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Chemical Residues of some Pyrethroid Insecticides in Egg plant and Okra Fruits:
Effect of Processing and Chemical Solutions
1
SAJIDA ZAFAR, 1
ANWAAR AHMED*, 2,3
RIAZ AHMAD, 4
MUHAMMAD ATIF RANDHAWA,
1
MUHAMMAD GULFRAZ, 1
ASIF AHMAD AND 1
FARZANA SIDDIQUE
1
Department of Food Technology, PMAS-Arid Agriculture University, Rawalpindi, Pakistan.
2
Department of Soil Science & Soil Water Conservation, PMAS-Arid Agriculture University,
Rawalpind, Pakistan.
3
Director Quality Enhancement Cell, PMAS-Arid Agriculture University, Rawalpindi, Pakistan.
4
National Institute of Food Science & Technology, University of Agriculture, Faisalabad, Pakistan.
anwaarft@hotmail.com*
(Received on 26th
September 2011, accepted in revised form 13th
December 2011)
Summary: We investigated residual levels of cypermethrin [(RS)-α-cyano-3-phenoxybenzyl
(1RS,3RS;1RS,3SR)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate],deltamethrin [(S)-
α-cyano-3-phenoxybenzyl(1R,3R)-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane car- boxylate]
and lambda cyhalothrin [(RS)-α-cyano-3-phenoxybenzyl (Z)-(1RS,3RS)-3-(2-chloro-3,3,3-
trifluoroprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate] in eggplant and okra fruit and their
dissipation patterns during different processing operations viz washing, peeling, cooking and
blanching, and dipping in acidic and alkaline solutions. The samples were extracted in ethyl acetate
and cleaned up with activated charcoal. After concentration on rotary evaporator the samples were
analysed on High Performance Liquid Chromatograph (HPLC). Mobile phase was methanol:water
(85:15) and separation of insecticides was achieved on C-18 cloumn at 202 nm with UV-VIS
detector. The samples of eggplant and okra fruit were collected from supervised fields as well as
from farmer’s field. In the supervised field trials, the higher levels of residues of each of the three
insecticides were found in the raw samples of eggplant and okra fruit. In raw eggplant fruit the
residues were in the order of cyhalothrin (1.40±0.15 mg kg-1
) > cypermethrin (1.13±0.01 mg kg-1
) >
deltamethrin (0.65±0.1 mg kg-1
), whereas in raw okra fruit these were in the order of cypermethrin
(1.17±0.1 mg kg-1
) > deltamethrin (1.08±0.12 mg kg-1
) > cyhalothrin (0.48±0.03 mg kg-1
). Among
all the processing operations, cooking was found the most effective for reduction of the insecticide
residues followed by blanching, peeling, washing and dipping in the tap water in both vegetables.
Treatments with acidic solutions were more effective than with alkaline solutions for reduction of
residues of insecticides in these vegetables. These results have implications for assessing dietary
exposure limits of these insecticides.
Keywords: dissipation, cypermethrin, deltamethrin, cyhalothrin, washing, peeling, cooking
Introduction
Food production in the world, particularly in
the third world countries, has to keep pace with the
rapid increase in population and this would be
impossible without the use of pesticides. Out of
various methods recommended from time to time, the
chemical method at present is considered the most
effective to combat the insect pests of crops and
vegetables effectively and economically. Synthethic
prethroid insecticides are now increasingly used on
many crops because of their some interesting
properties, such as greater photostability, effective-
ness at low concentrations, low environmental
persistence, and easy breakdown as compared to
organo- chlorine and organophosphorus insecticides
[1, 2]. They constitute approximately 25% of
worldwide insecticide market for plant protection and
have been in use for more than 40 years [3].
Among vegetables, brinjal (Solanum
melongena L.) and ladyfinger (Abelmoschus
esculentus L.) are common and important staple
vegetable crops of Pakistan. However, these are
badly affected by a number of insect-pests attack
during their growing periods. These include fruit and
shoot borer, fruitworm and cutworm [4]. The brinjal
suffers badly at fruiting stage due to attack of shoot
and fruit borer causing up to 70% damage to the crop
making it totally unfit for human consumption [5]. To
overcome these losses, use of pyrethroid insecticides
viz cypermethrin [(RS)-α-cyano-3-phenoxy-
benzyl(1RS,3RS;1RS,3SR)-3-(2,2-dichlorovinyl)-2,2-
dimethyl cyclo propane carboxylate], deltamethrin
[(S)-α-cyano-3-phenoxy -benzyl (1R,3R)-3-(2,2-
dibromovinyl)-2,2-dimethylcyclopropanecarboxy-
late] and cyhalothrin [(RS)-α-cyano-3-phenoxy-
benzyl(Z)-(1RS,3RS)-3-(2-chloro-3,3,3-trifluoroprop-
1-enyl)-2,2-di-methylcyclopro-pane-carboxylate] is
very common in this region and they are repeatedly
applied, even at the fruiting stage. However,
indiscriminate and haphazard use of these chemicals,
particularly at fruiting stage, leads to their
accumulation in the vegetables which consequently
cause hazards to human beings through food chain
[6]. However, there is no regular testing of food
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residues in Pakistan. However, the mass media has
created awareness about the risks involved in the use
of pesticides in agriculture. This has created a havoc
regarding the accumulation of residues of these toxic
chemicals in daily food.
The aim of present study was to evaluate
residual levels of three commonly used pyrethroid
insecticides, cypermethrin, deltamethrin and
cyhalothrin, in eggplant and okra fruits and to assess
the effect of different chemicals and household
processes: such as washing, peeling, blanching and
cooking on the fate of residues of these pyrethroids.
Results and Discussion
Effect of Processing on Pyrethroid Residues in
Eggplant and Okra Fruits
The data given in Table-1 and 2 show that
the residues of the insecticides were significantly
high in unprocessed (raw) eggplant and okra fruits.
They ranged between 1.13 mg kg-1
and 1.17 mg kg-1
for cypermethrin, 0.65 mg kg-1
and 1.08 mg kg-1
for
deltamethrin and 1.40 mg kg-1
and 0.48 mg kg-1
for
cyhalothrin in eggplant and okra fruit, respectively.
