Tebuconazole and Azoxystrobin Residue Behaviors and Distribution

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Tebuconazole and azoxystrobin residual behaviors and distribution of field and cooked peanut Fan Hou, Pei pei Teng, Fengmao Liu, and Wen zhuo Wang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 12 May 2017 Downloaded from http://pubs.acs.org on May 13, 2017

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Journal of Agricultural and Food Chemistry

Tebuconazole and azoxystrobin residual behaviors and distribution of field and cooked peanut Fan Hou†, Peipei Teng†, Fengmao Liu†,*, Wenzhuo Wang† †

Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, 100193, P.R. China *Corresponding author: Prof. Fengmao Liu, Pesticide Residue and Environmental Toxicology Lab, Department of Applied Chemistry, College of Science, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing, 100193 China. Tel.: +86 10 6273 1978 Fax: +86 10 6273 3620 E-mail address: [email protected] (Fengmao Liu*)

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1 ABSTRACT: Residue behaviors of tebuconazole and azoxystrobin in field condition and 2 the variation of their residue levels during the boiling process were evaluated. The 3 terminal residues of peanut kernels were determined by modified QuEChERS method 4 (quick, easy, cheap, effective, rugged, and safe) by means of the optimization of the novel 5 purification

procedure

with

multi-walled

carbon

nanotubes

(MWCNTs)

and

6 Fe3O4-magnetic nanoparticle (Fe3O4-MNP) under the presence of external magnetic field, 7 and the results were all at trace level at harvest time. The residues in shells were detected 8 as well to investigate the distribution in peanuts. Tebuconazole and azoxystrobin residue 9 levels varied before/after boiling in kernels and shells to different degrees due to various 10 factors, such as the mode of actions and physico-chemical properties of pesticides. The 11 residues have been transferred from peanut into the infusion during boiling with the 12 higher percentage of azoxystrobin as its lower logKow. The processing factors (PFs) for 13 tebuconazole and azoxystrobin after processing were less than 1, indicating that home 14 cooking in this study could reduce the residues level in peanut. Risk assessment showed 15 there was no health risk for consumers. 16 17 KEYWORDS: Tebuconazole, Azoxystrobin, Peanut, Terminal residue, Home cooking, 18 Risk assessment 19 20 21 22

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23 INTRODUCTION 24 Peanut (Arachis hypogaea L.), as one of the most important leguminous plants and major 1

25 oil-bearing crops, is widely cultivated throughout the world , among which China is one 26 of main peanut production and export nations. Being abundant in protein, unsaturated 27 fatty acid, minerals, vitamins and anti-fibrinolysis enzymes, peanut is of great agricultural 2

28 and economic significance . Most agricultural products are consumed freshly, while a 3

29 majority of raw agricultural commodities (RAC) are thermal processed before consumed . 30 Taking peanut as an example, it can be eaten as its raw state in recipes, and also it can be 31 processed into various forms, such as peanut sauce, peanut butter, peanut pickle and many 4

32 other snack products are available in the market . According to Chinese customs and 33 traditions, peanut kernels are usually boiled with the shells together during thermal 34 processing, which is the most popular household cooking way for eating peanut. Peanut 35 shell, as a common agricultural waste from peanut cultivation, was produced about 5.0 5

36 million metric tons annually in China . It can be a source of animal feed rich in nutrition, 37 and also can be used as culture medium of the cultivation of edible fungi as well. Recently, 38 peanut shell is commonly introduced into the soy sauce production as raw material. 39

With the increase of the cultivation of peanuts on a large scale, pesticides are often

40 applied to reduce loss from weeds, pests and fungi. However, pesticide residues in 41 agricultural products resulting from the improper and abusive application could pose a 6

42 risk to animals and human beings . Excessive pesticides applied to peanut plants result in 43 residue partly remaining on peanut shells, which brings about risks to animals. Nowadays, 44 it is increasingly prevalent to apply multiple pesticide mixtures based on their respective

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45 effects in crop protection instead of individual pesticides. Recently, 29% of suspension 46 concentrate (SC) formulation of tebuconazole and azoxystrobin in combination we used 47 has been registered in China for preventative and curative purposes. Its active ingredients 48 of tebuconazole and azoxystrobin differ in their spectrum of activity and mode of action, 49 which could be complementary to each other when applied in combination. Tebuconazole 50 (Table.

S1

in

supplementary

material),

51 [(RS)-1-p-chlorophenyl-4,4-dimethyl-3-(1-H-1,2,4-triazol-1-ylmethyl) pentan-3-ol], is a 52 broad spectrum triazole fungicide that has effective control against many fungal 7

53 pathogens , such as powdery mildew, scab, head smut, leaf spot disease etc. of peanut, 54 maize, wheat, apple, banana and other crops. Tebuconazole in triazole group is one of the 55 most effective fungicides in inhibiting fungal growth and reducing late leaf spot 8

56 symptoms after initial infection . However, it has been classified as a potential human 57 carcinogen by the US Environmental Protection Agency (EPA), posing serious food safety 9

58 issues and health risks to consumers . Akin to the major control objects of tebuconazole, 59 azoxystrobin (Table. S1 in supplementary material) (methyl (E)-2-{2-[6-(2-cyanophenoxy) 60 pyrimidin-4-yloxy] phenyl}-3-methoxyacrylate), a developed systemic and protectant 61 foliar fungicide derived from the naturally-occurring strobilurins, is used for the control of 7

62 powdery mildew, downy mildew and sclerotinia in different crops . It has been registered 63 for use in more than 85 different crops around the world, ranking as the No. 1 agricultural 64 fungicide on the global fungicides market according to the data provided by McDougall 65 P.

