Identification and dissipation of omethoate and its main metabolite

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Food Safety and Toxicology

Identification and dissipation of omethoate and its main metabolite DMP in wheat determined by UPLC-QTOF/MS Lili Yu, Lina Wang, Yang Zhao, and Bujun Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06799 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Journal of Agricultural and Food Chemistry

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Identification and dissipation of omethoate and its main metabolite DMP in

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wheat determined by UPLC-QTOF/MS

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Running title: Omethoate and its metabolites identification using UPLC-QTOF/MS

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Lili Yu, Lina Wang, Yang Zhao, Bujun Wang *

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Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / Laboratory of

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Quality and Safety Risk Assessment for Cereal Products (Beijing), Ministry of

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Agriculture and Rural Affairs of the People’s Republic, Beijing, 100081, China

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* Corresponding author:

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Institute of Crop Sciences, Chinese Academy of Agricultural Sciences

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No.12 Zhongguancun South St., Haidian District, Beijing, China, 100081

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E-mail: [email protected]

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Telephone: +86-10-82-10-5798;

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Fax: +86-10-82-10-8742;

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ACS Paragon Plus Environment


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Abstract

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A study was carried out to evaluate the dissipation kinetics of field-applied

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omethoate during wheat storage. Both the identification and metabolic dynamics of

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omethoate metabolites were analyzed using UPLC-QTOF/MS. The presence of the

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metabolite dimethyl phosphate (DMP) was confirmed in wheat samples with applied

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omethoate. This might be because the group attached to the P atom of omethoate is

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replaced by a hydroxyl group through hydrolysis, thus leading to the formation of the

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specific metabolite DMP during wheat storage. Although the initial concentration of

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DMP in different doses were considerably lower than those of omethoate, the half-life

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values of DMP were 11.87-31.50 days, which were close to the half-life of the parent

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omethoate (11.85-30.94 days). This indicates that the potential health risks might be

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caused by dietary exposure to DMP and omethoate. Therefore, more importance

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should be given to the risk assessment for omethoate and its metabolite DMP in

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wheat.

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Keywords: omethoate; DMP; dissipation; metabolite; wheat; UPLC-QTOF/MS

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Introduction

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Organophosphorus (OP) pesticides are traditional pesticides that are commonly

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used during cultivation to protect wheat from pest and disease infestations.1 At

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present, OP pesticides have been used for more than 70 years, and there are more than

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150 kinds of OP pesticides that are commercially available worldwide. More than 30

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kinds of OP pesticides are commonly used in China, most of which are insecticides,

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and a few of them are fungicides, herbicides, etc.2 However, the phosphate portion of

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OP pesticides is readily substituted by dialkyl (dimethyl or diethyl) to form toxic

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metabolites. OP pesticides can be hydrolyzed or spontaneously hydrolyzed to produce

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dialkylphosphates (DAP) metabolites in organisms.3 It is reported that more than 75%

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of OP pesticides can yield one or more DAPs of the following ： diethyl

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dithiophosphate (DEP), diethyl thiophosphate (DETP), diethyl dithiophosphate

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(DEDTP), dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP) and dimethyl

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dithiophosphate (DMDTP).4 These DAPs were widely used as urinary biomarkers to

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measure occupational exposure to OP pesticides that may pose health risks to animals

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and humans. Therefore, due to the potential adverse effects of OP pesticides and their

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metabolites on the ecological environment and human health, the presence of harmful

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pesticides and their metabolites in wheat has caused a great concern among producers

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and consumers.5

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Omethoate is an effective insecticide that is widely used for pest control with

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vegetables, fruits and crops.6 In China, omethoate is one of the main pesticides used

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to control wheat aphids through contact and ingestion. However, the half-life of 3



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omethoate in an acidic environment exceeds 90 days. In addition, omethoate could

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inhibit the activity of acetylcholinesterase and cause the significant accumulation of

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acetylcholine that is released by cholinergic nerve endings. Therefore, the excessive

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use of omethoate presents high environmental pollution potential and human health

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risks.7,8 As a typical organophosphorus pesticide, the phosphate portion of omethoate

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is easily replaced by dimethyl groups to form DMP or DMTP. In addition, omethoate

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could produce the toxic intermediate O, O, S - trimethyl phosphorothioate (TMP)

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through in vitro hydrolysis, under the action of Aspergillus or through catalytic

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ozonation with Fe(III)-loaded activated carbon.9-12 Zhao et al. (2014)13 demonstrated

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that after degradation by surface discharge plasma, dimethoate can be oxidized to

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omethoate and further oxidized to monomethyl phosphate (MMP), dimethyl

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phosphonate (DPN), DMP, etc. These findings give strong support to the fact that

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omethoate could be degraded into phosphorus-containing, small molecular weight

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intermediates under complex environmental conditions, which are still biologically

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toxic.

