)}\times 100\%$$

(2)

where NEDI denotes the national estimated daily intake of each agent (mg kg⁻¹); FI denotes the intake of a certain food type (kg); STMR denotes the supervised trials median residue (mg kg⁻¹); bw denotes the average body weight of Chinese adult residents (which is generally 63, kg); RQ denotes the risk quotient; ADI denotes the acceptable daily intake of the agent (mg kg⁻¹, bw).

2.5.3. Dissipation Dynamics

The first-order kinetic equation was used to calculate the dissipation and half-life of the pesticides in spinach as follows [22,23]:

$${\mathrm{C}}_{\mathrm{t}}={\mathrm{C}}_0\times {\mathrm{e}}^{-\mathrm{kt}}$$

(3)

$${\mathrm{DT}}_{50}=\frac{\ln 2}{\mathrm{k}}$$

(4)

where Ct (mg kg⁻¹) denotes the agent residue over time (days), C₀ denotes the original deposition amount (mg kg⁻¹), and k denotes the dissipation coefficient (day⁻¹).

2.5.4. Matrix Effect

The matrix effect (ME) was evaluated using the differences between the slopes(S) of the matrix standard solution and the pure solvent standard solution, as shown in the equation as follows [24,25,26]:

$$\mathrm{ME}(\%) =\frac{{\mathrm{S}}_{\mathrm{matrix} -{ \mathrm{S}}_{\mathrm{solvent}}}}{{\mathrm{S}}_{\mathrm{solvent}}}\times 100\%$$

(5)

where S_matrix denotes the slope of the matrix standard curve, while S_solvent denotes the slope of the solvent standard curve.

2.6. Method Validation

Standard solution of the chemical compounds (0.02, 0.5, and 5 mg kg⁻¹ for methoxyfenozide, chlorantraniliprole, lufenuron, chlorfenapyr, tralopyril and 0.02, 0.5, and 3 mg kg⁻¹ for indoxacarb) was added to the blank solutions of the spinach samples. Each level was repeated 5 times, followed by an additional recovery test. The addition recovery rate and the relative standard deviation were calculated, and the lowest addition level was used as the LOQ. Moreover, the working solution (0.01–5 mg L⁻¹) was prepared with acetonitrile and blank spinach extract. The standard external method was used for quantification, and the standard curve was plotted using the concentration as the independent variable and the peak area as the dependent variable.

3. Results and Discussion

3.1. Optimization of Purification

The impurities in the spinach samples were removed by dispersive solid phase extraction. It is well known that spinach contains higher chlorophyll levels, which can cause a significant matrix effect that interferes with the detection results, increasing the maintenance cost of the instrument [27,28,29]. Different amounts of adsorbents significantly impact the purification and recovery rates of pesticide extracts during the pretreatment process. Therefore, it is important to select the appropriate adsorbent dosage during the experiment [30]. This study compared the effects of different amounts of the three commonly used purification agents (C18, PSA, and GCB) on the purification and recovery of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, chlorfenapyr, and tralopyril from acetonitrile and spinach extracts (Figure 1). After purification with different doses of C18, the recovery rate of the six target substances was between 85–103%. The recovery rate of tralopyril decreased, while that of the other agents was above 90% without obvious adsorption, with the increase in PSA dosage. GCB had strong adsorption for chlorantraniliprole, tralopyril, and lufenuron but less adsorption for methoxyfenozide, indoxacarb, and chlorfenapyr. This may be due to the strong adsorption ability of GCB to planar structure compounds [19,31,32,33]. Compared with acetonitrile extracts, the recovery rates of chlorantraniliprole, tralopyril, and lufenuron from the spinach extract were between 73–78% after treatment with 20mg of GCB purification agent (Figure 2). This suggested that GCB can adsorb the pigment and other impurities present in spinach extract, thereby reducing the adsorption of the three pesticides. However, the recovery rate of the three agents rapidly decreased with increasing GCB dosage, which is consistent with previous reports [10]. Although the recovery rates of the target substances were higher after purification with C18 and PSA, the spinach samples still contained many pigments after purification, which may cause problems with instrument maintenance. Although GCB has an efficient removal effect on pigments [28,34,35], the recovery rate of the three compounds significantly decreased with the dosage increase in GCB in the present study. Therefore, GCB dosage should be reduced as much as possible during purification to ensure an efficient purification effect. In this study, 20 mg of GCB was the suitable dosage efficient purification effect.

