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American-Eurasian J. Agric. & Environ. Sci., 6 (5): 513-519, 2009ISSN 1818-6769 IDOSI Publications, 2009

Corresponding Author: Sajjad Ahmad Baig, Institute of Quality and Technology Management, University of the Punjab, Lahore 54590, Pakistan Tel: +92 3004781314

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Determination of the Organophosphorus Pesticide in Vegetables by High-Performance Liquid Chromatography

Sajjad Ahmad Baig, Niaz Ahmad Akhtera, 1 1Muhammad Ashfaq and Muhammad Rafique Asi2 3

Institute of Quality and Technology Management, University of the Punjab, Lahore 54590, Pakistan1

Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad38000, Pakistan2

Department of Entomology, University of Agriculture, Faisalabad 38000, Pakistan3

Abstract: In the existing study, an effort has been made to evaluate and determine residues of three pesticidesin vegetables, belonging to Organophosphate group, which was extensively used in the Southern part ofPunjab province in Pakistan. Triazophos, profenofos and chlorpyrifos Organophosphorus insecticides, werecommonly used on the summer vegetables like: egg plant, Pumpkins and okra cultivated in study area.Vegetables were analyzed for pesticide residues using multiresidue analysis by High-Performance LiquidChromatography (HPLC) equipped with UV detector. It was observed that 33.0% of the samples werecontaminated with any of the above three pesticides. The results showed that 8% of the samples testedcontained residues higher than the MRLs. Based on observations made in these studies; it is proposed thatmore extensive monitoring investigations covering all vegetables from different agro-climatic regions of thestate be carried out to find the exact position of pesticide residues.

Key words: HPLC Organophosphate pesticides Vegetables Pesticide Residues

INTRODUCTION A huge amount of pesticide is used on crops as well

Agriculture is the single largest sector of Pakistanis becomes the reason of accumulation of pesticide residueseconomy with 21 percent contribution to GDP and in the primary agriculture products. The problem ofemploying about 44 percent of the workforce. More than residues accumulation needs more attention intwo-thirds of Pakistans population lives in rural areas vegetables because most of time these are consumedand their livelihood continues to revolve around either raw or without much storage time [4]. Currentlyagriculture and allied activities [1]. Organophosphorus pesticides (OPs) enjoy wide use in

Vegetables are important ingredient of our food the world for the pest control with Organochlorinehaving a high nutritional value. Vegetables, like, okra, pesticides [5, 6], which leads to increased world foodegg plant, spinach, cauliflower, tomato, pumpkin, carrots, production. These Organophosphorus pesticides are alsoturnips etc. are produced in the country for local widely used in Pakistan for controlling the agricultureconsumption as well as for export purposes [1]. For better pests. In spite of the toxicological problem its efficiencyproduction and aesthetic value, farmers are using a large has been demonstrated in fruit flies, aphids and leafamount of insecticides during the entire period of growth miners The continued use of organophosphorusof vegetables, even at fruiting stage and sometimes pesticides increases the possibility that residues of thesefarmers also ignored the recommended waiting period compounds could be found in some vegetables [7]between the harvest and last spray. Owing to this and affecting the alimentary security; that is the reason for theother injudicious practice related to pesticide usage, commitment between the public health and the sanitarypesticides become the inner part of vegetable, in the defense.shape of residues, which could be used by consumers For this reason, it is urgent to develop programsthus creating health hazards [2, 3]. related to the monitoring of pesticide residues in food to

as on vegetable and their irrational and continual use

Am-Euras. J. Agric. & Environ. Sci., 6 (5): 513-519, 2009

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secure consumers. These programs could determine the Profenofos is a broad-spectrum Organophosphorusamount of the contamination problem and recognise away to solve the situation. Pesticide (Organophosphorus)residue monitoring studies have been reported in manycountries of the world on fruits and vegetables [8-13].Effects of pesticides have been reported in Pakistan invegetables, fruits, feed, cottonseed and fish meal atdifferent intervals [15-19] The above mention studies inPakistan showed that most of the samples werecontaminated by chlorinated pesticides; some of themwere exceeding the maximum residue limits, which can bethreatening for the ultimate consumers. Although, a highvariation of pesticide residues was observed in fruits andvegetables that could be due to change in the climaticzone (hot, humid and cold) and variation among differentspecies of plants. No study has been carried out inPakistan to analyze the OPs residues in vegetables, alsothe study on pesticide residues in vegetables was alsonever carried out in the Southern Punjab, Pakistan.