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The maximum residue limits (MRLs) of omethoate in wheat are 0.01, 0.05 and

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0.1 mg/kg in the European Union (EU), Australia and Japan, respectively,14-16 while

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omethoate is regarded as a pesticide that cannot be detected in wheat in the United

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States (USA) and at the Joint FAO/WHO Meeting on Pesticide Residues (JMPR)

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based on the Codex Alimentarius Commission (CAC).17-19 In China, the MRLs for

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omethoate as a pesticide in wheat are 0.02 mg/kg.20 Although MRLs are reliable

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means for enforcing the acceptable use of pesticides, some of the metabolites might 4


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be more toxic than the parent compounds. However, there are few studies that are

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focused on the establishment of MRLs for pesticide metabolites in foods. It is,

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therefore, of great research significance to investigate the mechanisms of omethoate

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metabolites in wheat.

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A quadrupole time-of-flight mass spectrometer (Q-TOF/MS) coupled to a

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chromatographic system has been proven to be a reliable and novel technique for

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target and nontarget compound identification.21,22 Q-TOF/MS has the advantage of

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providing more exact mass information through its high resolution capabilities and

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high sensitivity in its full scan acquisition mode.23 In addition to determining the

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chromatographic retention time and accurate mass of the analytes, Q-TOF/MS can

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also determine the exact molecular weights of the parent and fragment ions by further

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optimizing the collision energy. In addition, Q-TOF/MS can offer structural

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elucidation tools such as diagnostic ions, adduct profiles, isotopic matches, and

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collision cross sections that can be applied for metabolites’ qualitative and

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quantitative analyses.24,25

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Q-TOF/MS has become the most commonly used technique for multiple

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pesticide analysis and the metabolite screening of fruits, vegetables and crops

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currently. Sánchez-Hernández et al. (2016)26 found the contents of thiamethoxam,

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clothianidin and imidacloprid and the different metabolic products of these

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neonicotinoid insecticides in honey and pollen from sunflower and maize crops using

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UPLC-QTOF/MS. Bauer et al. (2018)24 investigated the degradation pathways and

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distribution profiles of thiacloprid, azoxystrobin and difenoconazole and their main 5



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metabolites in Brassica species pak choi and broccoli using ultrahigh-performance

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liquid chromatography travelling wave ion mobility quadrupole time-of-flight mass

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spectrometry (UPLC-TWIMS-QTOF/MS). In another study, Yang et al. (2018)5

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developed a multiresidue method for the identification and quantification of 50

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pesticides in minor fruits using UPLC-QTOF/MS. However, few studies have focused

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on the dissipation behavior of omethoate and metabolite screening during wheat

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storage using Q-TOF/MS. Hence, elucidating the dissipation of omethoate and its

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metabolites during wheat storage is essential to the risk assessment of wheat-based

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food residues to consumers.

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The objective of this study was to investigate the dissipation regularity of

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omethoate and to identify its possible metabolites during wheat storage using the

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UPLC-QTOF/MS technique. Furthermore, in the present study, the development of a

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powerful method for omethoate quantification and its metabolite identification is

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described.

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Materials and methods

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Chemicals and reagents. The standard pesticide solution of omethoate (1,000 mg/L)

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was obtained from the Agro-Environment Protection Institute at the Ministry of

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Agriculture and Rural Affairs of China (Beijing, China). The analytical standard for

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the omethoate metabolite dimethyl phosphate (purity 97%) was purchased from

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Toronto Research Chemicals (Brisbane Road, Toronto, Canada). The commercial

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pesticide omethoate (40% emulsifiable concentrate (EC)) was purchased from

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Shandong Dongtai Agrochemicals Co., Ltd. (Shandong, China). The organic solvents, 6


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including acetonitrile, methanol, ethyl acetate, ammonium acetate, and formic acid,

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that were used for both sample extraction and analysis were of HPLC/MS grade and

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purchased from Thermo Fisher Scientific Corporation (Shanghai, China). Ultra-pure

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water was obtained from a Milli-Q system (Millipore, Billerica, MA, USA).

