Article pubs.acs.org/JAFC
Chlorpyrifos Residual Behaviors in Field Crops and Transfers during Duck Pellet Feed Processing Rui Li,† Wei Wei,† Liang He,§ Lili Hao,# Xiaofeng Ji,† Yu Zhou,*,† and Qiang Wang*,† †
State Key Laboratory Breeding Base for Zhejiang Sustainable Plant Pest Control, Agricultural Ministry Key Laboratory for Pesticide Residue Detection, Zhejiang Province Key Laboratory for Food Safety, Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, 310021 Hangzhou, Zhejiang, China § National Shanghai Center for New Drug Safety Evaluation and Research, 201203 Shanghai, China # College of Life Science and Technology, Southwest University for Nationalities, 610041 Chengdu, Sichuan, China ABSTRACT: Chlorpyrifos is a widely used organophosphorus pesticide in agricultural crops (including food) and animal feeds in China, resulting in heavy contamination. Many studies have focused on the food-processing effects on chlorpyrifos removal, but sufficient information is not observed for feed-processing steps. Here, chlorpyrifos residual behaviors in field crops and its transfers in duck pellet feed-processing steps were evaluated. In field trials, the highest residues for rice grain, shelled corn, and soybean seed were 12.0, 0.605, and 0.220 mg/kg, respectively. Residues of all rice grain and about half of shelled corn exceeded the maximum residue limits (MRLs) of China, and five soybean seeds exceeded the MRL of China. Chlorpyrifos residue was reduced 38.2% in brown rice after the raw rice grain was hulled. The residue in bran increased 71.2% after milling from brown rice. During the squashing step, the residue reduced 73.8% in soybean meal. The residues reduced significantly (23.7−36.8%) during the process of granulating for rice, maize, and soybean products. Comparatively, the grinding process showed only limited influence on chlorpyrifos removal (0.1 mg/kg) for animal feeding. Chlorpyrifos residues were removed significantly by processing steps of pellet feeds, but the residue of raw materials was the determining factor for the safety of duck feeding. KEYWORDS: chlorpyrifos, pesticide residue, field corps, feed processing, pellet feed
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treatment.12,13 For example, the study of Cogburn et al. indicated that the processing of parboiling reduced pesticide residues (malathion and chlorpyrifos methyl) of brown rice and hulls, but increased the residues of other obtained fractions.14 Different squashing methods sometimes exhibited quite different effects on the pesticide transfer. The majority of chlorpyrifos residues transferred from soybean to soybean oil by means of gold pressing; however, the chlorpyrifos residues reduced to 25.3−49.7% in soybean oil (compared to unprocessed soybean), and more than half of the chlorpyrifos residues were transferred to soybean meal (or removed) by means of hot pressing. The final residues of soybean oils were positively correlated to residual levels of raw materials and squashing methods.15 Several studies indicated that pesticide residues (including chlorpyrifos) were removed significantly from fresh vegetables, apples, and other agricultural products by means of home preparation and commercial processing.16−18 Therefore, the pesticide residual behaviors on field crops and the commercial or home processing factors should equally be considered when pesticide exposure is assessed. Although many studies have examined the effects of pesticide residues (including chlorpyrifos) in cereals and other agricultural products using home preparation and commercial processing
INTRODUCTION A large number of pesticides are used in agricultural crops and lead to serious pesticide residues in animal products through feeds from contaminated raw agricultural crops and byproducts.1,2 Internationally, maximum residue limits (MRLs) have widely been set for various pesticides in animal commodities.3 In China, duck meat, eggs, and other derived products play an important role in traditional diet, and more than half of the worldwide duck meat is produced and consumed in China.4 Through animal feeds, as the byproduct of agricultural crops, pesticide residues could transfer to animal tissues and contaminate animal commodities.1,5 Therefore, the security of feeds is the key controlling factor for the human consumption safety of duck meat and derived products. Chlorpyrifos is considered as one of the most popular broadspectrum pesticides, and the pesticide has the largest production capacity in the world (especially in China).6 Chlorpyrifos inhibits pest acetyl cholinesterase in an irreversible manner, which shows potential damage to nontarget organisms including vertebrates.7 Chlorpyrifos frequently contaminates water, soil, fruits, and vegetables.8−11 However, scarce information could be obtained on the occurrences or residual behaviors of the pesticide in cereals. The main uses of cereals are human consumption and animal feedstuff production after commercial or home processing. The processing steps (e.g., washing, milling, and baking) are the important factors leading to removal of pesticide residues from the products of cereals and other agricultural materials during harvest or postharvest © XXXX American Chemical Society
Received: May 9, 2014 Revised: September 15, 2014 Accepted: October 2, 2014
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for foods, only a few studies have focused on feed-processing effects on pesticide persistence. The pellet feeds that originate from cereals are the most frequently used feedstuffs for ducks and other poultry.19 Therefore, chlorpyrifos residual behaviors in field crops (i.e., rice, maize, and soybean) were determined according to the “Guideline on Pesticide Residue Trials” of China, and in the results of field trials, processing steps of duck pellet feed were mimicked to investigate chlorpyrifos persistence in these steps. Finally, the potential exposure risks of chlorpyrifos in duck feeding were evaluated, under the conditions of domestic ducks fed processed pellet feeds that originated from the heaviest contaminated raw materials of this study.
