Dissipation behavior and dietary exposure risk of pesticides in Brussels sprout evaluated using LC–MS/MS

In this study, the dissipation behavior and dietary exposure risk of eight pesticides in Brussels sprout were evaluated under greenhouse conditions. Brussels sprout samples were collected 0, 7, 14, and 21 days after the last pesticide treatment. Ultra-high performance liquid chromatography with tandem mass spectrometry was used for sample analysis. Recovery rates at different concentrations of pesticides (0.01 and 0.1 mg/kg) were in the range of 70.2–104.5%, and the relative standard deviations were ≤ 10.6%. The pesticide residues in Brussels sprouts were determined for each treatment. For acephate, etofenprox, imidacloprid, indoxacarb, alpha-cypermethrin, zeta-cypermethrin, fludioxonil, and oxytetracycline, the half-lives were, respectively, 11.3, 9.8, 11.3, 15.8, 10.6, 13, 9.1, and 8.2 d and the dietary intake rates were, respectively, 2.90%, 0.81%, 0.7%, 1.19%, 0.06%, 0.24%, 0.05%, and 0.36% of the acceptable daily intake. The findings of this study provide important insights into the establishment of maximum residue limits in the Republic of Korea and pesticide control measures for Brussels sprout.

Field experiments. The test field was located in Eumseong-gun (GPS coordinates; 36.947296, 127.469195, Chungcheongbuk-do, ROK). The total field size for each pesticide treatment was 30 m (length) × 5.5 m (width). To eliminate the variation in plant growth and climate change, each field was divided into four plots, and samples on each field were harvest on the same day (Table 1). A control plot that was located in an area that was separated from the treated areas. In the cases of acephate, etofenprox, imidacloprid, indoxacarb, alpha-cypermethrin, and zeta-cypermethrin, each plot was treated with pesticide twice before harvest (Fig. 1a). For fludioxonil and oxytetracycline, each plot was treated with the pesticide either twice or thrice before harvest (Fig. 1b). Each plot Sample preparation and extraction. All plant samples were rapidly transferred to the laboratory after harvest. Brussels sprout samples were homogenized with dry ice and then stored at − 20 °C in polyethylene bags.
Other pesticides. The EN-QuEChERS kit was used to extract the pesticides 31 . Homogenized Brussels sprout (10 g) was weighed in a 50-mL falcon tube. A total of 10 mL of acetonitrile was added to the tube. The QuECh-ERS kit components (4 g ofMgSO 4 , 1 g of NaCl, 1 g of sodium citrate, and 0.5 g of disodium citrate sesquihydrate) were added to the falcon tube. The tube was shaken (1200 rpm) for 1 min. After centrifugation (4000 rpm, 5 min), 1 ml of the supernatant was filtered using a 0.2-μm polytetrafluoroethylene syringe filter.

LC-MS/MS analytical conditions. All samples were analyzed using Shimadzu LC-MS-8045 with
UHPLC Nexera X2 (Kyoto, Japan). Chromatographic separation was performed using a Kinetex C18 column (2.1 × 150 mm; 2.6 μm particle size; Phenomenex, USA) maintained at 40 °C. Information regarding the mobile phase, gradient program, and injection volume for all analytes are presented in Supplementary Tables S3a and  S3b. A triple quadrupole (QqQ) mass spectrometer (Shimadzu) with a positive electrospray ionization (ESI) source was used for all the analytes, except fludioxonil (negative ESI; Supplementary Table S4). Supplementary  Table S4 shows the multi reaction monitoring conditions for all analytes.
Method performance. The method was validated in terms of linearity, accuracy, precision, and limit of quantitation (LOQ). Linearity was assessed using a pure standard solution and homogenized pesticide-free Brussels sprout based on the data obtained using five concentrations: 5, 10, 20, 50, and 100 ng/mL. The lowest concentration among the chromatograms that produced a signal-to-noise ratio of > 10 was selected as the LOQ.
To determine the recovery rate, the pesticide standards were added to the homogenized pesticide-free samples at two different concentrations: 0.01 and 0.1 mg/kg. Each test was performed in triplicate.
Definition of pesticide residues in Brussels sprout. The individual compounds were analyzed for eight pesticides. The information regarding the residue definitions was provided by ministry of drug and food safety 16 . The parent substance only was evaluated except for acephate residues. Acephate residue in plants is the sum of acephate and methamidophos, which is a metabolite of acephate. The molecular weights of acephate and methamidophos are 183.2 and 141.1, respectively. To evaluate the total acephate in Brussels sprout, the sum of acephate residue concentration was calculated as follows: Statistical analysis. The dissipation rate was calculated based on the initial residue concentration (average residue level on plot 4, mg/kg) and that measured 21 day after pesticide treatment. The dissipation rate was calculated as follows: The dissipation patterns of the eight pesticide residues in Brussels sprout over time were expressed using the following first-order kinetics equation (Eq. 3), and the half-lives were calculated using Eq. (4) 32 : (1) www.nature.com/scientificreports/ where C 0 is the initial residue concentration (mg/kg) in plot 4, C t is the residue concentration (mg/kg) in plots 1, 2, and 3, t is the days after pesticide treatment, and k is the rate constant of dissipation. The initial residues were calculated using the MRLs for Brussels sprout in the EU and those for cabbage in the ROK, which is applied to Brussels sprout.

