Article pubs.acs.org/JAFC
Insecticide Residues in Head Lettuce, Cabbage, Chinese Cabbage, and Broccoli Grown in Fields Miao-Fan Chen,*,† Jung-Fang Chen,§ Jing-Jing Syu,# Chi Pei,# and Hsiu-Pao Chien# †
Department of Biotechnology, National Formosa University, 64 Wunhua Road, Huwei Township, Yunlin County 63201, Taiwan Department of Agronomy, National Chung Hsing University, 250 Kuo Kuang Road, Taichung City 402, Taiwan # Division of Residue Control, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, 11 Guangming Road, Wufeng, Taichung City 403, Taiwan §
ABSTRACT: The residues of four insecticides belonging to different families were studied on head lettuce (Lactuca sativa L. var. capitata L.), cabbage (Brassica oleracea Linn. var. capitata DC.), Chinese cabbage (Brassica pekinensis Skeels), and broccoli (Brassica oleracea var. italica) after pesticide application. To reduce application variability, a tank mix of acetamiprid 20% SP, chlorpyrifos 22.5% EC, deltamethrin 2.4% SC, and methomyl 40% SP was applied at recommended and double doses. Initial deposits of all pesticides on head lettuce were higher than those of the other three crops. The residues of chlorpyrifos and deltamethrin were higher than the maximum residue limits (MRLs) at recommended preharvest intervals (PHIs) on head lettuce and Chinese broccoli treated with higher doses. The residues of methomyl on head lettuce also showed the same phenomenon. KEYWORDS: insecticides, residue, analysis, vegetables
■
INTRODUCTION High temperature and humidity contribute to the quick spread of pests so insecticides are widely required for vegetable protection in Taiwan. The integrated pest management strategies require spraying insecticides with different modes of action during the cultivation of vegetables. Consequently, studies of pesticide residues in vegetables have become essential to ensure food safety. Although many studies have been carried out for assessing residues of one pesticide in one crop, the differences between residues of different pesticides in similar types of vegetables is largely yet unknown. The purpose of this study was to compare the dissipation of four different insecticides, namely, acetamiprid, chlorpyrifos, deltamethrin, and methomyl, in one kind of leafy vegetable group in Taiwan.1 To facilitate comparison of pesticide residues in crops, four widely used pesticides with significantly different physicochemical properties were selected to test. These insecticides are commonly used for controlling aphids, diamond-back moths (Plutella xylostella), and striped flea beetles (Phyllotreta striolata) on vegetables in Taiwan. Some physicochemical properties of the four pesticides are summarized in Table 1. Acetamiprid is a neonicotinoid insecticide, which has a relatively low log POW of 0.80 and a relatively high water solubility of 4250 mg/L. Chlorpyrifos is a nonsystemic organophosphate insecticide, which has the log POW of 4.7 and a lower water solubility of 1.4 mg/L. Deltamethrin is a contact pyrethroid insecticide, which has a log POW of 4.6 similar to that of chlorpyrifos but a very low water solubility of 95%) of acetamiprid, chlorpyrifos, deltamethrin, and methomyl were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Stock standard solutions were prepared with acetonitrile in a concentration of 500 or 1000 μg/ mL. Working solutions were prepared by dilution of the stock solution Received: Revised: Accepted: Published: 3644
December March 26, March 31, March 31,
17, 2013 2014 2014 2014
dx.doi.org/10.1021/jf405555w | J. Agric. Food Chem. 2014, 62, 3644−3648
Journal of Agricultural and Food Chemistry
Article
Table 1. Summary of the Physicochemical Properties of the Four Insecticides Studied