However, the residues significantly reduced after
they were washed, peeled, blanched or cooked (Fig. 1
and 2). Among all the processing techniques cooking
was found the most effective in the elimination of
residues of the three insecticides.
The results showed that washing under tap
water dislodged pyrethroid residues in eggplant and
okra fruit significantly. As the pyrethroids are non-
systemic in nature, therefore most of the residues
remain as microparticles on the surface and are easily
removed by the mechanical stirring with water during
washing. Thus washing had a significant effect on the
removal of residues in eggplant and okra fruit
because of less/no penetration of the chemical into
the cuticle layer of the plant surface and resulting in
deposits removable by washing. These results agree
with those obtained by several researchers [7-10]
who reported 50-60% removal of the residues by the
washing operation. A reduction of 10-30% of
alphamethrin residues in tomato and eggplant have
been reported [11].
0
10
20
30
40
50
60
70
80
90
100
Washing Tap water
dipping
Peeling Blanching Cooking
Processing Techniques
%Reduction
Cypermethrin Deltamethrin Cyhalothrin
Fig. 1: Percent reduction of pyrethroid residues in
eggplant fruit after processing.
0
10
20
30
40
50
60
70
80
90
100
Washing Tap water dipping Blanching Cooking
Processing Techniques
%Reduction
Cypermethrin Deltamethrin Cyhalothrin
Fig. 2: Percent reduction of pyrethroid residues in
okra fruit after processing.
Table-1: Effect of processing on the residue levels of the pesticides applied to egg plant fruit.
Pesticides
Raw
mg kg-1
± SD
Washing
mg kg-1
±SD
Tap water dipping
mg kg-1
±SD
Peeling
mg kg-1
± SD
Blanching
mg kg-1
± SD
Cooking
mg kg-1
± SD
Cypermethrin 1.13±0.01 0.45±0.01 0.79±0.01 0.23±0.002 0.20±0.02 0.06±0.005
Deltamethrin 0.65±0.1 0.39±0.06 0.44±0.07 0.18±0.03 0.19±0.03 0.10±0.14
Cyhalothrin 1.40±0.15 0.97±0.09 1.19±0.11 0.55±0.05 0.49±0.05 0.35±0.06
SD= Standard deviation of the mean
Table-2: Effect of processing on residue levels of the pesticides applied to okra fruit.
Pesticides
Raw
mg kg-1
± SD
Washing
mg kg-1
± SD
Tap water dipping
mg kg-1
± SD
Blanching
mg kg-1
± SD
Cooking
mg kg-1
± SD
Cypermethrin 1.17±0.13 0.59±0.07 0.81±0.09 0.23±0.03 0.12±0.01
Deltamethrin 1.08±0.12 0.72±0.08 0.78±0.09 0.54±0.06 0.19±0.01
Cyhalothrin 0.48±0.03 0.37±0.03 0.33±0.03 0.19±0.07 0.08±0.02
SD= Standard deviation of the mean
Uncorrected proof
Peeling with a knife is common household
practice for most fruits and vegetables. Eggplant is
eaten normally without their peel. Pyrethroid
insecticides are applied directly to the crops and
show very limited movement or penetration into the
cuticle. Therefore, the residues of these materials are
confined to the outer surfaces where they are
amenable to removal by peeling, hulling or trimming
operations. Cypermethrin, deltamethrin and
cyhalothrin are non-systemic insecticides. Therefore,
when peel was removed, great fractions of insecticide
residues were also removed. Peeling reduced 80%
residues of cypermethrin, 66% of cyhalothrin and
70% of deltamethrin in eggplant in the current study.
Other studies carried out [12] also showed that most
of the residue concentration was located in or on
peel. Awasthi [12] reported that peeling off the
mango fruit skin completely removed the residues of
various pesticides including cypermethrin, reflecting
the accumulation of residues in the fruit pericarp only
and no further transfer of pesticide into the fruit pulp.
In another study, 73% reduction in chlorpyrifos, 82%
reduction in lindane level, 75% reduction in
cypermethrin residues and 77% reduction in ethyl-
enebisdithiocarbamates after peeling of asparagus
was reported by Chavarri et al., [7]. Thus, removal of
the peel may reduce a large fraction of residues,
leaving little in the edible portions. This is especially
important for fruits which are not eaten with their
peels. However, the peel from commercial peeling
processes is sometimes used as animal feed or for the
production of essential oils or pectin (citrus, apple
etc.). For such industrial processes, it is imperative to
consider that non-systemic surface residues are often
concentrated in the peel.
Tap water dipping was found relatively less
effective in the reduction of residues as compared to
washing and peeling with reduction in the residues
ranging from 27 to 31%. However, the results are in
line with those reported for mango fruit, where the
reduction in residues of fenvelerate and cypermethrin
ranged between 21 and 27%, 66 and 68% for
dimethoate and fenthion, rspectively [12].
Cooking/boiling was found the most
effective among all processing operations. Cooking
eliminated 95%, 86% and 78% residues of
cypermethrin, deltamethrin and cyhalothrin,
respectively in eggplant, where as for okra fruit these
were 92%, 82% and 85%, respectively. Processes
involving heat can enhance volatilization of the
chemicals and their hydrolysis thus reducing the
residue levels [13]. Hotellier [14] reported that
deltamethrin residues reduced appreciably by
cooking. However, Kumari [15] found 37, 40 and
42% reduction in synthetic pyrethroids in eggplant,
cauliflower and okra fruits, respectively. Similarly 19
to 40% reduction in deltamethrin residues was
observed in vegetables in a study by Randhawa et al.,
[10].
