SOLID-PHASE EXTRACTION AND CHROMATOGRAPHIC DETERMINATION OF PESTICIDES IN FOOD AND WATER SAMPLES Muhammad Rafique Asi INSTITUTE OF CHEMISTRY UNIVERSITY OF THE PUNJAB LAHORE, PAKISTANSOLID-PHASE EXTRACTION AND CHROMATOGRAPHIC DETERMINATION OF PESTICIDES IN FOOD AND WATER SAMPLES: A thesis submitted to the University of the Punjab In fulfillment of requirement for the Degree of Doctor of Philosophy In Chemistry By Muhammad Rafique Asi* M. Se. ~ roy Vay = IN ‘UTE OF CHEMISTRY ~ UNIVERSITY OF THE PUNJAB_ LAHORE, PAKISTAN 2003 *Corresponding Address Scientific Officer Nuclear Institute for Agriculture and Biology Jhang Road P. O. Box No. 128, Jhang Road, FAISALABAD, PAKISTAN Phone No: — 00-92-41-654221 - 30 Fax No: 00-92-41-654213 E. Mail: asi_niab(@ hotmail.comTABLE OF CONTENTS TITLE CERTIFICATE ACKNOWLEDGEMENT ABSTRACT LIST OF FIGURES LIST OF TABLES LIST OF CONTENTS CHAPTER-I. Introduction CHAPTER-2. Review of Literature CHAPTER-3. Materials and Methods CHAPTER-4. Results and Discussion CHAPTER-S. References PUBLICATIONS vii xiii 37 59 166 192APPROVAL CERTIFICATE It is certified that this thesis is based on the results of experiments carried out by Mr. Muhammad Rafique Asi and it has not been previously presented for a higher degree. Mr. Muhammad Rafique Asi has done this research work under our supervision. Ie has fulfilled all the requirements and is qualified to submit this thesis for the degree of Doctor of Philosophy in Chemistry. fens eo De Ys ” Supervisor Co-supervisor Dr, Jamil Anwar Chudhary Dr. Altaf Hussain Director, Instiwute of Chemistry Chief Scientific Officer (retd.) University of the Punjab Advisor to Member Biosciences Lahore, Pakistan. NIAB, Faisalabad.ACKNOWLEDGEMENTS Praise be to ALMIGHTY ALLAH, the omnipotent, the omnipresent, the most merciful and the most compassionate who blessed me with sound health, realistic thinking and opportunity for higher studies and his Holy Prophet, MUHAMMAD (Peace be upon him) the most perfect and exhaled among ever born on the earth. I deem it utmost pleasure to avail myself of this opportunity to express the heartiest gratitude and deep sense of obligation to my reverend supervisor, Prof. Dr. Jamil Anwar Chaudhary, Director, Institute of Chemistry, University of the Punjab, Lahore and co-supervisor, Dr. Altaf Hussain, Chief Scientific Officer (retd.) and Advisor to Member Biosciences, PAEC, Islamabad. Their skilful guidance, leamed patronage, untiring help at all time, masterly advice and inspiring attitude made it very easy for me to undertake this work and to write this manuscript. I have the honour to express my deep sense of gratitude and profound undebtness to ever affectionate Dr. Mohsin Iqbal, Director, NIAB, Faisalabad, Mr. Zafar Iqbal, Mr. Rashid Ahmad, S. Tahir Mehmood, Mr. Kamran Mirza and other colleagues in Biological Chemistry Divisions, for their great help, valuable guidance and ever encouraging atlitude throughout the course of this investigation. There is a special pleasure in recording my heartfelt thanks to Mr. Saeed Ahmad for tracing all the chromatograms, Mr. Shabbir-ud-Din Ahmad for their precious time and moral support This work was partly supported by IAEA grant through the Research Contract No, 9907/RB which is gratefully acknowledge. Sincere thanks are due to my adoring parents and family members for their moral support, unforgettable sacrifices and offering countless prayers throughout my career. Finally, as is customary, the errors that remain are mine alone and [ apalogise if | caused offence to anybody. (Muhammad Rafique Asi)SOLID-PHASE EXTRACTION AND CHROMATOGRAPHIC DETERMINATION OF PESTICIDES IN FOOD AND WATER SAMPLES ABSTRACT Modem agriculture has and is producing thousand of tonnes of food for domestic and foreign market. The tool of the trade includes many different chemical compounds that are used for a wide variety of pest problems. Pesticide use has improved both the efficiency of growing crops and the quality of food produced. However, alongwith the benefits emerged the potential effect of trace amounts of pesticide residues remaining on food commodities and there has been great concern by consumer groups demanding assurance from the agriculture community that the food we eat is indeed safe. The use of contaminated fruits and vegetables for long time may cause illness and other serious diseases in man and wildlives. Different analytical tools like solid-phase extraction (SPE), thin layer chromatography (TLC), gas chromatography (GC) and high performance liquid chromatography (HPLC) were used to assess the pesticide residue level in water, fruits and vegetable samples. Ethyl acetate, acetone and methanol proved to be good solvents for successive elution of pesticides adsorbed on the cartridge bed. The recovery percentage of each pesticide recovered from SPE cartridge (ENVI-Carb) showed excellent behaviour (>95%). From the results it is revealed that 200 ml sampie volume and 2 minutes equilibrium time before the elution of pesticides gave high recovery of every type of pesticide. Waters of different aquifers behaved in the same way but that water, which had soil particles, showed some adsorption of pesticide which could not be eluted completely. SPE cartridges were used but ENV1-Carb found good among others with salient features for the pre-concentration of pesticide residues from surface and ground waters. It showed consistent recovery behaviourafler successive elution with organic solvents and did not develop any backpressure. The use of this technology for the extraction and preconcentration of pesticide residues for aquifer samples did not pose any risk to the environment due to the less use of solvent. The extracted/eluted water samples by SPE cartridges were analyzed by GC and HPLC, The water samples from cotton-belt contained maximum pesticide residues as compared to rice and vegetable areas. Well water was found highly contaminated than canal and handpump waters whereas least residues were found in tubewell samples. High residues of endosulfan were found in aquifers of cotton-belt area. Residues of insecticides were found in greater proportion than the herbicides and fungicides in water samples. No fungicide was detected in water samples of cotton area but chlorothalonil was detected in surface water of Gujranwala, surface and ground waters of Stalkot and Sheikhupura but no residue was detected in Faisalabad areas. Water samples of vegetable areas were analyzed and it was found that alpha- endosulfan, lindane and p, p-DDT were present in all samples except tubewell water, Alpha-endosulfan was in high concentrations in water samples following lindane and p, p-DDT. Most water samples were found contaminated with fungicides and their concentrations in canal water samples were not same in the three provinces, The concentration of isoproturon was higher in NWFP water samples as compared to Punjab and Sindh, Mcthamidophos, monocrotophos and phosphamidon residues were found higher in canal water in Punjab, Sindh and NWEP but these compounds have lower residues in tubewel] waters in these provinces. Pesticide residues in vegetables and fruits were determined by GC, HPLC and HPTLC, High residue of mancozeb was detected in tomato and potato. A very high concentration of carbendazim was found in cauliflower, tomato and potato samples belonging to farm areas. Benomy| was detected in ladyfinger, bittergourd, brinjal and pumpkin whereas chlorothalonil was found only in bittergourd andbrinjal. Imidacloprid was detected in most vegetable samples but high concentration was found in tomato. Chlorpyriphos and methamidophos were also found in most vegetables but high residues of chlorpyriphos and methamidophos were detected in cauliflower, spinach, tomato, potato and tadytinger respectively. Pyrethraids were also found in afl vegetable samples but their residues were higher in farm samples as compared to city area samples. High residues of bifenthrin were found in spinach, cauliflower, tomato, ladyfinger and green peas whereas cypermethrin and cyhalothrin were detected in brinjal and green peas. Decamethrin was only found in potato, Endosulfan, heptachlor and DDT’ were also found in most vegetables. Mancozeb, triforin, thiophanate-methyl, fenarimol and carbendazim were detected in most fruits and higher concentrations of these compounds were detected in farm samples. High residue of carbendazim was found in apricot samples. Endosuifan, DDT and heptachlor were also found in fruit samples. Melon had high residues of endosulfan and DDT than other fruits whereas no residues of endosulfan and DDT were detected in guava and apple samples. Heptachlor was also found in pear, banana, plum, apricot and grapes in substantial concentrations, High residues of cypermethrin, bifenthrin, fenvalerate, cyhalothrin and fenpropathrin were found in pear, Orange, banana, plum, apricot, melon, guava, mango and apple samples, Imidacloprid was only found in pear, orange and banana whereas dimethoate and monocrotophos in apple and mango samples.SOLID-PHASE EXTRACTION AND CHROMATOGRAPHIC DETERMINATION OF PESTICIDES IN FOOD AND WATER SAMPLES List of Figures Figure No. 41 42 43 44 45 4.6 4.7 Contents Comparison of recovery (%) of pesticides in water samples obtained from different sources Standard chromatogram of organochlorine pesticides (0.05 ig/ml) with ECD 1, lindane, 2. heptachlor, 3..a-Endosulfan, 4. p, P'-DDE, 5. p, p'-DDD, 6. p, p’-DDT Chromatogram of standard pesticides in NPD-P made (SE-S4, 30 m x 0.53 mm ID, column 120 °C (3 min) to 300 °C @ 5 °C min.", injector 250 °C, detector 310 °C, flow rate 1.5 ml min”, injection 1 yul, splitless, Peaks; 1. dichlorvos, 2. mevinphos, 3. ethoprop, 4. monocrotophos, 5. phorate, 6, dimethoate, 7. diazinon, 8. phosphamidon, 9. parathion-methyl, 10. fenitrothion, 11. malathion, 12, chlorpyriphos, 13. fenthion, 14. chlorfenvinphos Chromatogram of standard pesticides analyzed by reverse-phase HPLC in gradient programme; UV 214 nm, mobile phase acetonitrile/water, flow rate 1.5 ml min.", column temperature 30 °C (Peaks 1-11; 1, oxamyl, 2. methomyl, 3. aldicarb, 4. simazine, 5. cyanazine, 6. carbofuran, 7. atrazine, 8. carbaryl, 9. diuron, 10. linuron, 11. ehlorpropham) Chromatogram of pyrethroids analyzed by RP-HPLC in isocratic programme; UY 206 nm, mobile phase acetonitrile: water (75:25), flow rate | m! min, column temperature 30 °C (Peaks 1-6; 1. fenpropathrin, 2. cyfluthrin, 3. A-cyhalothrin, 4. c- cyhalothrin, 5. deltamethrin, 6, fenvalerate) Chromatogram of standard mixture of 0,p-DDT and it metabolites, UV 240 nm, flow rate 0.5 ml min’, mobile phase 100% HPLC grade methanol Chromatogram of standard mixture of p,p'-DPT and it metabolites, UV 240 um, flow rate 0.5 ml min", mobile phase 100% HPLC grade methanol Page 67 73 76 79 80 81 8I vii4.8 4.9 4.10 4.11 4.12 4.13 4.4 4.21 4.22 4.23 4.24 4.25 4.26 4.27 4,28 4.29 4.30 43 Chromatogram of standard chlorinated pesticides by GC-ECD; 1. o-BHC, 2. B-BHC, 3. lindane, 4. heptachlor, 5. aldrin, 6. heptachlor epoxide, 7. ct-Endosulfan, 8. dieldrin, 9. p, p’-DDE, 10. endrin, 11. B-endosulfan, 12. p, p’-DDD, 13. Endosulfan sulphate, 14. p, p’-DDT Chromatogram of standard pesticides (mixture of OP’s, OC’s, carbamates and pyrethroids analyzed by GC-ECD); 1. carbofuran, 2. dichlorvos, 3. phosphamidon, 4. fenvaierate, 5. dimethoate, 6. lindane, 7. diazinon, 8. parathiommethyl, 9. fenilrothion, 10. malathion, 11, @-Endosulfan, 12. azinphos- methyl, 13. a-Cypermethrin, 14, deltamethrin Pesticide concentration in canal and well water samples in the Punjab Pesticide residues in canal water of rice area Fungicide residues in canal water samples of vegetable grown areas Organophosphorus pesticide residues in canal waters in vegetable areas of three provinces Pyrethroid residues (yyL"') in surface and ground waters in vegetable grown areas of three provinces Pesticide residue concentration in canal waters collected from cotton and vegetable area Response of marker compounds in o-loludine + KI method Response of marker compounds in silver nitrate + UV exposition method Response of marker compounds in Hill reaction method Response of marker compounds in fungi spore inhibition method Response of pesticides in enzyme inhibition with cow liver extract and B-naphthyl acetate method Response of pesticides in enzyme inhibition with horse blood serum and acetylthiocoline iodide substrate method «-Endosulfan residues (mg ky") in vegetables Organophosphorus pesticide residues (mg kg”) in vegetables Fungicide residues (mg kg"') in vegetables Pyrethroid pesticide residues (mg kg") in vegetables Fungicide residues (mg kg’!) in fruits «t-Endosulfan residues (mg kg") in fruits Organophosphorus pesticide residues (mg kg!) in fruits Pyrethroid pesticide residues (mg kg") in fruits Dimethoate residue (mg kg") in fruits Pyrethroid and fungicide residues (mg kg ') in fruits 84 85 90 92 93 34 95 103 107 107 108 108 109 viii4.32 4.33 4.34 4.35 4.36 4.37 Comparison of pesticide residues in vegetables from farm and city Comparison of pesticide residues in fruit samples collected from orchard and city Residues of fungicides in fruits and vegetables Pyrethroid residues (mgkg”) in vegetable samples Triforin and isoproturon residues in vegetable samples Mean pesticide recovery (%) from fruit and vegetable samples 146 147 147 155 155 156SOLID-PHASE EXTRACTION AND CHROMATOGRAPHIC DETERMINATION OF PESTICIDES IN FOOD AND WATER SAMPLES List of Tables Table No. Contents Page Materials and Methods 3.1 Sampling sites of vegetables 40 3.2 Sampling sites of fruits 43 Results and Discussion 41 Specification of different cartridges used in the study 60 4,2 Effect of solvents on the recovery (%) of dilferent pesticides 61 from 200 mJ of double-dislilled de-ionized water 43 Recovery (%) of pesticides from 200ml of double-distilled 62 deionized water (DDDW) spiked with 1 pry of each pesticide by ODS cartridges eluted with different volume of acetone: ethyl acelate; metfianol and equilibrium 44 Effect of sample volume on recovery (%) of pesticides spiked 63 with 1 pg 45 Recovery percentaye and precision obtained with DDDW, 65 canal, tubewell, handpump and well water samples 4.6 Efficiency (recovery %) of different solid-phase extraction 68 cartridges 4.7 Comparison of calculated and standard values of system 74 suitability test 4.8 Validation data of GC-ECD for the determination of pesticide 75 residues 49 Validation data of GC-NPD for the determination of pesticide 77 residues 4.10 Pesticide residues (ugL") in canal, handpump and tubewell of 86. districts (Mukan, Vehari, Lodhran, Bahawalpur, Sadiqabad, Bahawalnagar, Jhang, Hasilpur, Khanewal and Rajanpur) 4.1 Pesticide residues (\gL"') in surface and ground waters in the 87 province of Sindh (Nawabshah, Sanghar, Larkana, Sukkur, Jacobabad) 4.12 Pesticide residues in rice-belt’ (Gujranwala, Sialkot, 88 Sheikhupura and Faisalabad)89 4.13 Pesticide residues (jigL’') in water samples from vegetable area 4.14 Pesticide residues (\1gL"') in water samples collected from 102 cotton-belt and vegetable area 415 Ry and MDQ values of standard pesticides by TLC methods 106 4.16 Pesticide residues in spinach by HPTLC methods 110 417 Pesticide residues in cauliflower by HPTLC methods ut 418 Pesticide residues in tomato by HPTLC methods ML 419 Pesticide residues in potato by HPTLC methods 112 4.20 Pesticide residues in ladyfinger by HPTLC methods 112 4.24 Pesticide residues in bittergourd by HPTLC methods 113 4.22 Pesticide residues in brinjal by HPTLC methods 113 4.23 Pesticide residues in pumpkin by HPTLC methods 14 4.24 Pesticide residues in green peas by HPTLC methods 14 4.25 Pesticide residues in cucumber by HPTLC methods 5 4.26 Pesticide residues in apple by HPTLC methods 119 4.27 Pesticide residues in mango by HPTLC methods 120 4.28 Pesticide residues in guava by HPTLC methods 120 4.29 Pesticide residues in yrapes by HPTLC methods 121 4.30 Pesticide residues in melon by HPTLC meshods 121 431 Pesticide residues in apricot by HPTLC methods 122 4.32 Pesticide residues in plum by HPTLC methods 122 4.33 Pesticide residues in banana by HPTLC methods 123 4.34 Pesticide residues in orange by HPTLC methods 123 4.35 Pesticide residues in pears by HPTLC methods 124 4.36 Pesticide residues in apple samples 132 4.37 Pesticide residues in oranges samples 132 4.38 icide residues in mango samples 133 4.39 Pesticide residues in melon sampte 133 4.40 Pesticide residues in guava samples 134 44 Pesticide residue in grapes samples 134 4.42 Pesticide residue in apricot samples 135 4.43 Pesticide residue in plum samples 135 4.44 Pesticide residue in banana samples 136 4.45 Pesticide residue in pears samples 136 4.46 Pesticide residue in spinach samples 137 4.47 ide residue in cauliflower samples 137 4.48 ide residue in tomato samples. 138 4.49 icide residue in potato samples 138 4.50 Pesticide residue in ladyfinger samples 139 451 Pesticide residue in bittergourd samples 139 4.52 Pesticide residue in brinjal samples 140 4.53 Pesticide residue in pumpkin samples 140 454 Pesticide residue in green peas samples 141 4.85 Pesticide residue in cucumber samples 141144 4.56 Pesiicide residues (mgkg") in fruit samples collected from farm, wholesale and city areas 4.57 Pesticide residues (mgkg’) in vegetable samples collected from 145 farm, wholesale and city areas 4.58 Recovery (%) of pesticides from fruits and vegetables 152 4.59 Pesticide residues (mgkg'') in fruits 153 4.60 Pesticide residues (mgkg'') in vegetables 454 xiiSOLID-PHASE EXTRACTION AND CHROMATOGRAPHIC DETERMINATION OF PESTICIDES IN FOOD AND WATER SAMPLES List of Contents CHAPTER LIST OF CONTENTS PAC 1 INTRODUCTION 1 Ml Introduction 1 12 Objectives 6 2 REVIEW OF LITERATURE 7 2 Need for pesticides 7 2.2 Problems caused by pesticides 8 23 More food and a clean environment 10 24 Pesticide in water 12 2.5 Pesticide Residue in fruit and vegetables 24 2.6 Concluding remarks 36 3 MATERIALS AND METHODS 37 3A Water samplings 37 311 Hand pump water sampling 37 3.1.2 Tube well water sampling 38 3.1.3 Well water sampling 38 3.14 Canal water sampling 39 Vegetables sampling a9 Fruit sampling a2 Methodologies Employed for Pesticides Residue Analyses 44 Extraction of pesticides residues 45 Extraction of pesticides from water 45 Extraction of pesticides residues from vegetables 46 Extraction of pesticides residues from fruits 46 Analytical techniques used for pesticides residue analysis 48 Mulliresidue analysis 48 ‘Thin layer chromatography (TLC) 49 O-Toludine + Potassium iodide (oT + KI) method 50 xiii3.4.2.2.6 3.4.2.3 3.4.2.4 CONTENTS Aluminium Oxide G incorporated with silver nitrate and UV exposition Photosynthesis inhibition method (Hill reaction) Fungi spore (aspergillus niger) inhibition method (FAN) Enzyme inhibition with cow fiver extract and B-naphthyi- acetate substrate (EBNA) Enzyme inhibition with horse blood serum and acetylthiocoline iodide substrate (Eacl) Gas chromatography (GC) High performance liquid chromatography (HPLC) RESULTS AND DISCUSSION Validation of extraction and chromatographic techniques Solid-phase extraction Selection of sample volume for better recovery Evaluation of the procedure Efficiency of different solid-phase extraction cartridges Gas Chromatography (GC) System Suitability Test Validation of GC-ECD High Performance Liquid Chromatography (HPLC) HPLC-Standardization 4.2 Pesticide residue analyses in water by gas chromatography 4.2.1 Pesticide residues in water samples collected from cotton-belts 4.2.2 Pesticide residues in water samples analyzed by liquid chromatography 43 Pesticide residues in fruits and vegetables 431 Validation of thin layer chromatographic (TLC) methods 43.2 Behaviour of marker compounds in TLC methods 43.3 Analysis of vegetables and fruits by TLC methods 43.4 Analysis of pesticide residues by gas chromatography in vegetables and fruits 43.5 Comparison of farm, wholesale and city area samptes 43.6 Analysis of pesticide residues by liquid chromatography in fruits and vegetables SUMMARY PAG 50 31 53 34 56 57 58 59 59 59 63 67 R nR 74 3 aE) 83 102 105 105 107 110 131 144 152 159 xvOVERALL CONCLUSION 16E 5 REFERENCES 166 PUBLICATIONS 192introduction Chapter | SOLID-PHASE EXTRACTION AND CHROMATOGRAPHIC DETERMINATION OF PESTICIDES IN FOOD AND WATER SAMPLES INTRODUCTION Pesticides have a long history of use against insect and other pests. In the beginning these were either inorganic chemicals or compounds extracted from plant and animal sources. However, around World War II, the production of synthetic organic pesticides increased tremendously. Initially, the more persistent class of organochlorine pesticides was favoured, but it did not take long to realise the side effects of pesticides including losses of wildlife