10, 11

, while in China, registration for azoxystrobin covers with plenty of crops, such as 12

66 soybeans, peanut, rice, cereals, vegetables and fruit . Being a systemic and protectant

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67 foliar fungicide, it commonly exhibits its effective control against those diseases above 68 through crop leaf surfaces to the leaf tips and growing edges by inhibiting the germination 13

69 and pre-penetration growth of the certain fungi , which can be complementary to 70 tebuconazole. Although azoxystrobin is considered to have low acute and chronic toxicity 14

71 to humans, mammals, birds and bees , recent study has clearly indicated that this kind of 72 fungicide was classified as acutely toxic to aquatic organisms and has potential 73 endocrine-disrupting activity after the toxicity data gap has been identified by European 74 Food Safety Authority (EFSA), potentially causing long-term adverse effects in the 10,14

75 environment 76

.

The food processing techniques focused on commercial or home processing of

77 agricultural products, included washing, blanching, boiling, peeling, cooking, roasting, 78 frying and so on. Some of the food processing techniques have been found to significantly 15

79 reduce the pesticide residues in agriculture products in several studies . Although there 80 has been extensive research carried out on the pesticide residues behavior during 81 processing in fruits and vegetables, to the best of our knowledge, however, there are scare 82 reports paying attention to the research of residue behavior and distribution of 83 tebuconazole and azoxystrobin during peanut boiling. Moreover, tebuconazole and 84 azoxystrobin, as systemic fungicides, could be moved easily into the edible portion 85 (peanut kernel) during cooking, which may pose a high risk to people based on their 86 potential toxicity properties. Therefore, taking the edible method of peanut (fresh or 87 cooked) into account, it is more essential, meaningful and worthy to have good 88 knowledge of the residue behavior of tebuconazole and azoxystrobin before and after

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89 Chinese traditional boiling in peanut, which could be served as a direction to evaluate the 90 human exposure to pesticides and could be monitored the residue level in peanut shells 91 when using as animal feeds. 92

Therefore, in the study, tebuconazole and azoxystrobin residual behaviors in peanut

93 field conditions were investigated according to the “Guideline on Pesticide Residue 94 Trials” of China and the terminal residues level in kernels and shells were determined by 95 modified QuEChERS method (quick, easy, cheap, effective, rugged, and safe) with high 96 performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. 97 Based on the results of field trials, peanut boiling was carried out to mimic Chinese 98 household practice in order to investigate the possible residue behavior and distribution in 99 peanut kernels and shells during the processing operation, and then throw light on the 100 processing factors (PFs). Finally, this study intended to investigate the potential dietary 101 risk assessment of tebuconazole and azoxystrobin for different groups of people in China 102 based on the residue levels. 103 MATERIALS AND METHODS 104

Chemicals and Reagents. Pesticides of analytical standard grade tebuconazole (99.1%

105 purity) and azoxystrobin (97.5% purity) were both obtained from National Research 106 Center for Certified Reference material, China. HPLC-grade acetonitrile, being sufficient 107 quality for pesticide residue analysis, was purchased from Honeywell (Burdick & 108 Jackson). The water used was purified with a Milli-Q water purification system from 109 Millipore, USA. Dispersive solid phase extraction (d-SPE) adsorbents including 110 multi-walled carbon nanotubes (MWCNTs, 5-10 nm) with average outer diameters of

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111 10~20 nm, primary secondary amine (PSA) and graphitized carbon black (GCB), were all 112 provided by Bonna-Agela Technologies, Ltd., China. Three types of magnetic 113 nanoparticles (MNPs) (i.e. Fe3O4 (100-300 nm), γ-Fe2O3 (20 nm) and Fe3O4 (20 nm)) 114 were obtained from the Aladdin Reagent Corporation (Shanghai, China). Analytical 115 reagent grade anhydrous sodium chloride (NaCl) and anhydrous magnesium sulphate 116 (MgSO4), which were used to remove the water content in the extraction and subsequently 117 to reinforce the partition of the matrix interfering compounds on adsorbents during 118 clean-up procedure, were obtained from Beijing Chemical Reagents Company, China. 119

Apparatus. A TARGIN VX-III Multi-Tube Vortexer was used to swirl the Teflon

120 centrifuge tubes during sample preparation. Centrifugation was performed in two different 121 instruments respectively: A RJ-TDL-40B low-speed desktop centrifuge equipped with a 122 bucket rotor (8 × 100 mL) was purchased from Jiangsu Ruijiang Co., Ltd., China, and a 123 SIGMA 3K15 microcentrifuge equipped with angular rotor (24 × 2.0 mL) was provided 124 by Jiangsu Qilinbeier Co., Ltd., China. 125

Stock Solution Preparation. Standard stock solutions were prepared by dissolving

126 accurately weighed 12.8 mg tebuconazole and 12.6 mg azoxystrobin standard in 25 mL 127 volumetric flask with chromatographic grade acetonitrile to obtain a final concentration of 128 500 mg/L, separately. Both of the solutions were stored at –20°C. 129

The working standard solutions required for fortification and calibration were freshly

130 prepared from stock solution by diluting with acetonitrile. With regard to calibration 131 solutions, the solvent calibration solutions were prepared by diluting the stock solutions 132 with acetonitrile, while similarly, the matrix-matched ones were achieved by diluting the

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133 stock solutions with blank extracts of all matrices. 134