with acetone, acetonitrile, or methanol. Stock solutions were diluted with proper solvent to make standard solutions in the range of 0.01−5 mg/L to prepare calibration curves. All of the solvents were of pesticide grade (TEDIA, USA) or analytical grade and purchased from Merck Co. (Darmstadt, Germany). Anhydrous Na2SO4 was purchased from Merck (Taipei, Taiwan). Commercial acetamiprid 20% SP, chlorpyrifos 22.5% EC, deltamethrin 2.4% SC, and methomyl 40% SP were purchased from Great Victory Chemical, Xinnong, Bayer, and Du Pont, respectively, Taiwan. Extraction Procedures. A 20 g portion of vegetable sample was weighed in a glass jar of a blender and homogenized with 60 mL of acetone for 1 min. The macerate was then filtered under vacuum through a funnel using Advantec no. 2 filter paper (Toyo Roshi Kaisha, Japan). The volume of filtrate was adjusted to 100 mL with the same solution. For cabbage and Chinese cabbage before solid phase extraction (SPE), a 10 mL portion of filtrate solvent was transferred into a separatory funnel and partitioned twice with 50/30 mL of ethyl acetate. The organic phase was collected, filtered through 20 g of anhydrous Na2SO4, and evaporated to dryness under reduced pressure. For all vegetables, the residue was dissolved in 3.5 mL of a solution of n-hexane/acetone (90:10, v/v) and loaded to the Florisil (1000 mg/6 mL, JT Baker) cartridge (×2) for SPE. The combined cartridges were previously conditioned with 10 mL of n-hexane, followed by sample loading and collecting with 3.5 + 7 mL of n-hexane/acetone (90:10, v/ v) and collecting with 8 mL of n-hexane/acetone (50:50, v/v) after washing with 10 mL of n-hexane/acetone (90:10, v/v). The combined eluents were evaporated to dryness under nitrogen and reconstituted to 2 mL of acetonitrile for GC/μ-ECD analysis to determine acetamiprid, chlorpyrifos, and deltamethrin. A 1 mL portion of acetonitrile was reconstituted to methanol for the HPLC-FLD (OPA post reaction) analysis to determine methomyl. GC/μ-ECD. An Agilent 6890 (Taipei, Taiwan Ltd.) gas chromatograph equipped with a 63Ni μ-electron-capture detector (μ-ECD) and a fused silica capillary column DB-608 (30 m × 0.25 mm i.d., 0.25 μm film thickness) was used. The operating conditions were as follows: initial temperature, 200 °C (5 min), increased at 10 °C min−1 to 220 °C for 2 min and increased at 20 °C min−1 to 280 °C for 10 min; injector temperature, 270 °C; N2 carrier gas; column average linear velocity (μ = 40 cm/s) operated in the splitless mode; injection volume, 1 μL; detector temperature, 300 °C; makeup gas, N2. Identification of acetamiprid, chlorpyrifos, and deltamethrin was
carried out by comparing their individual retention times on GC/μECD. The amounts of standards in the test solution were calculated from the peak areas. If the amount corresponding to the peak areas was larger than that of the maximum amount from the calibration curve, the test solutions were diluted to an appropriate concentration. HPLC with Postcolumn Derivatization and Fluorescence Detection (FLD). The system consisted of an Agilent model 1100 HPLC quaternary pump, a model G1313A autoinjector, and a model G1321A fluorescence detector. All runs were acquired and processed using the Agilent ChemStation software. Chromatographic separations were carried out on a Merck LiChroCart 250 × 4.0 mm i.d. cartridge column, packed with LiChrosphere 100 RP select B (5 μm), in conjunction with a 4.0 × 4.0 mm i.d. guard column, packed with LiChrosorb 100 RP-18. Isocratic analysis was performed with a methanol/water (60:40) mobile phase at a flow rate of 0.8 mL min−1. The postcolumn reaction system consisted of a Pickering PCX 5000 postcolumn reaction module. The first pump added a 0.05 M NaOH solution through a vortex mixing chamber (1.2 μL