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MATERIALS AND METHODS
Figure 1. Flowchart of duck pellet feed processing in southern China.
Materials. Commercial seeds of rice, maize, and soybean for field trials were purchased from Zhejiang Wuwangnong Seeds Co., Ltd. (Hangzhou, China). Stock solution of chlorpyrifos (100 μg/mL in acetonitrile) was obtained from the Agro-Environmental Protection Institute, Ministry of Agriculture (Beijing, China). Commercial 40% chlorpyrifos emulsifiable concentrate (EC, w/v) was the product of Zhejiang Xinnong Chemical Co., Ltd. (Hangzhou, China). NPropylethylenediamine (PSA) and graphitized carbon black (GCB) were provided by Bonna-Agela Technologies, Inc. (Tianjin, China). Analytical grade acetonitrile, ethyl acetate, MgSO4, and NaCl were obtained from East China Pharmaceutical Group Co., Ltd. (Hangzhou, China). Chromatography grade acetonitrile and methanol were products of Honeywell International Inc. (Morris Plains, NJ, USA). Ultrapure water was the product of a Milli-Q system (Bedford, MA, USA). A paddy husker machine and a cereal grain milling machine were products of Tianyang Co., Ltd. (Shangdong, China). A poultry pellet miller was purchased from Gemco Machinery Co., Ltd. (Henan, China). A screw oil press machine was the product of Weichang Machinery Co., Ltd. (Rizhao, China). Field Trials. The field trials for chlorpyrifos residual behaviors on rice, maize, and soybean were performed in Danzhou (109.5 E, 19.5 N), Hangzhou (102.1 E, 30.2 N), and Haerbin (126.3 E, 45.5 N), and the field trials strictly complied with the “Guideline on Pesticide Residue Trials” (NY/T788-2004) in China.20 Each field trial consisted of triplicate test plots and a control plot (30 m2 for each plot), and each plot was separated by a buffer area. Chlorpyrifos 40% EC (w/v) was selected as the tested formulation in field residue trials. The recommended dosages (0.864 kg ai ha−1 for rice, 0.720 kg ai ha−1 for maize, and 0.240 kg ai ha−1 for soybean) and 1.5 times recommended dosages (1.296 kg ai ha−1 for rice, 1.080 kg ai ha−1 for maize, and 0.360 kg ai ha−1 for soybean) were chosen for field trials. The pesticide was sprayed on fields of mid- and late-planting periods two or three times at intervals of 7−14 days (7 days for rice, 10 days for maize, and 14 days for soybean) for each time during 2011 and 2012. Pesticide was sprayed on fields by a knap-sack hand sprayer (product of Shandong Shenglu machine works, Shandong, China), and there was no precipitation during the spraying period. Field samples were collected from each plot on days 7 and 14 for rice, days 10 and 20 for maize, and days 14 and 28 for soybean after the last chlorpyrifos spraying. After harvest, all samples were dried in the shade under ambient conditions. Afterward, the samples were transported at 4 °C in darkness by labeled polyethylene bags to the laboratory and stored at −20 °C until analysis. Feed Processing for Chlorpyrifos Persistent. As we know, pellet feeds are the most used feedstuff for poultry (e.g., ducks and goose) feeding.19 Generally, pellet feed processing steps include paddy rice hulling, milling, grinding, shelled corn grinding, soybean seed squashing, and granulating (Figure 1). In this study, the raw crop materials (paddy rice, shelled corn, and soybean seed) with the highest chlorpyrifos residues obtained from field trials were selected for pellet feed processing works to evaluate the chlorpyrifos persistent. Rice grain was hulled to yield brown rice and hull, and the brown rice was further milled to polishing rice and rice bran. The soybean seed was
roasted at 120 °C for 0.5 h to remove moisture. The dried soybean seed was squashed to produce soybean meal and soybean oil by screw press. As ingredients of duck pellet feeds, brown rice, rice bran, shelled corn, and soybean meal were further ground to powder, mixed, and granulated to pellet feeds. During the feed processing, raw materials and intermediate products (i.e., rice grain, rice hulls, rice bran, brown rice powder, rice bran powder, maize seed, maize powder, soybean seed, soybean meal, and soybean meal powder) in each step were collected, and the chlorpyrifos residues were determined. On the basis of the residual results of each step, chlorpyrifos persistence in feed processing works was evaluated, and final residues in different duck pellet feeds were calculated on the basis of two typical duck feed formulas in southern China (Table 1).