Field Trials-the Design of Terminal Residue Experiment. According to “Guideline

135 on Pesticide Residue Trials” issued by the Ministry of Agriculture, the People’s Republic 136 of China (NY/T 788-2004) and the recommendations of the pesticide labels, the field 137 experiments, including the residue dynamic and terminal residue experiments in 138 supervised field trials, were conducted in two consecutive years at three sites: Beijing, 139 Shandong and Anhui. Each experiment field was comprised of three replicate plots and a 2

140 control plot both with area of 30 m separated by irrigation channels. 1 m distance was set 141 up as a buffer area to separate each plot with different treatments in the same field. On the 142 basis of the climatic conditions in different regions of China, the field experiment began 143 in June in Beijing and in July in Shandong and Anhui. 144

The terminal residue experiment at supervised field trial was carried out with the -1

-1

145 recommended dosage of 174 g a.i. ha and a higher dosage of 261 g a.i. ha (1.5 times of 146 the recommended dosage). 29% of SC formulation, which was composed of 11% 147 azoxystrobin and 18% tebuconazole, was sprayed 3 and 4 times with an interval of 7 days 148 between each application both at low and high levels. Untreated blank control plots were 149 also set for comparison, which were with the same size and were sprayed with water only. 150 Each treatment was comprised of three replicate plots and one control plot. Representative 151 peanut samples (ca. 5 kg) were randomly collected at pre-harvest interval (PHI) of 7, 14 152 and 21 days from several points in each plot after the last spraying. And then, the peanut 153 samples collected were divided into small amount using quartering before being 154 homogenized for analysis.

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155

All the samples were stored at -20℃ for further analysis.

156

Sample Processing and Preparation. Fresh peanut samples with and without

157 pesticides were all collected from the supervised field described above. An appropriate 158 amount of peanut samples that pesticides were applied to at a rate 1.5 times higher than 159 the recommended commercial dosage without processing was used for providing the 160 initial deposits on peanut kernels and shells, and the rest of intact peanut was used to boil 161 processing. The processing procedure imitated traditionally Chinese household peanut 162 boiling that involved only three steps. As shown in Fig.1, firstly, the samples was placed 163 in a plastic colander, rinsed under the running tap water (1 L/min, 15 °C) for 1 min to 164 wash away the dust. Subsequently, the samples were immersed in 2 L of tap water (15 °C) 165 in a covered pot, followed by boiling for 30 min on the induction cooker (2500 W). 166 Finally, part of the boiled peanut samples were detached into kernels and shells. Peanut 167 samples, including the intact peanut, the detached kernels and shells with the weight ratio 168 of 7:3 (m:m), were comminuted respectively with a triturator for homogenization. All 169 crushed samples were put into the seal sample bags separately, and then stored at -20 °C 170 before analysis. 171

A certain amount of thoroughly homogenized samples of peanut kernel (10 g), shell (5

172 g) and the whole of them (5 g) were weighed accurately into 50 mL Teflon centrifuge 173 tubes. The QuEChERS method, originally developed for fruits and vegetables which 174 contained plenty of water, had been modified for dry food products in order to increase 175 the water content so as to make dry samples apt to the extraction solvent. Therefore, 5 mL 176 of 0.1 % formic acid water was added. The samples were allowed to stand for 10 min

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177 before 10 mL of acetonitrile was added to extract. The centrifuge tube was capped and 178 vortexed vigorously for 3 min with Multi-Tube Vortexer to ensure that the sample 179 interacted with the extraction solvent sufficiently. After 1 g anhydrous sodium chloride 180 and 4 g anhydrous magnesium sulfate were added to the mixture, the vortexing step was 181 repeated for 3 min and then the tube was centrifuged for 5 min at 3800 rpm (3420 rcf). 182

For d-SPE cleanup, an aliquot of 1.0 mL supernatant was transferred into a 2 mL

183 centrifuge tube containing pre-weighed different kinds of sorbents (5 mg MWCNT and 30 184 mg Fe3O4-MNP for peanut kernel, 10 mg MWCNT for shell and the whole). The mixture 185 was shaken vigorously by Multi-Tube Vortexer for 1 min and then centrifuged for 1 min at 186 10,000 rpm (8800 rcf) with a microcentrifuge. Particularly for peanut kernel, due to the 187 unique magnetic of Fe3O4-MNP, they can easily be separated out of sample solution by 188 applying an external magnetic field instead of centrifugation after shaking. The 189 acetonitrile layer was transferred into a vial for instrumental analysis after filtered through 190 a 0.22 µm polypropylene filter membrane. 191

HPLC-MS/MS Analytical Condition. Chromatographic separation of tebuconazole

192 and azoxystrobin was achieved using an Agilent 1200 HPLC equipped with a 193 reversed-phase column (ZORBAX SB-C18 analytical column (50 mm × 2.1 mm i.d., 1.8 194 µm particle size; Agilent, USA) at the constant column temperature of 25°C. A binary 195 mobile phase in isocratic elution mode system composed of HPLC grade acetonitrile and -1

196 water (0.1% formic acid) (80: 20, v: v) was adopted. A flow rate of 0.3 mL min and an 197 injection volume of 5 µL were used.