3.2. Method Validation

The accuracy and precision of the methods used were characterized by a recovery test. The results of the recovery test, the linear relationships, and the matrix effect are shown in

Table 2. Performance characteristic of the method for six compounds in spinach.

Compounds	Spiked Level (mg·kg−1)	Average Recovery (%) (n = 5)	RSD (%)	Matrix		Acetonitrile		ME (%)	LOQ (mg·kg−1)
				Regression Equation	r	Regression Equation	r
Methoxyfenozide	0.02	96	5	Y = 22,356X + 4262	0.9966	Y = 25,757X + 5715	0.9952	−13	0.02
	0.5	98	2
	5	91	5
Chlorantraniliprole	0.02	84	5	Y = 8006X + 1654	0.9961	Y = 9990X + 1901	0.9961	−20	0.02
	0.5	98	4
	5	92	2
Indoxacarb	0.02	98	7	Y = 3536X + 485	0.9962	Y = 3598X + 754	0.9925	−2	0.02
	0.5	96	3
	3	96	2
Lufenuron	0.02	99	11	Y = 91,324X + 6542	0.9985	Y = 112,601X + 1730	0.9997	−19	0.02
	0.5	89	6
	5	91	2
Chlorfenapyr	0.02	97	3	Y = 97,232X + 3569	0.9997	Y = 69,382X + 2531	0.9996	40	0.02
	0.5	95	2
	5	95	3
Tralopyril	0.02	92	4	Y = 249,504X + 25,412	0.9949	Y = 254,563X + 30,967	0.9931	−2	0.02
	0.5	99	7
	5	98	6

. The average recovery rates of the six compounds ranged from 84% to 99% in spinach, and their RSDs were less than or equal to 11%. The six compounds had LOQs of 0.02 mg kg⁻¹ each, indicating that the accuracy, precision, and sensitivity of the methods used met the requirements for pesticide residue detection. Moreover, the standard curves were well fitted in the different matrices in the range of 0.01–5.0 mg L⁻¹, and the linear correlation coefficients of the six compounds were greater than 0.9925. The calculation of the ME using the method mentioned in Section 2.5.4 suggested that the ME of chlorfenapyr was enhanced by 40%, while that of methoxyfenozide, chlorantraniliprole, and lufenuron was weakened by 13, 20, and 19%, respectively, in spinach. No evident MEs of indoxacarb and tralopyril were observed. In order to ensure the universality of the method, we used the matrix matching the external standard calibration to quantify the concentrations of the six target compounds in spinach.

3.3. Residue Dissipation Dynamics

Understanding the residue dissipation of pesticides in crops is vital for their safe usage. In this experiment, the residue dissipation of five pesticides was studied in spinach under different planting conditions (open and greenhouse fields). The results showed that the residues of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr gradually decreased in spinach with time, and their dissipation conformed to the kinetic first-order dissipation model (correlation coefficient > 0.766) (

Table 3. Dissipation dynamics parameters of the five compounds in spinach at the four sites.