Dietary risk evaluation.
To estimate the human health risk from the pesticide residues present in Brussels sprout, hazard quotient (HQ)was calculated based on the estimated daily intake and ADI using the following Eqs. (5-7): In case of the acephate and its metabolite (methamidophos), because their ADIs were different, HQs were calculated separately. The daily dietary intake for Brussels sprout was 7.17 g, which is the daily food intake for vegetables, and the average body weight in the ROK is 59 kg 33 .

Results and discussion
LOQ, calibration curve, and recovery. Standard curves of all pesticides showed good linearity in samples of Brussels sprout (Supplementary Table S5). The range was between 0.005 and 0.1 mg/L of standard solution. Method LOQ for all pesticides was 0.01 mg/kg. The accuracy and precision were determined based on the recovery rate and relative standard deviation (RSD) at different concentrations (0.01 and 0.1 mg/kg). Supplementary Table S6 shows the results of the recovery tests. For all pesticides, the range of recovery rates was 70.2-101.9% at low concentrations and 71.5-104.5% at high concentrations. The RSDs for all the pesticides were < 11%.
Pesticide residues in Brussels sprout. Pesticide residues were not detected in control samples; the pesticide residue results obtained from the field trials are presented in Table 2. The dissipation rate was calculated by comparing the results obtained from plots 1 and 4. Normalization was performed by calculating the residual concentration (mg/kg) compared with the spraying amount (g). The normalized values (NVs) of etofenprox and indoxacarb showed the highest initial concentration values of 1.37 and 1.31, respectively (Table 3). Although oxytetracycline and imidacloprid had low vapor pressure, initial NVs showed the lowest values of 0.24 and 0.45, respectively. The initial NVs were affected by vapor pressure, pesticides' physicochemical properties, minor substances, and formulation types 20,22,33,34 . For etofenprox, the NV was 1.37 in Brussels sprout; this value has been reported to be 0.06 in squash 22 , 4.37 in squash leaf 22 , 2.09 in Chinese cabbage 34 , and 4.50 in spring onion 20 . The NVs of squash leaf, Chinese cabbage, and spring onion were higher than the value of Brussels sprout because of their surface area and texture. Squash leaves have a large surface area, and the fruit has a slippery surface. The Chinese cabbage studied was the loose-head type vegetable, and spring onion has a greater surface area than the other aforementioned crops; therefore, more pesticides can adsorb to its surface. Regarding indoxacarb, the calculated NVs were 1.31 in Brussels sprout; this value has been reported to be 0.39 in cucumber 35 as they have different surfaces and properties.
Half-lives of pesticides on Brussels sprout. The half-lives of the eight pesticides studied vary across crops and are influenced by many factors such as class of target and chemical, plant species, field conditions, chemical and microbial decomposition, dilution by plant growth, volatilization, acidity, photodecomposition, temperature, surface washoff, and spatial variability 36 . Figure 2 shows the regression curves and half-lives of the eight pesticides in Brussels sprout. The half-life for acephate was 11.3 days. Acephate in Brussels sprout decreased slowly compared with acephate in brinjal (2.13 days) 17 , green chili fruit (4.02 days) 18 , and mango (5 days) 19 . The half-life of etofenprox was 9.8 days in Brussels sprout, which is similar to that noted in spring onion (9.5 days) 20 and higher than that noted in