Environ Monit Assess (2011) 176:465–472 DOI 10.1007/s10661-010-1597-y
Monitoring of pesticide residues in market basket samples of vegetable from Lucknow City, India: QuEChERS method Ashutosh K. Srivastava · Purushottam Trivedi · M. K. Srivastava · M. Lohani · Laxman Prasad Srivastava
Received: 9 March 2010 / Accepted: 23 June 2010 / Published online: 16 July 2010 © Springer Science+Business Media B.V. 2010
Abstract The study was conducted on 20 vegetables including leafy, root, modified stem, and fruity vegetables like bitter gourd, jack fruit, french-bean, onion, colocassia, pointed gourd, capsicum, spinach, potato, fenugreek seeds, carrot, radish, cucumber, beetroot, brinjal, cauliflower, cabbage, tomato, okra, and bottle gourd. Forty-eight pesticides including 13 organochlorines (OCs), 17 organophosphates (OPs), 10 synthetic pyrethriods (SPs), and eight herbicides (H) pesticides were analyzed. A total number of 60 samples, each in triplicates, were analyzed using Quick, Easy, Cheap, Effective, Rugged, and Safe method. The quantification was done by GC-ECD/NPD. The recovery varies from 70.22% to 96.32% with relative standard deviation (RSD) of 15%. However the limit of detection ranged from 0.001–
A. K. Srivastava · P. Trivedi · M. K. Srivastava · L. P. Srivastava (B) Pesticide Toxicology Laboratory, Indian Institute of Toxicology Research, (Council of Scientific and Industrial Research Govt. of India), P.O. Box 80, Mahatma Gandhi Marg, Lucknow 226 001, India e-mail:
[email protected] M. Lohani Department of Biotechnology, Integral University, Lucknow, India
0.009 mg kg−1 for OCs, SPs, OPs, and H, respectively. Twenty-three pesticides were detected from total 48 analyzed pesticides in the samples with the range of 0.005–12.35 mg kg−1 . The detected pesticides were: -HCH, Dicofol, -Endosulfan, Fenpropathrin, Permethrin-II, β-cyfluthrin-II, Fenvalerate-I, Dichlorvos, Dimethoate, Diazinon, Malathion, Chlorofenvinfos, Anilophos, and Dimethachlor. In some vegetables like radish, cucumber, cauliflower, cabbage, and okra, the detected pesticides (-HCH, Permethrin-II, Dichlorvos, and Chlorofenvinfos) were above maximum residues limit (MRL) (PFA 1954). However, in other vegetables the level of pesticide residues was either below detection limit or MRL. Keywords QuEChERS · Pesticides · Vegetables · Maximum residues limit (MRL)
Introduction Vegetables are the important ingredient of the human diet for the maintenance of the health and prevention of disease in the Indian sub continent. The total Indian meal constitutes about 150– 250 g of vegetables per day (Mukherjee and Gopal 2003). A wide range of pesticides are globally used for crops protection during the cultivation of
466
vegetables due to heavy pest infestation throughout the season crop and food (Agnihotri 1999; Kalara 2003), Literature reveals that vegetables contain the residues of pesticides above their respective maximum residue limit MRL (Taneja 2005) may pose health hazards to consumers (Elliion et al. 2000; Mukherjee and Gopal 2003). Monitoring of pesticides is conducted globally to assess the environmental load of their residues. Currently pesticides (OPs, SPs, and H) enjoy wide use in the world as an alternative pest control replaying persistent organochlorines (Lyton et al. 1996; Subhani et al. 2001; Toan et al. 2007). Because of wide spread use of pesticides, the presence of their toxic residues (Torres 2004) have been reported in various environmental component/commodities (Cox et al. 1999; Kumari et al. 2002, 2003a, b, 2004, 2005, 2006; Kumari and Kathpal 2008; Srivastava et al. 2000, 2001, 2006; Wang et al. 2008), These pesticide residues find their way into the human body through food, water, and environment. Thus, analysis of pesticide residues in food and other environmental commodities like soil, water, fruits, vegetables, and total diet have become essential requirement for consumers, producers, and foodquality control authorities. In view of the above and to assess the present environmental load of the pesticide residues, it is imperative to determine the amount of pesticide residues in vegetable samples in and around Lucknow, Uttar Pradesh, India. The study also includes the application of Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) methods (Anastassiades et al. 2002; Aysal et al. 2007) for the estimation of 48 pesticides comprising 13 organochlorines (OCs), 17 Organophosphates (OPs), 10 Synthetic Pyrethriods (SPs), and eight Herbicides (H) in 20 vegetables.