Compounds	Dosage (g a.i. ha−1)	Location	Regression Equation	Correlation Coefficient (r)	Half-Life (day)	Initial Residue (mg kg–1)
Methoxyfenozide	50.4	Shandong	C = 1.25e−0.228T	0.992	3.0	1.2
		Anhui	C = 4.58e−0.611T	0.899	1.1	2.8
		Guizhou	C = 4.64e−0.569T	0.963	1.2	3.3
		Guangdong	C = 5.97e−0.497T	0.998	1.4	3.8
Chlorantraniliprole	22.5	Shandong	C = 2.02e−0.175T	0.982	4.0	2.1
		Anhui	C = 3.62e−0.238T	0.984	2.9	3.1
		Guizhou	C = 4.23e−0.267T	0.891	2.6	3.5
		Guangdong	C = 5.71e−0.253T	0.766	2.7	4.4
Indoxacarb	40.5	Shandong	C = 1.05e−0.166T	0.920	4.2	0.91
		Anhui	C = 2.20e−0.408T	0.942	1.7	1.7
		Guizhou	C = 1.66e−0.321T	0.926	2.2	1.2
		Guangdong	C = 1.84e−0.295T	0.991	2.3	1.6
Lufenuron	22.5	Shandong	C = 0.811e−0.17T	0.899	4.1	1.1
		Anhui	C = 0.739e−0.156T	0.936	4.4	0.80
		Guizhou	C = 1.57e−0.204T	0.963	3.4	1.4
		Guangdong	C = 1.63e−0.197T	0.981	3.5	1.5
Chlorfenapyr	49.5	Shandong	C = 3.12e−0.163T	0.992	4.3	3.0
		Anhui	C = 4.95e−0.239T	0.986	2.9	4.2
		Guizhou	C = 4.87e−0.248T	0.960	2.8	3.7
		Guangdong	C = 3.78e−0.213T	0.994	3.3	3.5

). The degradation half-lives of the five pesticides in spinach were 1.2–1.4 d, 2.6–2.7 d, 2.2–2.3 d, 3.4–3.5 d, and 2.8–3.3 d under open field planting conditions, and were 1.1–3.0 d, 2.9–4.0 d, 1.7–4.2 d, 4.1–4.4 d, and 2.9–4.3 d under greenhouse planting conditions. Previous studies have shown that these agents have short half-lives in vegetables such as asparagus [4,36], mustard [6], chieh-qua [7], cabbage [4,9], leek [4], chive [4], flowering Chinese cabbage [8], pak choi [10], cauliflower [11], tomato [13], pigeon pea [14], and tea [37], which is consistent with our present results. The great differences in temperature, humidity, and light conditions between protected and open fields resulted in the varied residue status observed between the two field conditions. Zhang et al. [38] showed great differences in the original deposition and residue dissipation of iprodione and procymidone between tomatoes grown in the greenhouse and open fields. Tang et al. [10] also found differences in the residue and dissipation of methoxyfenozide and lufenuron in pak choi grown in open and greenhouse fields. In this study, the dissipation experiments of five compounds in spinach were carried out in Shandong, Anhui, Guizhou, and Guangdong in open and greenhouse fields. The results showed that the five pesticides rapidly degraded, and their half-lives were not significantly different under the two field conditions. This may be due to crop types, climatic conditions, and the physicochemical properties of the pesticides. Spinach is a leafy vegetable with a high surface area, making it easy for sprayed applications to cause a higher original deposition of the chemicals being sprayed [39]. This study showed that, although the original deposition amount of spinach is high, the pesticides rapidly degrade with time. This may be mainly related to the rapid growth of spinach, thus indicating that growth dilution also plays an important role in reducing pesticide residues in crops. The original deposition of the five pesticides was slightly different at different test sites, and this might be attributed to the growth state and planting pattern of spinach during pesticide application.