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Preparation of standard solutions. The standard stock solutions of omethoate and

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DMP (100 mg/L) were diluted with methanol and stored at -20°C. The working

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standard solutions of omethoate and DMP (0.001, 0.02, 0.05, 0.1, 0.2, 0.5 and 1

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mg/L) were prepared by diluting the stock solution. Correspondingly, the

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matrix-matched standard solutions of omethoate and DMP (0.001, 0.02, 0.05, 0.1, 0.2,

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0.5 and 1 mg/L) were prepared by diluting the working matrix standard solutions.

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These solutions were stored in the dark at 4°C.

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Field trials and storage conditions. A wheat field at Shunyi Farm that was located in

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the northeast of Beijing, China (E116°33’, N40°13’) was divided into a control plot,

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which was sprayed with water, and treatment plots that were sprayed with 3 different

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concentrations of omethoate. Plot #1 was used as the control. Plots #2-#4 were

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sprayed with the commercial pesticide omethoate 40% EC at the recommended

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dosage (400 mL/hectare), two-fold the recommended dosage (800 mL/hectare) and

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ten-fold the recommended dosage (4000 mL/hectare), respectively. Considering that

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if the initial concentration of the parent pesticide is too low, most of the omethoate

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will be degraded under the natural conditions in the field, and the metabolites may not

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be detected when transported to the laboratory for storage testing. Therefore, in the

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absence of phytotoxicity, we designed a high concentration dose group (ten-fold the 7



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recommended dosage). Each plot was sprayed twice at 14 and 7 days before harvest.

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The plots were randomly arranged, and each treatment was replicated 3 times. The

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wheat samples were harvested, placed in polyethylene bags and transported to the

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laboratory for the next stage of the study.

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The wheat samples harvested from plots #1-#4 were threshed, stored in 4

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separate individual polyethylene bags and transported to the laboratory for the next

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wheat storage experiments at ambient temperatures (18-26°C). The wheat samples

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were collected from each plot on days 0, 1, 3, 5, 7, 10, 14, 21, 30, 60, 90, 120 and 180

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of storage to conduct the dissipation behavior and metabolite screening studies. The

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samples were stored at -40°C until analysis.

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Sample extraction and cleaning-up of omethoate and DMP. The extraction and

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clean-up procedure of omethoate were carried out following the QuEChERS method.

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Five grams of homogenized sample were inserted into a 50-mL polypropylene

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centrifuge tube and then extracted with 20 mL of acetonitrile (50:50, v/v) for 30 min

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using an automatic shaker. Afterwards, 4 g of magnesium sulfate (MgSO4), 1 g of

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sodium chloride (NaCl), 1 g of sodium citrate dihydrate and 0.5 g of sodium hydrogen

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citrate sesquihydrate were added and shaken vigorously for 2 min, and the sample was

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centrifuged for 5 min at 6000 rpm. Then, for the clean-up, dispersive solid-phase

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extraction (d-SPE) was conducted by adding 5 mL of the supernatant phase to a

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15-mL centrifuge tube that contained 900 mg of MgSO4, 150 of mg PSA and 150 of

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mg C18. The sample was immediately vortexed for 1 min and centrifuged for 5 min at

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6000 rpm. Then, 2 mL of the supernatant-cleaned extract was evaporated to dryness 8


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in a nitrogen evaporator with a water bath at 60°C. The dry residue was then

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dissolved in 1 mL of methanol, which was followed by filtering through a 0.22-μm

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nylon syringe filter (Jinteng, Tianjin, China). After that, it was ready for analysis.

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The extraction and clean-up procedure of DMP occurred as follows. 5 g of

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homogenized samples were inserted into a 50 mL polypropylene centrifuge tube.

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Then, 20 mL mixture of acetonitrile and ethyl acetate (50:50, v/v), 10 mL of saturated

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sodium chloride solution and 0.5 mL of 12 mol·L-1 hydrochloric acid were added to

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the tube. The tube was shaken for 30 min using an automatic shaker. After being

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centrifuged at 10000 rpm for 5 min, 4 mL of the supernatant-cleaned extract was

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transferred to the Oasis Prime HLB (Waters Corp, Milford, MA, USA) for

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purification. It was then evaporated to dryness in a nitrogen evaporator with a water

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bath at 45°C. Then, 1 mL of the dry residue (methanol) was filtered with a 0.22-μm

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nylon syringe filter (Jinteng, Tianjin, China) for HPLC-QTOF/MS analysis.

