Fate of chlorpyrifos, omethoate, cypermethrin, and deltamethrin during wheat milling and Chinese steamed bread processing

Abstract To investigate the fractioning of chlorpyrifos, omethoate, cypermethrin, and deltamethrin during wheat milling and the fate of four pesticides during Chinese steamed bread (CSB) processing, wheat samples, which were sprayed twice with chlorpyrifos, omethoate, cypermethrin, and deltamethrin at three levels of concentrations during the grain‐filling stage, were milled, and wheat flour was processed to CSB. The residues of four pesticides in the milling products, kneaded dough, fermented dough, and CSB were determined with GC‐MS/MS. The concentrations of chlorpyrifos, omethoate, cypermethrin, and deltamethrin in bran were 1.46–1.57, 1.85–2.13, 1.27–1.86, and 1.63–2.33 times higher than those in wheat, respectively, while the residues of the four pesticides in shorts decreased approximately 27.97% to 57.02% for chlorpyrifos, 6.22% to 44.77% for cypermethrin, and 13.13% to 61.15% for deltamethrin compared with the residues in wheat (p < .05); however, omethoate levels approximately doubled in the ten‐fold treatment group in shorts compared with those in wheat (p < .05). The residues of the four pesticides in flour were significantly lower than those in wheat, ranging from 38.68% to 98.04%. Chlorpyrifos and omethoate levels showed a slight decrease during the kneading and fermentation process, and further decreases of 2.46%–29.51% for chlorpyrifos and 14.22%–71.11% for omethoate were found in CSB; however, most of the groups of cypermethrin and deltamethrin showed various degrees of increases in kneaded and fermented dough and steamed bread compared with flour. The mechanism of this increase is unknown and needs further research.

pollution not only to the crop products but also to the ecological environment, such as soil, air, and water, thus damaging the ecological balance. These pollutants may enter and accumulate in the human body through the food chain, thus endangering human health.
To ensure the safe use of pesticides, relative regulations have been established throughout the world. The maximum residue limits (MRLs) of wheat have been established by many countries and organizations, such as the European Union (EU), Codex Alimentarius Commission (CAC), and USA. In EU, Australia, USA, Japan, and CAC, the MRLs of chlorpyrifos in wheat are 0.05, 0.1, 0.5, 0.5, and 0.5 mg/kg, respectively; the MRLs of cypermethrin in wheat are 2, 0.2, 0.2, 0.2, and 0.2 mg/kg, respectively; and the MRLs of deltamethrin in wheat are 2, 2, 1, 2, and 2 mg/kg, respectively. The MRLs of omethoate in wheat are 0.01, 0.05, and 0.1 mg/kg in the European Union (EU), Australia, and Japan, respectively (Kim, Huang, Zhu, & Patricia, 2009;Liu et al., 2010Liu et al., , 2018, while omethoate is regarded as a pesticide that cannot be detected in wheat in the United States (USA) and at the Joint FAO/ WHO Meeting on Pesticide Residues (JMPR) based on the Codex Alimentarius Commission (CAC). In China, the MRLs for chlorpyrifos, omethoate, cypermethrin, and deltamethrin as pesticides in wheat are 0.5, 0.02, 0.2, and 0.5 mg/kg, respectively (2016).
Although MRLs are reliable means for enforcing the acceptable use of pesticides, they do not permit the human health risk assessment from residue intake unless accurate knowledge about the fate of residues in wheat processing and the distribution of residues in the final wheat products is available (Fleurat-Lessard, Chaurand, Marchegay, & Abecassis, 2007). Moreover, there are currently no MRL standards for pesticide residues in postharvest wheat samples including wheat milling samples and final wheat products. It is, therefore, of great research significance to understand the transfer mechanisms of pesticide residues during wheat milling, as well as further processing of wheat products.
Chinese steamed bread (CSB), a traditional staple food, has been consumed for nearly 2,000 years in China, representing more than 40% of Chinese wheat consumption (He, Liu, Javier Peña, & Rajaram, 2003). Generally, the manufacturing process of CSB begins with the mixing of wheat flour, water, and yeast, followed by fermentation (38°C) and steaming (100°C) (Liu et al., 2018). CSB is regarded as a healthy food due to the absence of toxic Maillard reaction products, such as acrylamide and furan, as well as the lower oil and sodium contents. Furthermore, the lower steaming temperature (100°C) during CSB processing may better maintain various endogenous and exogenous nutrients compared with baked bread (Zhu, 2014).
Little research has been reported on the fate of pesticide residues during the CSB-making process. The objective of this study was to investigate the dissipation regularity of chlorpyrifos, omethoate, cypermethrin, and deltamethrin during wheat milling and the CSB-making process, which will contribute to the establishment of MRLs in wheat products and the risk assessment of wheat-based food consumers to residues.