Effect of Chemical Treatment on Pesticide Residues
in Eggplant and Okra Fruit
The levels of insecticide residues in eggplant
and okra fruit after chemical treatments are given in
Table-3 and 4. The residues of each three of
pyrethroids significantly decreased as compared to
those in raw samples when the vegetables were
treated with 4%, 8%, 12% each of NaOH, and acetic
acid solutions. From these results it is clear that these
washing solutions alone are highly effective in
reducing the insecticide residues. Solution of 4%
NaOH decreased 27 and 40% in insecticide residues
in eggplant and okra fruit, respectively; 8% NaOH
resulted in a reduction of 55 and 60%, respectively;
where as 12% NaOH showed maximum reduction of
75% in residues in the vegetables (Fig. 3 and 4).
0
20
40
60
80
100
120
4% 8% 12% 4% 8% 12%
NaOH solution Acetic Acid solution
Chemical Treatment
%Reduction
Cypermethrin Deltamethrin Lamda Cyhalothrin
Fig. 3: Percent reduction of pyrethroid residues
in eggplant fruit by chemical treatment
0
20
40
60
80
100
120
4% 8% 12% 4% 8% 12%
NaOH solution Acetic Acid solution
Chemical Treatment
%Reduction
Cypermethrin Deltamethrin Cyhalothrin
Fig. 4: Percent reduction of pyrethroid residues in
okra fruit by chemical treatment.
Acetic acid solution was found more
effective than NaOH in the dissipation of
cypermethrin, deltamethrin and cyhalothrin residues.
Reduction in the pyrethroid residues increased with
increase in concentration of the washing solution.
There are some reports which show efficacy of
chemical washing to dislodge the pesticides from
crops. Pesticides treated tomato samples on washing
with different levels of acetic acid solution gave,
Uncorrected proof
47.0%, 33.7%, 91.5%, 86.0% and 93.7% loss in
lindane, DDT, dimethoate, profenofos and pirimip-
hosmethyl, respectively. The pesticide residues
reduction increased with increase in concentration of
solutions [16, 17]. The chemical solution causes
dissipation of the pesticides through pH change. Dip
treatment of fruits in water, NaCl solution, HCl,
acetic acid, NaOH solutions and potassium
permanganate removed 50 to 60% of surface residues
of pyrethroids compared to 40 to 50% removal by
hydrolytic degradation with NaOH and a detergent
solution removed 50 to 60% residues [18].
Experimental
Material
All solvents and other reagents used were of
purity compatible with pesticide residue analysis.
Analytical grade reference insecticide standards of
cypermethrin, deltamethrin and lambda cyhalothrin
(>95% purity) were purchased from Dr. Ehrenstorfer
GmbH, Germany. Chemical structures of the
insecticides are given in Fig. 5. Commercial
formulation of insecticides was purchased from open
market. Acetic acid, sodium hydroxide, anhydrous
sodium sulphate, sodium chloride, acetone, activated
charcoal and glass wool were purchased from Merck,
Pakistan, where as HPLC grade chemicals/solvents
like water, methanol and ethyl acetate were from
Sigma, Pakistan.
Cypermethrin
Deltamethrin
Lambda Cyhalothrin
Fig. 5: Chemical structures of the pesticides.
Sample Collection
A survey was conducted at three different
locations in the adjoining villages of Rawalpindi and
Islamabad regarding the use of pesticides by the local
farmers. Eggplant and okra fruit samples were
collected from ten different fields. The farmers were
allowed to use their traditional methods of pesticide
application on these vegetables. At maturity the
samples of eggplant and okra fruit were collected
from the farmer’s fields and brought to the laboratory
and stored at -4o
C. In the supervised field trial the
okra and eggplant fields were selected and sprayed
with cypermethrin (Arrivo 10 EC) @ 250 mL Acre-1
,
lambda cyhalothrin (Karate 2.5 EC) @ 300 mL/acre
and deltamethrin (Decis 2.5 EC) @ 250 mL Acre-1
twice at the insect pest infestation. Each insecticide
was dissolved in 100 liter water and sprayed with
Knap-sack sprayer. Both the vegetable crops received
normal agronomic practices.
Treatment of Contaminated Vegetables
To determine the effects of household
processes, the collected samples were divided into
lots. They were subjected to washing under tap water
(~25 ⁰C), peeling, blanching and cooking till softness
as described elsewhere [19]. Moreover, the
contaminated samples were soaked for 10 min in tap
water, acetic acid solutions at concentrations of 4, 8
and 12% and aqueous solutions of sodium hydroxide
at concentrations of 4, 8 and 12% prior to the
extraction, cleanup and analysis of each sample.
Extraction and Cleanup
The insecticide residues in the vegetable
samples were extracted and cleaned up following the
method of [20] with some modification. Briefly, 1 kg
of the vegetable was chopped into small pieces and
blended in a Warring Blender (Anex, Germany) to
make a fine paste. A representative sample of 50 g
was taken and extracted with 50 mL ethyl acetate,
20g anhydrous sodium sulphate and 10 g NaCl on
mechanical shaker (IKA, UK) for 1 h at 150 rpm.
The extract was filtered through Whatman No. 4
filter paper. Glass wool was placed at the bottom of
column used for cleanup. The column was then filled
with activated charcoal that has been previously
activated at 300 ⁰C for 3 h with a top layer of
anhydrous sodium sulphate. The column was washed
with acetone before loading the sample to the
column. The cleaned up extract was collected in 100
mL flask and was then concentrated using rotary
evaporator (Eyela, USA) at 60 ⁰C. The concentrated
sample was transferred to sampling vials and the
solvent was evaporated by gentle stream of nitrogen
to just dryness. The dried samples were dissolved in
Uncorrected proof
500 µL methanol prior to analysis and filtered
through 0.45 µm nylon membrane filter (Millipore,
Germany) before injection to High Performance
Liquid Chromatography (HPLC) in order to remove
any particulate material present in the sample.