Table 1. Formula Ingredients of Two Typical Duck Pellet Feeds in Southern China feed A
feed B
ingredient
content (%)
ingredient
content (%)
maize rice bran soybean meal fish meal tricalcium phospahte limestone salt DL-methionine trace mineral premix vitamin premix
59.50 10.00 21.30 7.20 0.35 0.40 0.25 0.07 0.50 0.43
brown rice rice bran soybean meal fish meal tricalcium phospahte limestone salt DL-methionine trace mineral premix vitamin premix
60.00 5.00 25.00 8.00 0.35 0.40 0.25 0.07 0.50 0.43
Sample Preparation and Chlorpyrifos Determination. Extraction and cleaning procedures of chlorpyrifos from raw materials and the intermediate products were performed according to the methods of Qu et al. with minor modifications.21 An aliquot of 5.0 g of homogenized sample was transferred to a 50 mL Teflon centrifuge tube with 10 mL of ultrapure water and immersed for 30 min. Afterward, 25 mL of acetonitrile acidified with 1 M HCl (99:1, v/v) was added and shaken for 15 min by an oscillator, followed by centrifugation for 5 min at 966g (room temperature). The supernatant was transferred into a 100 mL glass measuring cylinder with a graduated stopper. The extraction steps were repeated again, the extracts were pooled, and then 8 g of NaCl was added. The extracted mixture was shaken vigorously for 1 min and left to stand for 30 min. An aliquot of 10 mL of organic extract was concentrated using a rotary vacuum evaporator at 40 °C and dried with a gentle stream of nitrogen. The residue was redissolved with 1.0 mL of ethyl acetate in a 2.5 mL microcentrifuge tube with a mixture of 150 mg of MgSO4, 25 mg of GCB, and 50 mg of PSA. The tube was shaken vigorously for 1 min and centrifuged for 5 min at 9030g (room temperature). The B
dx.doi.org/10.1021/jf502192c | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Total ion chromatograms obtained from fortified samples of paddy rice (A), brown rice (B), rice bran (C), shelled corn (D), soybean seed (E), and soybean meal (F). extract was filtered through a 0.22 μm PVDF filter before analysis. Chlorpyrifos determination by GC-MS/MS was performed following the method of Zhao et al.15 TSQ Quantum gas chromatography (GC, Thermo Fisher Scientific, USA) with a GC system TRACE GC Ultra and a triple-quadrupole mass spectrometer Quantum (mass range from m/z 10 to 3000) was used throughout the determination. Samples were injected with an AS3000 autosampler into a split/ splitless injector. The Zebron ZB-MultiResidue-2 capillary column (30 m × 0.25 mm i.d, 0.25 μm film thickness, Guangzhou FLM Scientific Instrument Co., Ltd., Guangzhou, China) was applied for analyte separation. The precursor ion was selected by analyzing the 20 μg/L chlorpyrifos standard solution under the full scan mode in the GC-MS system, and the highest intensity ion (m/z 314) was used for subsequent fragmentation to create product ions (m/z 286 and 258). The retention time of chlorpyrifos was approximately 9.22 min.