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198

For the MS/MS analysis, a triple quadrupole mass spectrometer (6410 TripleQuad),

199 which was equipped with an ESI source, was employed for analysis of tebuconazole and 200 azoxystrobin in positive ionization mode. The gas temperature was set at a temperature of -1

201 350°C with the gas flow (N2) rate of 8.00 L min . The capillary voltage was 4000 V and 202 the pressure of the nebulizer gas was 35 psi. Based on the selection of the precursor ions 203 in full scan mode, two product ions with the highest sensitivity and optimal selectivity 204 were selected for the qualification of each compound in the multiple reaction monitoring 205 (MRM) parameters. The ion with higher response was used for quantification. Afterwards, 206 the most abundant fragmentor (V) and the collision energy (eV) values were optimized in 207 MRM condition. MS/MS parameters including MRM transitions, retention time, 208 molecular mass, and collision energies were optimized individually for the two target 209 compounds, summarized in Table S1 in supplementary material. 210

GC-MS Condition. The gas chromatography-mass spectrometer (GC-MS) instrument

211 was used to confirm whether the possible metabolites of the target compounds existed or 212 not after boiling process by detecting samples in full scan mode and searching every peak 213 in comparison with the NIST 12.0 library. It was carried out on the Agilent 6890N 214 Network GC system (Agilent Technologies, USA) equipped with a 5975B inter mass 215 spectrometer. Samples were injected with a 7683B Series splitless auto-injector. Agilent 216 Technologies Capillary Column HP-5MS phenylmethyl siloxane fused-silica capillary 217 analytical column (30 m length×0.25 mm i.d.×0.25 µm film thickness) was used for GC -1

218 separation. Helium (99.99 % purity) at a constant flow rate of 1.2 mL min

was used as

219 carrier gas. The column temperature was set at 80℃ (hold for 1 min), which increased

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-1

-1

220 initially at a rate of 30℃ min to 150℃, then at 3℃ min to 210℃, and finally increased to -1

221 290℃ at a rate of 10℃ min , holding for 12 min. The temperature of the injector port was 222 250℃ and a volume of 1 µL extract was injected in splitless mode. 223

Statistical Analysis. The residue level of tebuconazole and azoxystrobin might be

224 reduced after boiling process, which can be expressed as removal rate using the following 225 equation: 226

removal rate (%) =

Cpre − Cpost × 100% Cpre

227 During peanut boiling, pesticide residue might be transferred from peanut into the boiled 228 water. The transfer percentage of tebuconazole and azoxystrobin from the intact peanut 229 into infusion was calculated by the following equation: 230 transfer percentage (%) =

Caq × Vaq ×100% Cpre × M

231 Taking the variation of residue levels during processing into consideration, Joint 16

232 FAO/WHO Meeting Pesticide Residue (JMPR)

and Organization for Economic

3

233 Cooperation and Development (OECD) considered and calculated PFs as follows: 234

PFs =

Cpost Cpre -1

235 , where Cpre and Cpost are the residue levels (µg kg ) in the raw product and in processed 236 product, respectively, corresponding to each portion of peanut samples; Caq is the 237 concentration of tebuconazole and azoxystrobin in the boilng infusion; Vaq is the volume 238 (L) of the infusion; M is the weight (g) of peanut sample. 239 RESULTS AND DISCUSSION 240

Optimization of Clean-up Procedure for Kernels (edible portion). In this work, due

241 to the existence of the different potential interferences from matrices, the kind of

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242 dispersive sorbents were selected to achieve the best purifying effect according to 17

243 different matrices’ features. In our previous investigation research , we found that 244 MWCNTs showed more brilliant cleanup performance in respect of removing lipids 245 interferences than the other sorbents. Therefore, for the peanut kernel samples in our study, 246 5 mg MWCNT was used in the d-SPE clean-up to reduce the matrix effects (MEs). 247

Traditionally, in the QuEChERS method, adsorbents can be separated from the

248 extracted solution by centrifugation. However, in recent years, MNP has been gradually 249 drawing the scientific attention as a novel and interesting material due to its distinct 18

250 superparamagnetism feature . With the combination of MNPs and d-SPE adsorbents, the 251 present method could eliminate the necessary centrifugation procedure when achieving 252 two-phase separation readily under the presence of external magnetic field, which has 253 been greatly simplified. Recently, MNPs were usually modified and synthesized with 19

254 coating material or active groups as sorbents to analyse pesticide residues in samples . Li 18

255 Y. F., et al

reported that the mixture of bare MNPs with PSA and GCB have excellent

256 function as adsorbent when purified, and that it is better to be separated from the extract 257 solution. In this work, MNPs mixing with sorbents endowed themselves with magnetic 258 operational convenience. The mixture could be dispersed in a sample solution by votexing 259 and the phase separation could be rapidly and conveniently conducted by supplying an 260 external magnetic field after adsorbing matrix impurities in the sample solution, which is 261 beneficial to guarantee simplicity and rapidity of the method. 262

To investigate the cleanup efficiency, pesticides’ recoveries were studied for peanut -1

263 kernel samples spiked with the pesticides at 10 µg kg

(n=5). The spiked extracts were

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264 purified using different kinds and different amounts of MNPs mixing with 5 mg 265 MWCNTs. In terms of the kinds of MNPs, three types of MNPs (i.e. Fe3O4 (100-300 nm), 266 γ-Fe2O3 (20 nm) and Fe3O4 (20 nm)) were examined, and the results were illustrated in 267 Fig. 2a. As obviously shown in the figure, the addition of Fe3O4 (100-300 nm)-MNP could 268 achieve better recoveries for both tebuconazole and azoxystrobin. Taking both residue 269 recoveries and cleanup efficiency into account, the Fe3O4 (100-300 nm)-MNP was 270 selected for further study. 271

Furthermore, the amount of MNPs also has an influence on the extraction recovery of

272 tebuconazole and azoxystrobin. The amount in the range of 20~50 mg was investigated to 273 evaluate the influence on recoveries of the pesticides in peanut kernel samples. The results 274 (Fig. 2b) indicated that the recoveries and the response value increased with the increasing 275 amounts of MNPs from 20 to 30 mg. When the amount increased to 40 mg, the recoveries 276 had no obvious difference, which was most likely due to the excess MNPs not being 20

277 effectively desorbed, lowering the concentration of analytes in the sediment phase . And 278 the recoveries leveled off at 40 mg. Thus, the amount of Fe3O4 (100-300 nm)-MNP was 279 set at 30 mg. 280

Method Validation. In order to decrease the matrix effects in the quantitative analysis,

281 the linearity of peanut kernel, shell and the whole samples were performed by 282 matrix-matched standard solutions of at least five concentrations. The matrix-matched 283 calibration curves, constructed by plotting the peak area (y) against the analyte 2

284 concentrations (x), were of good linearity with a determination coefficient of R >0.9971 285 (Table 1).