Instrumental Analysis and Method Validation
The cleaned extracts were analyzed on a
HPLC (Shimadzu Class-VP V6.13 SPI) equipped
with variable-wavelength ultraviolet detector. The
column used was reverse phase C-18 (25 mm x 4.6
mm id). Mobile phase was methanol and water
(85:15) at a flow rate of 1 mL min-1
. The injection
volume was 20 µL and the detector was set at 220 nm
and run time was 15 minutes. Retention time
obtained for different insecticides are shown in
Table-5. Three metabolites of cypermethrin were
identified and residues of cypermethrin are expressed
as sum of the metabolites. A chromatogram of
unknown okra sample is shown in Fig 6a. A high
degree of resolution for separation of different
components of sample (insecticides) was achieved
under the conditions used in the study. When the
same sample of okra was spiked with known
quantities of each of the three insecticides the same
degree of resolution was achieved as shown in Fig
6b. The spiked samples confirmed the presence of
insecticides in the okra sample.
Fig. 6a: Chromatogram of okra sample showing
residues of lambda cyhalothrin, deltamethrin
and cypermethrin.
Fig. 6b: Spiked chromatogram of okra sample
showing residues of lambda cyhalothrin,
deltamethrin and cypermethrin.
Table-3: Effect of chemical solutions on pesticide residues in eggplant fruit (mg kg-1
±SD).
NaOH solution Acetic acid solution
Pesticide Raw
4% 8% 12% 4% 8% 12%
Cypermethrin 1.13±0.01 0.67±0.01 0.51± 0.01 0.39± 0.01 0.83±0.01 0.53±0.01 0.02±0.01
Deltamethrin 0.65±0.10 0.53±0.08 0.37±0.06 0.23±0.03 0.49±0.07 0.40±0.06 0.26±0.04
Cyhalothrin 1.4± 0.15 1.34± 0.14 1.05±0.1 0.79± 0.07 1.26± 0.12 0.65± 0.06 0.36±0.03
SD= Standard deviation of the mean
Table-4: Effect of chemical solutions on pesticide residues in okra fruit (mg kg-1
±SD).
NaOH solution Acetic Acid solution
Pesticides Raw
4% 8% 12% 4% 8% 12%
Cypermethrin 1.17±0.13 0.59±0.06 0.47±0.05 0.29±0.03 0.86±0.10 0.55±0.07 0.04±0.04
Deltamethrin 1.08±0.12 0.89±0.10 0.75±0.08 0.45±0.05 0.82±0.09 0.59±0.06 0.24±0.02
Cyhalothrin 0.48±0.04 0.39±0.03 0.27±0.02 0.21±0.02 0.39±0.03 0.30±0.02 0.14±0.02
SD= Standard deviation of the mean
Table-5: Linear range, recovery and retention time of insecticides.
Insecticides Retention time (min) Recovery(%) Spike level (µg) Linear range (µg) R2
Lambda Cyhalothrin 5.15 97 30 1.11 to 71 0.999
Deltamethrin 5.45 98 30 0.91 to 58 0.999
Cypermethrin
(sum of metabolites)
5.73, 6.06, 6.37 95 30 1.17 to 75 0.998
Uncorrected proof
The method used for determination of
pyrethriod insecticide residues was validated before
analysis of actual samples. For this purpose 30 µg
each of three insecticides were added in the 50 g
blended okra (from supervised field where no spray
was done) and extracted as described above. Cleanup
and concentration was also performed by following
the same set of conditions. Reproducible recoveries
of > 94% were obtained for the insecticides used in
this study (Table-5). Linear range of determination
for each of the insecticide was constructed by using
step wise dilution analysis of each insecticide on
HPLC. The standard curve showed that linear range
was found to be 1.17 to 75 µg, 0.91 to 58 µg and 1.11
to 71 µg for cypermethrin, deltamethrin and lambda
cyhalothrin, respectively. The analytical method was
found to be highly sensitive to fulfill quality criterion
for insecticide residue analysis. The R2
value for
standard curve of each insecticide is also shown in
Table 5. Then concentration of unknown sample was
quantified by using the same standard curves,
respectively for each of the insecticide (Fig 7).
Fig.7: Standard curves for deltamethrin, cype-
rmethrin and lambda cyhalothrin.
Conclusion
Residual effect of cypermethrin,
deltamethrin and lambda cyhalothrin in eggplant and
okra fruits and their dissipation patterns during
different processing operations viz washing, peeling,
cooking and blanching and dipping in chemical
solutions was studied. The results revealed that
processing and chemical treatments were effective in
reduction of the insecticide residues (cypermethin,
deltamethrin and lambda cyhalothrin) in eggplant and
okra fruits. Though washing with water is important
to decrease the concentrations of insecticide residues,
cooking of vegetables helps greatly to eliminate
major fractions of the pesticide residues. Simple
dipping of vegetables in acidic or alkaline solutions
can also reduce the insecticide residues efficiently.
The acceptable maximum residue limits established
by the FAO/WHO Codex Alimentarius Commission,
or other international bodies refer to the whole
vegetables and therefore are appropriate for assessing
compliance with good agricultural practices.
However, these limits may be of limited significance
for assessing dietary exposure to pesticides from the
vegetables such as eggplant and okra, which are
consumed after household processing.
Acknowledgements
The authors are grateful to the National
Institute of Food Science and Technology University
of Agriculture, Faisalabad, for providing facilities to
carry out a part of the research project.
References
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Association of Official Analytical Chemists
International, 77, 738 (1994).
2. G. F. Pang, C. L. Fan, Y. Z. Chao and T. S. Zhao,
Journal of Chromatography, 67, 348 (1994).
3. T. J. Shafer, D. A. Meyer and K. M. Crofton,
Environmental Health Perspectives, 113, 123
(2005).
4. A. Saleem and Ibrar-ul-Haq, Pesticides
standardized in Punjab - dose(s), crop(s) and
target pest(s). Directorate of Agricultural
Information Government of the Punjab,
Agriculture Department, Lahore (2003).
5. B. Duara, A. Baruah, S. C. Deka and N. Burman,
Pesticide Research Journal, 15, 43 (2003).
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8. S. Jayakrishnan, A. K. Dikshit, J. P. Singh and D.
C. Pachauri, Bulletin of Environmental
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M. S. Randhawa, Food Chemistry, 103, 1016
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Randhawa, A. Ahmed, U. Farooq, M. Ibrar and
M. A. Randhawa, Journal of Chemical Society of
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13. P. T. Holland, B. Hamilton, B. Ohlin and M. W.