3.4. Final Residue

The five pesticides were applied once to plants under the two field conditions by spraying at the recommended dosage. The spinach samples were collected at 5 and 7 d after application, and the detection results of the final residues at the eight test sites are shown in Figure 3. The residues of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr were 0.02–0.76 mg kg⁻¹, 0.65–1.7 mg kg⁻¹, 0.28–1 mg kg⁻¹, 0.16–0.32 mg kg⁻¹, and 0.14–4.1 mg kg⁻¹, respectively, in the spinach samples with a harvest interval of 5 d. The pesticide residue in the spinach samples with a 7 d harvest interval was significantly lower than that with a 5 d harvest interval. These samples had methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr residues of 0.02–0.46 mg kg⁻¹, 0.055–0.80 mg kg⁻¹, 0.086–0.64 mg kg⁻¹, 0.086–0.23 mg kg⁻¹, and 0.061–2.0 mg kg⁻¹, respectively. Currently, China has formulated the maximum residue limit (MRL) values of 3 mg kg⁻¹ for indoxacarb on spinach and 20 mg kg⁻¹ for chlorantraniliprole on leafy vegetables; however, the MRLs of methoxyfenozide, chlorfenapyr, and lufenuron on spinach are yet to be formulated [40]. The MRLs for pesticides are set by the regulatory authorities in each country and are the limits and standards that ensure the safety of agricultural products and food. Different countries and regions have slightly different methods of developing MRL and risk assessment. For example, the OECD adds the mean to four times the standard deviation (SD) of all residue values to obtain an unrounded MRL, from which the proposed MRLs are derived [41]. Then, based on the proposed MRL, chronic consumer risk assessment upon exposure to the pesticide was determined. A slight difference concerning the OECD is that both China and the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) use the median value for risk assessment. The MRLs of the five pesticides on spinach and leafy vegetables formulated by other countries and organizations are listed in

Table 4. Acceptable daily intake (ADI) of the five compounds and the maximum residue limit (MRL) for spinach.

Compounds	MRL (mg kg–1)							ADI mg kg–1 (bw)
	China	CAC	The United States	Australia	Korea	European Union	Japan
Methoxyfenozide					20	4	30	0.1
Indoxacarb	3		14 *	5 *	3 *	2		0.01
Chlorantraniliprole	20 *	20 *	13 *	15 *	5	20	20	2
Chlorfenapyr					10			0.03
Lufenuron					5			0.01

Note: * MRL for Leafy vegetables, not spinach.

. The final residues obtained from the eight test sites were all within the corresponding MRL range formulated by China and other countries or regions. However, spinach intake differs in different countries and regions due to dietary habit differences. Therefore, whether the MRLs of the pesticides on spinach formulated by different countries and regions are suitable for China needs further assessment based on dietary intake.

3.5. Long-Term Dietary Risk Assessment

In this study, both international and national (China) risk assessment methods were conducted. The corresponding supervised trial median residues of the target compound residues in the registered crops in China (Table S3) should be considered in order to accurately calculate the national estimated daily intake (NEDI). If the median residual value cannot be obtained, the established MRL should be used for the calculation, and the long-term dietary intake risk should be assessed in combination with the dietary structure in China. The selection of crops whose MRL values have not been established in China should be in the following order: CAC > USA > EU > Australia > Korea > Japan [42]. Our results showed that five days after the application, the standard residue median values of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr in spinach were 0.34, 1.1, 0.48, 0.22, and 1.7 mg kg⁻¹, respectively. The average daily intake of dark vegetables among the Chinese urban and rural residents was 0.0915 kg, and the ADI values of the above five pesticides were 0.1, 0.01, 2, 0.01, and 0.03 mg kg⁻¹ (bw), respectively. According to the equation (1) and (2), methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr had the NEDI values of 0.009597, 0.010983, 0.010831, 0.003998, and 0.012907 mg kg⁻¹ (bw) and RQ values of 9.6, 0.55, 108.3, 40.0, and 43.0%, respectively, among the general population (

Table 5. Long-term dietary risk assessment of compounds in all of the registered crops in China.