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286

The limit of detection (LOD) was calculated by the lowest concentration that produced

287 a signal-to-noise (S/N) ratio of 3. The limit of quantification (LOQ) was set at the lowest 288 fortified concentration level of the analytes which could give a response quantified with 289 satisfactory relative standard deviations (RSDs). The specific LOD and LOQ in peanut 290 samples were listed in Table 1 respectively, which were sufficient to verify the compliance 291 of products with the legal tolerance. 292

Recoveries with RSDs of tebuconazole and azoxystrobin in each peanut matrix were

293 determined at three fortification concentration levels at least with five replicates to 21

294 validate and evaluate the accuracy, precision and reproducibility of the method . The 295 recoveries and RSDs were shown in Table 1. The results demonstrated that the mean 296 recoveries for tebuconazole and azoxystrobin ranged from 77 to 105% and 86 to 110% 297 respectively, both with RSDs lower than 7%. In general, the validation data of the 298 methods for studied pesticides was in line with the EU guidelines for pesticide residue 22

299 analysis, reflecting good method performance . The representative HPLC-MS/MS 300 chromatograms of tebuconazole and azoxystrobin for matrix matched standards were 301 shown in Fig. 3. 302

The occurrence of matrix effects is regarded as a signal suppression or enhancement of

303 the analyte due to the co-elution of matrix components, playing an important role in 23

304 quality of the quantitative data generated by the method . It may result in poor analytical 305 accuracy, linearity and reproducibility compared to those produced by solvent standards of 24

306 the target analyte . Matrix effects were examined by comparing the slopes obtained in the 307 calibration from matrix-matched standards with those obtained from solvent-based

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308 standards, calculating the values for the two pesticides in the peanut matrices by the 309 formula: ME(%) = (slopematrix/slopesolvent − 1) × 100%. Matrix effects can be divided into 310 three different categories depending on the value of this percentage. The percentage 311 between -20% and 20% could be considered as no matrix effect. A mild matrix effect 312 would occur when the percentage was between -50% and -20% or between 20% and 50%. 313 The percentage value below -50% or above 50% was indicative of a strong matrix 25,26

314 effect

. As presented in Table 1, after the sample treatment was performed, the

315 calculated ME(%) values were between -76% to 30% for tebuconazole and azoxystrobin. 316 Strong matrix suppression effects was observed for tebuconazole in peanut kernel and 317 shell samples, which were probably on account of the different substances or compounds 318 in the peanut kernel and shell samples. With regard to azoxystrobin, it can be seen a little 319 bit higher matrix enhancement effect for peanut shell samples. For more accurate results, 320 validation experiments and pesticide residue concentrations were performed in this study 321 with matrix-matched standards to compensate for matrix effects. 322

Terminal Residues in Field Trials. The developed methods as described in sample

323 preparation in section 2.5.1 for peanut kernel and shell were applied in routine analysis of 324 pesticide residues in samples collected from the supervised field in Beijing, Shandong and 325 Anhui. The terminal residue concentrations of tebuconazole and azoxystrobin in peanut 326 kernel and shell samples were detected after spraying the SC formulation directly on the 327 peanut plants thrice and four times at levels of recommended dosage and 1.5 times 328 recommended dosage in three experiment sites respectively. Residue results on the harvest 329 time at different intervals of 7, 14, 21 days in kernel and shell matrices were presented in

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330 Table S2 in supplementary material. It was shown that there were only traces of -1

331 tebuconazole and azoxystrobin residues in shells (9.05~471.77 µg kg

for tebuconazole

-1

332 and 6.85~499.41 µg kg for azoxystrobin) when the SC formulation was applied under the 333 designed experiment. The terminal residue concentrations of tebuconazole and 334 azoxystrobin in peanut kernels at the different intervals of 7, 14, 21 days were in the range 335 of 0.34~6.94 µg kg

-1

and ＜0.1~2.87 µg kg-1. Although some residues have been

336 determined in peanut kernel and shell samples irrespective of application dosage and 337 times in three experimental spots, the trace residue result manifested that the spraying 338 application of tebuconazole and azoxystrobin formulation on peanut crops some days after 339 sowing may be considered safe from the view point of toxic residues. 340

MRLs Setting. The maximum residue limits (MRLs), as a crucial parameter, is the

341 safety limit of pesticides in agricultural products and food. The MRL established by 342 Codex, China and European Union for tebuconazole in peanut kernels is 0.05, 0.1, 0.05 -1

343 mg kg respectively, while for azoxystrobin, in Codex and European Union, the MRL is -1

344 set both at 0.2 mg kg , and there is no Chinese MRL for azoxystrobin in peanut kernels. 345

The terminal residues of tebuconazole and azoxystrobin in peanut kernel following the

346 recommended dosage and 1.5 times recommended dosage (NY/T 788-2004) were far less -1

347 than the Codex MRL (0.05 mg kg

for tebuconazole and 0.2 mg kg-1 for azoxystrobin,

348 respectively). Meanwhile, compared with the Codex MRL established for tebuconazole -1

