Residual dynamics and dietary exposure risk of dimethoate and its metabolite in greenhouse celery

Test materials

Celery was used as the test crop. The field test was carried out in the vegetable production base in the Liaozhong district of Shenyang City. No extreme weather events, such as heavy rain and hail, were observed during pesticide applications, and the climatic conditions were normal. The test pesticide was 40% dimethoate EC, Hebei Zhongtian Bangzheng Biologic Science Co., Ltd.. The maximum recommended dose is 600 g a.i./ha in China. The formulation of dimethoate was analyzed before application in this study. The content of dimethoate met the requirements, and no omethoate was detected in it.

Instruments

Waters UPLC TQ Ultra Performance Liquid Chromatography-Tandem Mass Spectrometer, Waters, USA; Zhongjia HC 3514 High-Speed Centrifuge, Anhui USTC Zonkia Scientific Instrument Co., Ltd.; Ding Haoyuan RS-1 Vortex Mixer, Beijing Ding-Hao Yuan Technology Co., Ltd.; Pine-tree ultra-pure water machine, Beijing Xiangshunyuan Technology Co., Ltd.; JACTO HD400 Backpack Sprayer, JACTO Agricultural Machinery Co., Ltd.; 0.22 μm needle filter, 50 mL polypropylene plastic centrifuge tube, Xinkang Medical Equipment Co., Ltd.

Reagents

Methanol, acetonitrile (chromatographically pure), Merck. Wondapak QuEchERS extraction and separation kit, Shimadzu Kojima (Shanghai) Trading Co., Ltd. Dimethoate and omethoate were purchased from the Environmental Quality Supervision and Testing Center of the Ministry of Agriculture (Tianjin).

Standard solutions

Standard stock solutions (100 mg L⁻¹) of dimethoate and omethoate were diluted with acetonitrile to make the working standard solution of different concentrations (0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 mg L⁻¹). Additionally, celery samples cultivated in control plots were used as blanks. The stock solution was diluted with the clean control extract to generate the matrix standard solution (0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 mg L⁻¹). Standard solutions were stored in the dark at −20 °C. Blanks with a dimethoate and omethoate solution at three concentration levels (0.01, 0.1 and 1 mg kg⁻¹) were employed for the recovery assay. The analytical method’s performance parameters, such as linear ranges, limit of quantification (LOQ), and limit of detection (LOD), were determined in addition to recovery rates.

Field test design

According to Guideline’s requirements on pesticide residues trials (NY/T 788-2004, 2004), the test plot was designed with a plot area of 30 m², a buffer zone of 2 m, and three repeat plots, which were randomly arranged. A control area of 30 m² without pesticide application was also set up to collect control samples.

Dissipation dynamics

Dimethoate was sprayed at 900 g a.i./ha (1.5 times the maximum recommended dose) using a knapsack sprayer on the surface of celery in the middle growth stage, and the experiment was repeated on three plots. The samples were collected two hours, 1, 3, 5, 7, 10, 14, 21, 28 and 42 days after pesticide application.

Final residual dynamics: The pesticide application dose was 600 g a.i./ha (the maximum recommended dose) and 900 g a.i./ha (1.5 times the maximum recommended dose), respectively. Dimethoate was sprayed once using a knapsack sprayer on the soil in the seedling stage, and ripe celery samples were collected 145 days after the pesticide application. Dimethoate was sprayed once using a knapsack sprayer on the surface of celery in the transplanting stage, and ripe celery samples were collected at 90 days after the pesticide application. Dimethoate was sprayed once using a knapsack sprayer on the celery surface in the middle growth stage, and samples of ripe celery were collected at 45 days after the pesticide application. Besides, dimethoate was sprayed using a knapsack sprayer on the surface of celery two and three times in the harvesting stage with intervals of 7 days between applications, and the experiment was repeated on three plots. The samples were collected at 3, 5, 7, 10, 14 and 21 days after the last pesticide application.