Materials and methods Chemicals All solvents like n-hexane, acetone, and ethyl acetate (HPLC grade) were purchased from Sigma– Aldrich Co., USA, Spectrochem Pvt. Ltd. India and were glass distilled before use. Acetone was
Environ Monit Assess (2011) 176:465–472
refluxed over potassium permanganate for 4 h and then distilled. Sodium chloride (NaCl), anhydrous sodium sulfate (Na2 SO4 ), and anhydrous magnesium sulfate (MgSO4 ) procured from Himedia Pvt. Ltd. India. Before use anhydrous sodium sulfate (Na2 SO4 ) and anhydrous magnesium sulfate (MgSO4 ) were purified with acetone and baked for 4 h at 600◦ C in muffle Furnace to remove possible phthalate impurities. Primary secondary amine (PSA) bondasil 40 μm part 12213024 of Varian was used for sample preparation. Pesticide Standards were procured from Supelco Sigma– Aldrich USA, Fluka Sigma–Aldrich Schweis, and Rankem Pvt. Ltd. New Delhi, India. Sample collection Twenty different vegetables including leafy, root, modified stem, and fruity vegetables: bitter gourd, jack fruit, French bean, onion, colocassia (arbi), pointed gourd (parval), capsicum, spinach, potato, fenugreek seeds (methi), carrot, radish, cucumber, beetroot, brinjal, cauliflower, cabbage, tomato, okra, and bottle gourd were collected in year 2009 from local market basket samples of Lucknow, Uttar Pradesh, India. Three samples of each vegetable were analyzed. Extraction and cleanup The collected fresh vegetable sample (100 g) was washed, cleaned, chopped, and grind in warring blander. 10 g macerated sample of each vegetables in triplicate was taken for multi-pesticide residue analysis by QuEChERS method. Ten grams of macerated sample was mixed with 10 ml ethyl acetate, 4 g of anhy. MgSO4 , 1.0 g activated NaCl, and shaken for 10 min at 50 rpm on rotospin test tube mixture. The extract was centrifuged for 10 min at 10,000 rpm. One milliliter aliquot of vegetable extract was cleaned with the mixture of 50 mg PSA, 150 mg anhy. MgSO4 , and 10 mg activated charcoal. The extract was again shaken for 10 min at 50 rpm on rotospin and centrifuged for 10 min at 10,000 rpm. The supernatant was collected in 2 ml vial and mixed with 5 μl acidified ethyl acetate (ethyl acetate acidified with 5% formic acid). One microliter of clean
Environ Monit Assess (2011) 176:465–472
467
Fig. 1 GC-ECD chromatogram of organochlorines and synthetic pyrethroids pesticides
extract was used for the multi pesticide (OCs, SPs, OPs, and H) residues analysis on gas chromatography (GC).
1 μl nitrogen flow rate 0.79 ml/min and makeup 30 ml/min with split ratio 1:10; using carrier gas (N2 ) 99.5%; Injector port temperature 280◦ C; detector temperature 300◦ C (Figs. 1, 2, 3).