volume) to the column eluent. Hydrolysis took place in a PTFE knitted tube (5.1 m × 0.25 mm i.d.; 1.0 mL volume), kept at a temperature of 100 °C. After the hydrolysis reaction coil, o-phthalaldehyde/thiofluor reagent (0.05 M) was added through the second mixing chamber. The flow rate of both reaction solutions was 0.3 mL min−1. The excitation and emission wavelengths for fluorescence detection were 330 and 460 nm, respectively. Recovery Test. The specificity of the analytical method was confirmed by analysis of blank vegetable samples in triplicate and by processing through the extraction procedures described above. No interference peak was observed on the chromatograms from the blank samples. The accuracy and precision of the analytical method were confirmed by the recovery test of acetamiprid, chlorpyrifos, deltamethrin, and methomyl. Field Experiments. The field experiments on head lettuce (Lactuca sativa L. var. capitata L.), cabbage (B. oleracea Linn. var. capitata DC.), Chinese cabbage (B. pekinensis Skeels), and broccoli (B. oleracea var. italica) were conducted in a field in Changhua, Taiwan, from September 15, 2008, to January 14, 2009. The experimental design was a split-plot arrangement of a randomized complete block with three replicates. The tank mix of acetamiprid 20% SP, chlorpyrifos 22.5% EC, deltamethrin 2.4% SC, and methomyl 40% SP was applied at the recommended (label) dose (0.2, 1.5, 1.0, and 0.8 3645
dx.doi.org/10.1021/jf405555w | J. Agric. Food Chem. 2014, 62, 3644−3648
Journal of Agricultural and Food Chemistry
Article
Table 2. Mean Recoveries (R) and Relative Standard Deviations (RSD) of Acetamiprid, Chlorpyrifos, Deltamethrin, and Methomyl in Four Vegetables cabbage
Chinese cabbage
broccoli
spike levels, n = 3 (ppm)
%R
% RSD
%R
% RSD
%R
% RSD
%R
% RSD
acetamiprid
0.125 0.25 0.5
75.9 76.9 76.3
1.1 3.0 2.9
77.4 91.4 80.9
4.5 2.6 3.9
89.2 90.0 90.9
1.5 2.7 2.3
80.9 85.8 82.3
5.9 4.8 0.9
chlorpyrifos
0.25 0.5 1
77.4 82.4 84.1
0.7 0.3 0.2
78.1 81.3 80.2
5.2 2.7 2.2
80.1 84.1 82.7
4.2 1.2 1.9
83.1 83.8 81.9
6.7 2.6 1.3
deltamethrin
0.25 0.5 1
72.3 83.9 82.5
1.5 1.6 1.4
76.3 83.1 77.8
5.0 2.6 3.2
90.8 92.1 100.8
3.5 1.5 1.3
85.9 85.7 80.4
9.6 3.2 4.2
methomyl
0.25 0.5 1
83.5 80.5 83.4
0.4 2.0 1.7
86.2 88.4 93.8
9.5 5.2 5.2
109.0 97.0 92.6
3.4 4.6 1.1
86.4 94.2 84.8
2.1 3.7 0.3
L/ha) and at 2 times the rates (0.4, 3.0, 2.0, and 1.6 L/ha), respectively. They were applied with a hand-driven knapsack sprayer. One application was conducted at 65 days (for Chinese cabbage and head lettuce), 80 days (for cabbage), and 98 days (for broccoli) after seeding of the respective vegetables. Sampling and Storage. Sample (12 plants at least) collection was initiated 2−3 h after foliar application of a tank mix of four pesticides. The process of collection was repeated at 3, 6, 9, 12, 15, 18, and 21 days afterward to determine the residues of four pesticides in/on vegetable samples. Meteorological conditions were continuously recorded. During the period of pesticide application and sampling, daily total rainfall amounts ranged from 0 to 18.7 mm/day. The minimum and maximum temperatures were 10.1 and 31.1 °C, respectively. The stability of pesticides in vegetable samples during frozen storage was evaluated using 20 g aliquots of blank vegetable samples. The prepared blank samples in triplicate were spiked at levels of 0.25, 0.5, and 1 ppm (μg/g) of the four pesticides. The residues in blank samples were determined as described above.
■
head lettuce
pesticide
Figure 1. Dissipation of acetamiprid in different wrapped leaf vegetables treated with higher doses. MRL: standards for pesticide residue limits of acetamiprid in the four vegetables, Food and Drug Administration, Ministry of Health and Welfare, Taiwan.