Selection of Pesticide: However, nowadaysorganochlorine (OC) pesticides were not used primarilydue to their persistence in the environment [20]. At thistime, the use of OPs and CMs glided due to theiravailability and quick degradation in the environmentOrganophosphorus pesticides have been widelymanufactured and used in the world. And they replacedthe chlorinated pesticides. A survey of the study area i.e.southern part of the Pakistani Punjab, was carried out toassess the extent of widely used pesticides beforesample collection and analysis. In the survey, 240 farmersfrom four districts (Layyah, Muzaffargarh, Multan andKhanewal) were interviewed to through a well structuredquestionnaire to judge the extent of pesticide use,practices related to the a pplication of the pesticidesand equipments and safety measures. Results of thesurvey showed that many Ops pesticides were usedarea but three insecticides (Triazophos, Profenofosand Chlorpyrifos) were widely used in the area on thevegetable crops.

Triazophos (C H N SPO ) is a common name12 16 3 3for O,O-diethyl-O-1-phenyl-1H-1,2,4-triazol-3-ylphosphorothioate). Triazhphos is a moderately toxicchemical that is a broad spectrum and no systemicOrganophosphorus pesticide that is used in largequantities throughout the world [21]. It is used to controlaphids, bollworms, red spiders, fruit borers, leaf hoppersand cutworms on a variety of crops, such as fruits, cotton,maize, wheat, cereals and vegetables and is particularlyeffective in the control of plant nematodes [22].

pesticide. Profenofos (Ensedan1), O-(4-bromo-2-chlorophenyl)O-ethyl S-propyl phosphorothioate, wasdeveloped for those insect strains which developedresistant to other Organophosphorus pesticides It isselectively toxic to insects compared to mammals due todifferent metabolism of the propylthiol group [22].

Chlorpyrifos is an organophosphate pesticide usedthroughout the world around the world and Chlorpyrifosis currently registered as insecticide with the used in awide variety of crops and vegetables The primarydegradant of chloropyrifos is 3, 5, 6-trichloro-2-pyridinol,a base hydrolysis product [23]. As compare to otherorganophosphate pesticide, Chlorpyrifos is fairly stableand persistent member of organophosphate pesticides

MATERIALS AND METHODS

In this section, all the materials used in the study andthe methods of analysis have been discussed. Thissection has been divided into three sub-sections, whichhave been discussed in following text.

Chemicals: Ethyl acetate (HPLC grade), Methanol (HPLCgrade), Sodium chloride (HPLC grade), acetonitrile (HPLCgrade), potassium permanganate (GR 99%)were allobtained from Merck, Germany)grade. Distal water wasobtained with the help of glass-distilled and furtherpurified with the help of a Millipore Milli-Q water purifier.Germany. Analytical standard of profenofos, chlorpyrifosand triazophos was obtained from Dr Ehrenstorfer Ltd(Augsburg, Germany). Stock standard solution (1mg/mL)was prepared by dissolving standard in acetonitrile andstored in the dark at 4C

Sample Collection Procedure: In this portion of thechapter sample collection and preparation methodologieshas been discussed. This section is further divided intotwo parts;

Sample Collection from Farmers Fields: Sampling wascarried out, from July 2008 to the last week of August 2008from, the farmers fields of four districts in SouthernPunjab., i.e., Layyah, D.G. Khan, Muzafargarh and Multan.Some parts of the study area, were suffering from water-logging and salinity, problems. Different depths of water-tables were found in the study area. Normally, in themonth of January, the temperature is low and theevaporation is slow. The month of June and July, are the