349 and azoxystrobin in peanut fodder (both 30 mg kg ), the application of the SC 350 formulation followed by good agricultural practice (GAP) conditions could not pose a risk 351 for animal as fodders according to the terminal residue result in peanut shells. Therefore,

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352 based on all the terminal residue data, it would be acceptable to recommend the PHI for 353 peanut as 7 days following the recommended dosage and the MRL for azoxystrobin as 0.2 354 mg kg

-1

in accordance with Codex MRL. Furthermore, this work would be useful for

355 Chinese government to establish MRL of azoxystrobin in peanut kernel and to provide 356 guidance on the safe and proper use of this pesticide. 357

Distribution of Pesticide Residues in Fresh and Cooked Peanut. Figure 4 showed

358 the residue magnitudes of tebuconazole and azoxystrobin in different peanut portions 359 before and after the boiling process. Processes involving washing and heating can increase 27

360 volatilization, hydrolysis or other chemical degradation and thus reduce residue levels . 361 As shown in Fig. 4, after the combined process, the residue level varied from 0.52 to 0.34 -1

-1

-1

362 µg kg , 0.26 to 0.24 µg kg in peanut kernels and 24.3 to 5.94 µg kg , 20.52 to 2.33 µg 363 kg

-1

in peanut shells for tebuconazole and azoxystrobin, respectively. The mean residue

364 reduced in peanut kernel, shell and the intact peanut by 7%, 76%, 44% for tebuconazole 365 and 15%, 84%, 74% for azoxystrobin, respectively. During 30 min boiling process, the 366 transfer rate of tebuconazole and azoxystrobin residues from the intact peanut to infusion 367 were 48% and 93%. The residue of tebuconazole reduced more significantly than that of 368 azoxystrobin after processing. 369

Firstly, the reduction of tebuconazole and azoxystrobin might be partly on account of

370 the mode of action of the pesticides in boiling procedure. As systemic fungicides, 371 tebuconazole and azoxystrobin can penetrate the epicuticular wax of the crops, 372 subsequently the cuticle layer, and finally inside the pathogenic fungi by means of 28

373 absorption paths . Contact pesticides remaining on the surface residues are easier to be

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29

374 washed away, whereas systemic residues present in tissues will be little affected . 375 Therefore, washing step might be contributed less to the reduction of tebuconazole and 376 azoxystrobin in peanut. Secondly, processes relating to heat may increase evaporation or 377 thermal degradation, so it was assumed that vaporization might have contributed to the -3

378 decrease of pesticide residues, as the vapor pressure of tebuconazole (1.7×10 mPa) is -7

379 higher than that of azoxystrobin (1.1×10

mPa)30. That was in accordance with the

380 research conducted by Rasmusssen who found that compared with other pesticides, 381 fenitrothion and tolylfluanid reduced significantly during apple boiling with their relative 31

382 higher vapor pressure . 383

Furthermore, the concentrations of both of the pesticides in the boiled water showed

384 that the residue transfer of azoxystrobin was higher than that of tebuconazole. So it was 385 assumed that more azoxystrobin residues with lower logKow of value 2.5 were transferred 386 from peanut into the water than tebuconazole (logKow of value 3.7). Generally, under a 387 certain temperature, the logKow value represents the ratio of the amount of a compound 388 distributed in octanol solvent (organic phase) and water (aqueous phase). The lower 389 logKow value of a chemical substance indicates that it has the strong capacity to remain in 32

390 the aqueous phase . Therefore, as a major determinant of the transfer rate in this work, 391 different logKow values resulted in different residue concentrations in boiled water 392 between azoxystrobin and tebuconazole. 393

Since thermal processing might concentrate or convert residues to more toxic 33

394 by-products or metabolites in food , more attention should be paid to pesticides’ 395 decomposition or even possible toxic metabolites converted from the certain pesticides. In

19



396 order to confirm whether the possible metabolites of the target compounds existed or not 397 after boiling process, all the matrix samples were detected by GC-MS in full scan mode 398 and searched every peak one by one in comparison with the NIST 12.0 library. As a result, 399 no possible metabolites were monitored, which indicated that there were no degradation 400 compounds existing after boiling process. 401

It is well known that food processing can affect the level of pesticide residues and result 34

402 in different behavior of pesticides that were present in food . The JMPR report evaluates 403 food processing data on residue behavior when significant residues are detected in plant or 16

404 plant products which are processed into food . PFs used to estimate the dilution and 405 concentration of residues, are usually adopted to assist dietary assessment of pesticide 35

406 intake from processed commodities . 407

A PF value 1 (=concentration factor) indicates the concentration effect 36

409 of the processing procedures . The calculated PFs for tebuconazole and azoxystrobin 410 after boiling process were showing Table 2. Results of this study showed that the PF 411 values of tebuconazole and azoxystrobin for three portions of peanut in processing step 412 were generally less than 1.0, which indicated that the residue magnitudes of the two 413 pesticides

decreased after processing. The processing made tebuconazole and

414 azoxystrobin remarkably reduced in peanut shell with the PF values of 0.24 and 0.11, 415 manifesting that the continuous heating during boiling was more conducive to the 416 evaporation or thermal removal of pesticides. However, with regard to the peanut kernel 417 matrix, PF values of the two pesticides were close to 1.0, which showed that the residue

20


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418 levels of the two pesticides in peanut kernels varied not as dramatically as shells. That 419 might be on account of the peanut kernel shielded by the shell, thus suffered little effect 420 from the processing surrounding conditions. 421

Health Risk Assessment. Public awareness on food safety has increasingly enhanced,

