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Dissipation and residue of fenpropidin in wheat and soil under field conditions
Ecotoxicology and Environmental Safety 77 (2012) 52–56
Contents lists available at SciVerse ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Dissipation and residue of fenpropidin in wheat and soil under field conditions Huiyu Zhao, Jiaying Xue, Naiwen Jiang, Wei Peng, Fengmao Liu n College of Science, China Agricultural University, Beijing 100193, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2011 Received in revised form 9 October 2011 Accepted 12 October 2011 Available online 9 November 2011
The residue levels and dissipation rate of fenpropidin in wheat and soil were investigated by LC–MS/MS and GC–MS, respectively. The dissipation rates of fenpropidin were described using first-order kinetics and its half-life ranged from 3.1 to 3.3 days in wheat plants and 13.4–16.5 days in soils. During harvest time, the terminal residues of fenpropidin in wheat were below the EUs maximum residue limit (MRL, 0.5 mg kg 1) when collected 20 days after the final application, which suggested that the use of this fungicide was safe for humans. The residues persistence varied between two geographically separated experimental sites, indicating that it might be influenced by climate, soil properties and growth dilution factor. These results would be helpful in setting MRL guidance of fenpropidin in wheat in China. Crown Copyright & 2011 Published by Elsevier Inc. All rights reserved.
Keywords: Fenpropidin Pesticide residue Dissipation Wheat Soil
1. Introduction Wheat (Triticum aestivum Linn.) ranks third leading crop in China after rice (Oryza sativa) and maize (Zea mays), which is cultivated throughout the country. It is one of the major crops in Chinese daily life (Wang et al., 2009). The wheat was ground into flour to make bread, biscuits, cakes, noodles, dumplings and so on. Wheat can be fermented into beer, alcohol or biofuel and even the wheat straw can be used as fertilizer and feedstuff. Fenpropidin, 1-[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropyl]-piperidine, is a piperidine fungicide, which belongs to ergosterol biosynthesis inhibitor by inhibition of steroid reduction (sterol-D14-reductase) and isomerisation (D8 to D7-isomerase). It is particularly effective against powdery mildew (Erysiphe graminis), rusts (Puccinia spp.) and leaf spots such as Rhynchosporium secalis (Food and Environment Act 1985 Part 3). Fig. 1 shows the chemical structure of fenpropidin. The analytical methods for fenpropidin were reported in recent years. A method of accelerated solvent extraction (Sch¨afer et al., 2008) was used to determine fenpropidin and other 4 polar pesticide residues in some small streams. But the recovery of fenpropidin was only 56%. A gas chromatography with mass spectrometric detection (GC–MS) method was carried out for determination of 500 pesticides including fenpropidin residues in fruit and vegetables (GB/T 196482006). Hiemstra and Kok (2007) developed a multi-residue method for the analysis of 171 pesticides including fenpropidin in crops using liquid chromatography–tandem mass spectrometry and acetone,
n
Corresponding author. Fax: þ86 10 62733620. E-mail addresses: [email protected], [email protected] (F. Liu).
dichloromethane, petroleum were used as extraction solvents. Pizzutti et al. (2009) compared the acetonitrile and acetone extraction efficiencies for the analysis of fenpropidin in soya grain by liquid chromatography–tandem mass spectrometry. The results demonstrated that the LOQ of the method with acetonitrile extraction was 0.05 mg/kg. Fenpropidin had been registered in Italy, France, Denmark and Switzerland on wheat, barley, grape and banana. To ensure the safety of food for consumers and to regulate international trade and legislation, some countries have established maximum residue limits (MRLs) for fenpropidin in some commodities. In EU, the MRL was set at 0.5 mg/kg in wheat (Commission Directive 2004 EU No 600/2010). In New Zealand, the MRL was set at 0.02 mg/kg in wheat grain. However, to our knowledge there is no paper reported the residue dissipation of fenpropidin in wheat under field condition. No residue limit has been established of fenpropidin in wheat in China. In this work, simple LC–MS/MS and GC/MS methods were established to detect the residue of fenpropidin in wheat and soil, respectively. A field study was done to investigate the dissipation of fenpropidin in wheat plant and soil. This work would help the government to establish the MRL of fenpropidin in wheat and to provide guidance on proper and safe use of this pesticide.