RESULTS AND DISCUSSION
Recoveries of Pesticides. The recoveries of analytical methods were 72.3−109.0% (levels, 0.125−1.0 mg/kg; RSD, 0.3−9.6%; n = 3) for the residue determination of four insecticides on head lettuce, broccoli, cabbage, and Chinese cabbage. Limits of quantification (defined as a signal-to-noise ratio >10) were 0.01 mg/kg (Table 2). Comparative Dissipation of Pesticides on Vegetables. Residues of four pesticides in/on four vegetable samples collected after pesticide application at double doses are shown in Figure 1−4. Whereas the initial deposits of all pesticides on head lettuce were higher than those of the other three crops at 0 days after application, the residues on all crops dissipated with time. On the four vegetables, acetamiprid residues declined to less than the tolerance (MRL), 2.0 mg/kg, at the recommended preharvest interval (PHI, 6−12 days, Figure 1). On head lettuce and broccoli, the concentrations of chlorpyrifos and deltamethrin were still higher than the tolerance at the PHI of 6−10 days (Figures 2 and 3). Only on the head lettuce were the residues of methomyl higher than 0.7 mg/kg at 6 days after application (Figure 4). Ripley et al. compared the residues of pesticides on different vegetables. The residues of five of the nine pesticides applied to cabbage, Chinese cabbage, and bok choi did not differ significantly from 3 to 14 days after application, but the
Figure 2. Dissipation of chlorpyrifos in different wrapped leaf vegetables treated with higher doses. MRL: standards for pesticide residue limits of chlorpyrifos in the four vegetables, Food and Drug Administration, Ministry of Health and Welfare, Taiwan.
residues of cypermethrin and three fungicides were significantly higher on Chinese cabbage and bok choi.8 Residues were generally significantly higher on bok choi than on the other cultivars.8 Ripley et al. found that structure significantly affects 3646
dx.doi.org/10.1021/jf405555w | J. Agric. Food Chem. 2014, 62, 3644−3648
Journal of Agricultural and Food Chemistry
Article
the influence of various factors such as differences in crop species, plant cultivation methods, and physicochemical properties of the pesticides.7 They found that the variability factors in the residue levels of both pesticides in cabbage were clearly higher than those in grapes.7 Yu et al. reported that both the hermetic environment of a greenhouse and season affected dissipation rates of chlorpyrifos on pakchoi (leafy vegetable).11 They found that chlorpyrifos declined more rapidly outside than inside the greenhouse.11 Lozowicka et al. monitored the presence of pesticide residues in broccoli, Brussels sprouts, cauliflower, and head and Chinese cabbages produced in northeastern Poland (2006−2009), which could not be considered a serious public health problem.12 The global food classification and crop group was revised by Codex and the International Crop Grouping Consulting Committee (ICGCC). Head lettuce was grouped within the leafy vegetables but not a member of bulb or cole vegetables according to the classification of Codex or ICGCC. Our results indicated that head lettuce contains higher residues than other vegetables. The major production of vegetables is cabbage in Taiwan. Residues of eight of the nine pesticides on head lettuce were lower than on the other lettuces 1 day after the last application.8 There are significantly different residue potentials and cultivated behaviors between head lettuce and the other leafy lettuces, so that head lettuce is not suitable for grouping within the leafy vegetables with small leaves.8 The present study demonstrated that the initial deposits of all pesticides in head lettuce were higher than in broccoli, cabbage, and Chinese cabbage. The concentrations of some pesticides were still higher than MRLs at the PHIs if treated with higher doses. Storage Stability. All treated samples in this study were stored frozen for no longer than 336 days between sampling and analysis. Fortified and unfortified samples were analyzed to evaluate the storage stability of insecticides in vegetable samples during storage at −20 ± 5 °C for up to 336 days. The results indicated that these insecticides were stable in samples under frozen condition for up to 336 days.
Figure 3. Dissipation of deltamethrin in different wrapped leaf vegetables treated with higher doses. MRL: standards for pesticide residue limits of deltamethrin in the four vegetables, Food and Drug Administration, Ministry of Health and Welfare, Taiwan.
■
Figure 4. Dissipation of methomyl in different wrapped leaf vegetables treated with higher doses. MRL: standards for pesticide residue limits of methomyl in the four vegetables, Food and Drug Administration, Ministry of Health and Welfare, Taiwan.