Recoveries of the analytical method were evaluated by spiked blank samples at three chlorpyrifos concentrations with six replicates. The spiked samples were kept at room temperature for 2 h to allow solvent evaporation before analysis. Matrix effects were evaluated by comparing the slopes of solvent and matrix calibration sets. Series 1 was prepared by diluting the stock standard solution in solvent at three concentrations of 5, 50, and 500 μg/kg and series 2 by diluting the stock standard solution in matrix extract that was obtained from pesticide-free sample. Statistical Analysis. Unless otherwise indicated, all tests were performed in three replicates, and the values are presented as means ± standard deviation (SD). Data were statistically evaluated by one-way ANOVA analysis with SPSS base 11.0 software. Once a significant difference was observed, the least significant difference (LSD) test was applied to validate the differences among means. C
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RESULTS AND DISSUSSION Analytical Method Validation. The GC-MS analytical results of chlorpyrifos extracted from fortified samples indicated that no interference peak was observed that might affect the analysis accuracy of the pesticide (Figure 2). At three fortified concentrations (0.01, 0.1, and 0.5 mg/kg), the chlorpyrifos recoveries ranged from 81.8 to 110.7% in different raw materials and intermediate products, and the relative standard deviations (RSDs) were from 3.2 to 11.6% (Table 2). The
was much higher than the MRL on corn forage and fodder set by the U.S. Environment Protection Agency (8.0 mg/kg).24 The chlorpyrifos residues of paddy rice showed positive correlations to the pesticide applied dosage and spraying times. The results indicated that chlorpyrifos residues were relatively high if the rice crops were treated at the recommended dosage more than two times, and the residues of this study definitely pose serious health risks in foods for human consumption and even as feed material for animal production. The chlorpyrifos residues based on “Guideline on Pesticide Residue Trials” in rice suggest that the pesticide (chlorpyrifos EC, 40%) should be applied fewer than two times at the recommended dosage (≤0.864 kg ai ha−1), and the harvest interval (from the last spraying to crop harvest) should be >14 days. Unlike rice, chlorpyrifos is not officially registered for maize and soybean by means of spraying in China, but dosages of 0.720 kg ai ha−1 with a PHI of 10 days for maize and 0.240 kg ai ha−1 with a PHI of 14 days for soybeans are recommended by the product (chlorpyrifos EC, 40%) handling instructions. In the 1.5× dosage trials, chlorpyrifos residues in shelled corn with three treatments and 10 days PHI all exceed the maize MRL of China (0.05 mg/kg, GB 2763-2012)23 (Figure 3C,D). About half of the shelled corn in two years’ field trials exceed the maize MRL of China, and the highest residue is 0.605 mg/kg. Similarly to paddy rice, chlorpyrifos residues of shelled corn showed positive correlations to pesticide applied dosage and spraying times. The field trial results by means of spraying indicated that the recommended dosage of 0.720 kg ai ha−1 (chlorpyrifos EC, 40%) should be applied on maize crop fewer than two times or with a PHI of longer than 20 days. In soybean trials, five soybean seeds (about 20.8%) exceed the soybean MRL of China (0.1 mg/kg, GB 2763-2012),23 and the highest residue was 0.220 mg/kg (Figure 3E,F). The residual results of soybean trials were obviously better than those of rice and maize crops, but under the recommended spraying conditions of this study, some residues still exceeded the soybean MRL of China. Field trials of soybean by means of spraying indicated that the recommended dosage of 0.240 kg ai ha−1 (chlorpyrifos EC, 40%) applied on soybean crops under the conditions of this study was slightly high, fewer treatments or a longer PHI being obligatory. Field trials of three crops indicated that the chlorpyrifos recommended dosages appeared to be high for rice, maize, and soybean application, and a revised GAP is required for crop production. For each crop, residual levels were moderately different from location to location and from year to year. These results were consistent with the previous study that pesticide residues were affected significantly by various environmental factors (e.g., temperature, humidity, soil characteristics, and climate), which are always diverse from different locations or years.25 Chlorpyrifos Transfers during Pellet Feed Processing. Chlorpyrifos residue of the raw rice grain selected for pellet feed processing was 12.0 mg/kg. After hulling, the chlorpyrifos residue in brown rice was 7.42 mg/kg and the transfer rate from rice grain to brown rice was 61.8%. Chlorpyrifos residue in rice bran was 12.7 mg/kg after milling processing (Figure 4A,B). The processing results showed that a considerable proportion of chlorpyrifos deposited on the skin of the rice grain, and the finding was similar to chlorpyrifos residual behaviors on vegetables and fruits.13,26 Brown rice and rice bran were manufactured to powder by grinding for approximately 30 s under a temperature lower than 50 °C, and the chlorpyrifos