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hottest months in the study area. Rainfall occurs mostly Extraction and Clean up: Vegetable samples werein the months of July and August, during the monsoon extracted by following the method of 24 with someperiod. Rabi and Kharif are the two cropping seasons, in modification made by 25 and 26. Ethyl acetate was usedthe area. Since the Kharif season covers most part of the for the extraction because of extraction efficiency, of ethylsummer and rainy periods, vegetables of Kharif season, acetate is to be equally good for the extraction of bothwere selected because of the maximum sprays were carried polar and non-polar pesticides in all primary agricultureout on them due to a favorable atmosphere for pests, to products [27] g of vegetable sample was grind and thenflourish. well-homogenized 50 g from the grind vegetable was

Samples of three vegetables i.e., Okra, (Abelmoschus selected and mixed with 20 g anhydrous sodium sulfate +.esculentus L. Moench), Pumpkins, (Cucurbita Pepo) 1 moler Nacl and 50 ml of ethyl acetate. The whole mixtureand Egg plant (Solenum melongena L.), which were was s haken in GFL shaker (Germany) at high speed forextensively grown vegetables in the study area, were 5 minutes and filtered. And then transferred onto a glasscollected. Information about farmers aptitude, type of column having 5 ml layer of anhydrous sodium sulfatepesticide used and agronomic practices adopted by the and 10 g activated charcoal for bleaching. The mixture wasfarmers, were also collected with the help of a well vacuum-filtered through a Whatman no. 6 filter paperstructured questionnaire. After filtering solution was shifted into 250 ml round flask

Samples from 108 farms; comprising 32of egg plant, and then evaporate and concentrate with a rotary32 of okra and 32 of pumpkins were collected. Composite evaporator at 55C. and made a volume up to 10 ml andsamples: consisted of 12 kgs of each vegetable were then filtered through a 0.45 m filter membrane collectedcollected from the field, when it was ready for sale. After elute was concentrated just to dryness under a gentleselecting the field location and considering all the factors, stream of nitrogen gas and re-dissolved in acetonitrile,which can affect the pesticide residues, the desired volume made up to 500 l for analyzing with HPLCvegetables, were collected in a defined way, such as, S or HPLC analyses were performed on isocratic systeman X in the field. During the sample-collection disposable using a Shimadzu Chromatograph including LC-10 ASgloves were used and changed every time before pumps, 20-ll Reodyne injector, SPD-10A UV detectorcollecting the next sample. Dust from the samples was operating at 208 nm and a Supelco C18 analytical columnremoved with light brushing, without washing prior to (25 cm _ 4.6 mm(i.d)). a mixture of water and acetonitrileplacing them into, the collection bags. Samples were (ACN) was used as mobile phase..For the removal of anydouble packed, in order to save from any other impurity, bubbles and dissolved oxygen, the double distalcontamination and transported from the field to the water and ACN was firstly passed through 0.45 lm filterlaboratory. During the transportation of samples, it was paper with filtration assembly and then sonicated forassured that these were protected against any alterations 10. The flow rate was kept at 1 ml/min.in the residue-situation. In the laboratory, samples were Before analyzing the samples of vegetables, thefreeze in the refrigerator and analyzed within a weeks time HPLC method was validated using chromatographicafter collection. Only edible parts were selected for the parameters including recovery, reproducibility and limitsanalysis of pesticide residues. of detection (LOD) as proposed by 29 and 30. For

Sample Collection from SupervisedFields: Three summer were spiked with selected pesticides at 0.5mg/kg andvegetables i.e. okra (Abelmosc hus esculentus L.), 1.5 mg/kg.pumpkin (Cucurbita Pepo) and egg plant (Solanum For each fortification level, five replicates weremelongena L.) were grown in the experimental field of, analyzed by HPLC using UV detector. The efficiency ofselective districts respectively. Each vegetable was grown the method was validated statistic with recoveries studies,in a separate plot, measuring 150 x 30 feet in four blocks. fortification samples without pesticides (organicEach plot was separated by fallow strip of 20 feet. The vegetables) by spiking the three different amounts ofvegetables, in each season, received normal agronomic pesticides. And then pesticides were extracted from thepractices, without the spraying of pesticides along with vegetables as described early. the reproducibly of allthe selective spray of pesticides. The samples collected pesticides were more than 85%.which was satisfactory.from supervised field trial were used for the spiking of These values were quite satisfactory and meet thevegetable. requirements of the European Commission 31, indicating