2. Chemicals and methods 2.1. Chemicals and reagents Fenpropidin standard (99.5% purity) was supplied from J&K Science Ltd.; sodium chloride (AR) was purchased from Beijing Chemical Reagents Company, China; Acetonitrile (HPLC grade) for chromatography was purchased from Thermo
0147-6513/$ - see front matter Crown Copyright & 2011 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2011.10.017
H. Zhao et al. / Ecotoxicology and Environmental Safety 77 (2012) 52–56
Fig. 1. Chemical structure of fenpropidin. Fisher Co., Ltd., America; HPLC grade water was made by purifying demineralized water in a Milli-Q Plus ultra-pure water system (Millipore, Molsheim, France); primary secondary amine (PSA) and graphitized carbon black (GCB) sorbent were from Tianjin Bonna-Agela technologies Ltd., China. 2.2. Field experiment design Experiments were conducted at two sites: Zhangjiakou (114.871E, 40.781N) and Changchun (125.411E, 43.811N), China. Each experimental plot was 30–50 m2 and each treatment was carried out in triplicates. A buffer zone was used to separate the plots of different treatments. The experiments were designed according to NY/T 788-2004 (Guideline on Pesticide Residue Trials) issued by Ministry of Agriculture, People’s Republic of China. During the experimental period, the number of rainfall events in Zhangjiakou and Changchun was 13 and 38, respectively; the average temperature was 23.5 and 22.2 1C, respectively; and the average relative humidity was 43.1% and 75.8%, respectively. The soil in Zhangjiakou field was sandy loam, with the organic matter 1.54 g kg 1, and pH 7.94; the soil in Changchun was sandy clay loam with the organic matter 2.94 g kg 1, and pH 6.10. To investigate the dissipation of fenpropidin in wheat plant and soil, fenpropidin formulation (275 g a.i./L of EC) was sprayed to the wheat and soil with the dosage of 495 g a.i. ha 1 (1.5 times the recommended dosage). To investigate the terminal residues of fenpropidin in wheat grain, straw and soil, the recommended dose (330 g a.i. ha 1, with two treatments: spray 2 times and 3 times, with three harvest intervals: 20, 30, 40 days) were conducted in separate plots. The application interval was 10 days. A plot with the same size but no fenpropidin application was as control. 2.3. Sample and storage Representative samples were collected from each plot at different time intervals. The sample of wheat plant and soil were collected at day 0, 0.5, 1, 3, 5, 7, 10, 14, 21, 30 and 40 after spraying to investigate the dissipation of fenpropidin. Wheat grain, straw and soil samples were collected at day 20, 30 and 40 after application to determine the terminal residue of fenpropidin. All the samples were stored at 20 1C prior to analysis. 2.4. Analytical procedure 2.4.1. Sample preparation Wheat: The wheat includes three different matrices: wheat plant, grain and straw. Each matrix was grounded into small pieces or powder with a vegetation disintegrator, and then stored in a deep freezer at 20 1C. Soil: The soil samples were sifted through a 40-mesh sieve, and then stored in a deep freezer at 20 1C. 2.4.2. Sample extraction The wheat plant (4 g), grain (10 g) or straw (2 g) was weighted into 50-mL PTFE centrifuge tube. Subsequently, acetonitrile (40 mL) was added, and samples were shaken in a reciprocating shaker for 1 h. Then, the samples were centrifuged at 3000 rpm for 5 min. A 1 mL aliquot of the supernatant was used for clean up. Five gram of soil was mixed with 5 mL of water and 10 mL of acetonitrile in 50-mL PTFE centrifuge tube and then shaken in a reciprocating shaker for 1 h. The samples were centrifuged for 5 min at 3000 rpm. A 1 mL aliquot of the supernatant was used for clean up. 2.4.3. Cleanup procedure The supernatant above was transferred to 2-mL centrifuge tube, and 50 mg of PSA was added to soil and 30 mg of GCB to wheat straw, grain and plant sample. Then the samples were mixed in a blender for 30 s, and centrifuged at 10000 rpm for 1 min. The supernatant solution was transferred to a HPLC sample vial for instrumental analysis after filtered through a 0.22 mm polypropylene filter.
53
2.4.4. LC–ESI–tandem MS/MS Agilent 6410 series LC triple-quadrupole mass spectrometry (Agilent Technologies, USA), equipped with an electrospray ionization interface, was operated in the positive ion mode (ESI þ). Eclipse Plus C18 column (2.1 mm 5.0 cm, 3.5 mm) (Agilent technologies, USA) was used to separate the target compound from the interferences at 30 1C. The mobile phase was acetonitrile–water containing 0.1% formic acid (7/3, V/V) at the flow rate of 0.3 mL/min. The injection volume was 5 mL. The conditions for MS detection were: desolvation gas temperature 350 1C; desolvation gas flow rate 8.0 L/min; nebulizer gas (N2) pressure 35.0 psi; MS1 and MS2 Heater temperature at 100 1C, capillary voltage 4000 V. The multiple reaction monitoring (MRM) was applied and fenpropidin was calculated using the primary transition m/z 274.3-147.1 (Fig. 2). For instrument control, mass hunter workstation software data acquisition for triple quad B.02.01 (B 2043.12) and qualitative analysis version B.03.01/build 3.1.346.0 were used for data acquisition and processing. 2.4.5. GC–MS condition An Agilent Technologies 6890N network GC system equipped with an Agilent 7683 series autosampler, mass spectrometry detector (MSD) model 5975 network, and a 30 m 0.25 mm i.d. 0.25 mm VF-5MS capillary column (Varian Scientific, USA). The injector was held at 280 1C and the carrier gas was helium at a flow rate of 1.0 mL/min. The oven temperature was initially at 120 1C for 1 min, then increased to 260 1C at the rate of 20 1C/min holding for 1 min, and finally increased at the rate of 10 1C/min to 280 1C, kept for 5 min. Ion source temperature of the instrument was 230 1C, quadrupole temperature of 150 1C, transfer line of 280 1C, and solvent delay was 4 min. The mass spectrometry was in the electron ionization mode and 1 mL of sample was injected. Ions 98, 117, 273, 326 and 325 were monitored in Selected Ion Mode (SIM). TPP was quantified at m/z 326, fenpropidin was quantified at m/z 98 (Fig. 3). 2.4.6. Calculations The degradation kinetics of the fenpropidin in the wheat plant and soil were determined by plotting residue concentration over time. The residual concentration and half-life of fenpropidin were calculated by the first-order kinetics equations CT ¼ C0e KT and T1/2 ¼ln2/K, respectively, where T is the time (days) after pesticide application, CT is the residue concentration of the pesticide at time T, C0 is an initial pesticide concentration after application (at T¼0), K is a dissipation coefficient and T1/2 is defined as the time required for the pesticide residue level to fall to half of the initial residue level after application.