Analysis GC-NPD GC-ECD The final extracts were analyzed on (Perkin Elmer Clares-500) GC equipped with fused silica capillary column DB-1 (30 mt × 0.25 mm id) coated with 1% phenyl-methylpolysiloxane (0.25 μm film thickness) using 63 Ni electron-capture detector (ECD) for OCs, SP, and H. General operating condition were as fallows: Column temperature program: initially 170◦ C for 5 min, increase at 4◦ C/min to 240◦ C hold for 15 min, then 280◦ C increase 7◦ C/min hold for 37 min; injection volume:
Fig. 2 GC-NPD chromatogram of organophosphate pesticides
The remaining extracts were analyzed on GC machine (Shimadzu GC-2010) equipped with fused silica capillary column, DB-1 (30 mt × 0.25 mm id) coated with 1% phenyl-methylpolysiloxane (0.25 μm film thickness) using Nitrogen phosphorus detector (NPD). General operating conditions were as follows; Injector port temperature: 250◦ C; detector temperature 280; using carrier gas nitrogen (N2 ); flow 1.46 ml/min; hydrogen (H2 ) makeup is 30 ml/min and zero air 60 ml/min, column temperature program: initially 95◦ C for
468
Environ Monit Assess (2011) 176:465–472
Fig. 3 GC-ECD chromatogram of herbicides
4 min, increase at 2.5◦ C/min to 170◦ C hold for 7 min, then increase 225◦ C/min hold for 10 min; injection volume: 1 μl split ratio 1:5.
Results and discussion The recovery, limit of detection (LOD) and retention time of 48 analyzed pesticides in 20 vegetables are shown in Table 1. The analyzed pesticides were α-HCH, β-HCH, γ-HCH, δ-HCH, Dicofol, Aldrin, o, p-DDE, p, p-DDE, o, p-DDD, p, pDDD, p, p-DDT, α-Endosulfan, β-Endosulfan, Fenpropathrin, λ-Cyhalothrin, Permethrin-I, Permethrin-II, β-Cyfluthrin-I, β-Cyfluthrin-II αCypermethrine, Fenvalerate-I, Fenvalerate-II, Deltamethrine, Dichlorvos, Phorate, Phoratesulfone, Phorate-sulfoxide, Dimethoate, Diazinon, Methyl-parathion, Chlorpyrifos-methyl, Fenitrothione, Malathion, Chlorpyrifos, Chlorofenvinfos, Profenofos, Ethion, Edifenphos, Anilophos, Phosalone, Atrazine, Dimethachlor, Fluchloralin, Dimethachlor, Alachlor, Pendimethalin, Butachlor, and Hexaconazole. LOD of following pesticides varied for 0.001–0.009 mg kg−1 . Similarly, the percent recovery of OCs, SPs, and H varied from 70.22–96.32% from the fortification level of 0.10 mg kg−1 . The percent recovery of OPs ranged from 70.22–90.50% from the fortification level of 0.50 mg kg−1 . The pesticides residue
recorded below the detection limit were considered as nondetectable (ND). Vegetable samples analyzed in triplicate for the presence of pesticides residues are given in Table 2. The level of pesticide residues in various vegetables were compared with their MRL fixed by Prevention of Food Adulteration Act (PFA), Govt. of India 1954 (Table 3). Presences of pesticides in vegetable like bitter gourd were Dicofol (below detection limit (BDL)—0.005 mg kg−1 ), -Endosulfan (0.174–0.189 mg kg−1 ), and Fenpropathrin (0.008–0.019 mg kg−1 ), and in French been -Endosulfan (ND–0.021 mg kg−1 ), but none of these pesticides was >MRL. However, in cucumber, three pesticide permethrin-II (ND–0.514 mg kg−1 ), β-cyfluthrin-II (ND– 0.219 mg kg−1 ), and anilophos (ND– 0.042 mg kg−1 ) were detected but only in one sample permethrin-II was >MRL (0.5 mg kg−1 , PFA). In okra, two samples contained -HCH (0.323–1.235 mg kg−1 ) and malathion (0.027– 0.425 mg kg−1 ) where one sample contained HCH > MRL (1 mg/kg, PFA). In leafy vegetables like spinach and cabbage, -HCH (BDL– 0.048 mg kg−1 ), dimethaclor (ND–0.025 mg kg−1 ), and dichlorvos (BDL–0.030 mg kg−1 ), malathion (ND–0.272 mg kg−1 ) were detected but none of these pesticides were >MRL. In cauliflower, only dichlorvos (BDL–0.157 mg kg−1 ) was detected and one sample showed its residues >MRL. In onion, fluchloralin (0.012–0.065 mg kg−1 ),