recovery experiments, untreated samples of vegetables

OP(OCH2CH3)2

SN

NN

OP(OCH2CH3)2

SN

NN

NCl

C l C l

OP(OCH2CH3)2

S


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that a method can be considered accurate and precisewhen the accuracy of data is between 70 and 110%, withrelative standard deviations (RSDs) not higher than 20%.

RESULTS AND DISCUSSION Fig 1: Structure of Triazophos

This research work was completed in two phases.In the first phase, a survey was conducted, fordetermining the nature and frequency of pesticidecurrently being used. Only those farmers wereinterviewed who have at least 3 acres for cultivation. As Fig 2: Structure of Profenofossuch structured questionnaire will developed to collectinformation from the farmers about their, knowledge of theadverse health effects of pesticides, work practices relatedto the pesticide use, use of protective measures and alsoabout an inventory of pesticides used on the farm Onthe bases of the questionnaire, the Organophosphorus Fig 3: Structure of Chlorpyrifospesticide for further study was selected.

It was found that 35 samples, out of 108, were found Okra: Out of a total, 36 samples of okra analyzed, 15 wereto be contaminated with some of the organophosphate found to be contaminated with the residues of selectedpesticides, studied. Profenofos was found to be the OP pesticides. The results of residues detected inmaximum in the analyzed samples of okra (25%), egg plant vegetable samples collected, from the farm gate are(17%) and pumpkins (5%), where as, Chlorpyrifos was presented through the Fig 4.2 and Table 4.3. The resultsfound to be the second most common op pesticide in the indicated that at raw stage the highest Profenofos residuevegetable. It was observed that frequency of pesticide was found in egg plant were 1.6 mg/kg (ND 1.6mg /kg);residues detection in the okra was directly related to their whereas the four samples were contained pesticideusage in the area. residues of Profenofos more than the MRLs and 7(19%)

Egg Plant: Out of a total of 36 samples of egg plant, the residues of chlorpyrifos, that the one sample wasanalyzed, 17 were found to be contaminated with the contained residues of chlorpyrifos exceeded from theresidues of pesticides; Chlorpyrifos was detected in a MRLs. Five samples of okra found to be contaminatedmaximum number of egg plant samples, as compared to with the residues of triazophos. The initial data collectedthe other two OP pesticides. It was determined that in the survey showed that the usage of profenofos was8 samples, out of 36 samples, were found to be polluted more on the okra crops as compared to that on others.with the chlorpyrifos -residues and out of these 8 During the survey it was also observed that, in manysamples, three samples contained pesticide-residues, more cases, okra was grown near the cotton field and thethan the MRLs The results of organophosphate pesticide farmers used same treatment of insecticide, withoutresidues, detected in vegetables, samples collected from differentiating the two crops.the farm gate, are, presented with the Fig 4 and Table 1.The results indicated that at a raw stage, the highest Pumpkins: A total of 36 samples of pumpkins, wereamount of chlorpyrifos residues found in egg plant was analyzed, out of which only 5 samples were found1.45 mg/kg (ND 1.45 mg /kg) and 17% of the egg plant to be contaminated with pesticide residues. Thesamples were seen contaminated with profenofos. residues of triazophos were not found, for the main reasonOne sample of the egg plant contained profenofos of this that no spray of triazophos was reported to beresidues more than the MRLs. Triazophos was detected carried out on pumpkins. In the area, most of thein a minimum number of egg plant samples, as in pumpkins were produced without the application of anycomparison to the other OPs. The initial data collected in pesticide sprays, which, if at all carried out, pesticide werethe survey showed that chlorpyrifos, was used more on carried out on early stage. No sample of the pumpkins wasegg plant, in the area, as in comparison to other found to contain more than one type of pesticide-organophosphates. residues.