3. Results 3.1. Linearity, recovery and detection limits To determine the linearity, a 100 mg/L stock solution was prepared, and working solutions were prepared by diluting the stock solution with acetonitrile. The linear range was 1–500 ng/mL for LC–MS/MS detection and 10–500 ng/mL for GC–MS detection, respectively. The results showed good linearity with a correlation coefficient (g) of 0.9996 (LC–MS/MS) and 0.9994 (GC–MS). The limit of quantifications (LOQs) defined as the minimum fortified level of recovery, were 0.01 mg/kg in soil, wheat plant, straw, and 0.02 mg/kg for grain, respectively. The limit of detection (LOD) was 1 10 2 ng for GC–MS and 5 10 3 ng for LC–MS/MS at a signal-to-noise ratio of 3. The fortified recovery experiment was set at three concentration levels with five replicates at each level. The fortified recoveries of wheat plant, straw, grain and soil samples ranged from 83.6–97.9%, 102.9–112.4%, 81.2–101.5% and 80.7–102.1%, respectively; and the relative standard deviations (RSDs) of the recovery data were 2.0–9.5% (Table 1). 3.2. Dissipation of fenpropidin in wheat plant and soil The initial concentration of fenpropidin in wheat plant was 59.4 and 35.7 mg/kg in Zhangjiakou and Changchun, respectively. The dissipation regressive equation could be described by the following equations: C¼37.262e 0.209T (g ¼0.9361) with the half life 3.3 days in Zhangjiakou, and C ¼21.727e 0.2265T (g ¼ 0.9543) with the half life 3.1 days in Changchun. More than 96% of
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H. Zhao et al. / Ecotoxicology and Environmental Safety 77 (2012) 52–56
Fig. 2. Chromatogram of LC–MS/MS: secondary fragmentation chromatogram (A), standard in wheat plant (0.1 mg kg 1) (B), standard in straw (0.01 mg kg 1) (C) and standard in grain (0.01 mg kg 1) (D).
the initial residue dissipated after 14 days. Fig. 4 shows the dissipation curve of fenpropidin in the wheat plant under field condition. The initial concentrations of fenpropidin in soil were 2.19 and 0.63 mg/kg in Zhangjiakou and Changchun. The dissipation regressive equation could be described by the following equations: C ¼1.2998e 0.0518T (g ¼0.9103) with the half life 13.4 days in Zhangjiakou, and C ¼0.7307e 0.042T (g ¼0.9598) with the half life 16.5 days in Changchun. More than 80% of the initial residue dissipated after 40 days. Fig. 5 shows the dissipation curve of fenpropidin in the soil under field condition. 3.3. Terminal residue levels The terminal residues of fenpropidin in wheat straw, grain and soil collected from the treated plots are summarized in Table 2. When the pesticide fenpropidin was sprayed at the recommended
dose (330 g a.i. ha 1) for 2 and 3 times, the terminal residue was below 0.21 mg kg 1 in grain, 0.06 to 0.30 mg kg 1 in soil and 0.78 to 3.22 mg kg 1 in straw.
4. Discussion The results showed the dissipation of fenpropidin in wheat plant was faster than that in soil. Take Zhangjiakou for example, the half-lives were 3.3 days in wheat plant compare with 13.4 days in soil. The situation was similar in Changchun where the half-lives were 3.1 days in wheat plant and 16.5 days in soil. Usually, besides the effect of some physical and chemical factors like light, heat, pH and moisture, growth dilution factor might have played a significant role in pesticides degradation in the plant (Tewary et al., 2005). However the factors that influence pesticide persistence in soil are climate, soil properties and the
H. Zhao et al. / Ecotoxicology and Environmental Safety 77 (2012) 52–56
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Fig. 4. Dissipation of fenpropidin residues in wheat plant (Changchun, Zhangjiakou).
Fig. 3. Chromatogram of GC–MS for soil sample: primary fragmentation chromatogram (A), standard (0.01 mg kg 1) (B).
Table 1 Fortified recoveries of fenpropidin and LOQ (n¼ 5). Sample type
Fortified level (mg/kg)
Soil
0.01 0.02 0.05
Wheat plant
Average recoveries (%)
RSD (%)
LOQ (mg/kg)
91.8 95.4 94.3
9.5 6.4 2.4
0.01
0.01 0.02 0.2
91.6 94.5 85.1
3.3 2.4 2.0
0.01
Straw
0.02 0.1 1
106.7 107.9 107.8
3.2 3.2 2.7
0.02
Grain
0.01 0.02 0.2
92.0 87.4 98.3
4.3 5.7 4.8
0.01
Fig. 5. Dissipation of fenpropidin residues in soil (Changchun, Zhangjiakou).