422 therefore, in this study, chronic exposure risk assessment for different groups of people 37

423 has been investigated, based on typical food (nuts) consumptions in China . Dietary 424 exposure assessment, as the critical step in the process of risk assessment, contains acute 38

425 and chronic exposure assessment . The acute dietary exposure assessment, estimating the 426 consumer health risk of intake of pesticides via food in short term, was calculated based 427 on the estimated short-term intake (ESTI) and the acute reference dose (ARfD). The 39

428 chronic dietary exposure assessment, considering the risk over the whole lifetime , was -1

429 calculated based on the estimated daily intake (EDI, mg kg , bw) and the acceptable daily -1

430 intake (ADI, mg kg , bw). The risk quotient (RQ) value indicates an unacceptable risk 431 when it is higher than 100%. The relevant equations were as follows. 432 ESTI =

HR × F mean body weight -1

433 , where HR means the highest residue (mg kg ) and F is the food consumption data (g -1

434 day ) corresponding to different ages and genders. 435 RQacute is acute risk quotient, expressed as the percentage of the ESTI to the ARfD: 436

RQacute =

437 EDI =

ESTI × 100% ARfD

STMR × F mean body weight -1

438 , where STMR means the supervised trials median residues (mg kg ). 439 The RQchronic is chronic risk quotient, which could be calculated from the following

21



Page 22 of 39

440 formula: 441

RQchronic =

EDI × 100% ADI

442 For risk assessment to tebuconazole and azoxystrobin residues in peanut samples, the -1

443 estimated daily intakes, ARfD (0.3 mg kg bw for tebuconazole) and ADI values (0.03 mg -1

-1

444 kg bw for tebuconazole and 0.2 mg kg bw for azoxystrobin) for the tested pesticides 40,41

445 were derived from JMPR report

. The fact was that the terminal residue levels is higher

446 in shorter PHI. Therefore, the risk should be low if the risk assessment of shortest PHI (7 447 day) was acceptable. At the PHI of 7 day, the HR and STMR of tebuconazole and -1

448 azoxystrobin in peanut kernel were all below the LOQ value (0.01 mg kg ). Results under 449 the LOQ of the analytical methods used for intake calculations were taken as LOQ values. 450

According to JMPR report in 201141, the ARfD for azoxystrobin was unnecessary

451 owing that the short-term intake of residues resulting from the use of azoxystrobin is 452 unlikely to present a public health concern. Therefore, the acute exposure of azoxystrobin 453 in peanut was not estimated in this study. The risk assessment for different groups of 454 people in China associated with tebuconazole and azoxystrobin residues in peanut was 455 shown in Table S3 in supplementary material. As shown in the Table S3, for short-term 456 risk assessment of tebuconazole, all RQacute values were below 1%, which meant that 457 there was a negligible short-term or acute risk with the exposure to the pesticides via 458 peanuts consumption. For long-term risk assessment, however, the RQchronic values of 459 tebuconazole ranged from 1.34% to 6.04%, which were notably higher than the RQacute 460 value. That may result from the relatively low toxicity (ADI) of tebuconazole, which 461 indicated that the chronic risk from pesticide exposure should be considered. As regard to

22


Page 23 of 39


462 azoxystrobin, all the RQs were significantly lower than 100%. 463

Generally, compared with other literature reports aiming at the risk assessment in fruits 42,43

464 and vegetables

, the health risk from pesticide residues in nuts of this study was

465 relatively tiny, which was in accordance with the work investigating the risk assessment of 44

466 multi-pesticides in nuts of China . These results indicated that for the recommended 467 application time, dose and PHI, both of the short and long-term risk exposure of 468 consumers to tebuconazole and azoxystrobin residues via peanuts consumption was 469 relatively insignificant. 470

In conclusion, the residual behaviors in field trials of peanut, the residue variation and

471 distribution during the combined process of washing and boiling were described. The 472 proposed analytical approach based on QuEChERS method with MNPs adsorbents was 473 applied to the determination of tebuconazole and azoxystrobin in peanut kernels 474 successfully. In order to investigate the distribution of the residues in kernels and shells, 475 the residues in the two matrices were all detected with the results of far less than the 476 codex MRL. Generally, the two pesticide residues distributed more in peanut shells than 477 in kernels. Meanwhile, the residue magnitudes before/aftere home cooking decrease to 478 varying degrees in peanut kernels and shells by 7%, 76% for tebuconazole and 15%, 84% 479 for azoxystrobin, respectively. During the boiling process, the transfer rate of 480 tebuconazole and azoxystrobin residues from the intact peanut to infusion were 48% and 481 93%. The variation of residues level in kernels and shells and the transfer percentage from 482 peanut to infusion might be affected by various factors including mode of actions and 483 physico-chemical properties of pesticide, such as vapor pressure and logKow constant

23



484 value. The PFs after processing were generally less than 1, indicating that home cooking 485 in this study could obviously reduce the pesticide residues level of peanut. From public 486 health point of view, the risk evaluation for different groups of people in China was 487 assessed based on the terminal residue results with RQ＜1, indicating that the observed 488 levels of tebuconazole and azoxystrobin residues in peanuts do not pose a serious health 489 risk to consumers. 490 ACKNOWLEDGMENTS 491 This work was supported partly by the Special Fund for Agroscientific Research in the 492 Public Interest of China (grant number: 201503107). 493 REFERENCES 494