and beneficial insects, residues in crops and food chain, health hazard to human and animals, etc. Moreover, many of the pests for whom these were designed to kill, developed resistant and pest resurgence became a common phenomenon. All along this time, the pesticide manufacture and consumption showed an exponential increase. The business of pesticide companies flourished thorough the introduction of other classes of pesticides and targeting different pests. The pesticide import in Pakistan started in 1954 through the Government of Pakistan. In a policy change, the pesticide business was handed over to the private sector in 1980. Since then, the pesticide consumption has been on the rise. The pesticide consumption figures for 2001-02 stands at 47592 metric tonnes (Anonymous, 2001). Insecticides comprise 85 % of the total pesticides and cotton crop is the major recipient of these chemicals. During the past decade research has indicated the presence of pesticide residues in a number of foods and feed items. In some cases the residues are presentIntroduction Analysis of crop soils at various sites has beyond the FAOAVHO pecinissible inits il, shown the presence of pesticides residues, which ave a potential danger (o s micro-fauna/tlora, and alter take up into plant can enter the food chain, At present the groundwater does not seem to be contaminated to & dangerous level, however, certain pesticides, if continuously wsed, say reach groundwater. ‘This is particularly true for herbicides, In the hilly area orchards of apples and decidwous fruits are ichment area for rainwater for rivers, the use extensively sprayed, These are the ci of chemicals in these areas is, therefore, dangerous. ‘The presence of pesticides in food items and their accumulation in tissues has direct Joxic effects on human and other non-target organisms. In situations where the safity measures are not applied properly these have direct effects on human and other workers (WHO, 1999). The health ey. on spray men, cotton picker cantinue and overuse of pesticides have also resulted in more serious problems by 1989, Rostad et al, 1989, Spadling et al., contiummated water supplies (Hail 1989) and degrading crop land sails The curred! translocation of tvod items grown with pestivitles is seen as a danger 10 human beings. even in areas where very little pesucides are ased (points to the reality that the pesticide problem does not merely concern the chemical sndasuy aid professional farmers, toresters- and appheators. oF one concerning only those who wish jo protect wildlife or those responsible for control of malaria and other vector born diseases, rather the pesticide problem concerns every person who wants food at a reasonable price, A number of consultant reports for improving environment related research in Pakistaw have appeared over the year but their recommendation are still far from implemented mainly que 1 lack of finances The need for an institution with bei appropriate set up for monitoring eavironmental aspects of pesticides is (elt even arate now a days. The mformation on fate of pesticide in the environment is a pre- reqursite for the registration af aay pesticide. It should include fate in soil, water, air,Introduction stability, side effects on non-target organisms, and beneficial insects, soil and aquatic organisms, and mammalian toxicity including dangers to human health, It is unfortunate that there is no establishment (hat can generate such information. At the moment al} of the pesticides are either imported as finished products or locally formulated from imported active ingredients, hence data obtained in the original country is accepted by default. Although the behaviour of pesticide may vary in different environment, in accepting the alien data there is a danger of overlooking the potential hazards to local ecosystem. Agriculture is producing thousand tonnes of food for domestic and foreign market. The too of the trade includes many different chemical compounds that are used for a wide variety of pest problems. Pesticides use in agriculture over the last several decades has proven to be great benefit to the production of our nation's food supply. Pesticide use has improved both the efficiency of growing crops and the quality of food produced. However, alongwith the benefits emerged the potential effect of trace amounts of pesticide residues remaining on food commodities and there has been great coneem by consumer groups demanding assurance from the agriculture community that the food we eat is indeed safe. Intemational trade in agriculture products and commodities expanded greatly in the 1990’s with an estimated value of US$38} billion in 1993 (Anonymous, 1998), A substantial portion of that imternational trade originates in developing countries. International trade in food and agricultural commodities is governed by agreement of the World Trade Organisation (WTO). The overall objective of the WTO agreement is to permit countries to take legitimate measures to protect the life and health of their consumers. To meet the requirement of WTO, it is necessary to develop Quality Contro! and Quality Assured (QC/QA) system in the laboratories. In recent years, both legislators and consumers have shown increasing interest in the safety of food products. Events such as the appearance of pesticide residues in fruitIntroduction and vegetables have impelled governments to set up monitoring programmes (Reed et al., 1987). These prograrnmes determine the contamination levels in food products and trace possible occasions in which residue tolerance levels were exceeded because of incorrect agricultural practice. To execute such monitoring programs efficiently, a sct of mulliresidue methods must be developed to detect as many pesticides as possible in all crop types available in the market. Pesticides, particularly insecticides, are used extensively in Pakistan, The import of pesticides (a.i.) during 1997 was 24150 metric tonnes with an increase of approximately 14% per year (Baloch and Haseeb 1995). As societies become more industrialized and information oriented, the challenge falls upon agriculture to produce more crops on less land without undue financial burden to the end consumer. Pesticide use in agriculture has produced certain benefits, including a decrease in the percentage of houschold income spen! on food and an increase in food quality. However, pesticide use also has created concem regarding its effect on the environment and the potentially toxic or carcinogenic residues remaining in the food chain (Page and French 1992). Environmental contamination is recognised as a worldwide problem. A part of this problem is caused by the application of pesticides that are used in agricuiture, horticulture and forestry as conservative against microbial infestations. Hundreds of different compounds are being used as pesticides, Intierently, they show a certain degree of toxicity and especially the less degradable, more persistent compounds pose a problem. Pesticides are released into the environment during the manufacture, transport, handling and application to the crops. Because of their widespread usc, pesticides residues have been found in human blood (Dua et al., 1996; Mes, 1992; Rosell et a/., 1993; Ansari ev al., 1997) drinking water (Fung and Mark, 2001; Hidalgo ef a/., 1997; Hartrik and Tekel, 1996), groundwater (Cohen et al., 1986), surface water (Tanabe ce «d., 2001), rain (Richards e7 al., 1987), fog waterIntroduction (Glotfelty ef al., 1987), vegetables (Page, and French, 1992), oils (Dikshith er al., 1989; Waheed, 1991), soil (Agarwal er a/., 1994 b; Hussain ef al., 1994; Hussain et al., 1984), and fruits being sold in the market (Kadenczki ef a/., 1992) which is a clear-cut indication of the pesticide contamination towards the environmental pollution. Groundwater pollution by agricultural biocides (Cohen ef af, 1984) has become growing concem in the world because 40-50% of domestic drinking water is pumped from groundwater resources. Water pollution is the most damaging and widespread environmental effect of agriculture production. There are some vulnerable sites, where the pesticide flux to groundwater is high. The sites usually have some combination of management, geological and climatic characteristics including high pesticide application rates of the more soluble and slowly degradable pesticides; high water percolation rates because of shallow or sandy soils, humid climates or imigation, low pesticide detention in soil by sorption because of low organic content or clay content, lower pesticide degradation or fixation rates in biologically less active soils. Much effort is therefore put in research and investigations concerning, development of new and improvement of existing methods for pesticide analysis. Already existing methods, generally used to measure pesticide residues, are radiometric method using radiomnelide as tracer (Hussain et al., 2001) and conventional techniques like HPLC (Steinheimer, 1993; Pinto and Jardim, 2000) and GC/MS (Lehotay, 2000) involving extraction of large volumes of samples, extensive purification, and often derivatization. As pesticide residues occur at low concentration, an extraction and concentration step is usually needed prior to the chromatographic analysis. Several methods for extracting organic residues from water and food commodities are currently available. Liquid-liquid extraction (LLE) procedures have been used successfully for a wide variety of compounds. However, they are time-consuming, laborious, and disposal of toxic and inflammable solvents and above all off-line (echniques, Solid-phase extraction (SPE) is relatively a newfatroduction approach for the isolation and concentration of the specified target compounds from water and food commodities. SPE has been widely used to extract organic chemicals and their derivatives from water, soi! and farm commodities (Basta and Olness, 1992; Newsome and Collins, 1989; Nash, 1990; Bogus ef al., 1990). This technique has gained popularity duc to rapid, cost effective, large reduction in the use of expensive and environmentally sensitive organic solvents; specialized glassware and manpower required in sample analysis compared with traditional liquid separation methods. As a consequence, attention has been directed to newer methods, and the solid phase extraction technique incorporation with GC/HPLC for the analysis of organophosphorus, pyrethroids, carbamates and organochiorines residues in water samples seems to be a good one for extraction, concentration and analysis of pesticides upto ppb and even ppt levels (Bucheli er a/., 1997), In a developing country like Pakistan, environmental pollution, especially with regards to pesticide residues contamination, is also a great problem. A variety of pesticides are being imported in Pakistan. In 2000-01, 47592 metric tonnes of pesticides have been sold with a market value of 7.741 billion rupees (Anonymous, 2001). The province of Punjab is mainly associated with agricultural activities. A wide range of pesticides js being heavily sprayed on variety of crops and their residues have certainly contaminated the whole environment exclusively, Some work has been done for the determination of pesticide residue levels in water, fruits and vegetables (Hussain ef al., 2002; Parveen and Masud 1988; Jabbar ef al., 1993). The present work was undertaken to study the levels of pesticide residues, both qualitatively and quantitatively, in water, fruits and vegetables,Review of Literature Chapter 2 REVIEW OF LITERATURE Pesticide use in agriculture in the second half of last century has produced certain benefits, including a decrease in the percentage of household income spent on food and an increase in food quality. However, pesticide use also has created concern regarding its effect on the environment and the potentially toxic or carcinogenic residues remaining in the food chain, Despite surveillance by national and local government agencies, consumer groups world-wide are demanding solid assurance as to the safety of agriculture products, Since a considerable amount of the applied pesticides frequently ends up in the soil, there are some chances that these pesticides will accumulate, These chemicals may then be leached into underground water supplies or rivers and lakes. In recent years, immense literature is available on the pesticides residues in water, fruits and vegetables but little information is available in Pakistan regarding the pesticide residue fevels in these farm commodities and aquifers. In the present investigation major emphasis was given ‘on the pesticide residues in fruits, vegetables and water (ground and surface water), while reviewing the subject. Nevertheless, indirect evidence was provided from pesticides in general to support some assertions. 2.1 Need for pesticides It has been estimated that the agricultural retum in the mid-1960s was approximately four time higher than the money spent on pesticides (Hesdley, 1968). It is more difficult to calculate benefits to the public health, however they are undoubiedly very high. It has been reported that field losses from pests average 35% for the world’s main food crops (Anonymous, 1980). In some places losses were far exceed this figure. Stadies in the United Kingdom have indicated that if no pesticides were usedReview of Literature on cereal crops losses would be 24% in the first year, but by the third year without the use of pesticides losses would be 45% (Anonymous, 1979). Pesticides have accounted for 20% of the increase in farm output in the United States. No-till production practices (crop production with little or no ploughing and cultivation), which are becoming increasingly popular to prevent erosion and reduce energy requirements, require effective weed control. This and the availability of effective herbicides have resulted in a dramatic increase in the use of herbicide over the last few years and this increase will continue. Globally, more herbicides are used than insecticides; although in developing countries herbicides are not used as extensively as in developed countries. Competition from weeds for limited supplies of soil, moisture, and plant nutrients often results in enormous losses to the cultivated crop. Crop reductions of $0 (0 70% resulting from competition by weeds are not uncommon (Rijin, 1979). Thus weed control is of great importance in developing countries. Control of vector-borne diseases of man and animals is based to a large extent on insect control, because there are very few vaccines available which can contro! those insect and harmful diseases (Anonymous, 1976). Thus insecticides play a major role in the control of malaria, filariasis, dengue and many other vector-borne diseases. 2.2. Problems caused by pesticides Despite the cnormous benefits derived from pesticides, these chemicals are not problem-free. Many pesticides are toxic to living organisms and interfere with specific biochemical systems. Since all living organisms have some similar biochemical systems, it stands to reason that a chemical, which kills a fly, may also kill a dog. Thus the problem that a chemical may be toxic to non-target organisms must be taken into account not only in developing the pesticides, but also in its use. The widespread and sometimes improper use of pesticides has led to resistant strains of insects, plant diseases, weeds, and rodents, which can no longer be controlled byReview of Literature certain pesticides, New pesticides must be developed to contro] these resistant pests. The increased cost of pesticide development (currently estimated at US$ 15 million to US$ 20 miflion per pesticide over a six to ten year period) has resulted in fewer new pesticides being developed in recent years (Goring, 1977). Pesticide residues in food are a potential hazard, which has received much attention during the past 20 years, Extensive regulatory agencies have been created in developed countries to deal with pesticide residues in food, In many developing countries acceptable quantities of pesticide residues in food (tolerances) have not been established, however the guidelines developed by Food and Agriculture Organization and the World Health Organization FAO/WHO) are generally followed. Because of the very small quantities of pesticide, which are permitted in food, elaborate analytical procedures are required. Some pesticides are relatively stable and since a considerable amount of the applied pesticide frequently ends up in the soil and in some cases bioaccumulation can occur to an extent, which causes damage to fish or birds (Abdallah er «,, 1990). Bioaccumulation will normally result only when the pesticide is stable in the environment; most new types of insecticides are not sufficiently stable to result in this type of problem. In view of current food production we must accept the fact that small quantities of pesticide residues will be present in our food supply. The significance of minute quantities of these chemical residues in food over an extended period of time is a much debated question and has become a problem for man ‘to face in controlling the total insult to his environment, Since some of the pesticides and their terminal products are reported to be carcinogenic, mutagenic, tumierogenic and teratogenic, therefore, these have aroused considerable concern for the authorities responsible for its check and control. Detection of large number of residues in food, water, soi] and air by developed countries have caused deep concem for everyone. To protect human and animal population from the toxic effects of these pesticidesReview of Literature and their breakdown products and to keep the environment free from them, strict surveillance and monitoring at national level and for export is urgently needed. Methods of application and formulations of pesticides are currently receiving much attention. Formulations of pesticides applied in or on the soil offer the potential of increasing the length of time over which they are effective, reducing potential residues in the crops being protected. Plant and animal foodstuffs exported from developing countries must mect the standards of the importing country. These standards include acceptable quantitics of pesticide residues. Thus, developing countries must establish and maintain research and regulatory organizations to ensure acceptable pesticide residues in exported commodities. 