samples of okra were observed to be contaminated with


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Table 1: Pesticide residues (mg kg_1) in fresh vegetables, collected from study area

Sr.No Vegetable Insecticide Analyses Contamination Range of Residues mg/kg MRLs

Okra 36 Profenofos 9(25%) Nd-.1.6 -

Triazophos 5(14%) Nd-.9 .5*

Chlorpyrifos 7(19%) Nd-1.7 1.5**

Egg plant 36 Profenofos 6(17%) Nd-1.45 1

Triazophos 4(11%) Nd.-.8 -

Chlorpyrifos 8(22%) Nd-1.34 1 **

Pumpkin 36 Profenofos 2(5%) Nd-.45 0.05*

Triazophos 1(2.5 %) Nd-.32 -

Chlorpyrifos Nd .2**

*Japanese MRLs ** FAOMRLs

Fig. 4: Occurrence of pyrethroid pesticide residues in fresh vegetables samples

Fig. 5: Organophosphate Residues in Vegetables

On the whole, 35 samples out of 108 were found to be residues level, did not care for the quality of their producecontaminated with selected type of (organophosphate) as they, were neither interested in ensuring the vegetablepesticide-residues. From the results, it was clear that safety, nor followed the good agricultural practices,although all selected vegetables were found to be because of their ignorance or lack of such a training.contaminated with the pesticide-residues, yet okra was It was further noticed that most of farmers did not fallowseen to be affected the most from the organophosphate the concept of Good Agriculture Practices. And theypesticides. Mostly, only one type of residues was found did not follow the with holding periods. A withholdingin the vegetable samples. But in some sample more than period (WHP) is the minimum time you must wait betweenone type of residues were detected. Out of 35 pesticide- applying an agrochemical and final harvesting or grazingcontaminated samples, 9 were contained residue more of the crop. Such a period allows enough time to decay tothan their MRLs an acceptable level [32]. Most of the vegetable samples

On a further investigation, from the farmers through were brought to the market, after first day of spraying,an open discussion, it was found that most vegetable contained residues, which exceeded their MRLs. Result ofgrowers, who were using toxic pesticides, on a high this study, were verified from the study of 33. Although,


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it was cleared that level of pesticides in the primary 7. Quintero, A., M.J. Caselles, G. Ettiene, N.G. deproducts were less as compared to the previous studies Colmenares, T. Ramirez and D. Medina, 2008.in Pakistan. Yet these results generally agree with the Monitoring of organophosphorus pesticide residuesother studies already carried out, locally [34] and in vegetables of agricultural area in Venezuela.international with 35 This was mainly due to the using of Bulletin of Environ. Contamination and Toxicol.,organophosphorus pesticides and new chemistry 81: 393-396.products, instead of organochlorines pesticide which are 8. Amoah, P., P. Drechsel, R.C. Abaidoo and W.J. Ntow,more persistent and have more chances of accumulation 2006. Pesticide and pathogen contamination ofin food [36] As an outcome of this study, it is suggested vegetables in Ghana's urban markets. Archives ofthat a wide-range of studies, on the monitoring of all Environ. Contamination Toxicol., 50: 1-6.summer vegetables and other crops grown in different 9. Mukherjee, I., 2003. Pesticides Residues inagro-climatic regions of Pakistan should be undertaken for Vegetables in and around Delhi. Environ. Monitoringdetermining the level of pesticide of pesticide residues and Assessment, 86: 265-271.and developing awareness about Good Agriculture 10. Gobo, A.B., M.H.S. Kurz, I.R. Pizzutti, M.B. AdaimePractices and R. Zanella, 2004. Development and validation of

ACKNOWLEDGEMENT organophosphorus pesticides in tomatoes. J.

The authors thank to university of Punjab, Pakistan 11. Martnez Salvador, I., A. Garrido Frenich, F. Egeaand also to HEC (Higher Education Commission) Pakistan Gonzlez and J. Martnez Vidal, 2006. Determinationfor providing financial aids for this project. of Organophosphorus Pesticides in Vegetables byLiterature Cited GC with Pulsed Flame-Photometric Detection and

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