| Chemicals and reagents
Pesticide standard solutions of chlorpyrifos, omethoate, deltamethrin, and cypermethrin (are all 1,000 mg/L) were obtained from
Correspondingly, matrix-matched standard solutions of chlorpyrifos, omethoate, cypermethrin, and deltamethrin (0.001, 0.02, 0.05, 0.1, 0.2, 0.5, and 1 mg/L) were prepared by diluting the working matrix standard solutions. These solutions were stored in the darkness at 4°C, and the working standard solutions were not observed to degrade over 3 months.

| Wheat sample preparation
A wheat field at Shunyi Farm, located in the northeast of Beijing, China (E116°33', N40°13'), was divided into control plot, which was sprayed with water, and treatment plots, which were sprayed with 4 kinds of pesticides at 3 concentrations. Plot #1 was used as the control. Plots #2-#5 were sprayed with commercial pesticides chlorpyrifos 45% EC, omethoate 40% EC, deltamethrin 25 g/L EC, and cypermethrin 45% EC at the recommended dosage (450 ml/hectare for chlorpyrifos, 400 ml/hectare for omethoate, 675 ml/hectare for cypermethrin, and 600 ml/hectare for deltamethrin). Plots #6-#9 were sprayed with the 4 pesticides at twofold the recommended dosage and plots #10-#13 at ten-fold the recommended dosage. Each plot was sprayed twice at 14 and 7 d before harvest. Plots were randomly arranged, and each treatment was replicated 3 times. Wheat samples were harvested, placed in polyethylene bags and stored in a −40°C freezer.

| Milling process
All wheat samples were wetted to 16.5% moisture content and left overnight before milling. Milling was carried out by a Chopin CD1 Laboratory Mill (Chopin, France) with two breaks and one stripping system. Three fractions, including bran, shorts, and flour, as well as wheat grain, were obtained and submitted for analyses of pesticide residues.

| Chinese streamed bread (CSB) processing
Chinese streamed bread was prepared according to the Chinese Standard (procedure 10139-93, Appendix A, 1993). The basic formula for making CSB consisted of 100 g of milled flour, 48 ml of distilled warm water (38°C), and 1 g of active dry yeast. The ingredients were mixed evenly by hand for 3 min, and then, dough was fermented in a fermentation cabinet (38°C, 85% RH) for 1 hr. After that, it was kneaded and shaped manually for 5 min into a round dough piece with a smooth surface. After resting at room temperature for 15 min, the dough was put in a steamer when the water was boiling and was steamed (100°C) for 20 min, followed by cooling at room temperature for 40-60 min. Three replicate samples at every stage (kneading, fermenting, and steaming) were taken. All representative samples were stored at −20°C until analysis.

| Sample extraction and cleaning up
The extraction and cleaning up procedure was carried out following the QuEChERS method. Five grams of homogenized sample were weighted in a 50-mL polypropylene centrifuge tube and then extracted with 20 ml of acetonitrile (50:50, v/v) for 30 min using an automatic shaker. Afterward, 4 g of magnesium sulfate (MgSO 4 ), 1 g of sodium chloride (NaCl), 1 g of sodium citrate dihydrate, and 0.5 g of sodium hydrogen citrate sesquihydrate were added and shaken vigorously for 2 min, and the sample was centrifuged for 5 min at 6,037 g. Then, a clean-up, dispersive, solid-phase extraction (d-SPE) was conducted by adding 5 ml of the supernatant phase to a 15-ml centrifuge tube that contained 900 mg of MgSO 4 , 150 of mg PSA, and 150 of mg C 18 . The sample was immediately vortexed for 1 min and centrifuged for 5 min at 6,037 g, and then, 2 ml of the supernatant-cleaned extract was evaporated to dryness in a nitrogen evaporator with a water bath at 60°C. The dry residue was then dissolved in 1 ml of acetone, followed by filtering through a 0.22-μm nylon syringe filter (Jinteng). After that, it was ready for analysis.