2.3 More food and a clean environment Pesticides are essential to improve man’s living conditions. The quantity of pesticides used throughout the world will continue to increase. To minimize the undesirable effects of pesticides while maximizing pesticide efficiency requires the use of radiolabelled pesticides to study pesticide metabolism, fate, residues and formulations. Economical pest contro! with minimal effects on non-target organisms will result in efficient food production and a clean environment. Pesticide related problems would continue to be identified, explored and solved, partially with the aid of nuclear techniques with supports from the Joint Food and Agriculture Organization/International Atomic Energy Agency (Joint FAOAAEA. Division) (Lindquist, 1981). For the foreseeable future, pesticide will remain the major weapon in the farmer’s armoury of defences against pests and diseases. Over the past thirty years, the developing technology of crop protection and pest control has become highly effective and now provides methods for preventing or reducing the damage caused hy most economically important pests and diseases. Application of this technology 10Review of Literature has proved very financially rewarding to farmers, and this is the main reason for wide spread use of pesticides and for the remarkably rapid growth of the pesticide industry. It has also made a major contribution to the substantial increases in yields per hectare of many crops, particularly of cereals, which have been achieved during that period. Human population is increasing steadily and is likely to go on increasing, but the amount of arable land available in cach country remains the same, or actually decreases as cities and towns are expanding to accommodate increased number of human beings, as agricuitural land is taken for motor ways, air ports, shopping plaza’s, parking lots, and as affluent societies demand that more land be reserved for their amenity and recreation. If these populations are to continue to be provided with their present variety and quantity of foodstuffs at reasouable prices and if they are not to be faced with the need to spend much higher proportion of their incomes on food, then yield per heclare wil! have to be increased still more and to achieve tI use of crop protection technology will have to be intensified, not restricted. With growing populations the human race simply cannot afford to allow substantial proportions of the food they grow to feed insects or nourish neither microorganisms nor they permit unwanted plants to compete with essential crops (Green er al., 1977). Good food is not only expected to look fresh and tasty but it must be free from all types of contaminants, The presence of pesticide residues in food products is a clear violation of this simple reasonable expectation. A recent poll conducted by the Food Marketing Institute, USA indicates that 75 per cent of today’s consumers consider pesticides a ‘serious hazard’ in supermarket food. The U.S. Environment Protection Agency (EPA) ranked pesticides in food as one of the nation’s most serious health and environmental problem. The use of pesticides is indispensable to reduce crop losses (35-40%) from pests. If these pesticides were neither poisonous nor harmful, then the question of residues would have been unimportant, However,Review of Literature in order to accomplish their purposes, these compounds must be lethal to pests. The residues of these, when consumed alongwith food, may prove harmful to man, animals, and the environment, the use of several organochlorine pesticides is cither restricted or banned in several developed countries. But due to various socio- economic factors, these chemicals are still widely used in agriculture and public in most of the developing countries, Although the per hectare use of pesticides in developing countries is less than that of developed countries, the problems caused by their ir-regulated use can be equally and sometimes even more dangerous. The use of pesticides in agriculture in developing countries is likely to increase; hence extreme precautions would be needed so that this prospective increase in the use of pesticides does not further degrade the quality of food and environment, 2.4 Pesticide in water Pesticide application has become an essential component of modern agriculture (Perry et al., 1988). The use of insecticides in the field of agriculture has proven very useful in protecting crops from pests and thereby significantly improving the yield, Besides agriculture, the role of insecticides in protecting human life by controlling the spread of epidemics and providing as healthy and hygienic environment is also well known, for example the use of DDT, which has saved milffions of human lives by successfuutly checking malaria, On the agricultural side too, the crop yield has been considerably increased by the use of DDT and the inventor, Paul H. Muller, of this insecticide was awarded Nobel Prize in 1948. The extensive use of insecticides became a cause of concern however, when some of their harmful side effects were observed, On both fronts, i.e. that of agriculture and human health, on the basis of quantitative results, it becaine cvident that in the long range, the use of insecticides is not in the overall interest of man. The pesticides presently in use are mostly synthetic organic compounds. The principal processes that influence their potential for loss from soil to groundwater are 12Review of Literature volatilization (and subsequent diffusion), decomposition, and retention by the soit and transport by water, Some organic pesticides however, are of little concem as ground water contaminations from pesticides use because they are relatively insoluble in water and are retained strongly by the soils. The principal mechanism by which pesticides are transported from soil to groundwater is downward percolation of water containing dissolved pesticides, United States Environmental Protection Agency (U.S. EPA) official has estimated that as nvany as 50 of the more than 1000 registered pesticides posses’ the potential for detection in groundwater under conditions conducive to downward movement. Cotton is the main crop of Pakistan and grown on vast area of Pakistan including Punjab, Sindh, Balochistan and NWEP provinces. Cotton production annually experiences economic damage from infestation of white fly and different other insects/pests. In an attempt to effectively control the pests and reduce the losses, cotton producers of the country are using multi-variety of synthetic chemicals. Agricultural use of insecticides for pests control has been recognized as one of non-point pollution sources in the United States (Lee, 1992), Insecticides utilized for cotton production are intensive and can contribute to water-quality problems (Crutchfield, er a/., 1992). Pesticides are used extensively in Pakistan to eradicate multidiversity of pests, insects and rodents, which damage a big share of crops al pre and post harvest stages. Pesticide losses from areas of application and contamination of non-target sites such as surface and ground water represent a monetary loss to the farmer as well as a threat to the environment. Certainly, pesticides have improved longevity and the quality of life, chiefly in the area of public health, bisect control programs have saved millions of lives by combating diseases such as malaria, yellow fever and typhus. Pesticide residues in water are significant in terms of human exposure, Since a considerable amount of the applied pesticides frequently ends up in the soil, there is some chance that these pesticides 13Review of Literature will accumulate. These chemicals may then be leached into underground water supplies or rivers and lakes (Stiegler er «l., 2000). Agro-chemicals are biologically active compounds. Their use for crop protection is essential to meet the needs of fast growing population. Their misuse poses a series of threat to the environment and the ultimate victim is the man itself: Contamination of natural water with pesticides from agriculture is still a problem of primary concern. In order to evaluate possible impacts of pesticides on aquatic ecosystems and drinking waters supplies, analytical methods for the routine simultaneous determination of several numbers of such compounds in water samples are required, Several methods based on gas chromatography (GC) have been developed for pesticide determination (Eisert and Levsen 1996). In recent years, immense literature is available on the pesticide residues in water, their clean-up by different techniques and analysis using different technologies but little information is available on the pesticide residues in water (ground water, surface water, canal water) of extensively sprayed areas in Pakistan. In this work major emphasis was given on the clean-up and analysis of pesticide residues in water, particularly applied in cotton, rice and vegetables grown in different agro ecosystems, while reviewing the present subject. Water from small loughs often used to irrigate the surrounding crops undergoes seasonal variations in its pesticide concentration, AS a result, it is necessary to emphasise the frequent presence of variable amounts of pesticides used for protecting the different surrounding crops. Pesticide nature differs considerably according to the type of crops and they belong to very different chemical families. So, it is necessary to have procedures that encompass a wide range of concentrations and can provide information on as many different pesticides as possible to evaluate their residues. A procedure for the determination of pesticide residues in water from * small loughs surrounded by different crops has been developed (Jimenez et al., 14,Review of Literature 1997). For this purpose, a solid-phase extraction procedure with octadecylsilane cartridges was used, optimizing the elution parameters as well as the breakthrough volume and the influence of the pesticide amount. The recoveries of the pesticide can be improved by about 10-20 %. Extracts were analyzed either by chromatography with electron-capture detection (ECD) and nitrogen-phosphorus detection (NPD) or high performance liquid chromatography (HPLC) with diode array detection to achieve a more reliable identification and determination of twenty-three pesticides from different chemical families, including triazines, phenyureas and organophosphorus compounds. In another study designed to evaluate the rapid analysis for target pesticides in water, Hartman er al. (2000) reported that the applicability of on fine trace enrichment with custom-made coated capillaries combined with tandem mass spectrometry for the target analysis of selected pesticide (mainly triazines, phenylureas and acetanilides) in water. The developed method allows rapid determination of several widely used plant protectants with a total analysis time of 11 minutes. Good linearity (120.995) was obtained for the selected pesticides in the range of 0,050-50 pgL. The relative standard deviation (RSDs) of the peak area was 3.8% for spiked Milli-Q water (SugL”). The RSDs obtained in analysis of spiked (IpgL") water samples (river water, sewage plant effluent) ranged from 2.9 to 6.8 %, indicating low influence of the matrix on enrichment and detection. The polyacrylate coating of the extraction capillary showed good stability in the presence of water and acetonitrile and allowed > 100 extractions with 1 capillary. Growing evidence indicates that trace amounts of pesticides are even present on non-agricultural land, in the atmosphere and in water, both in surface bodies and underground, far from the sites of pesticide application. Recently there has been increasing need for multiresiduc analytical methods for the trace level identification and quantification of pesticides in environmental water matrices, such as surface and ground water, fruits, vegetables and soils (Samuel ef a/., 1988). Isolation and pre- 1s.Review of Literature concentration via liquid-liquid extraction (LLE) and off-line and on-line solid-phase extraction (SPE) are generally used for this purpose (Cohen ef al., 1986; Holden, 1986 and Bernal ef al., 1996). SPE techniques usually provide better sample clean-up and recoveries than LLE techniques. For the isolation of pesticides from water samples the use of solid- phase extraction has been preferred for its countless advantages in terms of simplicity, robustness, use of small volumes of common solvents, requires very simple laboratory skills, does not require the use of highly specialized laboratory equipment, easy automation ad allows rapid sample clean-up in relation to liquid- liquid extraction (Hu et al., 1996). Potter e¢ al. (2000) used different SPE cartridges for the clean-up of series of surface run-off and drainage water samples from a cotton research plot which had been defoliated with a mixture of thidiazuron and dimethipin to get accurate and precise data. The method was used for analysis of cotton pesticide and harvest-aid chemicals in water using solid-phase extraction and GC-NPD, GC-MS and HPLC-DAD. Target compounds inclided the defoliants tribufos, dimethipin, thidiazuron; the herbicide diuron; and the insecticide methyl- parathion. Three solid phase extraction (SPE) media, octadecylsily! (ODS), graphitised carbon black (GCB) and divinylbenezene-N-vinyl pyrollidine copolymer (DVBVP) were evaluated. On GCB and ODS, recoveries varied depending on type of compound. Recoveries were quantitative for all the compounds on DVBVP, ranging from 87 to 115 % in spiked deionized water and surface run off. The method detection limit was less than 0.1 gl. SPE with DVBVP was applied to post- defoliation samples of surface run-off and the drainage from cotton research plot and surface run-off from a commercial field. The research plot was defoliated with a tank mixture of dimthipin and thidiazeron and the commercial field with tribufos. Dimethipin was detected (1.9-9.6 gL") in all research plot samples. In the commercial field samples, tribufos concentration ranged from 0.) to 1.35 gL’. An exponentially decreasing concentration was observed with each successive stormReview of Literature event (Potter ef al., 2000). Other research workers also have published analytical methods for the determination of diuron. tribufos and parathion-methy! in water (Lehman er al., 1983). Several authors (Balinova, 1996; Poole, 1996) indicated the need for a major simplification in the sample preparation accounting for a miniaturisation in scale, which will also result in reduction of time and solvent consumption (Wan and Wong, 1996). Solid-Phase Micro Extraction (SPME) appears to be a solvent-free extraction technique that presents some of the characteristics outlined before as primordial in new sample preparation strategies. The initial concepts on’ SPME application were published in 1989 by Belardi and Pawliszyn and the following rapid development resuited in first SPME device in 1990 (Arthur and Pawliszyn 1990). SPME was commercialised in 1993 by Supileo coated with polydimethylsiloxane (PDMS) and polyacrylate (PA), and that have now extended to other coatings as Carbowax- divinylbenzene, PDMS-divinylbenzene and Carboxen-PDMS. Since, _ its development, SPME has been applied to the determination of several organic compounds in gas, liquid and solid samples, paying special attention to determine of volatile compounds as benzene, toluene, ethylbenzene, xylenes (Langenfeld et al., 1996), volatile organic compounds (VOCs) (Bartell, 1997) and pesticide determination in water (Eisert and Levsen, 1996; Prosen and Kralj, 1999). Beltran e¢ af, (2000) provided an impressive and extensive review on the applications of solid-phase micro extraction (SPME) for sample preparation in pesticide residue analysis. Different approaches of this technique were used coupling to gas chromatography and high performance liquid chromatography for the analysis of pesticide residues (organochlorine, organophosphorus, triazines, thiocarbamates, substituted uracils, urea derivatives and dinitroanilines) in soil, water, food and biological uid samples. Bisert e¢ al, (1994) used PDMS (100pm) fibre for theReview of Literature extraction of six organophosphorus pesticides in Milli-Q and river water reaching detection limits in the range of parts per billion. Herbicides are used in agriculture to eliminate weeds that would otherwise compete with the desired crop (Dean ef al., 1996). The triazines herbicides form a wide group of substances used for pre- and post-emergence weed control. Today, mare than 30% of all agricultural herbicides are triazines (Pacakova et al., 1996). Symmetric triazine derivatives are among the most important selective herbicides. Pesticides, and their degradation products, are very toxic and highly resistant and survive many years ins the soi! (Pacakova et al., 1988). Atrazine has been classified as a possible human. carcinogen and has been banned in European countries and the European Union established a maximum admissible concentration of 0.1 ygL" for individual pesticide in drinking water (Knutsson ef al., 1996; Batinova, 1996)). Gas chromatography (GC) and high performance liquid chromatography (HPLC) are good options for monitoring triazines in water (Sherma, 1995). HPLC is favoured over GC for acidic pesticides, with high polarities, low volatilities and thermal instabilities because GC can only be used foifowing a prior derivatization step (Aguilar e¢ a/., 1996; Kim et af, 1997). Triazines herbicides strongly adsorb in the UV region 210-240 nm and they make excellent compounds for UV detection in liquid chromatography (Dean e7 a/., 1996; Liska and Stobodnik, 1996). Pinto and Jardim (2000) developed a method for determination of some triazine residues in water, The method involves concentration with Cg column with UV detection at 230 nm, a mobile phase of methanol-water (60:40, v/v) at pH 4.6 (phosphoric acid) and a flow-rate of 0.8 mimin™. After optimisation of the extraction and ‘separation conditions, the method was validated. The method can be used for determination of atrazine, simazine, cyanazine and metryn in water, within the international limits of O.1 pel", Rivers and canals are one of the most important water sources for agriculture as well as for drinking water. Therefore, pesticide contamination in canals and riverReview of Literature water has received much attention from environmental chemists. In rice-growing countries like Pakistan. India, Japan, Philippines and Bangladesh many pesticides have been used in the rice fields. Consequently, river and canal water may contain many pesticides and their metabolites. It is necessary to analyse pesticides and their metabolites in waters (river and canal) to assess their fate in the environment. Tanabe et al., 200] collected water samples from four sites on the Shinano River, Japan to investigate seasonal and spatial studies on pesticides residues in surface water. The water samples were passed through a Sep-Pak cartridge (styrene divinylbenezene copolymer). The contents were analyzed using a gas chromatogi hy-mass spectrometer. Among the total of 53 chemicals found, 22 were herbicides, 15 were insecticides, 11 were fungicides and 5 were metabolites. Herbicides were found primarily during July and August at all four sites. Insecticides and fungicides were found primarily during July and August at al four sites, The presence of pesticides in the river water correlated with the time of pesticide application to the rice fields near the river. One of the problems involved in multiresidue pesticide analysis in real samples is the tediousness and complexity of the procedures required for the extraction, clean up and pre-concentration of the matrix analytes, These prior sample treatment steps may indeed be the most laborious part of the whole analysis, especially when the matrices are complex. With the development of more selective extraction technique, such as supercritical fluid extraction (SFE) (Wheeler and MeNally, 1987; King and Zhang, 1998), microwave-assisted extraction, (MAE) (Lopez et al., 1994; Lopez ef al., 1996; Lord and Pawliszyn, 1998, Lehotay and Lee, 1997) and pressurized liquid extraction (PLE) analytical methods that are less affected by coextracted matrix components, clean up becomes less important than with many current methods.Review of Literature Liquid-liquid partitioning (Krause, 1985), absorption (Florisil) chromatography (Krynits ef al., 1988), gel permeation chromatography (GPC) (Balinova, 1998) and solid-phase extraction (SPE) (Schenck ev al., 1996) have been widely applied for the clean-up of extracts in residue analysis. Carabias ef al. (2000) used a new technique for the analysis of pesticide multiresidues in samples with high lipid contents, The sample was separated through membranes coupled to an HPLC system. The analytes (dichlorvos, dimethoate, Propoxur, paraoxen, picimicarb, atrazine, ametryne, terbutryne, azinphos-methyl, folpet) were subjected to prior extraction in a Soxhlet system, afler which the extract was introduced into the membrane separation device coupled to the HPLC system. This procedure provided clean chromatograms, hence considerably facilitating determination, and at the same time was efficient in removing macromolecular compounds. The recoveries of different analytes ranged from 60-98% and detection limits varied from 0.018mgkg" for dichlorvos to 0.002 mgky'! for atrazine. Insecticides are the most toxic forms of pesticides, followed by herbicides, acaricides and fungicides. These pesticides affect the nervous system, liver and reproductive systems and may cause allergy (Anonymous, 1989). In Pakistan, preharvest crop loses of 30-40%, post harvest losses 10-30% and total of 50% due to insects, weeds, diseases and rodents. In order to safeguard agricultural produce from the ravage of pests; the use of pesticide was the only solution. Hence, pesticides consumption has raised many folds during the last two decades. Due to such tremendous usage, pesticides can find their way to ground water through leaching, channelling (downward percolation), direct spillage and wind drift causing ground- water contamination, Pesticide residues in drinking and groundwater of developed countries like USA (Bushway ef al., 1992; Molto et al., 1991), France (Legrand et al., 1991), Australia (Ang et al., 1989) and Denmark (Felding, 1992) have been reported. In Pakistan pesticide residues in cattle drinking water in Karachi (Parveen and Masud, 1988), shallow groundwater in Samundri, Faisalabad (Ali and Jabbar, 20Review of Literature 1991), waste water samples from Lahore (Anonymous 1993) and groundwater of Mardan Division, NWFP (Ahad ef a/., 2000) have also been reported but there is hardly any report on the pesticide residues in ground water of rice-belt, vegetable area and cotton-belt. Hundreds of pesticides are