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Instrumentation and UPLC-QTOF/MS analytical conditions. The sample analysis

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was performed using an ultrahigh-performance liquid chromatography system

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(ACQUITY UPLC I-Class, Waters Corp, Milford, MA, USA) coupled with hybrid

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quadrupole time-of-flight mass spectrometry (VION IMS QTOF, Waters Corp,

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Milford, MA, USA). The sample separation was performed using a Waters

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ACQUITY UPLC HSS T3 (1.8 µm, 2.1 mm * 100 mm) column. The instrument

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conditions are as follows. Gradients of solvent A (methanol) and solvent B (10 mmol

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ammonium acetate in water) were prepared as follows: (i) 0.00 min (A:B, 2:98, v/v),

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(ii) 0.25 min (A:B, 2:98, v/v), (iii) 12.25 min (A:B, 99:1, v/v), (iv) 13.00 min (A:B, 9



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99:1, v/v), (v) 13.01 min (A:B, 2:98, v/v), and (vi) 17.00 min (A:B, 2:98, v/v). The

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flow rate was 0.45 mL/min. The injection volume was 5 µL and the column

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temperature was kept at 45 °C.

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The mass spectrometer was operated in the positive electrospray ionization mode

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(ESI＋) using the following parameters: a capillary voltage of 1.0 kV, a sampling cone

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of 40 V, a nitrogen gas-flow of the nebulizer of 50 L/h and for the desolvation gas of

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1000 L/h, a desolvation temperature of 550 °C, and a source temperature of 120 °C.

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The sample analysis was done using the mass spectrometer elevated (MSE)

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experiment mode in a full scan m/z of 50–1000 with a 0.2 s scan time. In the MSE

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function, the low collision energy spectrum was recorded at 6.0 eV. Then, the

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precursor ions from the low collision energy MS-mode were fragmented using high

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collision energy ramped from 10 to 45 eV. Leucine-enkephalin (m/z 556.2766 in the

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positive mode) was used as a real time reference lock-mass (200 pg/uL

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leucine-enkephalin

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0.05/49.925/49.925/0.1, v/v/v/v).

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Method validation. Recovery experiments were conducted by spiking untreated

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wheat samples at five different levels of 20, 50, 100, 200 and 500 ug/kg with

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omethoate and DMP working solutions in methanol. Triplicates of each concentration

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were analyzed. The limits of detection (LODs) and limits of quantitation (LOQs) for

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the two compounds were assessed at signal-to-noise (S/N) ratios of 3 and 10,

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respectively.

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Statistical analysis. The software UNIFI™ 1.8.1 (Waters Corp., Milford, MA, USA)

in

acetonitrile:water

with

10


0.1%

formic

acid,

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was used for data acquisition and quantitation. The data was processed with a

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scientific library that was created in-house containing a database of suspected

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omethoate metabolites (5 library entities) with information about the exact mass

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analysis of the precursor ions, the molecular structures, the characteristic fragment

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ions, the retention time and the adducts for each entry. The method conditions for the

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pesticide screening to establish the scientific compound library in this study were set

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according to Waters Corp. (Milford, MA, USA).27

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Dissipation studies of omethoate and its metabolite were performed using linear

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regression. Statistical analysis was performed using the PSAW Statistic 19.0 (SPSS,

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Chicago, IL) statistical software package. All data were subjected to a one-way

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analysis of variance (one-way ANOVA). The homogeneity of the variance was

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confirmed before ANOVA and the differences between the means were analyzed

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using Duncan’s multiple-range test. Considering that the moisture content of wheat

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samples in each treatment is different and fluctuates greatly and the measurement of

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moisture-based is incomparable, the data were shown and analyzed as micrograms per

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kilogram of matrix (µg/kg) on a dry matter basis in this study. The data were reported

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as the mean value ± the standard deviation (SD) of the 5 replicates.