Skidmore, Pure and Applied Chemistry, 66, 335
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14. M. L. Hotellier, Deltamethrin Momograph
Roussel Vcalf., Pp. 285-315 (1982).
15. B. Kumari, ARPN Journal of Agriculture and
Biological Sciences, 3, 46 (2008).
16. S. M. Ismail, A. Ali and R. A. Habiba, Journal of
Agricultural and Food Chemistry, 41, 610
(1993).
17. A. A. K. Abou-Arab, Food Chemistry, 65, 509
(1999).
18. M. D. Awasthi and A. Lalitha, Journal of
Entomology Research, 7, 139 (1986).
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Butt, A. Ahmed and M. S. Randhawa, Journal of
Scientific and Industrial Research, 66, 849
(2007).
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Gyorfi and G. Reese, Journal Association of
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Chemical residues of some pyrethroid insecticides in egg plant and okra fruits

  • 1. Uncorrected proof Chemical Residues of some Pyrethroid Insecticides in Egg plant and Okra Fruits: Effect of Processing and Chemical Solutions 1 SAJIDA ZAFAR, 1 ANWAAR AHMED*, 2,3 RIAZ AHMAD, 4 MUHAMMAD ATIF RANDHAWA, 1 MUHAMMAD GULFRAZ, 1 ASIF AHMAD AND 1 FARZANA SIDDIQUE 1 Department of Food Technology, PMAS-Arid Agriculture University, Rawalpindi, Pakistan. 2 Department of Soil Science & Soil Water Conservation, PMAS-Arid Agriculture University, Rawalpind, Pakistan. 3 Director Quality Enhancement Cell, PMAS-Arid Agriculture University, Rawalpindi, Pakistan. 4 National Institute of Food Science & Technology, University of Agriculture, Faisalabad, Pakistan. anwaarft@hotmail.com* (Received on 26th September 2011, accepted in revised form 13th December 2011) Summary: We investigated residual levels of cypermethrin [(RS)-α-cyano-3-phenoxybenzyl (1RS,3RS;1RS,3SR)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate],deltamethrin [(S)- α-cyano-3-phenoxybenzyl(1R,3R)-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane car- boxylate] and lambda cyhalothrin [(RS)-α-cyano-3-phenoxybenzyl (Z)-(1RS,3RS)-3-(2-chloro-3,3,3- trifluoroprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate] in eggplant and okra fruit and their dissipation patterns during different processing operations viz washing, peeling, cooking and blanching, and dipping in acidic and alkaline solutions. The samples were extracted in ethyl acetate and cleaned up with activated charcoal. After concentration on rotary evaporator the samples were analysed on High Performance Liquid Chromatograph (HPLC). Mobile phase was methanol:water (85:15) and separation of insecticides was achieved on C-18 cloumn at 202 nm with UV-VIS detector. The samples of eggplant and okra fruit were collected from supervised fields as well as from farmer’s field. In the supervised field trials, the higher levels of residues of each of the three insecticides were found in the raw samples of eggplant and okra fruit. In raw eggplant fruit the residues were in the order of cyhalothrin (1.40±0.15 mg kg-1 ) > cypermethrin (1.13±0.01 mg kg-1 ) > deltamethrin (0.65±0.1 mg kg-1 ), whereas in raw okra fruit these were in the order of cypermethrin (1.17±0.1 mg kg-1 ) > deltamethrin (1.08±0.12 mg kg-1 ) > cyhalothrin (0.48±0.03 mg kg-1 ). Among all the processing operations, cooking was found the most effective for reduction of the insecticide residues followed by blanching, peeling, washing and dipping in the tap water in both vegetables. Treatments with acidic solutions were more effective than with alkaline solutions for reduction of residues of insecticides in these vegetables. These results have implications for assessing dietary exposure limits of these insecticides. Keywords: dissipation, cypermethrin, deltamethrin, cyhalothrin, washing, peeling, cooking Introduction Food production in the world, particularly in the third world countries, has to keep pace with the rapid increase in population and this would be impossible without the use of pesticides. Out of various methods recommended from time to time, the chemical method at present is considered the most effective to combat the insect pests of crops and vegetables effectively and economically. Synthethic prethroid insecticides are now increasingly used on many crops because of their some interesting properties, such as greater photostability, effective- ness at low concentrations, low environmental persistence, and easy breakdown as compared to organo- chlorine and organophosphorus insecticides [1, 2]. They constitute approximately 25% of worldwide insecticide market for plant protection and have been in use for more than 40 years [3]. Among vegetables, brinjal (Solanum melongena L.) and ladyfinger (Abelmoschus esculentus L.) are common and important staple vegetable crops of Pakistan. However, these are badly affected by a number of insect-pests attack during their growing periods. These include fruit and shoot borer, fruitworm and cutworm [4]. The brinjal suffers badly at fruiting stage due to attack of shoot and fruit borer causing up to 70% damage to the crop making it totally unfit for human consumption [5]. To overcome these losses, use of pyrethroid insecticides viz cypermethrin [(RS)-α-cyano-3-phenoxy- benzyl(1RS,3RS;1RS,3SR)-3-(2,2-dichlorovinyl)-2,2- dimethyl cyclo propane carboxylate], deltamethrin [(S)-α-cyano-3-phenoxy -benzyl (1R,3R)-3-(2,2- dibromovinyl)-2,2-dimethylcyclopropanecarboxy- late] and cyhalothrin [(RS)-α-cyano-3-phenoxy- benzyl(Z)-(1RS,3RS)-3-(2-chloro-3,3,3-trifluoroprop- 1-enyl)-2,2-di-methylcyclopro-pane-carboxylate] is very common in this region and they are repeatedly applied, even at the fruiting stage. However, indiscriminate and haphazard use of these chemicals, particularly at fruiting stage, leads to their accumulation in the vegetables which consequently cause hazards to human beings through food chain [6]. However, there is no regular testing of food