extensively used for controlling weeds in agricultural crops throughout the world. They often contaminate the aquatic environment either by direct apptication to water bodies or through agricultural run- off and leaching from contaminated jand into the surface and ground water. Fung and Mark (2062) presented a new method for the determination of pesticides in drinking water by micellar clectro kinetic capillary chromatography. The sample was preconcentrated in two steps prior to separation by micellar electro kinetic capitiary chromatography for the determination of 14 pesticides such as aldicarb, carbofuran, isoproturan, chlorotoluron, metaiachlor, mecoprop, dichlorprop, MCPA, 2,4-D, methoxychlor, TDE, DDT, dieldrin and DDE in drinking water. Good recoveries of pesticides were obtained using SPE with sample pH adjusted to 2-3. Good linearity was obtained for all pesticides with detection limit down to 0.04-0.46 agin" and a working range of 0.1-40 ngmi'', Ali pesticides except dieldrin were found to be detected at concentrations at least 10 fold lower than the World Health Organization (WHO) guideline values. The analytical procedure developed offers an economic method for fast screening of multiple pesticide residues in drinking water for health protection. {( had been applied to determine carbofuran and MCPA in agricultural run-off water samples, giving satisfactory repeatability of 10-12%, respectively, with five numbers of samples for the determination of pesticides in contaminated water samples. The development and use of pesticides have played an important role in the increase of agricultural productivity. The majority of such substances are applied directly to soil or sprayed over crop field and hence released directly to the 21Review of Literature environment. For that, pesticides can enter as contaminants into natural waters either directly in applications or indirectly from drainage of agricultural lands. The amount and kind of pesticides in water of a given area depends largely on the intensity of production and type of crop. However, the transport of pesticides out of their area of application results in the presence of accumulation of these compounds in many parts of the hydrosphere. For example, atmospheric precipitation is an important route of transport of pesticides, resulting in contamination of environmental waters, far away from agricultural areas. Substantial amounts of pesticides have been found in ice and water of polar regions (Iwata ef al., 1994), lakes (Buser, 1990), seawater (Sehulz et al., 1995), rainwater (Bester et al., 1995; Baumann ef al., 1995; Albanis et al, 1998) or potable water (Chiron and Barcelo, 1993; Miyata er al., 1993), Vidal et al, (200). introduced a method for pesticide trace analysis using solid-phase extraction and gas chromatography with electron-capture and tandem mass spectrometric detection in water samples. They identified 12 pesticides in water samples. For this purpose, a solid-phase extraction procedure with Cyy cartridges was used, optimising the breakthrough volume and the saturation concentration. In GC- MS-MS, the lowest detectable concentrations for the pesticides were between 2 and 26 ngL", recoveries ranged from 70-133% in water samples spiked at 100 ngL” and the relative standard deviation were in the range 5.3 to 17.4%; the proposed analytical methodology was applied to analyse pesticides in wetland samples from Almeria, Spain. Though a lot of sensitive techniques are available for the analysis of pesticides at trace level in the presence of many interfering compounds. Evidently, the sensitivity is still not high enough to directly determine the trace amounts of pesticides in water samples at the level required by the EU Drinking Water Directive of 0.1 gl" for each pesticide and 0.5 gh" for the total amount of pesticides (Anonymous, 1988). Pablos-Espada ey al. (1999) described a method for the determination of dichlorvos, lindane, diazinon, chlorpyrifos-methy}, dichloflunid, chlorpyrifos, folpet, o- and P-endosutfan, endosuifan-suiphate, fenpropathrin and 22Review of Literature acrinathrin in water samples at the levels required by the EU afler solid-phase extraction (SPE). Contamination of natural waters with pesticides used for agriculture is still a problem of primary concern. Several methods based on gas chromatography and high performance liquid chromatography has been developed worldwide for pesticides determination (Vander Hoff and Van Zoonen, 1999; Eisert and Levsen, 1996). Moreover, high sensitivity of the analytical methods is compulsory for its use in environmental water monitoring, In order to decrease the analysis time, analytical procedures based on the on-line combination of liquid chromatography (LC) and mass spectrometry is often preferred for the analysis of pesticides and metabolites (Geerdink, 1996; Hogendoorm and Zoonen, 2000). Hernandez et al. (2001) proposed a very rapid, multi-residual, sensitive and specific procedure for determining 35 pesticides in environmental ground and surface waters. It is based on the use of solid-phase extraction (SPE) combined on-line with liquid chromatography (LC) electrospray (ES!) tandem mass spectrometry (MS-MS). Simultaneous target analysis of 29 pesticides (1 fungicide; 16 insecticides, 10 herbicides and 2 acaricides) and 6 metabolites with positive or negative ionization was reached by the direct injection of only 1.3 m! of filtered water sample, with a totai analysis time of 18 min. The SPE-LC-MS-MS method was validated, obtaining good results for all compounds at 0.5 and 0.1 gL”, Most of them could be correctly quantified at a concentration level as low as 25 ngL', Efficiency and applicability of this method was evaluated by the analysis of several samples included in a monitoring program). Many solvents can be used for the extraction of pesticides from different commodities by liquid-liquid extraction procedures such as hexane (Sala er ul., 1997), dichloromethane (Garcia-Repetto er ul., 1996), or solvent mixtures as hexane-acetone (Oliva et a/., 1999; Cabras et al., 1998), acetone-dichloromethane (Navarro ef al., 1999) and acctone-light petroleum (Cabras er ai., 1997), however, these methods are not free from drawbacks, such as the usc of toxic and expensive 23Review of Literature solvents, the difficulty of automation and the formation of emulsions. These problems can be overcome by using solid-phase extraction (SPE). The use of SPE has recently gained acceptance in the extraction and preconcentration of residues in aqueous and other environmental samples. A broad variety of organic chemicals has been enriched from drinking (De Carcia and Marchetti, 991; Davi ef af., 3992), ground (Brooks ef 2/,, 1990), waste (Feihn and Jekel, 1996; Lacorte and Barcelo, 1996), surface (Fernandez et al, 1998; Aguilar er al., 1999), estuarine (Chiron et al., 1994), coastal (Zhou et al., 1998) and marine waters (Utvik ef al., 1999) by SPE, The emphasis was placed upon organochlorine pesticides (Molto ef af., 1990), organophosphorus and nitrogen pesticides (Tolosa et al., 1996). The sorbent used in SPE includes graphitised carbon black (GCB) (Di Carcia et al., 1993), reverse phase material (modified silica gel) and polymeric materials, The most widely used RP material is the octadecyl (Cyg) phase but ethyl, butyl, cyclohexyl, octyl, phenyl, polyamino, dimethylaminopropy! and cyanopropyl reverse phase have also been applied (Font ef al., 1993; Benfenati ef al., 1990). 2.5 Pesticide Residue in Fruit and Vegetables Modern agriculture has and is producing thousand of tonnes of food for domestic and foreign market. The tool of the trade includes many different chemical compounds, which are used for a wide variety of pest problems. Use of pesticides is common in all countries and they are readily available. Pesticide use in agriculture over the last several decades has proven the efficiency of growing crops and the quality of food produced, However along with the benefits emerged the potential effect of trace amounts of pesticide residues remaining on food commodities and there has been great concern by consumer groups demanding assurance from the agriculture community that the food we eat is indeed safe. 24Review of Literature Pakistan is producing thousand tonnes of fresh, delicious and appealing fruit throughout the years. Many types of fruits like mango, apple, melon, banana, plum, guava, citrus and pomegranate are produced enormous}y in the country for local consumption as well as for export purposes. A diverse variety of insecticides and fungicides are extensively sprayed for the protection and production of fruit crop. ‘The minute amount of pesticide residues remain inv on fruit may cause deleterious als ahd birds, A little work has been done for the problems for human as well as ani determination of pesticide residues in fruits and vegetables in Pakistan (Hussain et al., 2002) but no intensive studies has been made so far on large scale, Europe, USA, Germany, Canada and other developed countries have set-up many analytical laboratories to monitor the pesticide residue level in fruits, In Canada, many institutions are involved in the research of pesticide residues in fresh fruit; analysis is performed by gas chromatography with mass-selective detection system. Sherma (1992) developed modem thin layer chromatographic methods for the analysis of pesticides using multiple development technique. High performance thin layer chromatographic plates coated with silica gel layer of 200 ym were used for sample and standard solutions. The plates were developed in a suitable solvent (methanol-dichloromethane) by horizontal, multiple or over pressured way. ‘The visible spots were measured by automated densitometric scanning at an optimal wavelength. High performance liquid chromatography/automated multiple development (HPTLC/AMD) procedure was applied to the simultaneous monitoring of phenylureas, carbamates and triazines according to European Economic Community drinking water regulations. The developed method revealed that changes in thickness and gradients increased the sensitivity and speed of analysis. The author further suggested that this method can be used in the laboratory for the analysis of pesticides and other analytes in all types of sample matrices. Thin layer chromatography is important in residwe analysis because of its simplicity and minimal cost, TLC can be used for screening pesticide residues in sample of 25Review of Literature unknown origin. Ambrus ef al. (1981) tested the applicability of TLC in pesticide residue analysis using general modes of detection: o-toludine plus KI, p- nitrobenezene-diazonium-fluoroborate, bioassay with fungi spores, enzyme inhibition, AgNO; plus UV radiation and p-dimethylamino-benzaldehyde. Single solvent system was used for the elution. Marker (indicator) compounds were used for controlling the proper conditions of detection. The detectability of 188 pesticide compounds was tested and minimum detectable amounts were determined. Sundararajan and Chawla (1983) described a simple, sensitive technique for detection and separation of halogenated synthetic pyrethroids by thin layer chromatography. Silver nitrate impregnated with alumina G plates was used for the routine detection and confirmation of the identity of halogenated synthetic pyrethroid insecticides. Maximum resolution was achieved by using n-hexane-benzene (45:55) and n-hexane-chloroform (60:40) solvent system, which gave distinct separations of the cis and trans isomers of permethrin and cypermethrin from fenvalerate and decamethrin. A minimum of 50 ng each of cis and trans-permethrin and cypermethrin isomers and fenvalerate and decamethrin can be detected. Minimum detectable limit of permethrin (0. ppm) in the cleaned extracts of tomato plant, fruit and soif samples can be determined using this detection method. This method is useful not only in screening samples containing synthetic pyrethroids but will also used for common chiorinated pesticides. Thin layer chromatography is also used for the determination of organophosphorus insecticides in biologica! materials, Ethyl parathion, methyl parathion and fenitrothion are widely used in agriculture, Patil and Shingare (1993) used thin layer chromatographic detection method for the determination of organophosphorus insecticides containing nitrophenyl group. The organophosphorus compounds were reduced using stannous chloride in HCI water (1:1) to amino derivatives, which were further diazotised and coupled with I- naphthylamine to give intense pink-orange spots. Metabolites of fenitrothion gave bluish to violet spot with this reagent after spraying p-nitrophenol. Murty ey a/. 26Review of Literature (1980) used another improved ammonium molybdate method for thin layer chromatographic detection of organophosphate residues. After development the plates were heated at 110°C for 2 firs and inorganic phosphate was treated with ammonium molybdate solution to form phosphomolybdate, which was further, reduced to a blue complex by ascorbic acid. This method can be used for the detection of 0.1-0.2 pg residues in any kind of sample. The method gave reproducible results under the described conditions in experimental layout. Lawrence (1980) developed a simple, sensitive and selective thin layer chromatographic technique for the detection of photosynthesis inhibiting herbicides. Triazines, phenylureas, phenylearbamates, uracils and acy! anilides were detected after spraying the TLC plates with spinach chloroplast and 2,6-dichloroindophenol. Minimum detectable levels of most urcas, tiazines and anilides were 100-500 pe/spot.. Carbamates, organophosphates and organochlorines gave no visible spots by this method. Atrazine (5-10 ppb) and linuron ( 7 days under appropriate conditions. Agriculture uses of pyrethroid insecticides have increased rapidly since the development of the first pyrethroid permethrin, which was approved for field use in the mid-1970s, Uses of pyrethroid insecticides also have expanded to other 29Review of Literature agricultural markets, including stored product pest control. The food and Agriculture Organization and the World Health Organization have recommended residue limits for some pyrethroid includés in agricultural and animals products (Anonymous, 1985), Analytical methods for pyrethroid insecticides were reviewed by Miyamoto, 1981; Papadopoulou-Mourkidou, 1983; Zweig and Shera, 1984; Sharp ef al., 1988 and Sherma and Cairns ef al., 1993, Gas chromatographic (GC) methods were reported for multiresidue determination of pyrethroid insecticides in fruits, vegetables and grains. (Bolygo et «l., 1983). Liquid chromatography (LC) is a fast growing separation technique suitable for analysis of technical and formulated materials of pyrethroid insecticides, especially when isomeric forms have to be analyzed (Doi er al., 1985; Stakok, 1990; Cayley and Simpson, 1986 and Chapman, 1983). Papadopoulou-Mourkidou (1985) and Faster and Akhtar (1981) applied LC to analyse fenvalerate in milk and chicken excreta respectively. Recently, thermospray mass spectrometry with selection monitoring was applied by Liu e¢ al. (1991) to analysis of fenevalerate residue in fruits and vegetables. Liquid chromatography with UV detection is a sophisticated too! used for the determination of pyrethroid insecticides in fruits and vegetables. Baker and Bottomley (1982) developed a method for determining bioresmethrin, cismethrim, cypermethrin, deltamethrim, fenpropatherin, fenvalerate, permethrin, phenothrin and resmethcin in fruits and vegetables. Pang et ai. (1995) developed a simple and rapid liquid chromatographic method for simultaneous determination of pyrethroids insecticides in fruits and vegetables. Residues are extracted from crops with methanol and partitioned with toluene. Extracts are cleaned-up by Florisil-Charcoal column chromatography. Liquid chromatographic separation is performed on a uBondapack Cig stainless steel column with acctonitrile-deionised water as mobile phase. The insecticides are detected at 206nm with 0.03 absorbance unit full scales. 30Review of Literature Recoveries of nine pyrethroid insecticides from three crops (apples, pears and peaches fortified at 0.5-5 mgkg’' were 62,7- 129.2 % and detection limits were about 0.05 mgkg". Benzoylphenylurea insecticides are used as non-systemic insect growth regulators for control of wide range of leaf-eating insects and their larvae in vegetables, pome fruit and mushroom, Benzoylphenylurea is effectively used to contro) insects because by interfering with the formation of chitin and, thus, blocking molting to the next larval stage, In this way life cycle of the insect is interrupted (Tomlin, 1994). Benzoytphenylurea have a sliht effect on the natural enemies of various harmful insects/species. These propertics and their acute low toxicity for mammals make them suitable for inclusion in integrated control programms. Hiemstra er al. (1999) used liquid chromatography with diode array detection for the estimation of residues of benzoylphenylurea insecticides diflubenzuron, teflubenzuron, triflumuron, hexflumuron, lufenuron, chlorfluazuron, flufenoxuron and flucycloxuron in pome fruit and fruiting vegetables. The general sample preparation and extraction for gas chromatography and LC multiresidue method scheme was used as a starting point. Crop samples were extracted with acetone and partitjoned into dichforomethane-petroleum ether. Solid phase extraction on amino propyl-bonded silica cartridges was used to separate the benzoylphenylurea (BPU) insecticides from major interfering sample components. LC separation was performed on a reverse phase column with acetonitrile- gradient as mobile phase, A diode array detector was used to monitor the insecticides at 260 nm and to acquire spectral information for additional confirmation. Recoveries and repeatability data were collected for benzoylphenylurea insecticides in mushroom, Chinese cabbage, apple and cucumber samples representing different commodity groups at one spike level. The limits of detection ranged from 20 to 50 pgkg" for all BPU insecticides studied and is dependent on the matrix-pesticides combination, A confirmation 31Review of Literature method for BPU insecticides based on GC-mass spectrometry (jon trap detector) of the characteristics degradation products is also reported by Hiemstra ef al. (1999). Fungicides are used commonly in fruits to protect them from a variety of fungus. Quinomethionate (6-methyl-1, 3-dithiolo-4, 5-6) quinoxalin-2-ore) is an acaricides and fungicide which has been commercially available for use on crops since 1960 s. Numerous countries permit its use on fruits and vegetables; however, its use is on fruits (Anonymous, 1981). The tolerance for quinomethionate on crops in United States (Anonymous, }981) ranges from 0.5 ppm oranges to 0.05 ppm on apples and pears, Various techniques have been published for determining guinomethionate residues in crops. Colorimetry (Havens ef a/., 1964), thin layer chromatography (Health er al., 1966) and GLC (Anderson, 1967) have been used in single residue method for determining quinomethionate. Previously the residue of this fungicide was determined by using a gas chromatograph equipped with a flame photometric detector operated in the sulphur mode. Argauer, 1980 reported the HPLC of quinomethionate on a cyano-bonded column with fluorescence detection of the cluted pesticide. Krause and Agust (1983) suggested the applicability of a muitiresidue method and high performance liquid chromatography for determining quinomethionate in apples and oranges. Quinomethionate was extracted with acetonitrile and coextractives were removed with liquid-liquid partitioning and Florisil adsorbent for non-fatty foods. The residue was determined on an HPLC octyl-bonded column and detected online with a fluorescence detector using 362 nm excitation and 395 nm emissions. Recovery studies were conducted and found in the range of 100 and 102% at 0.05 and 0.5 ppm level (Krause and August, 1983). Schattenberg and Hsu (1992) developed a statistical database for the pesticide residue level in farm produce of growers and brokers from which to purchase the produce to supply their stores. ‘The purpose of this study was to find the illegal levels of one or more residues is rejected from the warehouse and not allowed to 32.Review of Literature proceed to retail stores, Pesticide residue screening programme for 111 pesticides was performed on 6970 produce samples. Of the 81 varieties of samples, 2.4% contained illegal levels of pesticide residues and 13.3% contained levels within tolerable limits established by U.S. EPA. Pesticides