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Results and discussion

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Identification and confirmation of omethoate and its metabolites by

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HPLC-QTOF/MS. Table 1 summarizes the retention time, chemical formula,

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accurate mass, fragments and adducts of the omethoate obtained using

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HPLC-QTOF/MS in the full scan MSE mode. The retention time of omethoate is 2.77 11



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min, and the characteristic fragment ions of the parent compound at the identical

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retention time obtained by MS/MS analysis are m/z 109.0049, m/z 94.9892 and m/z

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78.9957, respectively. Figure 1 shows the total ion chromatogram (TIC) of omethoate

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(A 1) and the low collision energy channel data with omethoate adducts ([M + H]+,

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[M + Na]+ and [M + K]+) (B 1), in which the hydrogen adduct might be the main form

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of omethoate in the spectrum.

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We summarized all possible metabolites of omethoate and established a

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screening library in order to detect and identify the omethoate metabolites during

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wheat storage. In Table 2, the formula and exact masses of all possible metabolites of

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omethoate that were reported in above references were presented.4,9-13 The

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identification or diagnostic proposal of omethoate metabolites was performed

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according to the following criteria: (i) the exact mass analysis of precursor ions is < 2

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ppm mass error, (ii) the unique peaks in the treated sample are compared to the blank

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samples, (iii) there must be at least ≥1 characteristic fragments ions, and (iv) the

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retention time error of all samples should be < 0.1 min.24,26 This method for searching

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for small molecule metabolites and determining the structures of pesticides has been

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published in previous reports, and it can be quite helpful for unknown and nontarget

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analyses of pesticide metabolites using accurate mass data.24,26, 28-30

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Based on this efficient and accurate screening approach, we found the metabolite

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DMP of omethoate during wheat storage. Since a reference substance for DMP was

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commercially available, the further quantification of DMP in wheat samples was

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performed using HPLC-QTOF/MS. Figure 1 shows the TIC of DMP (A 2) and the 12


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low collision energy channel data with the omethoate adducts ([M + H]+ and [M +

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K]+) (B 2), in which the hydrogen adduct might be the main form of DMP in the

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spectrum.

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Method validation for omethoate and DMP in wheat. The methods for the

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determination of omethoate and DMP in wheat samples using UPLC-QTOF/MS were

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validated by applying a series of the omethoate and DMP standard solutions to the

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wheat samples. The correlation coefficients (R2), which show the correlation between

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the concentrations of pesticide residues and the detected areas in the wheat samples,

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were higher than 99.20%, demonstrating that the methods were sensitive and selective

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(Table 3). The mean recovery percentages of omethoate ranged from 85.00 to 92.00%

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with a relative standard deviation (RSD) lower than 6.11%, and the DMP recoveries

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ranged from 73.47 to 91.67%, respectively, with RSDs lower than 7.35%. The LOD

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of omethoate was 0.25 µg/kg and the LOQ was 0.8 µg/kg, which were below the

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maximum residue limits (MRLs) established by the EU, Australia, Japan and China.

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The LOD of DMP was 2.80 µg/kg and the LOQ was 9.50 µg/kg. As seen, the method

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was suitable for the determination of omethoate and DMP residues in wheat samples.

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Degradation kinetics and metabolic mechanism of omethoate in wheat during

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storage. Although omethoate is one of the most widely used organophosphorus

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insecticides for controlling insects during cereal growth, cereals are frequently stored

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for a long time at ambient temperatures before being processed.31 There is little

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literature that reports on the degradation kinetics of omethoate during wheat storage.

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For this reason, the effect of storage on the dissipation behavior of omethoate in 13



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wheat was examined at 13 time points during a half a year of storage in polyethylene

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bags at ambient temperatures (18-26°C). The UPLC-QTOF/MS analyses of the

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treated wheat samples from 0 to 180 days are presented in Figure 2A and Table 4. The

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degradation trends of omethoate in different dose treatments during wheat storage

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were fitted using a first-order kinetic equation: Ct = C0 e-kt (where Ct is the pesticide

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concentration at time t, C0 is the initial pesticide concentration, and k is the velocity

297

constant of the reaction (1/day)).32-34 Here, the fitting degree was high and the

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correlation coefficient ranged from 0.8675-0.9959. The degradation half-life (t1/2)

299

(days) was determined using the equation t1/2 = In (2)/k. As shown in Table 4, the

300

calculated half-lives and the velocity constant of the reaction of omethoate in

301

treatments 1, 2 and 3 were 11.85, 13.78 and 30.94 days and 0.0585, 0.0503, and

302

0.0224, respectively. These results indicated that a higher application concentration of

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omethoate could lead to a longer degradation half-life and a slower degradation rate