Environ Monit Assess (2011) 176:465–472
469
Table 1 The percent recoveries and retention time of fortified vegetable samples Pesticides
Classes
Fortification level (mg kg−1 )
Recovery (%)
Limit of detection (mg kg−1 )
Retention time
α−HCH β−HCH Lindane δ−HCH Dicofol Aldrin Op-DDE α−Endosulfan PP-DDE Op-DDD β−Endosulfan pp-DDD pp-DDT Fenpropathrin λ−Cyhalothrin Permethrin-I Permethrin-II β−Cyfluthrin-I β−Cyfluthrin-II α−Cypermethrin Fenvalerate-I Fenvalerate-II Deltamethrin Dichlorvos Phorate Phorate Sulfone Phorate sulfoxide Dimethoate Diazinon Methyl Parathion Chlorpyrifos methyl Fenitrothion Malathion Chlorpyrifos Chlorfenvinfos Profenofos Ethion Edifenphos Anilophos Phosalone Atrazine Fluchloralin Dimethachlor Alachlor Pendimethalin Fipronil Butachlor Hexaconazole
OC OC OC OC OC OC OC OC OC OC OC OC OC SP SP SP SP SP SP SP SP SP SP OP OP OP OP OP OP OP OP OP OP OP OP OP OP OP OP OP H H H H H H H H
0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
73.55 82.20 76.36 72.90 83.20 85.42 86.30 83.50 70.90 78.50 81.41 89.87 96.32 88.25 76.43 75.21 78.32 71.56 76.11 78.29 80.10 78.33 70.27 73.20 70.08 70.20 75.25 78.36 73.18 90.25 88.64 84.23 80.34 90.50 87.23 79.55 76.34 78.20 80.56 84.78 73.63 87.23 84.21 90.23 83.20 81.56 91.89 89.67
0.001 0.002 0.001 0.003 0.001 0.001 0.002 0.001 0.001 0.002 0.001 0.001 0.001 0.002 0.003 0.002 0.002 0.002 0.003 0.002 0.002 0.002 0.002 0.002 0.009 0.003 0.002 0.002 0.002 0.001 0.001 0.005 0.002 0.001 0.001 0.002 0.002 0.002 0.002 0.001 0.003 0.001 0.001 0.001 0.002 0.002 0.001 0.001
8.88 9.72 10.41 10.95 18.2 18.26 23.80 24.25 26.77 27.20 28.68 30.39 34.39 39.86 41.19 41.98 57.27 57.62 58.50 61.07 61.86 62.90 63.60 4.03 11.92 12.13 15.06 16.50 16.87 18.00 18.15 18.31 18.77 19.60 21.40 23.53 26.15 27.15 33.57 35.07 11.68 13.55 14.87 20.50 21.48 24.78 25.00 27.02
470
Environ Monit Assess (2011) 176:465–472
Table 2 Level of pesticide residues in vegetable sample Vegetable Bitter gourd
FrFrenchbean Onion
Spinach Radish
Cucumber
Beetroot
Cauliflower Cabbage
Okra
Pesticide Dicofol −Endosulfan Fenpropathrin −Endosulfan −HCH Fluchloralin Anilophos −HCH Dimethachlor −HCH Fluchloralin −Endosulfan Permethrin-II Fenvalerate-I Dimethoate Diazinon Anilophos Permethrin-II β−Cyfluthrin-II Anilophos Permethrin-II β−Cyfluthrin-II Fenvalerate-I Dichlorvos Dichlorvos Malathion Chlorofenvinfos −HCH Malathion
Number of sample
No of samples >MRLa
Mean
Analyzed
Detected
(mg kg−1 )
(residues range; mg kg−1 )
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
2 2 2 1 3 2 1 3 1 3 1 2 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 2 2
0 0 0 0 0 NA NA 0 0 2 NA 0 NA 0 0 0 0 1 NA 0 0 NA 0 1 0 0 1 1 0