results are presented both by commodity and category type. In this study, they pointed out that risk is nominal, however, all the produce under discussion contained pesticide residues and it may be tions from the past and the stability of the pesticide for due to the improper appli long time under the prevailing conditions. Traditionally, multiclass pesticide residue analysis is a time consuming process that often entails several post-extraction cleanup steps before analysis. Conventional wisdom holds that ruggedness and reliability of an approach suffer as shortcuts are taken in the sample preparation procedures. The use of selective detectors in gas chromatography (GC) reduces the amount of cleanup necessary for the removal of interfering coextracted components in the analysis. The use of mass spectrometry (MS), a universal selective detection approach, in GC analysis permits the detection and confirmation of a wide range of pesticides in complex extracts, Modern GC/MS instruments can achieve detection limits that approach those possible with traditional selective detectors and the use of large-volume injection (LVJ) in GC ean further help to reduce detection limits, but in these cases, cleanup of complex extracts becomes even more important to avoid contamination of the injection liner, capillary column and MS source with non-volatile matrix components. Lehotay (2000) used first time an injection technique which is commercially available as the ChromatoProbe. The sanipte cleanup is not necessary for the determination of pesticide residues in fruit and vegetables samples. The recoveries of the fortified samples were in the range of 92-109% for all pesticides. He used a direct sample introduction (DSI) or “dirty sample injection” for the determination of 22 diverse pesticide residues in mixed apple, green beans, and carrot extracts by benchtop gas chromatography/tandem mass _ spectrometry 33Review of Literature (DSVGC/MS-MS). The target pesticides, some of which were incurred in the samples, included chlorpyrifos, azinphos-methyl, parathion-methyl, diazinon, terbufos, p, p'-DDE, endosulfan sulphate, carbofuran, carbaryl, propergite, bifenthrin, dacthal, trifuralin, metalaxyl, pendimethalin, atrazine, piperony! butoxide, diphenylamine, —vinclozolin, —chlorothalonil, — quintozene and tetrahydrophtbalimide (the break down product of captam). The analyticai DSI method entailed the following steps: 30 g sarmple was blended with 60 ml acetonitrile for one minute in a centrifuge bottle, added 6 g sodium chloride and blended again for 30 seconds, centrifuged for 1-2 minutes, separated upper layer and took 5 ml ina vial and added 1 g anhydrous sodium sulphate and injected 11 yl into the DSV/GS/MS-MS instrument for analysis. Sample cleanup is not needed because GC/MS-MS is exceptionally selective for the target analytes, and non-volatile coextractive matrix components do not contaminate the injector or the GC/MS-MS. system. Average recovery of the pesticides were 103 + 7% with relative standard deviations of 14 + 5% on average, and limits of detection were < 2 ngg for nearly all pesticides studied. ‘The DSVGC/MS-MS approach for targeted pesticides is quantitative, confirmatory, sensitive, selective, nigged, rapid, simple and inexpensive Methods were developed that reliable and rapidly detect as many pesticides as possible in the most cost-effective manner, A rapid and efficient multiresidue method for analysis of 189 pesticides in fruit and vegetables by gas chromatography- mass-selective detection (GC-MSD) and for 10 pesticides by liquid chromatography (LC) with fluorescence detection was described by Fillion er a/, 1995. The pesticides were extracted using acetonitrile and coexiractives were removed with a miniaturized charcoal-Celitc column. Analysis was performed by gas chromatography with mass-selective detection in selective-ion monitoring mode. Two injections per sample were used to cover all compounds. Positive analytes were confirmed by retention time arti ion ratios, Carbamates were analysed by liquid 34Review of Literature chromatography with postcolumn reaction and fluorescence detection. Recoveries used to generate limit of detection (LOD) of the method were obtained by spiking three different crops (banana, carrots and pears) at 0.1-0.5 ppm depending on the sensitivity of the compound, Because recoveries of over 90% of the compounds in these three (3) matrices Were very similar, the recoveries were combined to generate LOD. In addition, the method demonstrated acceptable performance for analysis of ‘other crops such as apple, strawberry, orange, pineapple, asparagus, beet, cucumber, tomato, pepper, squash, green peas, potato and sweet polato. Salwa et al. (1999) work out a plan to monitor pesticide residues in Egyptian fruits and vegetables during 1995. Organophosphorus, dithiocarbamates and some synthetic pyrethroids pesticides, which were commonly used in Egypt for pest control, were monitored, as well as persistent organochlorines, which had been prohibited from use several years ago. Fruit and vegetable samples (397) were collected from 8 local markets and examined for 52 active ingredients. Of all analysed samples, 42.8% contained detectable residues, of which, 1,76% exceeded their maximum residue limits (MRL’s). The rates of contamination with the different pesticides were 0-86%, However, violation rates among contaminated products were very low ranging from 0 -4.6%. In general, organochlorines pesticide residues were not detected in most samples. Dithiocarbamate residues were found in 70.4% of 98 samples analysed for dithiocarbamates, but only one grape sample had residucs exceeding the MRL established by Codex Committee on Pesticide Residues. The most commonly detected residues were dithiocarbamates as well as dicofol (15.1% of 397 samples), dimethoate (6.8%), tetradifon (4.5%), Malathion (3.3%), profenofos (2.8%), omethoate (2.3%), chlorothalonil (2.0%) and chlozpyrifos-methyl (1.5%). Among all samples, 22 strawberry samples (5.32%) contained 10 pesticide residues, 65 grape samples (15.73%) contained 11 pesticides residues and 62 tomato samples (15.01%) contained 13 pesticide residues. [Cauliflower, onion and guava samples free {rom pesticides residues’ Samples ef carrot, and eggplant contained 35Review of Literature trace amounts of p, p’-DDT and p, p’-DDE residues. But in general, residues of DDT and HCH have disappeared almost completely from vegetables and fruits. Use of these pesticides in Egypt was completely prohibited by law in 1987. The national monitoring programs for pesticides residues are the key means of ensuring compliance with regulations. They also create a database to help assess the levels of pesticide residues and the levels of residue intake, The information is invaluable in assessing humtan exposure fo pesticide residues through the diet and assists in the country’s formulation of a pesticide strategy. Monitoring of pesticide residues also provides a check on compliance with good agricultural practice in the use of pesticides. Many countries in the world have established analytical laboratories to evaluate the actual contamination of food by pesticides and they check routinely the most common fruits and other food products received from agricultural origin consumed daily bases. (Dogheim er al., 1999; Dogheim et al., 1996). In recent years, both legislators and consumers have shown increased interest in the safety of food products, Events such as the appearance of pesticide residues in fruits and vegetables have impelled governments to set-up monitoring programs (Reed et al., 1987). 2.6 Concluding remarks So far no systematic research has been performed in Pakistan on monitoring pesticide residues in fruits, vegetables and waler. The present study was initiated to work on these lines and develops different methods for determining multiresidues in fruits, vegetables and water sarnples. 36Materials and Methods Chapter 3 MATERIALS AND METHODS Pesticides impart a vital role for the enhancement of food production according to the requirement of world population but the residues left in the food chain pose a lot of problems for human being, as wel as‘énvironment, In the present study water (surface and ground water), fruits and vegetables samples were collected to assess the concentrations of pesticide residues. 3.1 Water Sampling Worldwide pesticides are used for crop protection and production. Some polar pesticides have good tendency to leach down and run-off with rain water from field area and contaminated the fresh waters like canal, stream and well water, The resident at farm are using this contaminated water theirself and for animals. The attention was focused to do sampling from tube-well, hand pump or well installed near heavily sprayed field areas of cotton and rice. In order to assess possible impact of pesticides on aquatic ecosystems and drinking water supplies, water samples were taken from cotton belt (southern Punjab), rice area (Sheikhupura and Gujaranwala Districts). 3.1.1 Hand pump water sampling Different types of pesticides are sprayed on cotton and rice to protect crops from different infestations, Most residents at farm drink water from hand pumps or wells. It is a great concern to evaluate the quality of water and chéck the pesticide residue levels in drinking water (hand pump and wells). Water sampling was carried out 37Materials and Methods using the procedure of Knedel, 1999 and Tanabe ef. al., 2000. Before taking the water sample, the hand pump was run for $ minutes and then [ litre sample was taken in cleaned Pyrex glass botties in triplicate, All samples were taken from cotton, rice and vegetable areas, The samples were stored at ~4 °C before processing further. 3.1.2 Tube well water sampling Most area of Punjab is irrigated by canal water but to meet the requirements of irrigation, some farmer in central and southern Punjab installed tube wells having a range from 150-300 feet depending upon the level of water table. In ricy belt (Gujranwala District, Lahore Division and Sailkot District) farm owners irrigate their fields with tube wells due to shortage of canal water, Pesticides are extensively used to protect crops from different diseases and infestations in this area, so there is a great concern (to analyse the ground water (tube well and hand pump) to assess the present level of pesticide residues in waler, Tube well water samples were collected in polypropylene bottles, having capacity 1 litre and stored at —4°C before analysis. ‘The tube wells were tured on for 10 minutes and then the water samples were taken in triplicate after every 10 minutes intervals. 3.1.3 Well water sampling The water table (water level) in some parts of Pakistan is high ~ 30-50 feet, Considering the tremendous consumption of insecticides/herbicides (Anonymous, 2000), there is a great concer for water analysis both from human healt and from an environmental point of view. Well water was collected in borosilicate glass bottles marked properly for identification and stored at -4°C. Triplicate samples Were taken from each site. 38Materials and Methods 3.1.4 Canal water sampling Pesticide can contaminate the water reservoirs during spraying, transportation, and handling and by run-off waters from treated fields. Due to low literacy rate among the farmers community about the life hazards of pesticides, spraying machines are usually washed on the bank of canals, empty containers of pesticides are not properly dumped and above all the field force is not trained according to the good agricultural practices (GAP). Therefore, it is great of concern to evaluate/assess the canal water with respect to pesticide concentration. Pakistan has a very good walter channel system in-the-world to irrigate agriculture area, Maximum canals flow from Norther 10 Southern part of the country and irrigate vast area of agricutture land. Maximum pesticides are used in the cotton belt. It is important to check the water table, surface water and water supplies for pesticide contamination. Samples of surface water of canal were taken following the same procedure (section 3.1.1) in triplicate in glass bottfes, marked properly for identification and tractability. 3.2. Vegetable Sampling Pakistan's economy basically depends upon agriculture sector. More than 55% labour force is involved in the agriculture sector. Many types of vegetables are grown in Pakistan on 218 thousand hectares with a production of 2889.3 thousand tonnes (Anonymous, 2000). Pakistan is also caming substantial foreign exchange by exporting the good quality of vegetables to Middle East countries. To get more yield of good quality vegetables, farmers used pesticide to protect them from different infestations. Fresh vegetables were collected from farmer fields, wholesale markets and big shopping centres in triplicate. Alt samples were (Table 3.1) washed with tap water, rinsed with distilled water and dried for some time, packed in plastic bags and stored at — 4°C. 39Table 3.1 Sampling sites of vegetables Materials and Methods Vegetable Site/location Number of samples Total samples Spinach (Spinacia oleracea L.) Farm Field, Faisalabad 28 Jodhan, Faisalabad 4 Hyderabad Hafizabad AARI Sheikhupure Green Market, Faisalabad Lahore Cauliflower (Brassica oleracea L.) Green Market, Faisalabad [are City area, Faisalabad Chiniot ‘Haroonabad ‘Gujranwala Shang Hyderabad Lahore Peshawar Brinjal (Egg plant) (Solanum melongena L.) Green Market, Faisalabad 23 Gity area, Faisalabad Goyra Hasilpur Peshawar Karachi Nawabshah Rawalpindi Shahkot ‘Cucumber (Cucumis sativus L:) Lahore. Faisalabad Cay 24 Green Market, Faisalabad TT. Singh Thang Sialkot Rawalpindi Baden, Sindh Hafizabad ‘Sargodha Ladyfinger (ahelmaschus esculentus L.) Green Market, Faisalabad 23 Faisalabad City Jaranwala Sahiwal Hasilpur_ Haroonabad Gujranwala Hafizabad Hyderabad —[s[uleis[s[n[ejafets[e|n|spofelolotolaols)a)e eee a pal—|olalsloioje(apelealaloleln|olal 40Materials and Methods Bittergourd (Green Market, Faisalabad 25. (Momordica charantia L.) {Faisalabad Cit Jaranwala Gujranwala Hafizabad Peshawar ‘Dera Ismail Khan Liah ‘Tomato Green Market, Faisalabad 28 (Lycopersicon esculentum) [Faisalabad City Jaranwala Peshawar ‘Sheikhupura Sialkot ‘Supervised Trial, AART Potato ‘Green Market, Faisalabad 3h (Solanum tuberosum 1.) | Faisalabad Cit Supervised Trial Chiniot ‘Abottabad Peshawar Kundian Hyderabad Lahore. Haroonabad_ Green peas Supervised Trial, AARL 30, (Pisum sativum L.) Green Market, Faisalabad Faisalabad City Haroonabad Multan Khanewal { Gujranwala Sheikhupura Lah Peshawar Carrot Green Market, Faisalabad (Daucus carota L.) Faisalabad Cit Haroonabad Gujranwala Hyderabad Sheikhupura Lahore Rawalpiuds u)e[pfels|e[oleyals[eiolatolafsfofafajalaps|sya/elale|s)alulo|=|ejala)elelelola|olealo Hafizabad 4lMaterials and Methods 3.3. Fruit Sampling Agroclimatic environment of Pakistan is suitable for a variety of fruits like mango, apples, apricot, grapes, citrus, guava, melon and many others. Some fruits ate very delicate, delicious and having short shelf life and need a special attention to protect them from the start of carly flowering to maturity of the fruits. The owners of the gardens/orchards to protect the fruits from the very beginning so they spray the plants with different pesticides (insecticides, fungicides, herbicides and rodenticides) to control insect pest attack. Samples of different fresh fruits were collected from orchards/gardens, wholesale market, super markets and important fruit sale points. Fruits were washed with distilled water and stored the samples at 4 °C for pesticide residues analysis, ( Type of fruit, location and number of samplings are given in Table 3.2. wy eye as va feb, fer dene cttaoe, ty ada PtH wis : 4 ' eg at ter ar 7 42Table 3.2. Sampling sites of fruits Materials and Methods Fruit Siteflocation Number of samples “Total samples ‘Apple (Pyrus malus L.) Green Market, Faisalabad 20 Faisalabad City Lahore Rawalpindi Quitta ‘Peshawar Gujcanwala Mango (Mangifera indica Ly Green Market, Faisalabad 26 Faisalabad City Multan ‘Khanewal Sargodha ‘AARL Peshawar “Hyderabad ‘Orange (Citrus reticulata Blanco) Shahiwal, 30 ‘Sargodha Green Market, Faisalabad Faisalabad City Sheikhupura Lahore ‘AART Melon (Cucumis melo Lit ‘Green Market, Faisalabad 26 Faisalabad City Liah Peshawar Hyderabad Badeen Karachi Lahore “Apricot (Prunus armeniaca Linn.) Peshawar, 29 Green Market, Faisalabad Faisalabad City Lahore. Rawalpind! Gujranwala Abbotabad Banana (usa sapiennem Line.) | Hyderabad 3 Karachi Green Market, Faisalabad Faisalabad City Lahore Rawalpindi ‘Khanewat Rori Hafizabad ofa io)oJolelolelu|elalefe]elcfefohs|sis|alola|a)slolsfejujafelo|m|ololelalulajopelefehololol 43Materials and Methods Continued Table 3.2 Guava (Psidium guayaya Linn.) Green Market, Faisalabad 15 38 Faisalabad City — ‘Sheikhupura fb Kohat Lahore Sahiwal Multan Peshawar Plum (Brunus domestica) Green Market, Faisalabad 25 Faisalabad City Peshawar Kohat Banu Abbotabad_ Lahore Rawalpindi Grapes (Vitis vinifera) Green Market, Faisalabad 23 Faisalabad City Multan Peshawar Lahore Hafizabad Sheikhupura Rawalpindi Pear (Pyras communis Lint.) Green Market, Faisalabad 25 Faisalabad City Sargodha Sabiwal Gujranwala Haroonabad Lahore Faroogabad Rawalpindi ehsjusjelsfojoleloys[a(ojs|s]aalejelslelafelufefclalafafefaley 3.4 Methodologies Employed for Pesticide Residue Analyses Pesticides methods are continually being revised and improved with new and conventional techniques. A constant broadening range of chemical classes and increasing diversity of structure and properties made it difficult to develop methods that can determine all pesticides in different matrices. Many steps are involved to analyse pesticides in different selected fruits, vegetables and water samples. Extraction of pesticides residue with different solvents, concentration 44Materials and Methods and determination of various pesticide residues by different techniques are the major steps involved in this study. 3.4.1 Extraction of pesticide residues Different methods were used to extract the pesticides residues from water, vegetable and fruit samples collected from various localities. 3.4.1.1 Extraction of pesticides from water The water samples, which contain particulate matter, were passed through a glass fibre filter (1 um pore size) and pH, EC and turbidity of all collected water samples (hand pump, canal water, tube well and well water) were noted before the extraction of pesticides from water. Many solvent extraction methods were used in the past to collect afl the pesticides from water (Bucheli e a/. 1997; Lindley et al. 1996; Lagana et al. 1996; Vilchez et aj. 1996; Watts ed al., 1994 and Brauch and Schullerer 1991). Solid phase extraction cartridges were used for the isolation of multi-residues from water samples of different nature in this investigation. For the extraction and concentration of multiresidue of pesticides from different geological origin of water samples, Tanabi er af. (2000) method was followed. Furthermore, four different SPE cartridges (ENVI-Chrom P SPE Supelco, USA; LiChrolut® Cig Si-60 cartridges, Germany, SEP-PAK Cig cartridges, Waters Association, Millipore, USA and ENVI-Carb Cig Supelco, USA) were used in present study to evaluate their validity, accuracy and quality in respect of pesticide recovery. The cartridges were conditioned with 5 ml of each hexane, acetone, methanol and bi-distilled water in sequence and then the collected filtered water (Whatman 934-AH glass fibre filter) samples (200 ml) were passed under vacuum at 5 ml/min. flow rate. The cartridge was rinsed with methanol/water (1:1, v/v) dried 45Materials and Methods with air flowing for ] hour at maximum pressure. The target compoutids were eluted with acetone, hexane and ethyl acetate. 3.4.1.2 Extraction of pesticide residue from vegetables Many solvents like n-hexane, petroleum ether, methylene chloride and acetone or ethyl acetate were used as extraction solvents in the past (Steinwandter and Schluter, 1977, Kovacicova ef al., 1975, Balint and Gyorfi, 1978, Cairns er al., 1993). For the extraction of pesticide from vegetable samples Kadenczki et a/. (1992) method was followed with little variation because it is faster, less laborious, friendly to environment and less expensive. Ten gram homogenized sample was mixed with enough quantity of activated Florisil (8-12 g) depending upon the water content of sample to make it free-flowing powdered sample. The sample mixture was Uansferred onto the glass column having 5 mm layer of anhydrous sodium sulfate. The pesticide residue was eluted with 20 ml of methylene chloride-acetone or ethyl acetate at a flow rate of 5 ml min’. The extracts were concentrated on Kuderna- Danish Evaporator make under gentle stream of nitrogen to dryness and dissolved it in hexane. In the present study, 50 gram well-homogenized sample of vegetable was mixed with 20 gram anhydrous sodium sulfate + 10 gram sodium bicarbonate and 50 ml ethyl acetate was added in it. The whole mixture was shaken in GFL shaker, Germany at high speed for 1 hrs and filtered the solvent (25 ml) through glass fibre filter. The contents were evaporated to dryness and then dissolved in 250 st (cyclohexane:ethyl acetate; 1:1) and gel permeation chromatography was performed to clean the sample. 