304

during wheat storage. As seen from the degradation curves of omethoate (Figure 2A),

305

the degradation rate of omethoate is fast during the early stages of wheat storage since

306

more than 40-50% of the initial deposits of omethoate dissipated within 14 days. With

307

the prolongation of the storage time, the degradation rate of omethoate gradually

308

decreased. This phenomenon might be attributed to the characteristics of omethoate,

309

such as the chemical structure, volatility and adsorption ability to matrices. During the

310

early stage of wheat storage, most of the omethoate residues accumulated on the

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surface of the bran-coat, which led to the rapid degradation of omethoate; however,

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omethoate residues gradually penetrated into the wheat germ layers with time, which 14


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then were less likely to degrade. Furthermore, many environmental parameters,

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including the temperature, moisture content, light, and pH also impact the degradation

315

of omethoate.31,32,35

316

The metabolic behavior of omethoate in the natural environment is complex, the

317

metabolic intermediates are diverse, and some highly toxic metabolites and

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by-products can be formed in different organisms; however, the studies that have

319

focused on the degradation kinetics and metabolic mechanism of omethoate in plants

320

are scare. The present study investigated the degradation kinetics of the metabolite

321

DMP of omethoate during wheat storage at ambient temperatures (18-26 ℃ ). The

322

results were summarized and documented in Figure 2B and Table 4. Omethoate was

323

degraded to DMP during wheat storage, thus resulting in a considerable amount of

324

DMP residues remaining in wheat. As shown in Figure 2B, the behavior of DMP fit

325

the first order kinetics pattern with the calculated initial concentration of DMP in

326

treatments 1, 2 and 3 were 131.974, 193.983 and 416.227 ug/kg, respectively, which

327

were considerably lower than those of their parent omethoate residues (180.077,

328

333.845 and 1208.715 ug/kg, respectively). Surprisingly, the higher initial

329

concentration of omethoate could lead to less efficient degradation of DMP (73.29,

330

58.11 and 34.44%, respectively). This might be attributed to the fact that the

331

omethoate was applied under field conditions, which could be efficiently degraded to

332

DMP under natural conditions. However, the degradation rate of DMP in the

333

high-dose treatment is faster than that in the low-dose treatment at the initial

334

application stage, which led to the lower initial concentration of DMP after harvest 15



335

but before wheat storage. The half-life values of DMP for different doses were 11.87,

336

20.63 and 31.50 days, respectively, which were close to the half-life of the parent

337

omethoate, except for treatment 2 (Table 4). Similarly, the degradation rate of DMP

338

during the early stage of wheat storage is fast and gradually decelerates in the later

339

stages. At day 30, approximately 80% of DMP was degraded in treatments 1 and 2,

340

and a decrease of over 90% from the initial concentration of DMP was observed in

341

treatment 3 at day 120. Fortunately, DMP residues gradually decreased thereafter and

342

could not be detected in wheat at the end of storage.

343

Hydrolysis, photolysis and oxidation processes could result in the formation of

344

DAPs during the degradation of OPs in plants, animals and humans.36,37 Generally,

345

DMP has long been used as urinary biomarkers in animals and humans to assess their

346

occupational exposure to OP pesticides.38-40 There is little published data available on

347

the distribution of DMPs associated with their parent OP pesticides in plants known to

348

contain an OP residue. Zhang et al. (2008)37 demonstrated that the DMP, DMTP and

349

DMDTP metabolite residues were measured in both strawberry leaves and berries

350

after malathion application under field conditions, and DMP residues accounted for

351

87 mol % of all the metabolite residues in the berries by day 20. Li et al. (2012)36

352

reported that the DMP, DMTP and DMDTP metabolite residues increased

353

significantly during a 23 day period after malathion application on strawberries, and

354

the residues of all the metabolites declined much more slowly than that of the parent

355

malathion. An overarching conclusion of these studies is that the exposure potential of

356

metabolites is much greater than the exposure potential of parent pesticides 16


Page 16 of 34

Page 17 of 34


357

themselves due to the persistence of the metabolites in agricultural products. Our

358

study primarily concerns the dissipation of the OP pesticide omethoate and the

359

occurrence of its metabolite products during wheat storage. Similarly, we observed

360

that omethoate could be metabolized by wheat or degrade in the environment, thus

361

leading to the presence of the metabolite DMP in wheat. This phenomenon might be

362

attributed to the fact that the group attached to the P atom in the omethoate molecule

363

is replaced by a hydroxyl group to form the toxic metabolite DMP under the action of

364

hydrolysis through a variety of hydrolyzed esterases (Figure 3). Additionally, the

365

metabolic mechanism of omethoate in wheat can be affected by many abiotic and

366

biotic factors, such as the chemical structure of the parent pesticides, the time since

367

the pesticide’s application, and many environmental parameters including the

368

temperature, moisture content, pH, light and oxygen.31 These factors affect each other

369

and promote the migration and transformation of omethoate in wheat.