Table 2 Terminal residues of fenpropidin in wheat grain, straw and soil in Zhangjiakou and Changchun, China in 2010. Sample type
physical and chemical properties of the pesticide (Arias-Estevez et al., 2006; Pateiro-Moure et al., 2008). It has been reported fenpropidin was stable to UV light when in aqueous solution and stable to hydrolysis at 80 1C at pH 4, 7 and 10 (EFSA Scientific Report, 2007). It could be predicted that the different dissipation rates in wheat plant caused by the growth dilution factor. The initial residue levels of wheat plant and soil were 59.4 35.7 and 26.3 18.6 mg/kg, respectively. Although the initial residues of fenpropidin in two sites were different, the dissipation rate and half-lives were similar. According to the terminal residue results, the residue behavior of fenpropidin in grain, straw and soil under different treatments almost followed a trend in which higher fenpropidin doses and shorter harvest intervals led to more residual fenpropidin. The terminal residue levels in the order from high to low were straw, soil and grain. FAO/WHO, US have not established maximum residue limits (MRLs) for fenpropidin. In the EU, the fenpropidin MRL in wheat was 0.5 mg kg 1. From the residue results in field at two fields at harvest time, the maximum terminal residues in
Soil
Dosage Numbers of Days after (g a.i. ha 1) times spraying sprayed
Residue (mg/kg) Zhangjiakou
Changchun
330
20 30 40 20 30 40
0.15 70.011 0.22 70.028 0.21 70.032 0.29 70.035 0.307 0.062 0.29 70.031
0.06 70.003 0.11 7 0.006 0.07 70.003 0.07 70.003 0.06 70.002 0.08 70.002
20 30 40 20 30 40
0.107 0.009 0.047 0.002 ND 0.107 0.006 0.037 0.001 ND
0.16 7 0.007 0.07 70.004 0.08 70.001 0.21 7 0.010 0.19 7 0.010 0.09 70.012
20 30 40 20 30 40
2.407 0.015 1.25 70.011 0.78 70.020 2.29 70.057 1.33 70.096 1.077 0.011
2.61 7 0.177 1.28 7 0.018 1.05 70.076 3.22 7 0.410 1.52 7 0.061 1.20 70.084
2
3
Grain
330
2
3
Straw
330
2
3
Note: ND: not detected, r limit of detection (LOD).
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H. Zhao et al. / Ecotoxicology and Environmental Safety 77 (2012) 52–56
grain at interval of 20 days was below 0.5 mg kg 1. This result suggested that it was safe to harvest 20 days after applying the recommended dose of fenpropidin with an application interval of 10 days. However the terminal residue was dependent on the application methods. High amounts of residue were observed when more applications or shorter harvest intervals were used. We suggest that these extra application or insufficient harvest intervals should be prevented to ensure food safety.
5. Conclusion In this paper, the dissipation of fenpropidin residues in wheat plant and soil under field condition was investigated for the safe use of this pesticide and for the health of consumers. The results show that when fenpropidin was used under the experiment design, the maximum terminal residue in grain at interval of 20 days was below 0.5 mg kg 1. This result suggests that it is safe to harvest 20 days after applying the recommended dose of fenpropidin with an application interval of 10 days. The work could be a reference for the MRL establishment and safe use of fenpropidin. References Arias-Este´vez, M., Lo´pez-Periago, E., Martı´nez-Carballo, E., Simal-Ga´ndara, J., 2006. Carbofuran sorption kinetics by corn crop soils. Bull. Environ. Contam. Toxicol. 77, 267–273.
Commission Regulation (EU), No 600/2010 of 8 July 2010. Amending Annex IIIA to Regulation (EC) No 396/2005 of the European Parliament and of the Council as Regards Additions and Modification of the Examples of Related Varieties or other Products to which the same MRL Applies. Official Journal of the European Union L 174/18. European Food Safety Authority Scientific Report, 2007. Conclusion Regarding the Peer Review of the Pesticide Risk Assessment of the Active SubstanceFenpropidin. vol. 124, pp. 1–84. GB/T 19648-2006, Method for Determination of 500 Pesticides and Related Chemicals Residues in Fruits and Vegetables—GC–MS Method. Hiemstra, Maurice, de Kok, Andre´, 2007. Comprehensive multi-residue method for the target analysis of pesticides in crops using liquid chromatography–tandem mass spectrometry. J. Chromatogr. A 1154 (1–2), 3–65. Pateiro-Moure, Mirian, Arias-Este´vez, Manuel, Lo´pez-Periago, Eugenio, Martı´nezCarballo, Elena, Simal-Ga´ndara, Jesu´s, 2008. Occurrence and downslope mobilization of quaternary herbicide residues in vineyard-devoted soils. Bull. Environ. Contam. Toxicol. 80, 407–411. Pizzutti, Ionara R., de Kok, Andre´, Hiemstra, Maurice, Wickert, Cristine, Prestes, Osmar D., 2009. Method validation and comparison of acetonitrile and acetone extraction for the analysis of 169 pesticides in soya grain by liquid chromatography–tandem mass spectrometry. J. Chromatogr. A 1216 (21), 4539–4552. Pesticide Safety Directorate,1993. Food and Environment Protection Act 1985 (Part III) Control of Pesticide Regulations 1986, Issue No. 67. ¨ Schafer, Ralf Bernhard, Mueller, Ralf, Brack, Werner, Wenzel, Klaus-Dieter, Streck, Georg, Ruck, Wolfgang, Liess, Matthias, 2008. Determination of 10 particleassociated multiclass polar and semi-polar pesticides from small streams using accelerated solvent extraction. Chemosphere 70 (11), 1952–1960. Tewary, Dhananjay Kumar, Kumar, Vipin, Ravindranath, S.D., Shanker, Adarsh, 2005. Dissipation behavior of bifenthrin residues in tea and its brew. Food Control 16, 231–237. Wang, Fahong, He, Zhonghu, Sayre, Ken, 2009. Wheat cropping systems and technologies in China. Field Crops Res. 111 (3), 181–188.