| Pesticide determination by GC-MS/MS
Analyses of the pesticides were performed by a Bruker 450 GC system, equipped with an autosampler (Bruker CP 8400), that was coupled to a triple quadrupole mass spectrometer (Bruker 300 MS), operating in the electron impact ionization (EI) mode. The instrument conditions are as follows: the column was initially maintained at 60°C, and then, the temperature was increased to 250°C at a rate of 20°C/min and finally increased to 300°C at a rate of 5°C/min and maintained for 4.5 min. The total run time was 24 min. Argon collision gas flow was maintained at 1.8 m Torr pressure. Helium (purity > 99.999%) was employed as a carrier gas at 1.2 ml/min, and injections were carried out in split/splitless mode at 250°C using 1-μL injection volumes. Triplicates of each concentration were analyzed. The limits of detection (LODs) and limits of quantitation (LOQs) for the four pesticides were assessed at a signal-to-noise (S/N) ratio of 3 and 10, respectively.

| Statistical analysis
Statistical analysis was performed using the PSAW Statistic 19.0 (SPSS) statistical software package. All data were subjected to a one-way analysis of variance (one-way ANOVA). Homogeneity of variance was confirmed before ANOVA, and differences among the means were analyzed by Duncan's multiple range test. Data were shown and analyzed as micrograms per kilogram of matrix (µg/kg) on a dry matter basis. The differences were regarded as significant when p < .05, and data were reported as the mean value ± the standard deviation (SD) of five replicates.
The processing factor (PF) during different wheat milling fractions and CSB processing is calculated as follows: PF = C 1 /C 2 , where C 1 and C 2 are postprocessing and preprocessing pesticide residue levels (mg/kg), respectively.

| Method validation
The methods for the determination of chlorpyrifos, omethoate, cypermethrin, and deltamethrin residues in wheat grain, flour, bran, shorts, dough, and steamed bread using GC-MS/MS were validated by spiking a series of the pesticide standard solutions to the wheat products. Table 1 summarizes the retention time, formula, qualitative ion, quantitative ion, and collision energy of omethoate, chlorpyrifos, cypermethrin, and deltamethrin obtained using GC-MS/MS. The correlation coefficients (R 2 ), which show the correlation between the concentrations of pesticide residues and the detective areas in different wheat matrices, were higher than 99.10%, demonstrating that the methods were sensitive and selective (Table 2). Mean recoveries obtained ranged from 70.33% to 118.33% with a relative standard deviation (RSD) lower than 8.78%. The LOD for the four pesticides in different matrices ranged from 0.25 to 1.60 µg/kg, and the LOQ ranged from 0.80 to 5.00 µg/kg, which were below the maximum residue limits (MRLs) established by the EU, USA, and China.

| Fate of chlorpyrifos, omethoate, cypermethrin, and deltamethrin during wheat milling
Accurate knowledge about the fractioning of pesticide residues during the wheat milling process is important for the establishment of MRLs in grains and wheat-based food risk assessment. Many previous studies have indicated that most pesticide residues remained on the surface of grain and only a small part penetrated to the internal portions of wheat, and consequently residue levels in brain were consistently higher than that in wheat (Holland, Hamilton, Ohlin, & Skidmore, 1994;Kaushik, Satya, & Naik, 2009;Uygun, Koksel, & Atli, 2005;Uygun, Senoz, & Hamit, 2008). Malathion and fenitrothion were applied to wheat at toxicant concentrations of 8.89 and 24.50 mg/kg before processing into flour. During flour-making, only trace amounts (7.2% and 9.6% residues) were found in the flour, and levels of malathion and fenitrothion in bran can be 1.1 and 1.4 times greater than in wheat, respectively (Uygun et al., 2005).
Sgarbiero, Baptista, and Trevizan (Sgarbiero, Baptista, & Trevizan, 2002) demonstrated that 12 mg/kg pirimiphos-methyl was added into wheat grain and stored for 240 days. After the milling process, the pirimiphos-methyl level in bran was approximately 2.5 times that in wheat, but 60% of the residues were still found in white flour. Joia, Webster, and Loschiavo (Joia, Webster, & Loschiavo, 1985) reported that the highest concentrations of cypermethrin and fenvalerate were accumulated in bran, and the least, in endosperm. Reductions of 79%-84% for cypermethrin and 87%-88% for fenvalerate were present in flour.
As expected, in our study, chlorpyrifos, omethoate, cypermethrin, and deltamethrin levels varied significantly among the milling products (p < .05), in which the concentrations of the four pesticides in bran were significantly enhanced (p < .05), and in flour, they were significantly lower than in wheat (p < .05) ( Table 3). The concentrations of chlorpyrifos, omethoate, cypermethrin, and deltamethrin in bran were 1.46-1.57, 1.85-2.13, 1.27-1.86, and 1.63-2.33 times higher than that in wheat, respectively. Obviously, the PF values of four pesticides were all >1 during the malting process (Table 4).
Meanwhile, the residues of the pesticides in shorts decreased approximately 27.97% to 57.02% for chlorpyrifos, 6.22% to 44.77% for cypermethrin, and 13.13% to 61.15% for deltamethrin compared with residues in wheat (p < .05); however, the omethoate showed a different trend with a marked increase in shorts compared with in wheat (p < .05) in the ten-fold treatment group due to its strong systemic conductibility. Further degradation of four pesticide lev-  through amplified friction, crushing, rolling, or sieving steps during the milling process (Fleurat-Lessard et al., 2007). Furthermore, milling temperature and time also have an impact on the degradation of pesticides. They can be in interaction with the mechanical power during milling operations, which then leads to the structural changes of pesticides, thereby resulting in the degradation of pesticides in the final product.