  • 2. Uncorrected proof residues in Pakistan. However, the mass media has created awareness about the risks involved in the use of pesticides in agriculture. This has created a havoc regarding the accumulation of residues of these toxic chemicals in daily food. The aim of present study was to evaluate residual levels of three commonly used pyrethroid insecticides, cypermethrin, deltamethrin and cyhalothrin, in eggplant and okra fruits and to assess the effect of different chemicals and household processes: such as washing, peeling, blanching and cooking on the fate of residues of these pyrethroids. Results and Discussion Effect of Processing on Pyrethroid Residues in Eggplant and Okra Fruits The data given in Table-1 and 2 show that the residues of the insecticides were significantly high in unprocessed (raw) eggplant and okra fruits. They ranged between 1.13 mg kg-1 and 1.17 mg kg-1 for cypermethrin, 0.65 mg kg-1 and 1.08 mg kg-1 for deltamethrin and 1.40 mg kg-1 and 0.48 mg kg-1 for cyhalothrin in eggplant and okra fruit, respectively. However, the residues significantly reduced after they were washed, peeled, blanched or cooked (Fig. 1 and 2). Among all the processing techniques cooking was found the most effective in the elimination of residues of the three insecticides. The results showed that washing under tap water dislodged pyrethroid residues in eggplant and okra fruit significantly. As the pyrethroids are non- systemic in nature, therefore most of the residues remain as microparticles on the surface and are easily removed by the mechanical stirring with water during washing. Thus washing had a significant effect on the removal of residues in eggplant and okra fruit because of less/no penetration of the chemical into the cuticle layer of the plant surface and resulting in deposits removable by washing. These results agree with those obtained by several researchers [7-10] who reported 50-60% removal of the residues by the washing operation. A reduction of 10-30% of alphamethrin residues in tomato and eggplant have been reported [11]. 0 10 20 30 40 50 60 70 80 90 100 Washing Tap water dipping Peeling Blanching Cooking Processing Techniques %Reduction Cypermethrin Deltamethrin Cyhalothrin Fig. 1: Percent reduction of pyrethroid residues in eggplant fruit after processing. 0 10 20 30 40 50 60 70 80 90 100 Washing Tap water dipping Blanching Cooking Processing Techniques %Reduction Cypermethrin Deltamethrin Cyhalothrin Fig. 2: Percent reduction of pyrethroid residues in okra fruit after processing. Table-1: Effect of processing on the residue levels of the pesticides applied to egg plant fruit. Pesticides Raw mg kg-1 ± SD Washing mg kg-1 ±SD Tap water dipping mg kg-1 ±SD Peeling mg kg-1 ± SD Blanching mg kg-1 ± SD Cooking mg kg-1 ± SD Cypermethrin 1.13±0.01 0.45±0.01 0.79±0.01 0.23±0.002 0.20±0.02 0.06±0.005 Deltamethrin 0.65±0.1 0.39±0.06 0.44±0.07 0.18±0.03 0.19±0.03 0.10±0.14 Cyhalothrin 1.40±0.15 0.97±0.09 1.19±0.11 0.55±0.05 0.49±0.05 0.35±0.06 SD= Standard deviation of the mean Table-2: Effect of processing on residue levels of the pesticides applied to okra fruit. Pesticides Raw mg kg-1 ± SD Washing mg kg-1 ± SD Tap water dipping mg kg-1 ± SD Blanching mg kg-1 ± SD Cooking mg kg-1 ± SD Cypermethrin 1.17±0.13 0.59±0.07 0.81±0.09 0.23±0.03 0.12±0.01 Deltamethrin 1.08±0.12 0.72±0.08 0.78±0.09 0.54±0.06 0.19±0.01 Cyhalothrin 0.48±0.03 0.37±0.03 0.33±0.03 0.19±0.07 0.08±0.02 SD= Standard deviation of the mean
  • 3. Uncorrected proof Peeling with a knife is common household practice for most fruits and vegetables. Eggplant is eaten normally without their peel. Pyrethroid insecticides are applied directly to the crops and show very limited movement or penetration into the cuticle. Therefore, the residues of these materials are confined to the outer surfaces where they are amenable to removal by peeling, hulling or trimming operations. Cypermethrin, deltamethrin and cyhalothrin are non-systemic insecticides. Therefore, when peel was removed, great fractions of insecticide residues were also removed. Peeling reduced 80% residues of cypermethrin, 66% of cyhalothrin and 70% of deltamethrin in eggplant in the current study. Other studies carried out [12] also showed that most of the residue concentration was located in or on peel. Awasthi [12] reported that peeling off the mango fruit skin completely removed the residues of various pesticides including cypermethrin, reflecting the accumulation of residues in the fruit pericarp only and no further transfer of pesticide into the fruit pulp. In another study, 73% reduction in chlorpyrifos, 82% reduction in lindane level, 75% reduction in cypermethrin residues and 77% reduction in ethyl- enebisdithiocarbamates after peeling of asparagus was reported by Chavarri et al., [7]. Thus, removal of the peel may reduce a large fraction of residues, leaving little in the edible portions. This is especially important for fruits which are not eaten with their peels. However, the peel from commercial peeling processes is sometimes used as animal feed or for the production of essential oils or pectin (citrus, apple etc.). For such industrial processes, it is imperative to consider that non-systemic surface residues are often concentrated in the peel. Tap water dipping was found relatively less effective in the reduction of residues as compared to washing and peeling with reduction in the residues ranging from 27 to 31%. However, the results are in line with those reported for mango fruit, where the reduction in residues of fenvelerate and cypermethrin ranged between 21 and 27%, 66 and 68% for dimethoate and fenthion, rspectively [12]. Cooking/boiling was found the most effective among all processing operations. Cooking eliminated 95%, 86% and 78% residues of cypermethrin, deltamethrin and cyhalothrin, respectively in eggplant, where as for okra fruit these were 92%, 82% and 85%, respectively. Processes involving heat can enhance volatilization of the chemicals and their hydrolysis thus reducing the residue levels [13]. Hotellier [14] reported that deltamethrin residues reduced appreciably by cooking. However, Kumari [15] found 37, 40 and 42% reduction