Contents lists available at SciVerse ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Dissipation and residue of fenpropidin in wheat and soil under field conditions Huiyu Zhao, Jiaying Xue, Naiwen Jiang, Wei Peng, Fengmao Liu n College of Science, China Agricultural University, Beijing 100193, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2011 Received in revised form 9 October 2011 Accepted 12 October 2011 Available online 9 November 2011
The residue levels and dissipation rate of fenpropidin in wheat and soil were investigated by LC–MS/MS and GC–MS, respectively. The dissipation rates of fenpropidin were described using first-order kinetics and its half-life ranged from 3.1 to 3.3 days in wheat plants and 13.4–16.5 days in soils. During harvest time, the terminal residues of fenpropidin in wheat were below the EUs maximum residue limit (MRL, 0.5 mg kg 1) when collected 20 days after the final application, which suggested that the use of this fungicide was safe for humans. The residues persistence varied between two geographically separated experimental sites, indicating that it might be influenced by climate, soil properties and growth dilution factor. These results would be helpful in setting MRL guidance of fenpropidin in wheat in China. Crown Copyright & 2011 Published by Elsevier Inc. All rights reserved.
Keywords: Fenpropidin Pesticide residue Dissipation Wheat Soil
1. Introduction Wheat (Triticum aestivum Linn.) ranks third leading crop in China after rice (Oryza sativa) and maize (Zea mays), which is cultivated throughout the country. It is one of the major crops in Chinese daily life (Wang et al., 2009). The wheat was ground into flour to make bread, biscuits, cakes, noodles, dumplings and so on. Wheat can be fermented into beer, alcohol or biofuel and even the wheat straw can be used as fertilizer and feedstuff. Fenpropidin, 1-[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropyl]-piperidine, is a piperidine fungicide, which belongs to ergosterol biosynthesis inhibitor by inhibition of steroid reduction (sterol-D14-reductase) and isomerisation (D8 to D7-isomerase). It is particularly effective against powdery mildew (Erysiphe graminis), rusts (Puccinia spp.) and leaf spots such as Rhynchosporium secalis (Food and Environment Act 1985 Part 3). Fig. 1 shows the chemical structure of fenpropidin. The analytical methods for fenpropidin were reported in recent years. A method of accelerated solvent extraction (Sch¨afer et al., 2008) was used to determine fenpropidin and other 4 polar pesticide residues in some small streams. But the recovery of fenpropidin was only 56%. A gas chromatography with mass spectrometric detection (GC–MS) method was carried out for determination of 500 pesticides including fenpropidin residues in fruit and vegetables (GB/T 196482006). Hiemstra and Kok (2007) developed a multi-residue method for the analysis of 171 pesticides including fenpropidin in crops using liquid chromatography–tandem mass spectrometry and acetone,
n
Corresponding author. Fax: þ86 10 62733620. E-mail addresses: [email protected], [email protected] (F. Liu).
dichloromethane, petroleum were used as extraction solvents. Pizzutti et al. (2009) compared the acetonitrile and acetone extraction efficiencies for the analysis of fenpropidin in soya grain by liquid chromatography–tandem mass spectrometry. The results demonstrated that the LOQ of the method with acetonitrile extraction was 0.05 mg/kg. Fenpropidin had been registered in Italy, France, Denmark and Switzerland on wheat, barley, grape and banana. To ensure the safety of food for consumers and to regulate international trade and legislation, some countries have established maximum residue limits (MRLs) for fenpropidin in some commodities. In EU, the MRL was set at 0.5 mg/kg in wheat (Commission Directive 2004 EU No 600/2010). In New Zealand, the MRL was set at 0.02 mg/kg in wheat grain. However, to our knowledge there is no paper reported the residue dissipation of fenpropidin in wheat under field condition. No residue limit has been established of fenpropidin in wheat in China. In this work, simple LC–MS/MS and GC/MS methods were established to detect the residue of fenpropidin in wheat and soil, respectively. A field study was done to investigate the dissipation of fenpropidin in wheat plant and soil. This work would help the government to establish the MRL of fenpropidin in wheat and to provide guidance on proper and safe use of this pesticide.