| Fate of chlorpyrifos, omethoate, cypermethrin, and deltamethrin during CSB processing
In general, it is accepted that most processing techniques, such as cleaning, peeling, milling, extruding, cooking, baking, canning, and juicing, might reduce the pesticide residues in food (Abou-Arab, 1999;Sharma, Satya, Kumar, & Tewary, 2005;Soliman, 2001). In some special cases, however, some toxic metabolites and by-products can be formed, and the pesticide residues could increase during food processing (Bajwa & Sandhu, 2014). In the present study, the flours mixed by the break and reduction flours after milling were prepared to make CSB. The normal CSB-making process involves three major steps: kneading, fermentation, and steaming. As shown in Figure 1, the concentrations of chlorpyrifos, omethoate, cypermethrin, and deltamethrin showed significant changes compared with flour during the CSB-making process. Omethoate, cypermethrin, and deltamethrin concentrations in the kneaded dough of most treatment groups increased significantly (p < .05) compared with flour, while chlorpyrifos concentration showed a slight decrease during the kneading process. This could be contributed to the fact that the insufficient mixing of the flour caused inconsistent concentrations among the samples. Samples from the recommended dosage of omethoate and deltamethrin were too little to quantify because of their lower initial levels in flour. Additionally, in this study, the increased pesticide concentrations after kneading were mostly found in the recommended dosage and twofold of the recommend dosage groups. Hence, it is suggested that the enhanced matrix interference resulted in increased recovery of pesticides in the low level, and this was in accordance with the results that are shown in Table 2, in which the lower spiking levels (20 µg/kg) displayed a higher average recovery in different matrices.
Yeast-mediated fermentation is an essential process of wheat dough preparation in the traditional CSB-making process (Kim et al., 2009). It has been reported that many factors could affect the fate of pesticides during food fermentation, such as chemical structure, volatility, and adsorption ability to matrices of pesticides, biodegradation of microorganisms, and cleavage of extracellular enzymes (Aislabie & Lloyd-Jones, 1995;Azizi & A. Homayouni, 2009;Bayarri, Conchello, Ariño, Lázaro, & Herrera, 2015). These factors, themselves, are dependent on many environmental parameters, including processing temperature, moisture content, pH, and light (Aislabie & Lloyd-Jones, 1995;Azizi & A. Homayouni, 2009).
In the present study, we observed that the four pesticides showed different sensitivity to the applied dough fermentation. After fermentation (38°C, 1 hr), most groups of chlorpyrifos and omethoate showed lower pesticide concentrations compared with the flour (p < .05) (Figure 1). Similar results were reported by Sharma, Satya, Kumar, & Tewary (Sharma et al., 2005), in which yeast could degrade organophosphorus pesticides during the bread making progress, and yeast fermentation and baking removed 50.66%-88.35% of chlorpyrifos and 59.85%-79.04% of malathion compared with the wheat flour. Additionally, Zhou, Liu, and Zhao (2015) reported that organophosphorus pesticide concentrations decreased in ranges of 16.6%-26.6% to 23.4%-31.8% when added to 1.5% and 3.0% yeast F I G U R E 1 Pesticide residue levels during Chinese steamed bread (CSB) making process (µg/kg) after a fermentation time of 5 hr, depending on the fermentation time and yeast addition levels. These results proved that yeast had a clear ability to degrade some pesticides belonging to organophosphorus groups, and fermentation time played an important role in pesticide degradation. In our study, however, higher concentrations of cypermethrin and deltamethrin residues in fermented dough were observed than that in flour (p < .05). Obviously, pyrethroid groups showed a completely opposite trend compared with organophosphorus groups during fermentation, which might be partly governed by their chemical structure. Mao et al. (Mao et al., 2008) demonstrated that Candida tropicalis markedly affected the fate of organophosphorus pesticides, such as chlorpyrifos, dimethoate, isocarbophos, and triazophos (27.95%-62.28% reduction rates), during the fermentation of sweet orange peel, whereas no significant changes were observed in pyrethroid groups such as cypermethrin, deltamethrin, and bifenthrin during the fermentation process, indicating that the stability of pesticides might significantly affect the degradation rate of pesticides during fermentation. Furthermore, the observed increases in cypermethrin and deltamethrin levels in fermented dough are also likely due to a concentration effect or affinity for the lipid moiety of pesticides (Bajwa & Sandhu, 2014;Regueiro, López-Fernández, Rial-Otero, Cancho-Grande, & Simal-Gándara, 2015). Cypermethrin and deltamethrin are more strongly lipophilic pesticides than chlorpyrifos and omethoate, and they have strong affinities for the lipid moiety; thus, they are more easily combined with the lipid in flour during the CSB-making process, resulting in increased pesticide concentrations in dough and the final product.
In our study, reductions of 2.46%-29.51% for chlorpyrifos and 14.22%-71.11% for omethoate were observed during the steaming process (100°C, 20 min) compared with the initial levels in flour (Figure 1), and higher reductions were found during steaming with higher initial pesticide levels in flour. The results agreed with Sharma, Satya, Kumar, & Tewary (Sharma et al., 2005), who spiked four different concentrations (1, 2, 3, and 4 mg/kg) in wheat flours with six pesticides to make bread and found that the range of pesticide degradation was 47%-89%. Pal and Shah (Pal & Shah, 2008) found 32.5%-72.4% chlorpyrifos reduction during the bread making process. When flour was converted into white bread, degradation of the malathion and fenitrothion residues was approximately 81%-85% and 61%-88%, respectively (Uygun et al., 2005). Further, other food processing, such as cookie and pasta processing, can also lead to a significant reduction in organophosphorus pesticides in the final product (Uygun et al., 2008;Uygun, Senoz, Öztürk, & Koksel, 2009). These findings give strong support to the present results, which suggest that the heating process can lead to a large reduction in pesticides, which might be influenced by evaporation, codistillation, or thermal degradation, as well as the physicochemical properties of the pesticides, including the volatility, solubility, and hydrolysis rate constant (Holland et al., 1994;Kaushik et al., 2009;Sharma et al., 2005). Moreover, omethoate residue levels in CSB of the twofold and ten-fold treatment groups exceeded the MRLs (0.02 mg/kg), implying that the remaining omethoate in flour and CSB might endanger consumer health; thus, the use of omethoate in wheat should be strictly controlled. The cypermethrin and deltamethrin levels in CSB, however, were significantly higher than in flour (p < .05), increasing 1.16-fold to 1.43-fold compared to the flour, except for a decrease presented in the ten-fold treatment group (loss of 16.41% for cypermethrin and 21.69% for deltamethrin) (Figure 1).