in synthetic pyrethroids in eggplant, cauliflower and okra fruits, respectively. Similarly 19 to 40% reduction in deltamethrin residues was observed in vegetables in a study by Randhawa et al., [10]. Effect of Chemical Treatment on Pesticide Residues in Eggplant and Okra Fruit The levels of insecticide residues in eggplant and okra fruit after chemical treatments are given in Table-3 and 4. The residues of each three of pyrethroids significantly decreased as compared to those in raw samples when the vegetables were treated with 4%, 8%, 12% each of NaOH, and acetic acid solutions. From these results it is clear that these washing solutions alone are highly effective in reducing the insecticide residues. Solution of 4% NaOH decreased 27 and 40% in insecticide residues in eggplant and okra fruit, respectively; 8% NaOH resulted in a reduction of 55 and 60%, respectively; where as 12% NaOH showed maximum reduction of 75% in residues in the vegetables (Fig. 3 and 4). 0 20 40 60 80 100 120 4% 8% 12% 4% 8% 12% NaOH solution Acetic Acid solution Chemical Treatment %Reduction Cypermethrin Deltamethrin Lamda Cyhalothrin Fig. 3: Percent reduction of pyrethroid residues in eggplant fruit by chemical treatment 0 20 40 60 80 100 120 4% 8% 12% 4% 8% 12% NaOH solution Acetic Acid solution Chemical Treatment %Reduction Cypermethrin Deltamethrin Cyhalothrin Fig. 4: Percent reduction of pyrethroid residues in okra fruit by chemical treatment. Acetic acid solution was found more effective than NaOH in the dissipation of cypermethrin, deltamethrin and cyhalothrin residues. Reduction in the pyrethroid residues increased with increase in concentration of the washing solution. There are some reports which show efficacy of chemical washing to dislodge the pesticides from crops. Pesticides treated tomato samples on washing with different levels of acetic acid solution gave,
  • 4. Uncorrected proof 47.0%, 33.7%, 91.5%, 86.0% and 93.7% loss in lindane, DDT, dimethoate, profenofos and pirimip- hosmethyl, respectively. The pesticide residues reduction increased with increase in concentration of solutions [16, 17]. The chemical solution causes dissipation of the pesticides through pH change. Dip treatment of fruits in water, NaCl solution, HCl, acetic acid, NaOH solutions and potassium permanganate removed 50 to 60% of surface residues of pyrethroids compared to 40 to 50% removal by hydrolytic degradation with NaOH and a detergent solution removed 50 to 60% residues [18]. Experimental Material All solvents and other reagents used were of purity compatible with pesticide residue analysis. Analytical grade reference insecticide standards of cypermethrin, deltamethrin and lambda cyhalothrin (>95% purity) were purchased from Dr. Ehrenstorfer GmbH, Germany. Chemical structures of the insecticides are given in Fig. 5. Commercial formulation of insecticides was purchased from open market. Acetic acid, sodium hydroxide, anhydrous sodium sulphate, sodium chloride, acetone, activated charcoal and glass wool were purchased from Merck, Pakistan, where as HPLC grade chemicals/solvents like water, methanol and ethyl acetate were from Sigma, Pakistan. Cypermethrin Deltamethrin Lambda Cyhalothrin Fig. 5: Chemical structures of the pesticides. Sample Collection A survey was conducted at three different locations in the adjoining villages of Rawalpindi and Islamabad regarding the use of pesticides by the local farmers. Eggplant and okra fruit samples were collected from ten different fields. The farmers were allowed to use their traditional methods of pesticide application on these vegetables. At maturity the samples of eggplant and okra fruit were collected from the farmer’s fields and brought to the laboratory and stored at -4o C. In the supervised field trial the okra and eggplant fields were selected and sprayed with cypermethrin (Arrivo 10 EC) @ 250 mL Acre-1 , lambda cyhalothrin (Karate 2.5 EC) @ 300 mL/acre and deltamethrin (Decis 2.5 EC) @ 250 mL Acre-1 twice at the insect pest infestation. Each insecticide was dissolved in 100 liter water and sprayed with Knap-sack sprayer. Both the vegetable crops received normal agronomic practices. Treatment of Contaminated Vegetables To determine the effects of household processes, the collected samples were divided into lots. They were subjected to washing under tap water (~25 ⁰C), peeling, blanching and cooking till softness as described elsewhere [19]. Moreover, the contaminated samples were soaked for 10 min in tap water, acetic acid solutions at concentrations of 4, 8 and 12% and aqueous solutions of sodium hydroxide at concentrations of 4, 8 and 12% prior to the extraction, cleanup and analysis of each sample. Extraction and Cleanup The insecticide residues in the vegetable samples were extracted and cleaned up following the method of [20] with some modification. Briefly, 1 kg of the vegetable was chopped into small pieces and blended in a Warring Blender (Anex, Germany) to make a fine paste. A representative sample of 50 g was taken and extracted with 50 mL ethyl acetate, 20g anhydrous sodium sulphate and 10 g NaCl on mechanical shaker (IKA, UK) for 1 h at 150 rpm. The extract was filtered through Whatman No. 4 filter paper. Glass wool was placed at the bottom of column used for cleanup. The column was then filled with activated charcoal that has been previously activated at 300 ⁰C for 3 h with a top layer of anhydrous sodium sulphate. The column was washed with acetone before loading the sample to the column. The cleaned up extract was collected in 100 mL flask and was then concentrated using rotary evaporator (Eyela, USA) at 60 ⁰C. The concentrated sample was transferred to sampling vials and the solvent was evaporated by gentle stream of nitrogen to just dryness. The dried samples were dissolved in