2. Chemicals and methods 2.1. Chemicals and reagents Fenpropidin standard (99.5% purity) was supplied from J&K Science Ltd.; sodium chloride (AR) was purchased from Beijing Chemical Reagents Company, China; Acetonitrile (HPLC grade) for chromatography was purchased from Thermo
0147-6513/$ - see front matter Crown Copyright & 2011 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2011.10.017
H. Zhao et al. / Ecotoxicology and Environmental Safety 77 (2012) 52–56
Fig. 1. Chemical structure of fenpropidin. Fisher Co., Ltd., America; HPLC grade water was made by purifying demineralized water in a Milli-Q Plus ultra-pure water system (Millipore, Molsheim, France); primary secondary amine (PSA) and graphitized carbon black (GCB) sorbent were from Tianjin Bonna-Agela technologies Ltd., China. 2.2. Field experiment design Experiments were conducted at two sites: Zhangjiakou (114.871E, 40.781N) and Changchun (125.411E, 43.811N), China. Each experimental plot was 30–50 m2 and each treatment was carried out in triplicates. A buffer zone was used to separate the plots of different treatments. The experiments were designed according to NY/T 788-2004 (Guideline on Pesticide Residue Trials) issued by Ministry of Agriculture, People’s Republic of China. During the experimental period, the number of rainfall events in Zhangjiakou and Changchun was 13 and 38, respectively; the average temperature was 23.5 and 22.2 1C, respectively; and the average relative humidity was 43.1% and 75.8%, respectively. The soil in Zhangjiakou field was sandy loam, with the organic matter 1.54 g kg 1, and pH 7.94; the soil in Changchun was sandy clay loam with the organic matter 2.94 g kg 1, and pH 6.10. To investigate the dissipation of fenpropidin in wheat plant and soil, fenpropidin formulation (275 g a.i./L of EC) was sprayed to the wheat and soil with the dosage of 495 g a.i. ha 1 (1.5 times the recommended dosage). To investigate the terminal residues of fenpropidin in wheat grain, straw and soil, the recommended dose (330 g a.i. ha 1, with two treatments: spray 2 times and 3 times, with three harvest intervals: 20, 30, 40 days) were conducted in separate plots. The application interval was 10 days. A plot with the same size but no fenpropidin application was as control. 2.3. Sample and storage Representative samples were collected from each plot at different time intervals. The sample of wheat plant and soil were collected at day 0, 0.5, 1, 3, 5, 7, 10, 14, 21, 30 and 40 after spraying to investigate the dissipation of fenpropidin. Wheat grain, straw and soil samples were collected at day 20, 30 and 40 after application to determine the terminal residue of fenpropidin. All the samples were stored at 20 1C prior to analysis. 2.4. Analytical procedure 2.4.1. Sample preparation Wheat: The wheat includes three different matrices: wheat plant, grain and straw. Each matrix was grounded into small pieces or powder with a vegetation disintegrator, and then stored in a deep freezer at 20 1C. Soil: The soil samples were sifted through a 40-mesh sieve, and then stored in a deep freezer at 20 1C. 2.4.2. Sample extraction The wheat plant (4 g), grain (10 g) or straw (2 g) was weighted into 50-mL PTFE centrifuge tube. Subsequently, acetonitrile (40 mL) was added, and samples were shaken in a reciprocating shaker for 1 h. Then, the samples were centrifuged at 3000 rpm for 5 min. A 1 mL aliquot of the supernatant was used for clean up. Five gram of soil was mixed with 5 mL of water and 10 mL of acetonitrile in 50-mL PTFE centrifuge tube and then shaken in a reciprocating shaker for 1 h. The samples were centrifuged for 5 min at 3000 rpm. A 1 mL aliquot of the supernatant was used for clean up. 2.4.3. Cleanup procedure The supernatant above was transferred to 2-mL centrifuge tube, and 50 mg of PSA was added to soil and 30 mg of GCB to wheat straw, grain and plant sample. Then the samples were mixed in a blender for 30 s, and centrifuged at 10000 rpm for 1 min. The supernatant solution was transferred to a HPLC sample vial for instrumental analysis after filtered through a 0.22 mm polypropylene filter.
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2.4.4. LC–ESI–tandem MS/MS Agilent 6410 series LC triple-quadrupole mass spectrometry (Agilent Technologies, USA), equipped with an electrospray ionization interface, was operated in the positive ion mode (ESI þ). Eclipse Plus C18 column (2.1 mm 5.0 cm, 3.5 mm) (Agilent technologies, USA) was used to separate the target compound from the interferences at 30 1C. The mobile phase was acetonitrile–water containing 0.1% formic acid (7/3, V/V) at the flow rate of 0.3 mL/min. The injection volume was 5 mL. The conditions for MS detection were: desolvation gas temperature 350 1C; desolvation gas flow rate 8.0 L/min; nebulizer gas (N2) pressure 35.0 psi; MS1 and MS2 Heater temperature at 100 1C, capillary voltage 4000 V. The multiple reaction monitoring (MRM) was applied and fenpropidin was calculated using the primary transition m/z 274.3-147.1 (Fig. 2). For instrument control, mass hunter workstation software data acquisition for triple quad B.02.01 (B 2043.12) and qualitative analysis version B.03.01/build 3.1.346.0 were used for data acquisition and processing. 2.4.5. GC–MS condition An Agilent Technologies 6890N network GC system equipped with an Agilent 7683 series autosampler, mass spectrometry detector (MSD) model 5975 network, and a 30 m 0.25 mm i.d. 0.25 mm VF-5MS capillary column (Varian Scientific, USA). The injector was held at 280 1C and the carrier gas was helium at a flow rate of 1.0 mL/min. The oven temperature was initially at 120 1C for 1 min, then increased to 260 1C at the rate of 20 1C/min holding for 1 min, and finally increased at the rate of 10 1C/min to 280 1C, kept for 5 min. Ion source temperature of the instrument was 230 1C, quadrupole temperature of 150 1C, transfer line of 280 1C, and solvent delay was 4 min. The mass spectrometry was in the electron ionization mode and 1 mL of sample was injected. Ions 98, 117, 273, 326 and 325 were monitored in Selected Ion Mode (SIM). TPP was quantified at m/z 326, fenpropidin was quantified at m/z 98 (Fig. 3). 2.4.6. Calculations The degradation kinetics of the fenpropidin in the wheat plant and soil were determined by plotting residue concentration over time. The residual concentration and half-life of fenpropidin were calculated by the first-order kinetics equations CT ¼ C0e KT and T1/2 ¼ln2/K, respectively, where T is the time (days) after pesticide application, CT is the residue concentration of the pesticide at time T, C0 is an initial pesticide concentration after application (at T¼0), K is a dissipation coefficient and T1/2 is defined as the time required for the pesticide residue level to fall to half of the initial residue level after application.