| CON CLUS ION
This study investigated the fractioning and fate of chlorpyrifos, omethoate, cypermethrin, and deltamethrin during wheat milling and Chinese steamed bread processing. The levels of chlorpyrifos, omethoate, cypermethrin, and deltamethrin in bran were 1.46-1.57, 1.85-2.13, 1.27-1.86, and 1.63-2.33 times higher than those in wheat, respectively, while reductions of 27.97%-57.02% for chlorpyrifos, 6.22%-44.77% for cypermethrin, and 13.13%-61.15% for deltamethrin were observed in shorts compared with those in wheat (p < .05), with omethoate showing a different trend with a marked increase in the ten-fold treatment group in shorts compared with that in wheat (p < .05). Decreases in the four pesticides ranging from 38.68% to 98.04% were observed in flour.
To the best of our knowledge, this is the first detailed report focusing on the fate of chlorpyrifos, omethoate, cypermethrin, and deltamethrin in wheat milling regarding Chinese steamed bread processing. The results showed that chlorpyrifos and omethoate levels slightly decreased during the kneading and fermentation processing, and further decreases of 2.46%-29.51% for chlorpyrifos and 14.22%-71.11% for omethoate were observed in CSB compared with flour. The omethoate residue levels in CSB of the twofold and ten-fold treatment groups exceeded the MRLs

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.