The seedling stage was the day of sowing, the transplanting stage was 55 days after sowing, while the middle growth stage was 45 days after transplantation. Finally, ripe celery was collected 90 days after transplantation. The harvesting stage was 62–97 days after transplantation. The samples were collected 3, 5, 7, 10, 14 and 21 days after the last pesticide application. The growing stage, days of the pesticide application and sampling are shown in Fig. 2.

Sample analysis

Detection

Chromatographic conditions were as follows: Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm); column temperature, 25 °C; injection volume, 5 μL; flow rate, 0.38 mL min⁻¹; mobile phase A, water, and mobile phase B, methanol. Gradient elution conditions were as follows: 0–0.25 min, 90–5% A; 0.25–5.00 min, 5–90% A. Mass spectrometry conditions were as follows: electrospray ion source positive ion mode (ESI +); ion source temperature, 500 °C; capillary voltage, 1.0 kV; nebulizing gas flow rate, 900 L h⁻¹; and taper hole anti blow air flow rate, 50 L h⁻¹. The scanning method was the multiple reaction monitoring (MRM) mode. The other MS/MS parameters were separately optimized for each target compound and are listed in Table 1.

Dissipation dynamics

The first-order kinetic equation was used to express the dissolution dynamics of dimethoate and omethoate in celery over time.

(1) $$c_t=c_0{e}^{-kt}$$ (2) $$t_{1/2}={\displaystyle \frac{Ln2}{k}}$$where $t$ is time (day), $c_t$ is the concentration (mg kg⁻¹) at time t (days), $c_0$ is the initial concentration (mg kg⁻¹), $k$ is the degradation rate constant (day⁻¹) and $t_{1/2}$ is the half-life (d).

Final residue

The toxicological endpoints of dimethoate and its metabolite are the same; therefore, the sum of residues of dimethoate and omethoate should be considered together for both acute and chronic dietary intake. Omethoate is more toxic than dimethoate. The relative toxicity of omethoate compared to dimethoate following chronic and acute were found to be about ~3:1 and ~6:1, respectively (None, 2009).

Sum of dimethoate and 6×omethoate, expressed as dimethoate (for acute risk assessment);

Sum of dimethoate and 3×omethoate, expressed as dimethoate (for chronic risk assessment).

A World Health Organization (WHO) template for evaluating acute exposure (IESTI) is used to evaluate the risk of acute dietary exposure uses. In contrast, a WHO template to evaluate chronic exposure (IEDI) is used to evaluate the risk of chronic dietary exposure. (http://www.who.int/foodsafety/areas_work/chemical-risks/gems-food/en/).

The following formula (Geng et al., 2018) was used to calculate the risk of chronic dietary exposure of dimethoate and omethoate.

(3) $$NEDI=F\times STMR/bw$$ (4) $$RQ=NEDI/ADI$$where $NEDI$ is the country’s estimated daily intake (mg kg⁻¹ bw day⁻¹), $STMR$ is the median residue of the standard test (mg kg⁻¹), $F$ is the average food consumption (kg d⁻¹), $bw$ is the body weight (kg) and $ADI$ is the acceptable daily intake (mg kg⁻¹ bw day⁻¹). $RQ$ is chronic risk assessment. $RQ$ > 1 indicates that the chronic dietary intake risk is unacceptable. $RQ$ < 1 suggests that the chronic nutritional intake risk is acceptable; the smaller the risk quotient (RQ), the lower the risk.

The following formula was used to calculate the risk of acute dietary exposure of dimethoate (the single weight of unprocessed food was more than 25 gs and the edible portion’s available weight was more than or equal to the consumption by most individuals) (Geng et al., 2018).