3. Results 3.1. Linearity, recovery and detection limits To determine the linearity, a 100 mg/L stock solution was prepared, and working solutions were prepared by diluting the stock solution with acetonitrile. The linear range was 1–500 ng/mL for LC–MS/MS detection and 10–500 ng/mL for GC–MS detection, respectively. The results showed good linearity with a correlation coefficient (g) of 0.9996 (LC–MS/MS) and 0.9994 (GC–MS). The limit of quantifications (LOQs) defined as the minimum fortified level of recovery, were 0.01 mg/kg in soil, wheat plant, straw, and 0.02 mg/kg for grain, respectively. The limit of detection (LOD) was 1 10 2 ng for GC–MS and 5 10 3 ng for LC–MS/MS at a signal-to-noise ratio of 3. The fortified recovery experiment was set at three concentration levels with five replicates at each level. The fortified recoveries of wheat plant, straw, grain and soil samples ranged from 83.6–97.9%, 102.9–112.4%, 81.2–101.5% and 80.7–102.1%, respectively; and the relative standard deviations (RSDs) of the recovery data were 2.0–9.5% (Table 1). 3.2. Dissipation of fenpropidin in wheat plant and soil The initial concentration of fenpropidin in wheat plant was 59.4 and 35.7 mg/kg in Zhangjiakou and Changchun, respectively. The dissipation regressive equation could be described by the following equations: C¼37.262e 0.209T (g ¼0.9361) with the half life 3.3 days in Zhangjiakou, and C ¼21.727e 0.2265T (g ¼ 0.9543) with the half life 3.1 days in Changchun. More than 96% of
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H. Zhao et al. / Ecotoxicology and Environmental Safety 77 (2012) 52–56
Fig. 2. Chromatogram of LC–MS/MS: secondary fragmentation chromatogram (A), standard in wheat plant (0.1 mg kg 1) (B), standard in straw (0.01 mg kg 1) (C) and standard in grain (0.01 mg kg 1) (D).
the initial residue dissipated after 14 days. Fig. 4 shows the dissipation curve of fenpropidin in the wheat plant under field condition. The initial concentrations of fenpropidin in soil were 2.19 and 0.63 mg/kg in Zhangjiakou and Changchun. The dissipation regressive equation could be described by the following equations: C ¼1.2998e 0.0518T (g ¼0.9103) with the half life 13.4 days in Zhangjiakou, and C ¼0.7307e 0.042T (g ¼0.9598) with the half life 16.5 days in Changchun. More than 80% of the initial residue dissipated after 40 days. Fig. 5 shows the dissipation curve of fenpropidin in the soil under field condition. 3.3. Terminal residue levels The terminal residues of fenpropidin in wheat straw, grain and soil collected from the treated plots are summarized in Table 2. When the pesticide fenpropidin was sprayed at the recommended
dose (330 g a.i. ha 1) for 2 and 3 times, the terminal residue was below 0.21 mg kg 1 in grain, 0.06 to 0.30 mg kg 1 in soil and 0.78 to 3.22 mg kg 1 in straw.
4. Discussion The results showed the dissipation of fenpropidin in wheat plant was faster than that in soil. Take Zhangjiakou for example, the half-lives were 3.3 days in wheat plant compare with 13.4 days in soil. The situation was similar in Changchun where the half-lives were 3.1 days in wheat plant and 16.5 days in soil. Usually, besides the effect of some physical and chemical factors like light, heat, pH and moisture, growth dilution factor might have played a significant role in pesticides degradation in the plant (Tewary et al., 2005). However the factors that influence pesticide persistence in soil are climate, soil properties and the
H. Zhao et al. / Ecotoxicology and Environmental Safety 77 (2012) 52–56
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Fig. 4. Dissipation of fenpropidin residues in wheat plant (Changchun, Zhangjiakou).
Fig. 3. Chromatogram of GC–MS for soil sample: primary fragmentation chromatogram (A), standard (0.01 mg kg 1) (B).
Table 1 Fortified recoveries of fenpropidin and LOQ (n¼ 5). Sample type
Fortified level (mg/kg)
Soil
0.01 0.02 0.05
Wheat plant
Average recoveries (%)
RSD (%)
LOQ (mg/kg)
91.8 95.4 94.3
9.5 6.4 2.4
0.01
0.01 0.02 0.2
91.6 94.5 85.1
3.3 2.4 2.0
0.01
Straw
0.02 0.1 1
106.7 107.9 107.8
3.2 3.2 2.7
0.02
Grain
0.01 0.02 0.2
92.0 87.4 98.3
4.3 5.7 4.8
0.01
Fig. 5. Dissipation of fenpropidin residues in soil (Changchun, Zhangjiakou).