370

Although hydrolysis could reduce the toxicity of OP pesticide metabolites, DMP

371

was regarded as a potential source of human and animal urine biomarker exposure.

372

The results from our study indicated that the presence of DMP in urine potentially

373

results from the absorption of preformed DMP in plants that are used for food besides

374

the hydrolysis of OP pesticides.37 Accordingly, it is suggested that DMP, as the

375

metabolite of omethoate, should be taken into account in omethoate dissipation and

376

risk assessment in wheat.

377

In conclusion, our study is the first to reveal the individual metabolite formation

378

and metabolic mechanism of omethoate during wheat storage in addition to the 17



379

dissipation kinetics of the parent omethoate. The structure elucidation tools combined

380

with the HPLC-QTOF/MS technique were helpful for the determination of parent

381

pesticides and the tentative identification of their main metabolites. Considering the

382

potential health risks caused by dietary exposure to DMP through consuming

383

wheat-based foods containing omethoate residues, more awareness should be given to

384

the risk assessment for omethoate and its metabolite DMP in wheat. Furthermore, the

385

establishment of MRLs for pesticide metabolite residues in wheat should be

386

considered when conducting the potential risk assessment associated with the

387

consumption of wheat-based food containing parent pesticide residues.

388

Abbreviations used

389

OP, organophosphorus; DAPs, dialkylphosphates; DEP, diethyl dithiophosphate;

390

DETP, diethyl thiophosphate; DEDTP, diethyl dithiophosphate; DMP, dimethyl

391

phosphate; DMTP, dimethyl thiophosphate; DMDTP, dimethyl dithiophosphate;

392

TMP, O, O, S - trimethyl phosphorothioate; MMP, monomethyl phosphate; DPN,

393

dimethyl phosphonate; MRLs, maximum residue limits; UPLC-QTOF/MS,

394

ultrahigh-performance liquid chromatography system coupled with quadrupole

395

time-of-flight mass spectrometry; UPLC-TWIMS-QTOF/MS, ultrahigh-performance

396

liquid chromatography travelling wave ion mobility quadrupole time-of-flight mass

397

spectrometry; d-SPE, solid-phase extraction; ESI ＋ , electrospray ionization mode;

398

MSE, mass spectrometer elevated; LODs, limits of detection; LOQs, limits of

399

quantitation; TIC, total ion chromatogram; R2, correlation coefficients; and RSD,

400

relative standard deviation. 18


Page 18 of 34

Page 19 of 34

401


Acknowledgments

402

We would like to thank Yan Zhang and Juan Sun for their technical support in

403

the pesticide detection, and Huijie Zhang and Li Wu for their help in the English

404

editing.

405

Funding sources

406

This study was supported by the National Key Program on Quality and Safety

407

Risk Assessment for Agro-products (2018 GJFP2018001) and the Agricultural

408

Science and Technology Program for the Innovation Team on the Quality and Safety

409

Risk Assessment of Cereal Products, CAAS.

410

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549 550 551 552 553 554 25



555 556 557 558

Figure captions

559 560

Figure 1. HPLC-QTOF/MS extracted ion chromatogram and MSE spectra of

561

omethoate and DMP: (A1) omethoate standard at 100 ug/kg in wheat sample; (A2)

562

DMP standard at 100 ug/kg in wheat sample; (B1) the low collision energy adducts of

563

H+, Na＋, K＋ omethoate; (B2) the low collision energy adducts of H+ , K＋ DMP.

564 565

Figure 2. Degradation curves of omethoate and its metabolite DMP at different

566

applied dosages during wheat storage. (A) Omethoate, (B) DMP.

567 568

Figure 3. Hydrolyzed metabolic pathway of omethoate in wheat samples.