3.4.1.3 Extraction of pesticide residue from fruits A critical review of literature showed that different solvent were used in past for the extraction of pesticide residues from fruits such as petroleum ether, mixed hexane and benzene but all these solvents were lipophilic in nature (Hardin and Sarten, 46Materials and Methods 1962) As more polar pesticides, such as organophosphates, phenoxyacelic acid and triazines, came into use, more polar solvents, such as chloroform, acetone, acetonitrile and methanol found to be good (Luke ef al., 1981; Lee et al., 1990) Many workers used acetonitrile for the extraction of multiresidue of pesticides ftom fruits instead of methylene chloride, which is hazardous to environment (Cook et al., 1999; Krause and August, 1983), Fernandez-Alba er al. (2000), Kadenczki e¢ al, (1992) and Startin er al. (2000) found that ethyl acetate proved to be a good solvent as compared to other solvents for the extraction of residues of several pesticides from fruits and vegetables because its polarity is high, less volatile and thermally labile compound. No additional and tedious partitioning steps are avoided. In the present study, extraction of multiresidue of pesticides was performed with little modification according to Femandez ef al., 2000. 15 grams analytical portion of fruit sample (homogenised) was blended with 50 ml ethyl acetate and 15 gram sodium sulphate for 2 minutes in high blender (Braun, Germany). The solvent layer was decanted, fitered with fight suction through Buchner funnel fitted with Whatman glass fibre filter (934-AH) covered by 8 gram sodium sulphate. The above procedure was repeated with 20 ml ethyl acetate and the filter was rinsed with § ml solvent. Then 60 ml extract (10 g sample) was transferred to, round bottom flask and reduced the volume upto 1 ml using rotary evaporator (Buchi 011, Switzerland). The contents of the flask were transferred to a calibrated test tube and adjusted the volume upto 3 ml with ethyl acetate. The final volume was evaporated in water bath (40°C) with nitrogen stream to dryness and the residue was redissolved in 250 ull (acetonitrile: ethyl acetate) and GPC was performed for clean- up and then analysed the extract with different separation techniques. 47Materials and Methods 3.4.2 Analytical Techniques Used for Pesticide Residue Analysis Analytical methods employed for the qualitative and quantitative determination of pesticides and their metabolites in fruits, vegetables, food and biological samples play a significant role in evaluation and interpretation of the data. When sample analysis was conducted it is necessary to validate the analytical methods and provide appropriate validation information for the reliability of data, Different separation techniques were used for the determination of pesticide residues in fruits, vegetables and water samples in the present study. 3.4.2.1 Multiresidue analysis Pesticide residue analysis is used mainly for monitoring food and the environmental samples for “in house” quality control. At present, various biological and chemical technologies (Immunoassay, thin layer chromatography and capillary electrophoresis ele can be used efficiently to determine many pesticides in a variety of matrices, but by far the most popular methods for this type of analysis are gas chromatography (GC) and liquid chromatography (LC). During recent decades, capillary GC with different sensitive and selective detection systems, including mass spectrometry in multiresidue (MS) in the later years, has been the dominant method of analysis determination. Liquid chromatography combined with UV, dicdearray or fluorescence detection has also been used in most pesticide residue laboratories as a complementary technique to deterntine many of the new pesticides developed during. the last decade, which are characterised by high polarity and/or thermolability, In the present study techniques like HPTLC, gas chromatography and high performance liquid chromatography were used for the determination of pesticide residues in fruits, vegetables and water samples. Comparison was also made to check the robustness, efficiency and accuracy of different techniques. 48Materials and Methods 3.4.2.2 Thin layer chromatography (TLC) Methodologies used for the determination of pesticide residues are continuously being revised and improved with new and conventional technologies. Broadening range of chemicals and increasing diversity of structure/properties made it difficult to develop the methods that can determine all pesticides. Today many techniques like gas chromatography, liquid chromatography, mass spectroinetry, infrared spectroscopy and ELISA are used for monitoring pesticide residues in food, vegelables, fruits and water (Guo-Fang ef al., 1994). Of the various chromatographic (echniques, thin layer chromatography (TLC) has recently gained popularity not only because of advancements in instrumentation and improvement in efficiency but also because of its relatively low cost, simplicity and wide range of applications. This technique is equally used for the separation and determination of pesticide residues of al? classes, including insecticides, herbicides and fungicides in a wide variety of samples. The commission on pesticide chemistry has concluded that TLC is useful in multiresidue determination and quantification of the most important classes of pesticides (Batora ef al, 1981). Thin layer chromatography (TLC) originally developed in 1950’s and still widely practiced today for the separation of simple mixtures and qualitative identifications of samples. . By contrast modern TLC (usually termed high performance thin layer chromatography or HPTLC) is semi instrumental technique. The technique (HPTLC) is capable of producing fast, high- resolution separation and quantitative results with accuracy and precision with those obtained by gas chromatography (GC) and high performance liquid chromatography (LC). TLC is an off line process in which the various stages are carried out independently. Different validated HPTLC methods were used in the present study for the determination of pesticide residue in fruits, vegetables and water samples, 49Materials and Methods ‘TLC plates were prepared by mixing Aluminium oxide, Merck, Germany (45 g) with 90 ml 0.2% nitric acid in glass stoppered Erlenmeyer flask. The content was transferred into centrifuged tubes and centrifuged at 2500 rpm for 10 minutes. The acid layer was decanted and discarded. The aluminium oxide G layer was washed with 3 x 50 ml of distilled water for performing centrifuge. The washed aluminium oxide was transferred to a beaker (250 ml) and then 15 ml (1% AgNO) and 15 ml distilled water was added. The whole content was mixed with a glass rod and transferred the slurry to TLC spreader (Desaga, Germany) and coated four glass plates (20 x 20 cm) of 0.25 mm thickness, The plates were dried in air, activated at 75 °C and stored in dessicator. Thin layer chromatography was performed spotting actual samples with authentic standards, developed in ethyl acetate and put under UV light for 30 minutes for spot visualization. When spots became visible, distance of standard and sample spots was measured and R, values were calculated. The concentration of each pesticide present in the samples was determined following the procedure as mentioned in section 3.4.2.2.1, 3.4,2.2,3 Photosynthesis inhibition method (Hill reaction) This TLC detection method is very specific for the determination of herbicides, which inhibit the function of photosynthesis. Extraction of chlorophyll suspension Chiorophyil (of rice, wheat, spinach and mungbean leaves) is equally effective for the detection of herbicides in fruit and vegetable extracts but in the present study, wheat and rice leave extracts were used. Wheat and rice leaves (30 grant) were cul freshly and ground with pestle and mortar, When SlMaterials and Methods the leaves homogenized completely, 3 mt glycerine and 15 mt distilled water ‘vas added and the chlorophyll suspension was transferred to four-layer gauze knapsack over a conical flask. ‘The suspension was pressed with hand through thin cloth to get chlorophyll from leaves and flask was wrapped with aluminium foil and the extract was stored in refrigerator. Reagent solutions Borax buffer solution: Sodium borate (Merck, Germany) was prepared dissolving 9.5 g in 500 ml distilled water. 350 ml sodium borate solution was mixed with 150 ml 0.1 N HCI for borax buffer solution and stored in refrigerator. DCPIP solution: 200 mg of 2,6-dichlorophenol-indophenol Na-salt (Merck, Germany) was dissolved in 500 ml borax buffer solution and stored in refrigerator. Detecting reagent: Rice or wheat (10 ml) extract was mixed with 10 ml of DCPIP solution and then added drop-wise until the colour of mixture becomes bluish-green. This solution is enough for four plates of size 20 x 20 om. Methodology: Ready-made silica gel 60 plates were activated at 105 °C for 30 minutes. The plate was taken out from oven, fixed in spotting rack and spotted’ the plate With samples extract with authentic standards according to pre-written scheme. The plates were developed in a saturated tank with ethy! acetate. After drying the plate in fume hood, the plate was sprayed with reagent solution (DCPIP-chlorophyll) and put under light (60 W bulb) for 5 minutes for the maximum visibility of spots, Measurements of spot area were performed immediately as they disappeared after 10 minutes. The colour of the spot was bluish against the greenish background. Quantitative analysisMaterials and Methods was made comparing the average spot diameter of standards as mentioned in 3.4.2.2.1. 3.4.2.2.4 Fungi spore (4spergillus niger) inhibition method (FAN) This method is frequently used for the detection of fungicides in food, fruits and vegetables. Plant extracts did not interfere for the determination of pesticide residues. Production of fungi spore (Aspergillus niger) Preparation of Fungi culture media: Fresh potatoes were purchased from the tocal market and disinfected with Dettol for 10 minutes. The potatoes were washed with distilled water, peeled off and ground in high-speed blender (Multimix Braun, Germany). 50 g ground potato was boiled with 250 ml distilled water in 500 ml flask for 45 minutes, The concentrate was filtered through one layer gauze cotton cloth. Glucose (5 g) and agar-agar (5 g) were added to the filtrate and autoclaved at standard temperature (121°C) and pressure (15 Ibs). The autoclaved growth media was cooled down to 45 °C and pored into 10 sterilized petri plates and incubated for 48 hrs at 37 °C to see any contamination. The non-containinated petri dishes were selected and cultured with Aspergillus niger spores. The petri dishes were placed into the incubator for fungus multiplication and these were used later on for fungicide residues determination in samples. Preparation of fungi spore suspension 15 g glucose, 1.5 g agar-agar, 0.3 g KNOs, 1.5 g mall extract were added into a 250 mt flask with 70 mi distilied water and boiled for 15 minutes. The solution was allowed to cool down to 45°C and then kept at this temperature. The fungi spores were removed from the developed culture by adding 30 ml warm distilled water and added into the solution. The spores multiplied 53Materiais and Methods within 5 minutes and filtered it through 2 layer of thin cloth and kept it at 45°C. Methodology Glass plates 20 x 20 cin coated with silica gel 60, 0.25 mm thickness were activated at 105°C for 30 minutes. Each plate was fixed in spotting rack and spotted it with proper volume’ of sample extracts alongwith authentic standards (marker compounds). The plates were developed in pre-saturated tank with ethyl acetate upto the marked line and also recorded the elution time and solvent temperature. The extra solvent was evaporated in fume hood and sprayed the plates with prepared fungi suspension solution. The plates were put into a tank for 24 hours pre-saturated with water vapours at 37°C. White spots against black background appeared, the distance travelled by spots and solvent were noted and R; values were determined, 3.4.2.2.5 Enzyme inhibition with cow liver extract and B-naphthyl-acetate substrate (EBNA) This method is very specific for the detection of phosphoric, thiophosphoric acid esters and carbamate pesticides in food, fruits and vegetables, Preparation of enzyme solution Fresh cow liver was purchase from the market and cut into small pieces. Ten gram liver with 90 ml distilled water were homogenised in high speed blender. The content was transferred to centrifuge tubes and centrifuged it at 4009 rpm for 10 minutes. The supematant was collected in 20 m1 vials and stored in freezer. The dilution was made (enzyme: water; 1:5) before spraying the plate. 34Materials and Methods B-naphthyLacetate solution ‘The reagent (125 mg) was taken in volumetric flask and dissolved in 100 ml ethanol. The solution was stored in refrigerator at 4°C,_ and can be used for extended period, Echtblau-salt solution ([ast blue BB salt) 10 mg Fast blue BB salt (4-Amino-2,5-diethoxybenzanilide diazotated zinc double salt) was dissolved in 16 ml distilled water and used freshly for developing the plate. Substrate solption Mixed both solutions (B-naphthyl-acetate solution and Echtblau-salt solution) before spraying. Methodology Glass plates (Merck, Germany) 20 x 20 cm coated with silica gel 60, 0.25 mm thickness were activated at 105°C for 30 minutes. Each plate was fixed in spotting rack and spotted it with proper volume of sample extracts alongwith authentic standards (marker compounds). The plates were developed in pre-saturated tank with ethyl acetate upto the marked line and also recorded the elution time and solvent temperature. The extra solvent was evaporated in fume hood and treated the plates with bromine vapours in @ tank pre-saturated with bromine vapours for 15 minutes. The extra bromine vapours were removed by putting the plates in fume hood for 45 minutes, The plates were sprayed with liver enzyme solution and developed them in an incubator at 37°C for 30 minutes pre-saturated with water vapours, The plates were removed from the incubator and evaporated the extra water vapours with hot air stream and sprayed the plate with solution. White spots in pink background appeared. The distance travelled by spots and solvent was noted and Ry values were calculated. The Ry values were compared with authentic standards and calculated the concentration comparing the average spot area of standards as mentioned in section 3.4.2.2.1.Materials and Methods 3.4.2.2.6 Enzyme inhibition with horse blood serum and acetylthiocoline iodide substrate (Eacl) This method is very sensitive for the determination of organophosphate and carbamate pesticides in food, fruits and vegetables. Reagent solution 50 mg 2,6-dichlorophenol-indophenol Na-salt (0.5 mg/ml) was dissolved ia distilled water and stored in refrigerator. Enzyme solution Fresh horse blood was collected from Army Stud, Faisalabad, brought to laboratory, transferred (0 centrifuged tubes and centrifuged at 4000 rpm for 10 minutes, The serum was stored in freezer in 20 ml vials. 10 ml serum was diluted with 7ml tris buffer solution before use, Tris-buffer (0.05 molar) solution Tris (hydroxymethyl!) aminomethane (3.04 g) was taken in volumetric flask and dissolved in $00 ml distilled water and stored in refrigerator at 4°C. Substrate solution Acetyl thiocoline iodide (100 g) was taken in volumetric flask, dissolved in 100 ml distilled water and stored in refrigerator at 4°C. Methodology Glass plates (Merck, Germany) 20 x 20 cm coated with silica gel 60, 0.25 mm thickness were activated at 105°C for 30 minutes. Each plate was fixed in spotting rack and spotted it with proper volume of sample extracts alongwith authentic standards (marker compounds). The plate was 56Materials and Methods developed in pre-saturated tank with ethyl acetate upto the marked line. The extra solvent was evaporated in fume hood and treated the plate with bromine vapours in a tank pre-saturated with bromine vapours for 15 minutes. The extra bromine vapours were removed putting the plate in fume hood for 45 minutes, The plate was sprayed with horse blogd enzyme solution and developed it in an incubator at 37°C for 30 minutes pre-saturated with water vapours, The plate was removed from incubator and evaporated the extra water vapours with hot air stream. The plate was sprayed with substrate solution and incubated the plates again at 37°C for 15 minutes in incubator pre-saturated with water vapours. The plate was taken out ftom incubator and removed the excess water with hot air. The plate was then sprayed with reagent solution. Blue spots in white background appeared. The distance travelled by spots and solvent was noted and calculated the Ry values, The average spot area (vertical area + horizontal area) and concentration of each pesticide in extracts of vegetable and fruits were determined according to the procedure as mentioned in section 3.4.2.2.1, 3.4.2.3 Gas chromatography (GC) Though monitoring of pesticide residues is crucial for proper assessment of human exposure to pesticides through food. Effective monitoring of residues of pesticide in fruits, vegetables and water require suitable multiresidue methods to identify as many pesticides as possible. Generally, this is accomplished with capillary gas chromatography (GC) in combination with electron capture detector (ECD), nitrogen phosphorus detector (NPD), flame photometric detector (FPD) and mass spectrometric (MS) detection systems (Anderson and Ohlins, 1986; Luke ef al., 1981). For the under taken studies, fruits, vegetables and water extracts were analysed by gas chromatographic method using ECD and NPD detectors on capillary columns. Before analysing the actual samples, the instrument was checked by s7Materials and Methods performing system suitability test and then validated the different detectors using authentic standards. 3.4.2.4 High performance liquid chromatography (HPLC) Pesticide methodologies for the determination of residues are continually being revised and improved with new and conventional techniques. Today many sophisticated techniques are used for the determination of pesticide residues in vegetables, fruits, grains and water (Richter er al., 2001). Samples of vegetables, fruits and water were also analyzed by HPLC to compare the results of gas chromatography and thin layer chromatography following the methods of Ohlin, 1986 and DeKok and Hiemstra, 1992. In the present study, the analyscs were performed in isocratic and gradient system using reverse phase Cys column 25 em x 4.6 mm (id.). Different mobile phases were used to get optimum resolution and sensitivity with UV-Vis detection system. The contents of fruits and vegetables were dissolved in acetonitrile: ethyl acelale and GPC was performed to clean/remove the interfering compounds. Different UV ranges were used for different pesticide residue analysis and sensitivity of the scanned pesticides was noted. 58Results and Discussion Chapter 4 RESULTS AND DISCUSSION. 4.1 Validation of extraction and chromatographic techniques Validation is a prerequisite of any reliable process for extraction and chromatographic analysis. Many chromatographic and extraction parameters have been included in the validation process, such as linearity of the calibration curve, sensitivity and selectivity of solute detection, result reproducibility, instrumental precision, detection limit, quantitation limit, recovery percentage and ruggedness. 