Table 2 Terminal residues of fenpropidin in wheat grain, straw and soil in Zhangjiakou and Changchun, China in 2010. Sample type
physical and chemical properties of the pesticide (Arias-Estevez et al., 2006; Pateiro-Moure et al., 2008). It has been reported fenpropidin was stable to UV light when in aqueous solution and stable to hydrolysis at 80 1C at pH 4, 7 and 10 (EFSA Scientific Report, 2007). It could be predicted that the different dissipation rates in wheat plant caused by the growth dilution factor. The initial residue levels of wheat plant and soil were 59.4 35.7 and 26.3 18.6 mg/kg, respectively. Although the initial residues of fenpropidin in two sites were different, the dissipation rate and half-lives were similar. According to the terminal residue results, the residue behavior of fenpropidin in grain, straw and soil under different treatments almost followed a trend in which higher fenpropidin doses and shorter harvest intervals led to more residual fenpropidin. The terminal residue levels in the order from high to low were straw, soil and grain. FAO/WHO, US have not established maximum residue limits (MRLs) for fenpropidin. In the EU, the fenpropidin MRL in wheat was 0.5 mg kg 1. From the residue results in field at two fields at harvest time, the maximum terminal residues in
Soil
Dosage Numbers of Days after (g a.i. ha 1) times spraying sprayed
Residue (mg/kg) Zhangjiakou
Changchun
330
20 30 40 20 30 40
0.15 70.011 0.22 70.028 0.21 70.032 0.29 70.035 0.307 0.062 0.29 70.031
0.06 70.003 0.11 7 0.006 0.07 70.003 0.07 70.003 0.06 70.002 0.08 70.002
20 30 40 20 30 40
0.107 0.009 0.047 0.002 ND 0.107 0.006 0.037 0.001 ND
0.16 7 0.007 0.07 70.004 0.08 70.001 0.21 7 0.010 0.19 7 0.010 0.09 70.012
20 30 40 20 30 40
2.407 0.015 1.25 70.011 0.78 70.020 2.29 70.057 1.33 70.096 1.077 0.011
2.61 7 0.177 1.28 7 0.018 1.05 70.076 3.22 7 0.410 1.52 7 0.061 1.20 70.084
2
3
Grain
330
2
3
Straw
330
2
3
Note: ND: not detected, r limit of detection (LOD).
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grain at interval of 20 days was below 0.5 mg kg 1. This result suggested that it was safe to harvest 20 days after applying the recommended dose of fenpropidin with an application interval of 10 days. However the terminal residue was dependent on the application methods. High amounts of residue were observed when more applications or shorter harvest intervals were used. We suggest that these extra application or insufficient harvest intervals should be prevented to ensure food safety.
5. Conclusion In this paper, the dissipation of fenpropidin residues in wheat plant and soil under field condition was investigated for the safe use of this pesticide and for the health of consumers. The results show that when fenpropidin was used under the experiment design, the maximum terminal residue in grain at interval of 20 days was below 0.5 mg kg 1. This result suggests that it is safe to harvest 20 days after applying the recommended dose of fenpropidin with an application interval of 10 days. The work could be a reference for the MRL establishment and safe use of fenpropidin. References Arias-Este´vez, M., Lo´pez-Periago, E., Martı´nez-Carballo, E., Simal-Ga´ndara, J., 2006. Carbofuran sorption kinetics by corn crop soils. Bull. Environ. Contam. Toxicol. 77, 267–273.
Commission Regulation (EU), No 600/2010 of 8 July 2010. Amending Annex IIIA to Regulation (EC) No 396/2005 of the European Parliament and of the Council as Regards Additions and Modification of the Examples of Related Varieties or other Products to which the same MRL Applies. Official Journal of the European Union L 174/18. European Food Safety Authority Scientific Report, 2007. Conclusion Regarding the Peer Review of the Pesticide Risk Assessment of the Active SubstanceFenpropidin. vol. 124, pp. 1–84. GB/T 19648-2006, Method for Determination of 500 Pesticides and Related Chemicals Residues in Fruits and Vegetables—GC–MS Method. Hiemstra, Maurice, de Kok, Andre´, 2007. Comprehensive multi-residue method for the target analysis of pesticides in crops using liquid chromatography–tandem mass spectrometry. J. Chromatogr. A 1154 (1–2), 3–65. Pateiro-Moure, Mirian, Arias-Este´vez, Manuel, Lo´pez-Periago, Eugenio, Martı´nezCarballo, Elena, Simal-Ga´ndara, Jesu´s, 2008. Occurrence and downslope mobilization of quaternary herbicide residues in vineyard-devoted soils. Bull. Environ. Contam. Toxicol. 80, 407–411. Pizzutti, Ionara R., de Kok, Andre´, Hiemstra, Maurice, Wickert, Cristine, Prestes, Osmar D., 2009. Method validation and comparison of acetonitrile and acetone extraction for the analysis of 169 pesticides in soya grain by liquid chromatography–tandem mass spectrometry. J. Chromatogr. A 1216 (21), 4539–4552. Pesticide Safety Directorate,1993. Food and Environment Protection Act 1985 (Part III) Control of Pesticide Regulations 1986, Issue No. 67. ¨ Schafer, Ralf Bernhard, Mueller, Ralf, Brack, Werner, Wenzel, Klaus-Dieter, Streck, Georg, Ruck, Wolfgang, Liess, Matthias, 2008. Determination of 10 particleassociated multiclass polar and semi-polar pesticides from small streams using accelerated solvent extraction. Chemosphere 70 (11), 1952–1960. Tewary, Dhananjay Kumar, Kumar, Vipin, Ravindranath, S.D., Shanker, Adarsh, 2005. Dissipation behavior of bifenthrin residues in tea and its brew. Food Control 16, 231–237. Wang, Fahong, He, Zhonghu, Sayre, Ken, 2009. Wheat cropping systems and technologies in China. Field Crops Res. 111 (3), 181–188.