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29



ASSOCIATED CONTENT Supporting Information Tables S1-S3. AUTHOR INFORMATION Corresponding Author *(F.L.) E-mail: [email protected]. Phone: +86 10 6273 1978. Fax: +86 10 6273 3620. Funding We acknowledge the Special Fund for Agroscientific Research in the Public Interest of China (No. 201503107) for financial support. Notes The authors declare no competing financial interest. FIGURE CAPTIONS Fig.1 Flowchart for the home cooking of peanut used in this study. Fig.2 (a) The mean recoveries (n=5) of tebuconazole and azoxystrobin extracted from the peanut kernel matrix with various types of magnetic nanoparticles (A: Fe3O4 (100-300 nm), B: γ-Fe2O3 (20 nm), C: Fe3O4 (20 nm), D: without MNP; (b) The mean recoveries of tebuconazole and azoxystrobin extracted and purified from the peanut kernel matrix by 5 mg MWCNT with different amounts of Fe3O4 (100-300 nm)-MNP (n=3). Fig.3 Typical HPLC-MS/MS chromatograms in MRM acquisition mode of tebuconazole and azoxystrobin in peanut matrix matched standards: (A) kernel (10 µg L-1); (B) shell (10 µg L-1); (C) intact peanut (10 µg L-1); (D) real peanut kernel sample. Fig.4 Residue magnitudes of tebuconazole and azoxystrobin (µg kg-1) in individual portion of peanut before and after boiling process (n = 6).

30


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Page 31 of 39


TABLES Table 1. Average recoveries (%, n = 5), calibration curve with determination coefficients (R2), matrix effects (ME), LODs and LOQs for tebuconazole and azoxystrobin in peanut samples. Compound

Matrix

kernel

Tebuconazole

shell

whole

kernel

Azoxystrobin

shell

whole

Fortified level (µg kg-1)

Average recovery±RSD (%)

0.1

88±6

1

99±6

10

93±4

1

77±5

10

80±3

50

83±2

100

80±4

500

87±2

1

103±6

10

105±3

50

102±2

0.1

86±2

1

102±2

10

103±1

1

93±3

10

101±2

50

100±2

100

98±3

500

103±5

1

107±2

10

107±6

50

110±2

Linearity range

Calibration curve

R2

0.1 ~ 10

y= 9.4×105 x－394.39

1 ~ 500

-1

LOQ

LOD

ME

(µg kg )

(µg kg-1)

0.9987

0.1

0.04

-76%

y= 1.0×106 x＋3827.2

0.9998

1.0

0.28

-75%

1 ~ 50

y= 3.0×106 x－45092

0.9985

1.0

0.39

-75%

0.1 ~ 10

y= 3.0×106 x＋11283

0.9971

0.1

0.047

0

1 ~ 50

y= 4.0×106 x＋62095

0.9975

1.0

0.41

30%

1 ~ 50

y= 2.0×106 x－1751.6

0.9999

1.0

0.43

10%

(µg L )

31


-1


Page 32 of 39

Table 2. PFs of combined operations to tebuconazole and azoxystrobin residues in peanut kernel, shell and the whole peanut (mean values ± SD, n = 6). matrix

Processing factor (PF)

Tebuconazole

Azoxystrobin

kernel

0.65

0.92

shell

0.24

0.11

whole

0.56

0.26

FIGURE GRAPHICS

Figure 1.

Flowchart for the home cooking of peanut used in this study.

32


Page 33 of 39


Figure 2. a) The mean recoveries (n=5) of tebuconazole and azoxystrobin fortified at 10 µg kg-1 extracted from the peanut kernel matrix with various types of magnetic nanoparticles (A: Fe3O4 (100-300 nm), B: γ-Fe2O3 (20 nm), C: Fe3O4 (20 nm), D: without MNP; b) The mean recoveries of tebuconazole and azoxystrobin extracted and purified from the peanut kernel matrix by 5 mg MWCNT with different amounts of Fe3O4 (100-300 nm)-MNP (n=3).

Figure 3. Typical HPLC-MS/MS chromatograms in MRM acquisition mode of tebuconazole and azoxystrobin in peanut matrix matched standards: (A) kernel (10 µg L-1); (B) shell (10 µg L-1); (C) intact peanut (10 µg L-1); (D) real peanut kernel sample. 33



Figure 4. Residue magnitudes of tebuconazole and azoxystrobin (µg kg-1) in individual portion of peanut before and after boiling process (n = 6).

34


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For Table of Contents Only:

35



Figure 1. Flowchart for the home cooking of peanut used in this study. 249x215mm (150 x 150 DPI)


Page 36 of 39

Page 37 of 39


Figure 2. a) The mean recoveries (n=5) of tebuconazole and azoxystrobin fortified at 10 µg kg-1 extracted from the peanut kernel matrix with various types of magnetic nanoparticles (A: Fe3O4 (100-300 nm), B: γFe2O3 (20 nm), C: Fe3O4 (20 nm), D: without MNP; b) The mean recoveries of tebuconazole and azoxystrobin extracted and purified from the peanut kernel matrix by 5 mg MWCNT with different amounts of Fe3O4 (100-300 nm)-MNP (n=3). 299x121mm (150 x 150 DPI)



Figure 3. Typical HPLC-MS/MS chromatograms in MRM acquisition mode of tebuconazole and azoxystrobin in peanut matrix matched standards: (A) kernel (10 µg L-1); (B) shell (10 µg L-1); (C) intact peanut (10 µg L1); (D) real peanut kernel sample. 249x191mm (150 x 150 DPI)


Page 38 of 39

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Figure 4. Residue magnitudes of tebuconazole and azoxystrobin (µg kg-1) in individual portion of peanut before and after boiling process (n = 6). 197x159mm (150 x 150 DPI)


Tebuconazole and Azoxystrobin Residue Behaviors and Distribution

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