(5) $$IESTI=LP\times HR\times v/bw$$ (6) $$HQ=IESTI/ARfD$$where $IESTI$ is the estimated short-term intake (mg kg⁻¹ bw day⁻¹), $LP$ is the average food consumption (kg d⁻¹), $HR$ is the highest residue obtained in the test (mg kg⁻¹), $v$ is the variability factor assigned a value of 3 according to JMPR (Gao, Chen & Zhang, 2007), $bw$ is the body weight (kg), and $ARfD$ is the acute reference dose (mg kg⁻¹ bw day⁻¹). $HQ$ is an acute risk assessment. When $HQ$ < 1, which means that the risk of acute dietary intake is acceptable. When $HQ$ > 1, it indicates an unacceptable acute risk.

Method validation

The limits of detection (LODs) and the limits of quantification (LOQs) for dimethoate and omethoate were considered to be the concentrations produced at a signal-to-noise (S/N) ratio of 3 and 10, respectively. The LODs for the two target chemicals were 0.003 mg kg⁻¹, and the LOQs were 0.01 mg kg⁻¹. Good linear calibration curves were obtained over the concentration range of 0.005–0.5 mg L⁻¹ for dimethoate and omethoate and the correlation coefficient r was higher than 0.99 (Table 2). The sample concentrations outside the linear range were diluted to the appropriate analytical concentration. The matrix effect (ME) was calculated:

(7) $$\mathrm{M}\mathrm{E}(\mathrm{\%})=(\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{p}{\mathrm{e}}_{\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}}-1)\times 100\mathrm{\%}$$ (8) $$\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{p}{\mathrm{e}}_{\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}}=\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{p}{\mathrm{e}}_{\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{x}}/\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{p}{\mathrm{e}}_{\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{t}}$$where slope matrix and slope solvent are the calibration curve slopes of the celery and acetonitrile standard, respectively. The matrix effects (MEs) were –4% (Table 2), which caused the signal’s suppression. Thus, matrix-matched calibration solutions were used to compensate for errors associated with matrix-induced calibration.

The accuracy was evaluated by determining the recovery assay at three levels in celery. No dimethoate and omethoate were detected in the blanks. The mean recoveries were 83.4–92.9% and 80.4–94.6% for dimethoate and omethoate, with RSD in the range of 3.7–4.5% and 4.0–7.3%, respectively (Table 3). This finding indicated that the method of analysis was accurate and precise.

Dimethoate dissipation dynamics in celery

The results of dimethoate detection were expressed as average values of three repeat plots. As shown in Fig. 3 (when the safety interval was more than 28 days, and the concentration of dimethoate was lower than the LOQ), the degradation of dimethoate met the first-order kinetic equation, Ct = 4.0499e^{− 0.286t}, and the correlation coefficient r² was 0.9943. The half-life was 2.42 days, indicating that dimethoate was an easily degradable pesticide. Ten days later, the dissipation rate reached 94.6%, and the residual concentration of dimethoate decreased to less than 0.5 mg kg⁻¹ (the MRL of dimethoate on celery is 0.5 mg kg⁻¹), which was lower than the MRL. Furthermore, the dissipation rate reached 99% after 16.1 days.

Omethoate dissipation dynamics in celery

The results of omethoate detection were expressed as average values of three repeat plots. The dissipation data fitting is shown in Fig. 4 (when the safety interval was more than 42 days, the concentration of omethoate was lower than the LOQ). Before application, the formulation of dimethoate was analyzed, and no omethoate was detected in it. However, omethoate was detected in the celery. After the application of dimethoate, the concentration of omethoate increased to 0.19 mg kg⁻¹ on day 3 and gradually decreased after 3 days. This finding indicated that the levels of omethoate present in the celery were high due to dimethoate metabolism. After 28 days, the omethoate concentration was less than 0.02 mg kg⁻¹, which was lower than the allowable MRL of omethoate in celery. Hence, a 10 day safety interval was sufficient to ensure that the dimethoate concentration in celery declined to safe levels but was not enough for the omethoate concentration to reach safe levels. A safety interval of 28 days after dimethoate application is recommended based on the MRL (0.02 mg kg⁻¹) of omethoate in Chinese celery.