ND (BDL–0.005) 0.121 (0.174–0.189) 0.009 (0.008–0.019) 0.007 (ND–0.021) ND (ND–0.007) 0.026 (0.012–0.065) 0.011 (ND–0.033) 0.019 (BDL–0.048) 0.008 (ND–0.025) 4.46 (0.025–12.35) 0.175 (ND–0.525) 0.016 (0.013–0.035) 0.008 (ND–0.025) 0.005 (ND–0.013) 0.042 (ND–0.128) ND (ND–0.014) 0.014 (ND–0.042) 0.071 (ND–0.514) 0.073 (ND–0.219) 0.010 (ND–0.030) 0.067 (ND–0.201) 0.065 (ND–0.196) 0.151 (ND–0.452) 0.020 (BDL–0.157) 0.011 (BDL–0.030) 0.090 (ND–0.272) 0.005 (ND–0.056) 0.519 (0.323–1.235) 0.151 (0.027–0.425)
NA not available a Prevention of Food Adulteration Act (PFA), Govt. of India 1954
anilophos (ND–0.033 mg kg−1 ), in beetroot, permethrin-II (ND–0.200 mg kg−1 ), and βcyfluthrin-II (ND–0.196 mg kg−1 ) were detected, but none of these pesticide were >MRL. How-
Table 3 Maximum Residual Limit (MRL) of pesticide in vegetables Vegetables
Pesticides
MRLa mg kg−1
Reference
Radish Cucumber Cauliflower Cabbage Okra
−HCH Permethrin-II Dichlorvos Chlorofenvinfos −HCH
1.0 0.5 0.15 0.05 1.0
PFA PFA PFA PFA PFA
a Prevention of Food Adulteration Act (PFA) 1954, Govt. of India
ever, in radish -HCH (0.025–12.35 mg kg−1 ), fluchloralin (ND–0.525 mg kg−1 ), permethrin-II (ND–0.025 mg kg−1 ), fenvelrate-I (ND– 0.013 mg kg−1 ), dimethoate (ND–0.128 mg kg−1 ), diazinon (ND–0.014 mg kg−1 ), and anilophos (ND–0.042 mg kg−1 ) were detected showing one sample contained -HCH > MRL (1 mg kg−1 , PFA). It is interesting to note that some vegetables of Lucknow market like jack fruit, colocassia (arbi), pointed gourd (parval), capsicum, potato, fenugreek (methi), carrot, brinjal, tomato, and bottle gourd have not shown the presence any analyzed pesticides residue. However, the pattern of pesticide residues present are in following order: radish > bitter gourd > cucumber > beetroot > cabbage > okra > spinach > onion > frenchbean > cauliflower. It is obvious that the
Environ Monit Assess (2011) 176:465–472
presence of pesticide like 2/8 (H), 6/17 (OPs), 4/10 (SPs), and 11/13 (OCs) were noted among the vegetables. None of the vegetable samples have shown the presence of aldrin and DDT residues. However, the absence of these two pesticides in vegetables seems to be due to their banned or restricted use. The presence of pesticide residues in vegetables has become a global phenomenon. Authors have reported the residues of OCs, OPs and SPs, along with fungicide and herbicides in fruit and vegetables from India (Dikshit et al. 1990; Kumari et al. 2002, 2003a, b, 2006; Shahi et al. 2005; Bhanti and Taneja 2005) and abroad (Frank et al. 1987; Wang et al. 2008; Quintero et al. 2008). Further, the persistence and half-life period of many pesticides were found to be less in tropical countries (Rup et al. 1989).This could be one of the reasons for the presence of low level of pesticide residues in vegetables. Judicious use of pesticide with proper waiting period followed by farmers in vegetable crops may be another cause for obtaining low level of residues. Pesticides mainly OCs enters and accumulates in to the human body through the consumption of contaminated food commodities (meat, fish, milk, and milk products) and may produce toxicological hazards (Matsumura 1985; Hayge 1991).
Conclusion The low level of OC, OP, SP, and H residues in vegetables of the present study is an indicative of change in usage pattern of pesticides in India where shift has taken place from persistent OCs to the easily degradable groups like OPs and SPs since last decade. It has been observed that analyzed pesticide residues were either BDL or
