2. 466 Environ Monit Assess (2011) 176:465–472
vegetables due to heavy pest infestation through-
out the season crop and food (Agnihotri 1999;
Kalara 2003), Literature reveals that vegetables
contain the residues of pesticides above their re-
spective 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). Be-
cause of wide spread use of pesticides, the pres-
ence of their toxic residues (Torres 2004) have
been reported in various environmental com-
ponent/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 envi-
ronmental commodities like soil, water, fruits,
vegetables, and total diet have become essential
requirement for consumers, producers, and food-
quality control authorities. In view of the above
and to assess the present environmental load of
the pesticide residues, it is imperative to deter-
mine the amount of pesticide residues in veg-
etable samples in and around Lucknow, Uttar
Pradesh, India. The study also includes the appli-
cation 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 ac-
etate (HPLC grade) were purchased from Sigma–
Aldrich Co., USA, Spectrochem Pvt. Ltd. India
and were glass distilled before use. Acetone was
refluxed over potassium permanganate for 4 h and
then distilled. Sodium chloride (NaCl), anhydrous
sodium sulfate (Na2SO4), and anhydrous mag-
nesium sulfate (MgSO4) procured from Himedia
Pvt. Ltd. India. Before use anhydrous sodium sul-
fate (Na2SO4) 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 veg-
etable 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 mix-
ture 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 super-
natant 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
3. 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 chromatog-
raphy (GC).
Analysis
GC-ECD
The final extracts were analyzed on (Perkin Elmer
Clares-500) GC equipped with fused silica capil-
lary 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 in-
crease 7◦
C/min hold for 37 min; injection volume:
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; de-
tector temperature 300◦
C (Figs. 1, 2, 3).
GC-NPD
The remaining extracts were analyzed on GC ma-
chine (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 phos-
phorus detector (NPD). General operating condi-
tions 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
Fig. 2 GC-NPD chromatogram of organophosphate pesticides
4. 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 reten-
tion 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,p-
DDD, p,p-DDT, α-Endosulfan, β-Endosulfan,
Fenpropathrin, λ-Cyhalothrin, Permethrin-I,
Permethrin-II, β-Cyfluthrin-I, β-Cyfluthrin-II α-
Cypermethrine, Fenvalerate-I, Fenvalerate-II,
Deltamethrine, Dichlorvos, Phorate, Phorate-
sulfone, Phorate-sulfoxide, Dimethoate, Diazi-
non, Methyl-parathion, Chlorpyrifos-methyl,
Fenitrothione, Malathion, Chlorpyrifos, Chloro-
fenvinfos, Profenofos, Ethion, Edifenphos, Ani-
lophos, Phosalone, Atrazine, Dimethachlor,
Fluchloralin, Dimethachlor, Alachlor, Pendime-
thalin, Butachlor, and Hexaconazole. LOD of fol-
lowing 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 con-
sidered 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 pes-
ticides 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 Fen-
propathrin (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. How-
ever, 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
),
6. 470 Environ Monit Assess (2011) 176:465–472
Table 2 Level of pesticide residues in vegetable sample
Vegetable Pesticide Number of sample No of samples >MRLa Mean
Analyzed Detected (mg kg−1) (residues range; mg kg−1)
Bitter gourd Dicofol 3 2 0 ND (BDL–0.005)
−Endosulfan 3 2 0 0.121 (0.174–0.189)
Fenpropathrin 3 2 0 0.009 (0.008–0.019)
FrFrenchbean −Endosulfan 3 1 0 0.007 (ND–0.021)
Onion −HCH 3 3 0 ND (ND–0.007)
Fluchloralin 3 2 NA 0.026 (0.012–0.065)
Anilophos 3 1 NA 0.011 (ND–0.033)
Spinach −HCH 3 3 0 0.019 (BDL–0.048)
Dimethachlor 3 1 0 0.008 (ND–0.025)
Radish −HCH 3 3 2 4.46 (0.025–12.35)
Fluchloralin 3 1 NA 0.175 (ND–0.525)
−Endosulfan 3 2 0 0.016 (0.013–0.035)
Permethrin-II 3 1 NA 0.008 (ND–0.025)
Fenvalerate-I 3 1 0 0.005 (ND–0.013)
Dimethoate 3 1 0 0.042 (ND–0.128)
Diazinon 3 1 0 ND (ND–0.014)
Cucumber Anilophos 3 1 0 0.014 (ND–0.042)
Permethrin-II 3 1 1 0.071 (ND–0.514)
β−Cyfluthrin-II 3 1 NA 0.073 (ND–0.219)
Beetroot Anilophos 3 1 0 0.010 (ND–0.030)
Permethrin-II 3 1 0 0.067 (ND–0.201)
β−Cyfluthrin-II 3 1 NA 0.065 (ND–0.196)
Fenvalerate-I 3 1 0 0.151 (ND–0.452)
Cauliflower Dichlorvos 3 2 1 0.020 (BDL–0.157)
Cabbage Dichlorvos 3 2 0 0.011 (BDL–0.030)
Malathion 3 1 0 0.090 (ND–0.272)
Chlorofenvinfos 3 1 1 0.005 (ND–0.056)
Okra −HCH 3 2 1 0.519 (0.323–1.235)
Malathion 3 2 0 0.151 (0.027–0.425)
NA not available
aPrevention 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 β-
cyfluthrin-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 Reference
mg kg−1
Radish −HCH 1.0 PFA
Cucumber Permethrin-II 0.5 PFA
Cauliflower Dichlorvos 0.15 PFA
Cabbage Chlorofenvinfos 0.05 PFA
Okra −HCH 1.0 PFA
aPrevention 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 vege-
tables of Lucknow market like jack fruit, colo-
cassia (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 > beet-
root > cabbage > okra > spinach > onion >
frenchbean > cauliflower. It is obvious that the
7. Environ Monit Assess (2011) 176:465–472 471
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 vegeta-
bles 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 pes-
ticides 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 veg-
etable 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 commodi-
ties (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 indica-
tive 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
<MRL in approximate 90% vegetable samples of
Lucknow market. Vegetables form an important
food item and proper care should be taken to use
very safe pesticide for avoiding potential risk to
human. It is, therefore, suggested that the veg-
etables collected from in and around of Lucknow
city, India are comparatively safe from pesticide
residues. A periodical monitoring of pesticide
residues in other food commodities are the recent
need for the consumers as well as authorities of
food quality control.
Acknowledgements The authors are grateful to the Di-
rector Indian Institute of Toxicology Research (IITR),
Lucknow for his keen interest and providing research fa-
cilities. Authors also thank Miss Upasana, Shipra for their
technical support, Mrs. Shyamla Das for her assistance in
GC analysis and computer typing. The financial assistance
of CSIR net work project NWP-17 funded by Council
of scientific and industrial research New Delhi is also
acknowledged. IITR communication no. is 2821.
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