tomato (2.15 days) 21 and squash (3.5 days) 22 ; the differences are attributable to the variations in crop properties and enzymes. Imidacloprid's half-life (11.3 days) was longer in Brussels sprout than in mango (3.06 days) 23 and shorter in the former than in grape (16.6 days) 24 . Indoxacarb's half-life was 15.8 days and decreased slowly compared with indoxacarb's half-life in cabbage (2.88 days) 25 , tomato (3.21 days) 26 , and green chili fruit (3.85 days) 27 . Hypertrophy-fast crop growth in a short period-is reportedly a major factor in the half-life of pesticides 37,38 . Although cabbage and Brussels sprout have a similar wrapped-over form, the halflives are different because of the hypertrophy of cabbage. The half-life of fludioxonil in Brussels sprout (9.1 days) was similar to that noted in mandarin (8.7 days) 29 and longer than that noted in Chinese cabbage (4.0 days) 28 . Mandarins have a bumpy surface, and pesticides are, therefore, likely to adsorb to the surface. The half-lives of alpha-cypermethrin, zeta-cypermethrin, and oxytetracycline were 10.6, 13, and 8.2 days, respectively. In the aspect of chemical class of pesticides, dissipation half-lives range from 0.9 to 22.8 days for organophophates, 0.8-10.6 days for carbamates, 3.1-9.3 days for neonicotinoids, and 1.1-5 days for pyrethroids. Except acephate, most of dissipation half-lives were higher than previous reports 36 . In the aspect of plant species, dissipation halflives of pesticides were in the range of 0.9-13 days which is similar with this study. In this study, there was less correlation between dissipation half-lives and volatilization (vapor pressure). www.nature.com/scientificreports/ MRLs of pesticides in Brussels sprout. The initial residues (mg/kg) of the eight pesticides were compared with the domestic MRLs for cabbage and that in the EU for Brussels sprout. The initial acephate residue was 143% of the domestic MRL (5 mg/kg) and 71,600% of the EU MRL (0.01 mg/kg). In the case of etofenprox, the initial residue was 1.99 mg/kg, which was 995% of the domestic MRL (0.2 mg/kg) and 19,900% of the EU MRL (0.01 mg/kg). The initial imidacloprid residue (0.34 mg/kg) was 68% of the EU and domestic MRLs (0.5 mg/kg for both). The initial indoxacarb residue (0.98 mg/kg) was 490% of the domestic MRL (0.2 mg/kg) Table 2. Average residues and dissipation rates of eight pesticides in Brussels sprout. SD standard deviation, EU European Union, MRL maximum residue limit.     Table 4). Considering the daily consumption of Brussels sprout, the exposure risk to pesticides is considered to be low.

Conclusions
We evaluated the residue, dissipation pattern, and dietary risk of acephate, etofenprox, imidacloprid, indoxacarb, alpha-cypermethrin, zeta-cypermethrin, fludioxonil, and oxytetracycline in Brussels sprout. The half-lives of the pesticides in Brussels sprout were determined to be 11.3 (acephate), 9.8 (etofenprox), 11.3 (imidacloprid), 15.8 (indoxacarb), 10.6 (alpha-cypermethrin), 13 (zeta-cypermethrin), 9.1 (fludioxonil), and 8.2 (oxytetracycline) days. Pesticide residue is affected by various factors such as vapor pressure, pesticides' physicochemical properties, minor substances, formulation types, environment conditions, crop species, and growth dilution factors. Based on the %ADI values, it can be concluded that the intake of pesticide residues from Brussels sprout does not pose a significant health risk. These findings provide useful information for the establishment of MRLs in the ROK and pesticide control measures for Brussels sprout.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.