AUTHOR INFORMATION
Corresponding Author
*(M.-F.C.) Phone:+886-918-262605. E-mail mfchen99@nfu. edu.tw.
residue deposit and dissipation of pesticides on vegetables because of the higher exposed surface area-to-mass ratios; leafier crops had higher residue concentrations than head varieties.8 Their results showed that different PHIs might be necessary to achieve established MRLs for the different cultivars.8 Zhang et al. evaluated multiresidual dynamics of the pesticides in the spring (B. oleracea L. var. capitata) and autumn (B. chinensis L.) cabbages.5,6 Their results showed that the pesticide residues were closely related with the times and dosages of application and the weather after spraying.5,6 Raina and Raina studied the dissipation of chlorpyriphos on cauliflower following pesticide applications and recommend a safe preharvest interval of 6 days.9 Lots of variations of residue levels on different crops have been reported. Wang et al. studied pyrimorph residues in tomatoes and cucumbers and showed relatively similar dissipation rates, with half-lives of 5.8−7.7 days in tomatoes and 5.7−7.1 days in cucumbers.10 Fujita et al. considered the difference in the relative distribution of acetamiprid and cypermethrin between cabbage and grapes might be due to
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Dr. G. C. Li (former director of Taiwan Agricultural Chemicals and Toxic Substances Research Institute, gcli@tactri. gov.tw) for his valuable suggestions. We also thank the workers at the Taiwan Agricultural Chemicals and Toxic Substances Research Institute for their cooperation in the field experiments.
■
REFERENCES
(1) Food and Drug Administration, Ministry of Health and Welfare. Classification of Crops for the Pesticide Residue Limits in Foods; Standards for Pesticide Residue Limits in Foods. Taiwan; http://www.fda.gov. tw/EN/law.aspx, amended Sept 26, 2013 (accessed Sept 30, 2013). (2) Tomlin, C. D. S. A World Compendium, The Pesticide Manual, 14th ed.; British Crop Production Council: Surrey, UK, 2006. (3) Codex Classification of Foods and Animal Feeds. Pesticide Residues in Food; Codex Alimentarius, Joint FAO/WHO Food 3647
dx.doi.org/10.1021/jf405555w | J. Agric. Food Chem. 2014, 62, 3644−3648
Journal of Agricultural and Food Chemistry
Article
Standards Programme; FAO/WHO: Rome, Italy, 1993; Vol. 2, pp 147−157. (4) Chen, M. F.; Chien, H. P.; Wong, S. S.; Li, G. C. Dissipation of the fungicide azoxystrobin in Brassica vegetables. Plant Prot. Bull. 2004, 46, 123−130. (5) Zhang, Z. Y.; Zhang, C. Z.; Liu, X. J.; Hong, X. Y. Dynamics of pesticide residues in the autumn Chinese cabbage (Brassica chinensis L.) grown in open fields. Pest Manage. Sci. 2006, 62, 350−355. (6) Zhang, Z. Y.; Liu, X. J.; Yu, X. Y.; Zhang, C. Z.; Honga, X. Y. Pesticide residues in the spring cabbage (Brassica oleracea L. var. capitata) grown in open field. Food Control 2007, 18, 723−730. (7) Fujita, M.; Yajima, T.; Iijima, K.; Sato, K. Comparison of the variability in the levels of pesticide residue observed in Japanese cabbage and grape units. J. Agric. Food Chem. 2012, 60, 1516−21. (8) Ripley, B. D.; Ritcey, G. M.; Harris, C. R.; Denommé, M. A.; Lissemore, L. I. Comparative persistence of pesticides on selected cultivars of specialty vegetables. J. Agric. Food Chem. 2003, 51, 1328− 35. (9) Raina, A.; Raina, M. Dissipation of chlorpyriphos on cauliflower (Brassica oleracea L. var. botrytis). Pestic. Res. J. 2008, 20, 263−265. (10) Wang, J.; Zhao, L.; Li, X.; Jiang, Y.; Li, N.; Qin, Z.; Pan, C. Residue dynamic of pyrimorph on tomatoes, cucumbers and soil under greenhouse trails. Bull. Environ. Contam. Toxicol. 2011, 86, 326−330. (11) Yu, Y.-L.; Fang, H.; Wang, X.; Yu, J.-Q.; Fan, D.-F. Dissipation of chlorpyrifos on pakchoi inside and outside greenhouse. J. Environ. Sci. 2005, 17, 503−505. (12) Lozowicka, B.; Jankowska, M.; Kaczyński, P. Pesticide residues in Brassica vegetables and exposure assessment of consumers. Food Control 2012, 25, 561−575.
3648
dx.doi.org/10.1021/jf405555w | J. Agric. Food Chem. 2014, 62, 3644−3648