As shown in Fig. 5, the dissipation behavior of total residues of dimethoate and its metabolite omethoate conformed to the first-order kinetic equation, C_t = 3.7599e^−0.237t, the correlation coefficient r² was 0.9814. The half-life was 2.92 days, which was 20.1% longer than that of parent compound dimethoate. This finding indicated that omethoate should be taken into account in risk assessment as a metabolite of dimethoate.

Final residues following pesticide treatments in seedling, transplanting, and middle growth stages

The final residues of dimethoate and its metabolite omethoate after application in the seedling, transplanting, and middle growth stages of celery are shown in Table 4. The data showed that both residues of dimethoate and omethoate in celery were lower than the LOQ and the MRLs (the MRL of dimethoate is 0.5 mg kg⁻¹, and the MRL of omethoate is 0.02 mg kg⁻¹).

Final residues following pesticide treatments in the harvesting stage

The samples were collected at 3, 5, 7, 10, 14 and 21 days after the last pesticide application. The final residues of dimethoate and omethoate are shown in Tables 5 and 6. The data showed that, the residue of dimethoate in celery was lower than the allowable MRL of 0.5 mg kg⁻¹ after 10 days when two different dosages of dimethoate were sprayed two or three times. However, omethoate concentration was still higher than the allowable MRL of 0.02 mg kg⁻¹ after 21 days. Additionally, celery sprayed three times with the same dimethoate concentration had higher residues of dimethoate and omethoate compared with celery sprayed only twice, showing a cumulative effect of repeated pesticide application. However, whether the concentration of omethoate had declined to a level below the MRL by this stage could not be determined because a sample of celery 28 days after the final pesticide application was not collected.

Chronic dietary risk assessment

A safety interval of 28 days after dimethoate application is recommended based on the MRL of omethoate in Chinese celery. The dietary intake risk was not calculated at different times in this study. The standard trial median residue of the total of dimethoate and omethoate in celery is shown in Table 7. The allowable daily intake of dimethoate is 0.002 mg kg⁻¹ bw (GB 2763-2016, 2016). The daily consumption of vegetables is estimated based on the Chinese dietary structure (Wu, Zhao & Li, 2018; Liu et al., 2018). The daily intake of celery is lower than the total vegetable intake. Suppose the daily total vegetable intake replaces the celery intake. In that case the calculated dietary risk of the total residual of dimethoate and omethoate is acceptable in vegetable. The dietary risk of the total residual of dimethoate and omethoate in celery is acceptable.

The risk quotient (RQ) was calculated using the chronic dietary risk formulas 3 and 4. The results (Table 7) showed that on day 10, the RQs of dimethoate were both more than one, and therefore, the risks were unacceptable. On day 14, some RQs of dimethoate were more than 1 (2–12 years and 51–65 years/female), and the risks were unacceptable. After day 21, the RQs of dimethoate in celery were less than one, and the risk was acceptable.

Acute dietary risk assessment

The acute reference dosages (ARfD) of dimethoate is 0.01 mg kg⁻¹ bw (Geng et al., 2018; Utture et al., 2012). The HQ was calculated based on the dietary structures of different populations in China (Wu, Zhao & Li, 2018) using the acute dietary risk assessment formulas 5 and 6 to judge the level of acute dietary risk (Table 8). The results showed that the HQ range of dimethoate was 2.42–4.15 on day 10. On day 14, the HQ of dimethoate was 1.22–2.09. On day 21, the HQ of dimethoate was 0.67–1.14. On days 10 and 14, the HQs of dimethoate were more than one and the acute risks were unacceptable. On day 21, only children aged 2–7 years had an HQ of dimethoate more than 1 (an intolerable level of risk), while the acute dietary threats to other populations were within acceptable levels. As a precaution, it was recommended that large amounts of single types of food should not be included in for children aged 2–7 years in the short term to reduce acute dietary risk.