  • 5. Uncorrected proof 500 µL methanol prior to analysis and filtered through 0.45 µm nylon membrane filter (Millipore, Germany) before injection to High Performance Liquid Chromatography (HPLC) in order to remove any particulate material present in the sample. Instrumental Analysis and Method Validation The cleaned extracts were analyzed on a HPLC (Shimadzu Class-VP V6.13 SPI) equipped with variable-wavelength ultraviolet detector. The column used was reverse phase C-18 (25 mm x 4.6 mm id). Mobile phase was methanol and water (85:15) at a flow rate of 1 mL min-1 . The injection volume was 20 µL and the detector was set at 220 nm and run time was 15 minutes. Retention time obtained for different insecticides are shown in Table-5. Three metabolites of cypermethrin were identified and residues of cypermethrin are expressed as sum of the metabolites. A chromatogram of unknown okra sample is shown in Fig 6a. A high degree of resolution for separation of different components of sample (insecticides) was achieved under the conditions used in the study. When the same sample of okra was spiked with known quantities of each of the three insecticides the same degree of resolution was achieved as shown in Fig 6b. The spiked samples confirmed the presence of insecticides in the okra sample. Fig. 6a: Chromatogram of okra sample showing residues of lambda cyhalothrin, deltamethrin and cypermethrin. Fig. 6b: Spiked chromatogram of okra sample showing residues of lambda cyhalothrin, deltamethrin and cypermethrin. Table-3: Effect of chemical solutions on pesticide residues in eggplant fruit (mg kg-1 ±SD). NaOH solution Acetic acid solution Pesticide Raw 4% 8% 12% 4% 8% 12% Cypermethrin 1.13±0.01 0.67±0.01 0.51± 0.01 0.39± 0.01 0.83±0.01 0.53±0.01 0.02±0.01 Deltamethrin 0.65±0.10 0.53±0.08 0.37±0.06 0.23±0.03 0.49±0.07 0.40±0.06 0.26±0.04 Cyhalothrin 1.4± 0.15 1.34± 0.14 1.05±0.1 0.79± 0.07 1.26± 0.12 0.65± 0.06 0.36±0.03 SD= Standard deviation of the mean Table-4: Effect of chemical solutions on pesticide residues in okra fruit (mg kg-1 ±SD). NaOH solution Acetic Acid solution Pesticides Raw 4% 8% 12% 4% 8% 12% Cypermethrin 1.17±0.13 0.59±0.06 0.47±0.05 0.29±0.03 0.86±0.10 0.55±0.07 0.04±0.04 Deltamethrin 1.08±0.12 0.89±0.10 0.75±0.08 0.45±0.05 0.82±0.09 0.59±0.06 0.24±0.02 Cyhalothrin 0.48±0.04 0.39±0.03 0.27±0.02 0.21±0.02 0.39±0.03 0.30±0.02 0.14±0.02 SD= Standard deviation of the mean Table-5: Linear range, recovery and retention time of insecticides. Insecticides Retention time (min) Recovery(%) Spike level (µg) Linear range (µg) R2 Lambda Cyhalothrin 5.15 97 30 1.11 to 71 0.999 Deltamethrin 5.45 98 30 0.91 to 58 0.999 Cypermethrin (sum of metabolites) 5.73, 6.06, 6.37 95 30 1.17 to 75 0.998
  • 6. Uncorrected proof The method used for determination of pyrethriod insecticide residues was validated before analysis of actual samples. For this purpose 30 µg each of three insecticides were added in the 50 g blended okra (from supervised field where no spray was done) and extracted as described above. Cleanup and concentration was also performed by following the same set of conditions. Reproducible recoveries of > 94% were obtained for the insecticides used in this study (Table-5). Linear range of determination for each of the insecticide was constructed by using step wise dilution analysis of each insecticide on HPLC. The standard curve showed that linear range was found to be 1.17 to 75 µg, 0.91 to 58 µg and 1.11 to 71 µg for cypermethrin, deltamethrin and lambda cyhalothrin, respectively. The analytical method was found to be highly sensitive to fulfill quality criterion for insecticide residue analysis. The R2 value for standard curve of each insecticide is also shown in Table 5. Then concentration of unknown sample was quantified by using the same standard curves, respectively for each of the insecticide (Fig 7). Fig.7: Standard curves for deltamethrin, cype- rmethrin and lambda cyhalothrin. Conclusion Residual effect of cypermethrin, deltamethrin and lambda cyhalothrin in eggplant and okra fruits and their dissipation patterns during different processing operations viz washing, peeling, cooking and blanching and dipping in chemical solutions was studied. The results revealed that processing and chemical treatments were effective in reduction of the insecticide residues (cypermethin, deltamethrin and lambda cyhalothrin) in eggplant and okra fruits. Though washing with water is important to decrease the concentrations of insecticide residues, cooking of vegetables helps greatly to eliminate major fractions of the pesticide residues. Simple dipping of vegetables in acidic or alkaline solutions can also reduce the insecticide residues efficiently. The acceptable maximum residue limits established by the FAO/WHO Codex Alimentarius Commission, or other international bodies refer to the whole vegetables and therefore are appropriate for assessing compliance with good agricultural practices. However, these limits may be of limited significance for assessing dietary exposure to pesticides from the vegetables such as eggplant and okra, which are consumed after household processing. Acknowledgements The authors are grateful to the National Institute of Food Science and Technology University of Agriculture, Faisalabad, for providing facilities to carry out a part of the research project. References 1. G. F. Pang, C. L. Fan and Y. Z. Chao, Journal Association of Official Analytical Chemists International, 77, 738 (1994). 2. G. F. Pang, C. L. Fan, Y. Z. Chao and T. S. Zhao, Journal of Chromatography, 67, 348 (1994). 3. T. J. Shafer, D. A. Meyer and K. M. Crofton, Environmental Health Perspectives, 113, 123 (2005). 4. A. Saleem and Ibrar-ul-Haq, Pesticides standardized in Punjab - dose(s), crop(s) and target pest(s). Directorate of Agricultural Information Government of the Punjab, Agriculture Department, Lahore (2003). 5. B. Duara, A. Baruah, S. C. Deka and N. Burman, Pesticide Research Journal, 15, 43 (2003). 6. M. Nafees, M. R. Jan, Soil and Environment, 28, 113 (2009). 7. M. J. Chavarri, A. Herrera and A. Arino, International Journal of Food Science and Technology, 40, 205 (2005).
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