Table 2. Chlorpyrifos Analytical Recoveries and RSDs in Different Fortification Samples fortification level (mg/kg)
mean recovery (%)
RSD (%)
rice grain
matrix
0.5 0.1 0.01
93.6 94.2 89.3
4.6 5.3 6.8
brown rice
0.5 0.1 0.01
95.8 93.6 81.8
4.3 5.6 7.9
rice bran
0.5 0.1 0.01
90.8 83.5 90.2
3.2 4.6 8.8
shelled corn
0.5 0.1 0.01
91.5 86.4 82.6
3.5 6.5 4.9
soybean seed
0.5 0.1 0.01
85.3 108.6 93.2
4.8 5.6 9.5
soybean meal
0.5 0.1 0.01
91.2 96.4 110.7
4.6 10.5 11.6
LODs and LOQs of the determination were calculated on the basis of signal-to-noise ratios (S/N) of 3 (3:1) and 10 times (10:1) the background chromatographic noise, respectively. The LOD and LOQ were estimated to be 0.0006 and 0.002 mg/kg for paddy rice, brown rice, rice bran, and shelled corn, respectively. For the substrates of soybean seed and soybean meal, the LOD and LOQ were estimated to be 0.0009 and 0.003 mg/kg, respectively. Recoveries slightly greater than 100% were observed for some substrates (i.e., soybean seed and soybean meal), and this result indicated that the matrices may affect the pesticide recovery.22 Matrix effects of chlorpyrifos ranged from 92 to 118%, which indicated that the matrix effect was acceptable for quantitative analysis. Chlorpyrifos Final Residues in Field Trials. In China, chlorpyrifos is registered for rice at the recommended dosage of 0.864 kg ai ha−1 with a preharvest interval (PHI) of at least 7 days, followed by good agricultural practice (GAP) conditions. In the six field trials, which were performed following the “Guideline on Pesticide Residue Trials” in China at 1× (0.864 kg ai ha−1) and 1.5× (1.296 kg ai ha−1) the recommended dosage, the chlorpyrifos residues of harvest paddy rice treated two or three times with chlorpyrifos all exceed the MRL of rice grain in China (0.5 mg/kg, GB 2763-2012).23 (Figure 3A,B), and the results are consistent with the previous study.10 The highest chlorpyrifos residue in paddy rice is 12.0 mg/kg, which D
dx.doi.org/10.1021/jf502192c | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 3. Chlorpyrifos residues of rice grain, shelled corn, and soybean seed produced from six field trials.
of rice grain to rice pellet feeds (63.2% for pellet feed of rice bran; 39.0% for pellet feed of brown rice). The chlorpyrifos reduction in soybean meal was 73.8% during the squashing process, indicating that hot squashing led to a significant chlorpyrifos removal. In the grinding process, the loss of chlorpyrifos in soybean meal was 7.12%. The chlorpyrifos reduction of soybean pellet feed is 81.5% after several conventional processing steps. This might be attributed to the physical−chemical properties of chlorpyrifos including the solubility in oils. The high fat-soluble characteristic of chlorpyrifos (logarithm of partition coefficient P is 5.1) makes the pesticide transfer from soybean to the oil layer easy during the squashing processing.29 Chlorpyrifos residue was 0.0408 mg/kg after soybean meal manufactured to pellet feed by granulating, and the reduction was 23.7%, which was slightly lower than that of processed products of brown rice (30.9%) and maize (28.6%). Similar to the previous study, reduction of chlorpyrifos was significantly enhanced if the processing steps involved heat treatment.30 Chlorpyrifos residues in soybean intermediate product and pellet feed were reduced more significantly than those of rice and maize because the soybean processing steps involved two heating procedures (soybean roasting and pellet feed granulating) and oil pressing. Processing Factors and Feed Safety Assessment. Processing factors (PFs, pesticide concentration in processed product/pesticide concentration in preprocessing material) are usually adopted to assist dietary assessment of pesticide intake from processed commodities.31 On the basis of the residues and processing transfer rates in three cereal products in this
removal by grinding step was 0.1 mg/kg.3 For healthy aquaculture and food safety, the duck pellet feeds (feeds A and B) of this study definitely required further toxicological evaluation.
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AUTHOR INFORMATION
Corresponding Author
*(Y.Z. and Q.W.) Fax: 86-571-86401834. E-mail: microbesyu@ yahoo.com. Funding
This work was supported by grants from Zhejiang Provincial Science and Technology Project (2011C12024), International Cooperation Project of Ministry of Science and Technology (2013DFA31450), and International Cooperation Project of Zhejiang Province (2013C24021). Notes
The authors declare no competing financial interest.
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REFERENCES
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Figure 4. Three cereal-processing products, chlorpyrifos residues, and processing factors during duck pellet feed manufacture: (A) intermediate and processing products; (B) chlorpyrifos residues; (C) processing factors. (∗∗) P < 0.01; (∗∗∗) P < 0.001.
study, PFs were calculated and evaluated according to Joint Meeting on Pesticide Residues (JMPR).32 PF values 1.0 indicates a chemical residue rise in the processed products.33 Results of this study showed that the three cereals’ PF values in each processing step were generally