1 Solid-phase extraction Various pre-concentration methods have been used for the extraction and determination of pesticide residues in water samples. Some of the commonly used techniques are liquid-liquid extraction (LLE) and solid-phase extraction (SPE), which are extensively used for the extraction of residues from environmental and water samples (Barcelo ef al., 1993). Much interest has been developed in the field of pesticide chemistry to use solid-phase extraction for the isolation of organic pollutants from water samples instead of conventional techniques. Solid-phase extraction is a sample preparation technique and extensively used in analytical laboratories for the clean-up and concentration of the sample analyte before analysis. This technique reduces the use of organic solvents upto one third, reduces the associated risk, cost of use and is an effective and faster extraction technique than the conventional techniques. Water sainplings collected from different aquifers were 59Results and Discussion processed for the extraction and clean-up for multiresidue analysis. Currently, many different SPE cartridges are used for extracting pollutants from aqueous samples, As far as the best selection in respect of efficiency of SPE is concemed, these different types of cartridges were used in this study. The detail of the cartridges in the present study is given in Table 4.1, ‘Table 4.1 Specification of different cartridges used in the study ‘Type of cartridge ‘We. of adsorbent [Capacity vol. Surface area__| SEP-PAK Cig 500 mg, 6ml - (Waters Association, Millipore, USA) LiChrout® Cyy__-SI-60 500 mg om ~ Merck, Germany) ENVI-Chrom P SPE 500 mg 6m 800-950 m/g (styrene-divinylbenzene resin ) Supelco, USA ENVI-Carb Cis 500 mg 6ml 475 m‘/g Supelco, USA Efficiency of cartridges was determined after spiking the double distilled-deionized water with 1 yg of each pesticide in acetone solution. Cartridges were conditioned by successive elution of 15 ml methanol and 10 ml of water by means of gentle vacuum to avoid drying-out during the procedure, The sample volume was percolated at 5 ml min” and the cartridge was dried with nitrogen and eluted with ethyl acetate, hexane and methanol by force of gravity. The influence of an equilibrium (or soaking) time (2-4 niinutes) between the solvent and stationary phase, before eluting the cartridge with solvent (2, 3 and 4 ml) on the recovery was also studied. Volumes of 100, 200, 300 and 500 ml water spiked with 1 ig of cach pesticide were also used to obtain the breakthrough volume (Jimenez ef a/., 1997). 60Results and Discussion Results Table 4.2 shows the effect of solvent on the recovery (% age) of the pesticides. The qualitative analysis was preferentially done by GC-ECD, GC-NPD. Some compounds like metaxuron, isoproturon, chlortoluron, fenvalerate, cypermethrin, p, p-DDT, carbofuran, methomyl, carbaryl and atrazine were also confirmed by HPLC using UV detector, Table 4.2. Effect of solvents on the recovery (%) of different pesticides from 200 ml of double-distiiled de-ionized water (Cartridges mentioned in Tabte 4.1) Pesticide Ethyl acetate | Acetone Methanol Acetonitrile Ree." RSD**_ [Rec RSD. Rec. RSD Ree. RSD a-Endosulfan 85.5 3.6 | 89.9 42 81.2 {5.1 70.1 | 4.8 |p, p-DDT 90.1 5.3 | 95.7 57 89.2 13.5 87.6 13.5 Lindane 94.2 3.2) 96.1 47 92.8 14.3 90.9 | 5.2 Mcthamidophos | 99.6 5.2 102.8 | 5.0 971 15.9 92.2 [3.8 Dimethoate 89.7 43 94.0 2.8 95.3 13.9 96.4 | 4.2 Monocrotophos 101.2 | 3.9 98.2 3.2 97.2 143 OL [3.8 Parathion-m 98.3 41 99.7 4.0 95.6 {5.2 81.5 [4.5 Carbofuran 92.9 [33 [93.1 [53 [901 [47 [743 [5.2 Methomy! 99.1 [47 1973 [39 fo2s [38 [912 [3.1 Carbosulfan [100.2 [5.2 [95.7 [43 [908 [5.6 [80.1159 Cypermethrin [98.2 [3.9 [952 [53 [ais [37 [65231 | Deltamethrin [921 [4.9 [961 [42 [782 [57 {092 [5.8 | Cyhalothrin [90.3 [4.0 [942 [43 [871 [3.1 | 803 [48 Captan 963 158 [95.7 [59 [896 45 [772 [43 Thiophanate-m [918 [4.2 [932 [48 [885 [41 |724 [5.9 Carbendazim [95.9 [53 [892 [58 [832 [50 [684 [48 Avg. 94.08 [4.5 [9554 [4.58 [89.3 [452 [76.2 ]45 Data is mean of 3 replicates *Recovery **relalive standard deviation Data (Table 4.2) also show that the average recoveries obtained after eluting with 2 ml of different solvents for ethyl acctate, acctonc, methanol and acetonitrile were 94.0, 95.5, 89.3 and 76.2% respectively. Ethyl acetate, acetone and methanol wereResults and Discussion selected as eluting solvents due to their good elution power of pesticides. Acetonitrile was the worse choice for the nultiresidue analysis due to less recovery percentages, ‘The relative standard deviation (RSD) of the results ranged from 4.5 to 5.54%, The influence of equilibrium time (cartridge soaked for 2 min. prior to elution) and the eluant volume on the recovery was also checked (Table 4.3). Table 4.3 Recovery (%) of pesticides from 200ml of double-distilled deionized water (DDDW) spiked with | 1g of each pesticide by ODS cartridges eluted with different volume of acetone: ethyl acetate: methanol and equilibrium Pesticide Equilibrium tine 0 Equilibrium time 2 min Tint of each Imlofeach 2 mlofeach 3 ml ofeach Rec.* [ RSD** | Rec. [RSD | Rec. [RSD [Rec. [RSD a-Endosulfan | 92.5 [5.3 93.2 [3.8 [978 [3.2 [982 [5.2 |p. p-DDT 89.2 [47 52.1 752 [963 [38 [oor [34 Lindane 93.5 [56 951 [48 [972 [42 [981 [29 Methamidophos [94.3 | 3.8 96.2 [48 [97.9 [5 [992 “Tas Dimethoate 93.6 [4.3 94.2138 [96.2 [3.3 [971 [32 Monoerotophos | 95.4 [5.2 96.1 [43 [98.6 S51 [98.9 73.7 Parathion-m 96.1 13.4 968 13.9 [982 [5.2 [1012 [40 Carbofuran 92.8 [5.7 3.5 50 [958 133 [ori [3s | Methomyl 96.0 14.6 96.7 [3.3 [991 [43 [1021 [42 Carbosulfan 971 13.9 97.5 [3.5 [101.2 [a1 [103.4 [5.2 [Cypermethrin [90.2 [3.3 93.7 [40 [953 [3.8 [982 137 Deltamethrin [93.0 [4.0 41 [49 [966 [3.0 [96.9 [3.1 Cyhalothrin 91.2 [3.2 92.3132 [953 [3.7 [972 [40 Captan NT (38 92.5 [48 [961 [44 [992 [5.1 Thiophanate-m [92.1 | 4.7 92.8 [53 [952 [5.1 [956 [41 Carbendazim [94.5 [3.2 95.1 [5.0 [971 [a1 [973 [38 Data is mean of 3 replicates *Recavery **Relative standard deviation Data show that the equilibrium time between the stationary phase and solvent improved the recovery of various pesticides when the cartridge was eluted with 2 ml and 3 ml of cach cluting solvent as compared to | ml of solvent; for instance, the recovery increased 62Results and Discussion to 5% for a-Endosulfan, cypermethrin, cyhalothrin and also for captan, thiophanate- methyl and carbendazim. The recovery (%age) was not significant in case of 2 ml and 3 mi at 2 min equilibrium time. From the data, it is clear that solvent volume (2 ml of each) and equilibrium time (2 min) would be the best choice for the extraction of pesticide residues from water samples. 4.1.1.1 Selection of sample volume for better recovery Study was also performed for the breakthrough volume for getting good recovery of pesticide residues from SPE cartridges. Different volumes of double distilled deionized water (100, 200, 300, 400 & 500 nil) were spiked 1 jag of different classes of pesticides. The waters spiked were passed through the ODS cartridges and analyzed with gas chromatography, ‘The data is presented in Table 4.4. Table 4.4 Effect of sample volume on recovery (%) of pesticides spiked with | pg Pesticide 100 ml 200 ml 300 ml ] 400 ml 500 mt Rec.* | RSD** | Rec. | RSD | Rec. | RSD | Rec. | RSD | Rec. | RSD @-Endosul fan 96.3 [4.1 (986 429 1981 | 3.3 | 980/33 195.1149 p.p-DDT. 97.5 | 5.2 101.3 [4.2 |98.9 [4.1 | 98.5 |4.3 | 94.4 | 3.8 Lindane 96.2 | 3.8 99.2 [5.1 1985 [5.3 | 98.0 14.9 | 93.7 | 4.5 Methamidophos | 97%5_| 3.1 101.5 [4.3 |99.2 | 4.7 98.2 15.6 | 96.3 | 5.1 Dimethoate 95.3 | 4.1 98.2 ($4 197.8 | 5.0 1971 13.4 | 94.0 | 3.3 Monocrotophos | 96.4 _| 3.0 98.7 [3.7 |97.3 | 28 | 96.7134 | 95.2 | 4.4 Parathion-m 95.3 | 5.2 99.2 [4.7 |987 {5.4 | 98.0145 | 94.2 | 5.0 Carbofuran 96.9 14.9 98.5 {5.0 [98.0 14.7 |97.5 [4.1 | 95.1 | 5.3 Methomy! 97.2 | 3.8 101.9|2.9 | 100.1] 5.2 [99.5 [5.0 | 96.4 | 4.7 Carbosulfan 94.7 15.4 102.7 | 5.3 101.3] 4.9 | 99.0 | 3.8 | 93.3 | 3.7, Cypermethrin 93.1 | 4.0 98.9 14.5 [98.2 | 3.8 [98.0 49 |921 |} 4.1 4 Deltamethrin 95.5 15.4 971 754 [96.5 | 4.3 [96.1 13.8 | 90.6 | 3.5 Cyhalothrin 96.1 | 2.9 98.2 |3.8 [97.8 [3.5 197.3) 3.5 | 95.0] 3.9 Captan 94.8 | 5.2 99.5 [4.3 | 99.2 [3.9 | 984/49 | 91.1 15.4 Thiophanate-m_ | 95,3 | 4.7 96.1 15.1 [95.7 13.0 | 95.2 13.3 | 90.0 [4.2 2.9 976 [5.3 [97.2 |5.0 | 96.8 ]3.9 | 93.2 52_| Carbendazim. 96.1 Data is mean of 3 replicate *Recovery —_* *Relative standard deviation 63Results and Discussion From the data presented in Table 4.4, it is evident that the increase in sample volume affects the recovery of pesticides. Recovery (Yage) of deltamel captan, thiophanate-m and carbendazim were significantly affected in increasing the sample volume. When the volume increased from 100 to 500 ml, the recovery percentage was decreased by | to 5% but substantial decrease was observed between 200 ml to 500 ml. Lower variations were found among 200 to 400 ml volume and exhibited persistent recovery after triplicate analyses. Results were also subjected to analysis of variance (ANOVA) to test for statistical differences. The recoveries of p, p-DDT, lindane, methamidophos, dimethoate, parathion-m, methomyl, carbosul fan, cypermethrin, deltamethrin, captan, thiophanate-m and carbendazim showed significant recovery differences (P<0.05). From the results it is cleared that a volume of 200 ml was chosen as a compromise solution for the multiresidue analysis but the recovery percentage of any analyte was not increased while increasing the water volume (200-500 ml). 2 Evaluation of the procedure A comparison of recovery percentage was made among double distilled deionized water (DDDW), canal, hand pump, tubewell and well waters. Two hundred milliliter (200 mij of eaeh water sample was spiked with one microgram (1 1g) of the most extensively sprayed pesticides on cotton, rice and vegetable areas. 64Results and Discussion Table 4.5 Recovery percentage and precision obtained with DDDW, canal, tubewell, handpump and well water samples Pesticide | pDDWw* Canal water | Well water Handpump Tubewell R* [rsp |R ]RSD [R |RSD [R RSD [R [RSD e@Endosulfan [98.5 | 62 oat [39 [959 [62 [967 [38 joer [38 pp-DDT 101.2 [75 o24 [72 [971 156 [983 [65 [995 [68 Tindane 992 [61 932 152 [979 [e2 |985 ]71 [989 [37 | Methamidophios 1013 | 59 1002 {64a [99d [52 [001 !2s [i004 [72 Dimethoate 98.2 | 47 ost [54 [972 [as [978 [62 [oso [5.1 Monocrotophos | 98.7 | 8.2 974 [38 [975 [68 [979 [76 [ORT 1/30 [ Parathionm [99.2 [7.8 oa [oa [97 [74 [98s [83 [ows [aa Carbofuran 85 | 70 o29 [52 [952 [aa [972 [75 [oss [58 Methomyl 101.8 | 65 913 $39 [986 [75 [99.2 [os [997 172 Carbosulfan [102.5 ]43~ [951 [62 [975 [38 [987 [37 [99s [oa Cypenmethin [98.7 [45 o72 [3s 974 76s [98I [53 [98S [73 Deliametiein (97.3 | 34 965 [42 [966 [52 [969 [43 [O71 [39 Cyhalothrin [98.2 | 65 979 [sa [978 [38 [979 [33 [980 a3 Captan p94 [71 99.2 [42 [ost fas fore [43 [992 [53 Thiophanate-m [96.2 | 8.2 v6.1 [32 [925 [70 [952 [63 [950 [at Carbendazim 97.7 | 5.0 921 [72 [943 [52 [oae [50 [969 [73 Data is mean of 3 replicates “DDDW; double-distilled deionized water “**RSD; Relative standard deviation **R, means recovery percentage All the spiked water samples were passed through ODS cartridges at a flow rate of Smt min’, the pesticide residues were eluted with ethyl acetate, acetone and methanol (2ml of cach), concentrated to dryness, redissolved in n-Hexane and analyzed with chromatographic methods. obtained is given in Table 4.5. Recovery percentage of each pesticide 65Results and Discussion Data show the recoveries and precisions achieved through the proposed procedure used for the extraction and concentration of pesticide residues by ODS cartridges. Double-distilled deionized water (DDDW), handpump and tubewell waters gave comparable recoveries of cach pesticide while in case of canal and well waters the recoveries were comparable or lower than the more refined waters. The lower recovery may be due to the presence of organic matter in the real samples, which coukt affect the retention on the cartridges. However, higher recovery of pyrethroids (>95%) was obtained on canal and well waters. It may be associated with the organic matrix present in these samples which would favour the adsorption on ODS cartridges. The recovery percentage of each pesticide was same in double distilled deionized water and tubewell waters while the recovery of pesticide was comparable or lower in case of handpump water. Most pesticides showed more than 95% recovery in handpump and tubewell waters because these were more clean and filtered waters and had no organic carbon particles. In case of canal and well waters, some pesticides exhibited more than 95% recovery while fungicides showed least recovery (Fig 4.1). 66Results and Discussion Recovery {%) Miimiisin — Pratinns — Metonyt Qpemeten, Pesticide Fig. 4.1 Comparison of recovery (%) of pesticides in water samples obtained from different sources 4.1.1.3 Efficiency of different solid-phase extraction cartridges Solid-phase extraction cartridges of different Trade-mark were used to find the best cartridge in respect of its adsorption and desorption efficiency (Table 4.1), Two hundred milliliter double distilled deionized water was spiked with Ig of a-Endosulfan, p,p’- DDT, methamidophos, chlorpyriphos, carbofuran, methomyl, captan, thiophanate- methyl, isoproturon, chlortoluron, cypermethrin and fenvalerate. The treated water (triplicate) was passed through the cartridges at a flow rate of 5 ml min’ at pressure 20 psi. The spiked analytes were eluted with 2ml of each ethyl acetate, acetone and methanoi, concentrated and analyzed by gas chromatography. The results (recovery %) of different pesticides eluted from different cartridges are summarized in Table 4.6. 67Results and Discussion Table 4.6 Efficiency (recovery %) of different solid-phase extraction cartridges Pesticide SEP-PAK | LiChrolut] ENVI-Chrom P| ENVI-Carb a-Endosulfan 8825 9443 96 £2 971 p, p-DDT O13 9344 9543 96 £2 Methamidophos 9324 9545 9744 983 Chiorpyriphos Ot 3 96 £3 98 £2 98 +1 Carbofuran 8942 924 95+ 5 973 Methomyl 93x 4 9445 9643 9842 Captan 8943 934 9544 9643 Thiophanate-methyl 8846 907 94 +6 9544 Isoproturon 92 5 94 £8 9644 9742 Chlortoluron 9347 95 £6 975 9843 Cypermethrin TBE 5 9244 9443 96 £2 Fenvalerate 864 6 9345 95 £2 9743 Data is mean of 3 replicates + stundard deviation The ENVI-Carb and ENVI-Chrom P gave excellent recovery percentage of the pesticides as compared to SEP-PAK and LiChrolut cartridges. The pesticides eluted through ENVI- Carb cartridge gave 295% recovery but chlorpyriphos, methamidophos, methomyl and chlortoiuron showed 98% whereas in ENVI-Chrom P chlorpyriphos recovery was 98% but other compounds showed less as compared to ENVI-Carb cartridges. SEP-PAK and LiChrolut cartridges gave poor recoveries of the pesticides (Table 4.6). Discussion In the analyses of aqueous samples, the sample preparation step is frequently the most time consuming and is the primary cause of analyte loss from the matrix. Various preconcentration methods have becn and are currently used for the determination of pesticide residues in water samples. Solid-phase extraction (SPE) has widespread application for the isolation of trace levels of organic compounds present in aqueous solutions (Barcelo er al., 1993). Solid-phase extraction cartridges of different Trade-mark were used for the preconcentration of pesticides from surface and ground water samples, 68Results and Discussion ‘The results indicate that recovery percentage of the pesticides with the efution of 2 ml of ethyl acetate, acetone and methano! was significant as compared to the earlier reported work by Jimenez et al., (1997). They concluded in their study that recovery of pesticides with methanol elution was high. The same procedure was used in the present study but the results were not satisfactory (< 65%). The choice of different polar solvents in combined state or separately gave more than 95% recovery of the studied pesticides adsorbed on the stationary bed of SPE cartridge. Our findings are in line with the results reported by Castillo e¢ a/., (1999), They used different solid-phase extraction cartridges for the extraction of residues of eleven pesticides from the samples of aquifers used in banana grown territory. Ethyl acetate gave 60-101% recovery for the studied pesticides with high relative standard deviation (3.2-5.8%). ‘The influence of equilibrium time on the recovery of pesticides from cartridge showed 2 minutes of soaking prior to elution improved the recovery percentage but longer equilibrium time significantly reduced the pesticide recovery from cartridges (Hinckley and Bidleman, 1989). Sampling volume also plays an important role for the recovery of pesticides from water using solid-phase extraction column. The data from the present study show that most pesticides were recovered with significant percentage with 200 ml volume of water while Brauch and Schullerer (1991) mentioned that 2 L to 5 L of a water sample was taken for the analysis of different pesticides due to dilution of pesticides, which is contradictory to our results, ‘The recovery percentage and precision were also observed during the study. All the waters (double distilled deionized, canal, handpump, tubewell and well waters) were spiked at Jug” concentration, passed through the ODS cartridges at 5 mi min” flow rate and eluted the analytes with acetone, ethyl acetate and methanol (2 ml of each), The contents were analyzed and found that more than 95% recovery of each pesticide was obtained in all water samples while fungicides gave less recovery (<92%) as compared to other pesticides in canal water, It may be due to the adsorption of fungicide on the soil 69Results and Discussion particles or with the bed of cartridges (Aharonson and Kafkafi, 1975 b). All the pesticides showed good precision (RSD) afler eluting from the cartridges and the range of relative standard deviation was 4 to 8%. The results are in line with the findings of Pionke et al., 1988. Solid-phase extraction (SPE) is a growing area of research in the development of environmental sample preparation. Four types of SPE cartridges were used to evaluate the efficieney of the modem technology. All the cartridges gave excellent recovery of pesticides but ENVI-Carb and ENVI-Chrom P showed significant behaviour for the adsorption and clution of non-polar to polar pesticides. ‘The data in ‘Table 4.27 was analyzed statistically (ot =0.05) to highlight the best choice for cartridges. The analysis showed that ENVI-Carb gave significant response to every pesticide and did not cause any affect on the recovery of analyte, Our results are in line with the earlier studies (Hagan ef al., 1990; Senseman et a/., 1993. Mouvet and Jucker 1997). All the pesticides did not show much adsorption on the stationary phases of the used cartridges and hence better recovery from the cartridges. From the above results, it may be concluded that the ENVI-Carb was the best choice for the extraction and preconcentration of multiresidues trom surface and ground water samples. Conclusion Solid-phase extraction proved to be a good alternative technique to replace the time- consuming liquid-liquid extraction performed in separatory fumnels, which often require substantial amounts of costly, highly pure and environmentally sensitive solvents. Ethyl acetate, acetone and methanol (2m of each) proved to be good solvents for successive elution of pesticides adsorbed on the cartridge bed. The recovery percentage of each pesticide from SPE cartridge (ENV!-Carb) showed excellent behaviour (>95%). From the results it is cleared that 200 m! sample volume and 2 minutes equilibrium time before the elution of pesticides gave high recovery of each type of pesticide. Waters of different aquifers behaved in the same way but that water, which had soil particles, showed some adsorption of pesticide and thus not eluted compietely. Many SPE cartridges were used 70Results and Discussion but ENVI-Carb was found good among others with salient features for the pre- concentration of pesticide residues from surface and ground waters, It showed consistent recovery afer successive clution with organic solvents and did not develop any backpressure. The use of this technology for the extraction and preconcentration of pesticide residues for aquifer samples did not pose any risk to the environment due to less use of solvent. 