Food Classification	Dietary	Methoxyfenozide		Chlorantraniliprole		Indoxacarb		Lufenuron		Chlorfenapyr
	Amount (kg)	Reference Residue Limits (Sources)	NEDI(mg kg–1)/ RQ(%)	Reference Residue Limits (Sources)	NEDI (mg kg–1)/ RQ(%)	Reference Residue Limits (Sources)	NEDI (mg kg–1)/ RQ(%)	Reference Residue Limits (Sources)	NEDI (mg kg–1)/ RQ(%)	Reference Residue Limits (Sources)	NEDI (mg kg–1)/ RQ(%)
Rice and its products	0.2399	0.1 (China)	0.009597/ 9.6	0.5 (China)	0.010983/ 0.55	0.1 (China)	0.010831/ 108.3		0.003998/ 40.0		0.012907/ 43.0
Flour and its products	0.1385
Other cereals	0.0233			0.02 (China)				0.01 (CAC)
Tubers	0.0495			0.02 (China)				0.01 (CAC)
Dried beans and their products	0.016			0.05 (China)
Dark color vegetables	0.0915	0.34 (STMR)		1.1 (STMR)		0.48 (STMR)		0.22 (STMR)		1.7 (STMR)
Light color vegetable	0.1837	2 (China)		2 (China)		3 (China)		1 (China)		2
Pickies	0.0103
Fruits	0.0457	3 (China)		2 (China)				1 (China)		1
Nuts	0.0039
Livestock and poultry	0.0795
Milk and its products	0.0263
Egg and its products	0.0236
Fish and shrimp	0.0301
Vegetable oil	0.0327			0.3 (China)		0.1 (China)		0.05 (China)
Animal oil	0.0087
Sugar, starch	0.0044			0.05 (China)
Salt	0.012					5 (China)				20
Soy sauce	0.009	5 (USA)		0.02 (China)		0.01 (USA)				0.5

). This indicated that the residual level of indoxacarb is unacceptable to the health of the general population. However, the maximum value of indoxacarb in the spinach samples with a harvest interval of 5 d was 1.0 mg kg⁻¹, which is significantly lower than the formulated limit of 3 mg kg⁻¹ in China. The reason could be that the dietary data used for the general long-term dietary intake risk assessment is based on crop classification. Therefore, only the consumed crop categories are used, but certain agricultural products are not consumed alone. Thus, this assessment method maximizes the risk of dietary intake.

For international risk assessment, the total intake for methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr was calculated according to the dietary consumption data obtained from the global environmental monitoring system (GEMS)/Food Consumption Cluster Diets System. The supervised trial median residue (STMR) values of the five pesticides in various crops were obtained from the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) report. As shown in Table S4, the total intake of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr were 19.4–397.5, 54–403.6, 8.1–176.8, 5.8–52.2, and 17.14–115.6 μg/person, which accounted for 0.3–6.6, 0–0.4, 1–30, 0–4, and 1–6% of the ADI, respectively. These results indicated that the effects of the five pesticides on the health of the general population are at an acceptable risk level.

4. Conclusions

In this study, an analytical method for the determination of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, chlorfenapyr, and tralopyril in spinach was developed on the basis of QuEChERS using the LC-MS/MS (GC-MS/MS) technique. The method was satisfactory when considering linearity, selectivity, accuracy, and precision. Based on the method, the dissipation and terminal residues of five pesticides in spinach under field conditions were investigated.

The residue dissipation of methoxyfenozide, chlorantraniliprole, indoxacarb, chlorfenapyr, and lufenuron in spinach showed rapid degradation with a half-life range of 1.1-4.4 d under the two field experiment conditions and conformed to the first-order kinetic equation. The terminal residues of indoxacarb were below the MRL set by China. The long-term dietary risk assessment suggested that the risk of dietary intake for methoxyfenozide, chlorantraniliprole, chlorfenapyr, and lufenuron was acceptable, and spinach obtained under good agricultural practices conditions would not pose a threat to humans through their dietary intake. Finally, this work will be useful to establish the MRLs of methoxyfenozide, chlorantraniliprole, chlorfenapyr, and lufenuron in spinach and to provide guidance on their safe use.