569 570 571 572 573 574 575 576 26


Page 26 of 34

Page 27 of 34


577 578 579 580 581 582 583 584

Table 1 UPLC-QTOF/MS accurate mass measurements of omethoate and DMP in wheat samples

Compound Omethoate

DMP

Retention time (min) 2.77

Formula C5H12NO4PS

Observed neutral mass (Da) 214.0300

0.50

C2H7O4P

126.0082

585 586 587 588 589 590 591 592 593 594 595 596 597 27


Fragments

Adducts

182.9868 154.9920 124.9814 109.0049 94.9892 78.9957

+H, +Na, +K

+H, +K


Page 28 of 34

598 599 600 601 602 603 604 605

Table 2 List of reported metabolites of omethoate in references, their formula and calculated exact masses Compound

Formula

Exact mass

DMP

C2H7O4P

126.0082

DMTP

C2H7O3PS

142.1139

TMP

C3H9O3PS

156.1405

MMP

CH5O4P

112.0220

DPN

C2H7O3P

110.0490

606 607 608 609 610 611 612 613 614 615 616 617 28


(Da)

Page 29 of 34


618 619 620 621 622 623 624 625

Table 3 R2, recoveries, LOD and LOQ of omethoate and DMP in wheat samples Values (mean ± SD) in the same row.

Average recovery and standard deviations (%) LOD Spiking level (ug/kg) (ug/kg) 20 50 100 200 500 Omethoate 0.9998 85.00 ± 5.00 90.67 ± 6.11 92.00 ± 3.61 90.33 ± 4.25 89.13 ± 4.15 0.25 DMP 0.9922 77.57 ± 6.57 73.47 ± 7.35 81.38 ± 6.24 91.67 ± 2.93 85.19 ± 0.64 2.80 1.0 626 1.5 627 1.2 1.2 628 Compound

R2

629 630 631 632 633 634 635 636 637 638 639 640 641 29


LOQ (ug/kg) 0.80 9.50 3.00 5.00 3.60 3.60


Page 30 of 34

642 643 644 645 646 647 648 649

Table 4 Degradation kinetics of omethoate and its metabolite DMP at different applied dosages during wheat storage

Compound

Treatment

Omethoate

Treatment 1 Treatment 2 Treatment 3 Treatment 1 Treatment 2 Treatment 3

DMP

First-order kinetic equation Ct= 167.8364 e-0.0585t Ct= 361.6234 e-0.0503t Ct= 775.7975 e-0.0224t Ct= 118.3016 e-0.0584t Ct= 150.7093 e-0.0336t Ct= 382.8811 e-0.0220t

C0 (ug/kg) 167.8364 361.6234 775.7975 118.3016 150.7093 382.8811

650 651 652 653 654 655 656 657 658 659

30


R2 0.9649 0.9959 0.8675 0.8829 0.8685 0.9881

K (1/day) 0.0585 0.0503 0.0224 0.0584 0.0336 0.0220

t1/2 (days) 11.85 13.78 30.94 11.87 20.63 31.50

Page 31 of 34


660 661

A1

A2

662

B1

B2 [ M + H ]+

[ M + H ]+

[ M + K ]+

[ M + Na ]+ [ M + K ]+

663 664 665 666 667 668

Figure graphics Figure 1

31



669 670 671 672

Treatment1: recommended dosage Treatment2: Twofold of recommended dosage

200 180 160 140 120 100 80 60 40 20 0

A

140 Concentration (ug/kg)

Concentration (ug/kg)

Treatment3: Tenfold of recommended dosage

0

673

20

40

60

80 60 40 20 0

10

0

10

20 Time (Days)



300

40

250 200 150 100 50

200 150 100 50 0

20

40

60

80

100

20 Time (Days)

Time (Days) 1,400

30

40

500 Concentration (ug/kg)


30

250

350

0

674

1,200 1,000 800 600 400 200 0

678 679

100

Time (Days)

0

676 677

B

120

0

80

400

675

Page 32 of 34

0

50

100 Time (Days)

150

400 300 200 100 0

200

0

50

100 Time (Days)

Figure 2

680

32


150

Page 33 of 34


681 682 683

Hydrolysis

684 685

Figure 3

686 687 688 689 690 691 692 693 694 695 696 697 698 699

33


＋

H


700

Page 34 of 34

Graphic for table of contents

701 702 703

Field applied

Storage

704 705

34


Hydrolysis

Identification and dissipation of omethoate and its main metabolite

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