7Results and Discussion 4.1.2 Gas Chromatography (GC) ‘Today many sophisticated and sensitive techniques are used for the analysis of pesticide residues in different matrices, Among these techniques, gas chromatography is the real choice duc to its sensitivity; wide range of applications, quickness, cost-effective and most widely used for pesticide residues in fruits, vegetables, soil, biota and water. This technique has the ability to determine a significant number of pesticides and their metabolites in 2 variety of food matrices and environmental samples (Frost, 1996). Before performing the analysis of actual samples on gas chromatography, system suitability test was done. 4.1.2.1 System Suitabitity Test System suitability tests are an integral part of chromatographic methods (U.S. Pharmacopoeia, 1995). These tests are used to verify that the resolution and reproducibility of a chromatographic system are adequate for an analysis. These tests are on the base of concept that the equipment, electronics, analytical operations and sample constitute an integral system that can be evaluated as a whole (Krull and Swartz, 1998), By performing this test the following parameters were determined to ensure the performance of the system before or during the analysis of the unknown samples. i) Plate counts ii) Resolution iii) Retention factor (K’) iv) Asymmetry 72Results and Discussion Unretained peak (to) was received by injecting air or dichloromethane vapours (2 il). Standard sokition of organochlorine (lindane, heptachlor, a-Endosulfan, p,p'-DDE, p,p'-DDD and p,p’-DDT) was run on ECD (Fig 4.2) and calculated the parameters (Table 4.7) to ensure the performance of the instrument by using the following formulas: Ke retention factor y= retention time of solute l= retention time of unretained peak ge adjusted retention time of solute Retention factor: ¢ Ney number of effective plates Plate number: lay 5:545] 8. |? adjusted retention time of solute Wy= peak width at one-half peak height a thi = adjusted retention time of solute | Resolution: la = adjusted retention time of solute 2 Wai= peak width at one-half peak height Was peak width at one-half peak height b Asymmetry: ass? "ymnmeiry: a a= half width of a peak at 0.5 height b~ half width of a peak at 0.1 height Detector Response ° 5 10 1S 20 TIME (min ) Fig. 4.2 Standard chromatogram of organochlorine pesticides (0.05 jag/ml) with ECD I. lindane, 2, heptachlor, 3. o-Endosulfan, 4. p, p’-DDE, 5. p, p’-DDD, 6. p,p"-DDT 73Results and Discussion ‘Table 4.7 Comparison of calculated and standard values of system suitability test Parameter Calculated* Standard value Retention factor 5.71-27.48 K’>2 Plate number 17835-6542 N>2000 Resolution 8.2-16.5 R>2 ‘Asymmetry 25-286 07-18 *Values are mean of three injections 4.1.2.2 Validation of GC-ECD Standard solution of organochlorine and pyrethroids pesticides (endosulfan, heptachlor, lindane, p, p-DDT and its metabolites, bifenthrin, decamethrin, cypermethrin, cyhalothrin, (-cyhalothrin, B-cyfluthrin, fenpropathrin, benomyl, iriforine, thiophanate-methyl and chlorfenvinphos) at a concentration of 0,01, 0.05 and 0.1 jgmt! in n-Hexane was analyzed by GC-ECD. The detector response (signal to noise ration) was three times higher than the background noise. Regression analysis was performed and the values obtained were in the range of 0.989 to 0.995. The limit of detection (LOD), linear range, regression coefficient (R?) and relative standard deviation (RSD) of each analyte are given in Table 4.8. 4Results and Discussion ‘Table 4.8 Validation data of GC-ECD for the determination of pesticide residues. Pesticide MDQ, ng Linear range R RSD(%) ngmI! o- Endosulfan 0,005 0.01-0.1 0.992 45 Heptachlor 0.005 0.01-0.1 0.991 3.2 Lindane 0.002 0.01-0.1 0.995 5.1 p, p-DDT 0.005 0.01-0.1 0.994 54 p, p-DDE 0.005 0.01-0.1 0.992 61 p, p-DDD 0.005 0.01-0.1 0.995 65 Cypermetiirin 0.01 0.05-0.1 0.989 62 Bifenthrin 0.05 0.01-0.1 0.994 38 Decamethrin 0.05 0.01-0.1 0.995 5.0 Cyhalothrin 0.01 0.05-0.1 0.991 5.8 8-Cyfluthrin 0.05 0.01-0.1 0.994 3.5 Fenpropathrin 0.01 0.05-0.1 0.989 62 Fenvalerale 02 0.05-0.1 , 0.995, 3.8 Decametirin 001 0.05-0.1 0.989 30 Captan 0.01 0.05-0.1 0.994 45 Benomyl 002 | 005-01 6995 to ‘Triforin 0.02 0.05-0.1 0.995 54 Thiophanate-m 0.05 0.01-0.1 0.989 47 Chlorfenvinphos 0.05 001-01 0.992 61 Calculations were made on the basis of three replicates __- MDQ= Mininnum detectable quantity, Gas chromatograph equipped with electron captures detector showed linear behaviour to each standard compound. The precision level indicated that there is no significant difference in the linear range of individual standard. From the data (Table 4.8), iL is clear that the system is suitable for the determination of pesticide residues in unknown samples. For the identification of organophosphorus and carbamateResults and Discussion pesticides, gas chromatograph equipped with nitrogen phosphorus detector (NPD) was also validated. The analyses were performed on capillary column. Unrelained peak (te) was obtained by injecting 2 11 phosphine gas in P-mode of NPD and standard solution (chlorpyriphos, ethion, carbofuran, carbosulfan, carbaryl, quinalphos, imidacloprid, isoproturon, chlorbcomuron) was prepared in n-hexane and analyzed, The chromatogram is shown in Fig. 4.3. The minimum detectable quantity (MDQ), coefficient of regression (R’), linear range, and relative standard deviation of each compound are given in Table 4.9. Detector Response 20 30 TIME ( min ) Fig. 4.3 Chromatogram of standard pesticides in NPD-P mode (SE-54, 30 m x 0.53 mm ID, column $20 °C (3 min) t0 300°C @ 5 °C min.” injector 250 °C, detector 310 °C, flow rate 1,5 mi min, “, injection L ul, splitless. Peaks; 1. dichlorvos, 2. mevinphos, 3. ethoprop, 4. monocrotophos, 5. phorate, 6. dimethoate, 7. diazinon, 8. phosphamidon, 9. parathion-methy!, 10. fenitrothion, 11. malathion, 12. chlorpyriphos, 13. fenthion, 14. chiorfenvinphos 6Results and Discussion Table 4.9 Validation data of GC-NPD for the determination of pesticide residues. Pesticide MDQ, pg | Linear range Rr [| RsD@® | ng/ml Chlorpyriphos 0.10 0.01-0.1 0.992 03 Methamidophos | 0.05 0,05-0.1 0.987 34 Parathion-nt 0.10 0.01-0.1 0.996 28 Dimethoate 0.10 0.05-0,1 0.998 4.6 Moncerotophos | 0.10 0,05-0,1 0.994 5.2 Dichloryos 0.10 0.01-0.1 0.996 53 Profenophos 0.06 0.05-0.1 0.994 64 Ethion 0.10 0.05-0.1 0.992 72 Carbofuran a 0.05-0.1 0.997 69 Carbosulfan 0.06 0.05-0.1 0.992 45 Carbaryl 0.06 0.05-0.1 0,998 42 Quinalphos: 0.05 0.05-0.1 0.988 38 Imidacloprid 0.05 0.01-0. 0.983 35 Isoproturon 0.05 0.01-0.1 0.989 54 Chlorbromuron 0,06 0,01-0,1 0.992 44 Maiathion 0.10 0.01-0.1 0.996 33 Calculations were made on the basis of three replicates:Results and Discussion Data presented in Tables 4.8 and 4.9 show that the columns and parameters used for standards in both ECD and NPD are suitable and efficient for the analysis of pesticide residues in unknown sample with adequate sensitivity to determine pesticides at concentrations lower than the MRL. 4.1.3 High Performance Liquid Chromatography (HPLC) ‘A major application of high performance liquid chromatography (HPLC) has been in the separation of heat labile or non-volatile compounds, often following conversion to UV absorbing or fluorescing derivatives for the enhancement of detect ability (Barcelo e¢ al., 1993). A further advantage of HPLC may He in the needless extensive clean up than required with gas chromatography (GC) or thin layer chromatographic methods (Ambrus et al, 1981; ljaz, 1987). Reverse-phase (RP) chromatography has shown excellent promise for resolution of compounds of low and medium polarity (Bogus et al., 1990). HPLC was validated and used for the determination of residues present in fruits, vegclables and water samples using reverse-phase system with UV detector. 4.1.3.1 HPLC-Standardization HPLC system was standardized to check the performance of column and detector, suitability of mobile phase on resolution and linearity and ruggedness of the instrument, To evaluate the above paramcters, pesticide standard solutions were nin. Data showed that the instrument, column, detector and conditions were suitable for the analyses of fruits, vegetables and waters sample. Methanol/water and acetonitrile/water were used as mobile phase for validation of the instrument as well as for the analysis of pesticide residues in fruit, vegetable and water samples. The response was reproducible and voextracts in the sample showed less interfering peaks under UV detection. Chromatograms of standards are given in Fig, 4.4 - 4.7.Results and Discussion Detector Response 6 8 10 12 4 6 18 TIME ( min ) 2 4 fo] Fig. 4.4 Chromatogram of standard pesticides analyzed by Reverse-phase HPLC; UV 214nm, Mobile phase acetonitrile/water, flow rate 1.5 ml min."', column temperature 30 °C (PeaksI-I!; 1. oxamyl, 2. methomyl, 3, aldicarb, 4. simazine, 5. cyanazine, 6. carbofuran, 7, atrazine, 8. carbaryl, 9. diuron, 10. linuron, 11. chlorpropham) 79Results and Discussion TIME ( min) Fig. 4.5 Chromatogram of pyrethroids analyzed by RP-HPLC in isocratic programme; UV 206 nm, mobile phase acetonitrile: water (75:25), flow rate 1 ml min’, column temperature 30 °C (Peaks1-6; 1. phenpropathrin, 2. cyfluthrin, 3. A- Cyhalothrin, 4. a-Cyhalothrin, 5. deltamethrin, 6. fenvalerate) 80Results and Discussion Fig. 4.6 Chromatogram of standard mixture of 0, p-DDT and it metabolites, UV 240 nm, flow rate 0,3 ml min', mobile phase 100% HPLC grade methanol min Fig. 4.7 Chromatogram of standard mixture of p, p-DDT and it metabolites, UV 240 nm, flow rate 0.5 ml inin, mobile phase 100% HPLC grade methanol 8hResults and Discussion Conchiding remarks Today many sophisticated and sensitive techniques are used for the analysis of pesticide residues in different matrices, Among these techniques, gas chromatography is the real choice due to its sensitivity, wide range of applications, quickness, cost-effective and most widely used for pesticide residues. This technique has the ability to determine a significant number of pesticides and their metabolites in a variety of food matrices and environmental samples, High performance liquid chromatography (HPLC) also proved an efficient technique for the analysis of labile and non-labile compounds. ‘The analyses showed good repeatability (RSD) < 5 and linearity R? > 0.928, From the data obtained, it is clear that HPLC parameters used were good and showed high sensitivity for organophosphate, carbamate, pyrethroid and organochlorine pesticides. nth Me 82Results and Discussion 4.2 Pesticide residue analyses in water by gas chromatography Pesticide losses from area of application can contaminate the surface and ground waters by leaching or run-off, which could be a threat to farmer as well as to the environment (Rao et al., 2002). Groundwater pollution by agricultural chemicals has become growing concem in the world because 40-50% of domestic drinking water is pumped from ground water resources in USA (Hallberg, 1989). Solid- phase extraction was used for the extraction and analyses were performed using gas chromatography for the determination of pesticide residues in water samples. ‘The collected water samples of different aquifers were extracted and concentrated using ENVI-Carb Cig Supelco, USA (Section 4.1.1). Results Pesticide residues in water (ground and surface water) were determined by gas chromatographic methods after cleaned-up in order to assess the contamination level in heavily sprayed crop areas (cotton, rice and vegetables). 4.2.1 Pesticide residues in water samples collected from cotton-belts Samples of surface and ground waters were collected and analyzed by gas chromatographs equipped with electron capture and nitrogen phosphorus detectors. Standard mixture containing insecticides (organochlorines, organophosphates, carbamates and pyrethroids) was run before the actual samples. The chromatograms are shown in Big. 4.8-4.9. 83Results and Discussion Detector Response TIME ( min ) Fig. 4.8 Chromatogram of standard chlorinated pesticides by GC-ECD; 1, o-BHC, 2. B-BHC, 3. lindane, 4. heptachlor, 5. aldrin, 6. heptachlor epoxide, 7. o-Endosulfan, 8. dieldrin, 9. p, p"-DDE, 10. endrin, 11. B- endosulfan, 12. p, p"-DDD, (3. Endosutfan sulphate, 14, p, p’-DDT 84Detector response Results and Discussion Solvent peak Time (min) Fig. 4.9 Chromatogram of standard pesticides (mixture of OP's, OC's, carbamates and pyrethroids analyzed by GC-ECD); I. carbofuran, 2. dichlorvos, 3. phosphamidon, 4. fenvalerate, 5. dimethoate, 6. lindane, 7. diazinon, 8. parathion-methyl, 9. fenitrothion, 10. malathion, 11. a-Endosulfan, 12. azinphos- methyl, 13, a-Cypennethrin, 14. deltamethrin A total of 75 samples of surface and ground water were analyzed; all samples of water were contaminated with pesticides, The concentration of individual pesticide residue in water samples is presented in Table 4.10-4.13. RSResults and Discussion Table 4.10. Pesticide residues (tig) in canal, handpump and tubewell waters of districts; Multan, Vehari, Lodhran, Bahawalpur, Sadiqabad, Bakawalnagar, Jhang, Hasilpur, Khanewal and Rajanpur. [ Pesticides Canal water | Handpump water | Tubewell Well water o-Endosulfan | 0.16-0.130 ) 8.130-0.090 5.030-0050 | 0150-0150 Heptachlor 0.070-0.180 ] 0.090-0.160 0.040-0.050 | 0.060-0.160 Lindane Traces 0.080-0.220 0.050-0.070 | 0.070-0.090 Pp, p-DDT 0.005-0.010 | 0.006-0.012 - 0.008-0.009 P, P-DDE 0.002-0.007 | 0.001-0.005 Traces 0:008-0,006 | Methamidophos | 0.013-0.025 | 0.011-0.016 0.009-0.012 | 0.100-0.150 Monocrotophos | 0,090-0.140 | 0.050-0.170 0.004-0.090 | 0.080-0.130 Profenophos | 0.010-0.017 | 0.009-0.080 0.004-0.008 | 0.020-0.050 Chiorpyriphos | 0,110-0.170 | 0.080-0.120 0.001-0.009 | 0.120-0.160 Carbosulfan 0.070-0.110 | 0.100-0.280 0.009-0.050 | 0.160-0.290 Dichlorvos 0,100-0.120 [0.110-0.150 0,020-0.060|0.120-0.320 Dimethoate 0.005-0.009 | 0.003-0.090 0.002-0.004 | 0.050-0.090 Ethion 0.004-0.015 | 0.001-0.008 = 0.006-0.009 Diazinon 0.010-0.013 | 0.005-0.020 ~ 0.010-0.016 Deltamethrin | 0.008-0.012 | 0.003-6.009 - 0.005-0.018 Parathion-m —_| 0.002-0.025 | 0.004-0.007 Traces 0.007-0.012 Propergite 0.007-0.008 0.003-0.009 Traces, 0,004-0,013 Malathion 0.005-0.007 } 0.004-0.006 - 9.009-0.018 Quinalphos 0.015-0.025 | 0,009-0.018 0,002-0.007 | 0.012-0.035 Fenitrothion | 0.015-0.035 | 0.020-0.040 0.009-0.018 | 0.020-0.040 Imidacloprid | 0.009-0.020 [0.016-0.035 0,004-0.005 | 0.014-0.025 Acctamiprid | 0.016-0.024 | 0.025-0.032 0.005-0.017 | 0.018-0.031 Cypemethrin | 0.009-0.015 | 0.011-0.016 Traces 0.010-0.017 Fenvalerate 0.010-0.018 | 0.025-0.045 0.011-0.019 | 0.017-0.052 Cyhatothrin 0.008-0.012 | 0.012-0.018 0.003-0.006 | 0.014-0.024 [ Cyfluthrin 0.003-0.009 | 0,004-0.007 Traces 0.005-0.012 Bifenthrin 0.010-0.016 |0.012-0.018 ‘Traces 0.012-0.017 Data is in ranges 86Results and Discussion Table 4.11 Pesticide residues (\igL"!) in surface and ground waters in the province of Sindh (Nawabshah, Sanghar, Larkana, Sukkur, Jacobabad) Pesticides Canal water | Handpamp ‘Tubewell ‘Well water water, water a-Endosulfan | 0.120-0.150 | 0.060-0.100 | 0.020-0.040 | 0.130-0.160 Heptachlor 0.006-0.016 | 0.020-0.130 0.005-0.008 0.020-0.025 Lindane 0,002-0.009 | 0.005-0.012 0.004-0.006 0.006-0.008 p, p-DDT 0.008-0.012 | 0.007-0.014 0.004-0.007 0.010-0.012 p, p-DDE Traces 0.005-0.007 - 0.004-0.009 Methamidophos | 0.021-0.035 | 0.012-0.021 | 0.007-0.012 | 0.012-0.032 Monocrotophos | 0.012-0.018 | 0.020-0.180 | 0.009-0.011 | 0.010-0.013 Malathion: 0.007-0.012 | 0.005-0.009 Traces 0.009-0.015 Parathion-m 0.012-0.025 | 0.008-0.014 0.002-0.011 0.011-0.035 Chlorpyriphos 0.090-0.140 | 0.050-0.080 0.02-0.028 0.110-0,250 Carbofuran 0.120-0.250 | 0.090-0.150 | 0.008-0.012 | 0.110-0.180 Profenophos. 0.050-0.085 | 0.020-0.048 0,010-0.021 0,090-0,120 Carbosulfan 0.060-0.120 | 0.012-0.064 | 0.009-0.015 | 0.050-0.110_| Dichlorvos 0.120-0.240 | 0.060-0.180 0.009-0.080 0.100-0,160 Dimethoate 0.090-0.150 | 0.050-0.120 0.010-0.120 0.150-0.180 Diazinon 0.015-0.065 | 0.009-0.018 0.006-0.053 0.017-0.085 Quinalphos 0.012-0,025 | 0.007-0.016 0.008-0.012 0.120-0.240 Acetamiprid 0.012-0.120 [0.011-0.018 | 0.009-0.012 | 0.014-0.150 Imidacloprid 0.110-0,240 | 0,090-0,120 0.064-0.075 0.150-0.350 Fenitrothion: 0.120-0,250 | 0.060-0.085 0,009-0.018 0.180-0.320 Propergite 0.012-0.065 | 0,009-0.012 Traces 0.015-0.076 Ethion 0.110-0.240 | 0.090-0.150 0.040-0.050 0.120-0.340 Cypenmethrin 0.020-0.045 | 0.010-0.030 0.008-0.012 0.030-0.065 Fenvalerate 0,040-0.120 | 0.050-0.080 - 0.120-0.240 Cybalothrin 0.140-0.250 | 0.050-0.060 0.020-0.030 0.210-0.340 Bifenthrin 0.080-0.150 | 0.020-0.050 0.008-0.015 0.150-0.250 Cyfluthrin 0.090-0.130 | 0.020-0.080 0.010-0.030 0.120-0.360 87Results and Discussion Table 4.12. Pesticide residues (igh) in rice-belt (Gujranwala, Sialkot, Sheikbupura and Faisalabad) Paice Gujranwala Sakae Shelihapare Fatainbad Canal Mandpanp Tovett_ | Canat_ Handpu Canal Handpucap Tovel Hangpump well Caiboturan UBD [OW [oa piso [aI D360 [ae [waa POIROT O0eF Cap Tis | wre | woo | oreo sae fore fore fons [oso Chlorprripies [TRO | 0oR | Taf OAT | oxo oa Por | os foie Spore pons Tepracniae [005] OO | Tras | OT food foam Tore Pore | ome Oreo | THD | Tacs windoiation [OO] Cf = [oH [wow aad] oa 90s [oo | ORF aoTa BecDwT Dow TROT [Tacs | WHT] THD Pawo | oI | ToaT Poa | Oo | aver Citortewinphos | O01 | On| Boos [oso | Tare [omer foam oa eae foam | oor | noe Chiorpyriphos | 0035 | G2 food | oo foo [oom | 00 jos |— 0042 7 0.020 | a.007 Fen oor [oo |= | oop oar oo] ome [avis |= Panis Pore Metanidopos [02D UIT | oa [Os POI Poi | oasis fos | acu [oa | O10 | O00 Movecrorphos | 0160 [OID Foew ORO [0130 | ome aaa OTs oar | Ona | O1ee Toons Trinophos ORDO | ooaT | OO TTT Tease faa boxe ore | oa) | On TOOT] Carbowlfan 0.080 [0040 [ooo [0090 [0070 | Traces [0070 }0020 | — 0090 [00% [=] Aijhametiein | OFT [TOW [oom ors fom] ooo Po Powe f=] Disa [ooo = ‘Cyhalothrin 0.050 [0020 [0010 [0070 0030 | Traces [O08 ooI [O007 [0050 [0010 | Traces Decamemiia | TOI foo =| oow [wow awe | Toe | oo] -——f oos0 | aaa] — Gamat dos [woe poms [oui fous Poo | ous | ome | Boar om oH] = Quisalphos | TOT | oo foe Pou Ton Pome Fade aaa = oe oat | aaa ‘Chlorodhalonit ole = os Joo [oor | oo [oor = = = = Buiaehior CS a a a aResults and Discussion | Benomy! 0.018 | 0.009 | -- 0.022 | 0.012 | 0,009 | 0.026 | 0.015 | 0.008 p,p'-DDT 0.012 | 0.007 | -- BOLL 10.009 | -- 0.013 | 0.008 | -- Isoproturon 0.035 | 0.018 | 0.009 | 0.025 | 0.011 | 0.006) 0.038 | 0.021 | 0.007 Methamidophos [0.250 | 0.180 | 0.09 | 0.320 | 0.210 | 0.085 | 0.450 | 0.230 | 0,065 Triforin 0.012 } 9.008 | ~ 0.018 [ 0.009 | 0.002 | 0.016 | 0.010 | 0.006 Dichlorvos 0.064 [0.021 F0.011! 0.076 | 0.027 | 0.016 | 0.082 | 0.029 | 0.014 Monocrotophos ] 0.210 | 0.110 | 0.050 | 0.260 | 0.120 | 0.040 | 0.320 } 0.160 | 0.070 Phosphamidon 0.160 | 0.064 | 0.012 | 0.180 | 0.076 | 0.012 | 0.250 | 0.140 | 0,086 Bifenthrin 0.032 | 0.022 | 0.007 | 0.044 | 0.016 | 0,008 | 0.058 | 0.025 | 0.009 ‘Cyhalothrin. 0.040 | 0.025 | 0.010 | 0.056 | 0.024 } 0.011 | 0.064 | 0.027] 0.015 Cypermethrin 0.015 | 0.009 | - 0.012 | 0,007 | - 0.017 | 0.012 | 0.007 Carbendazim 0.045 } 0.005 | - 0.026 | - - 0.038 | 0.009 | - Chlorothaloni! 0.022 | 0.010 | - 0.018 | - - 0.028 | 0.012} - Data is mean of triplicate analysis HP; handpump, TW; tubewell Data presented in Table 4.10 ~ 4.13 indicate that higher concentrations of residues were detected in the water samples of Punjab and Sindh provinces belonging to cotton-belts. Maximum residues of o-Endosulfan, heptachlor, chlospyriphos, carbofuran, monocrotophos, carbosulfan and dichlorvos were found in canal and well waters as compared to handpump (HP) and tubewell (TW). Lindane was not detected in canal water but found in other ground water samples, Higher concentration of this compound was found in hand pump as compared to tubewell and well waters. Significantly higher amount of methamidophos (250 ngf."') was 89Results and Discussion present in well waters collected from District Bahawalpur. Maximum concentration of residues was observed in well water following canal, handpump and tubewell waters in the province of Punjab. Some pesticides like heptachlor, p, p-DDT, monocrotophos, chlorpyriphos, ethion and parathion-methyl were in lower concentration in well water as compared to canal water (Fig. 4.10). 02 Canal water mWell water ous ou vos Mepachlor ——pp-DDT—MenccrotwpiosCMlorpyriphos——Ehion—_~Parathion-methyl Fig, 4.10 Pesticide concentration in canal and well water samples in the Punjab Most of the samples containing pesticide residues showed higher concentration than the recommended MRL. No fungicide residue was detected in water sample. The reason of fungicide absence was due to non-utility of these fungicides reported in the area of cotton. A total of 27 pesticides belonging to four category were detected, out of which seven (a-Results and Discussion endosulfan, heptachlor, monocrotophos, profenophos, chlorpyriphos, carbosulfan and dichlorvos) had a very high concentration in water which were considered a main threat to animals and peoples belonging to this area (Yable 4.10). Water samples collected from cotton area of Sindh were also analyzed and the data is presented in Table 4.11, Maximum samples of this area were found to be contaminated with pesticides, Six to ten pesticides applications are given to cotton crop every year to protect it from virus, insects and weeds. A significant concentration of a-Endosulfan, chlorpyriphos, carbofuran, carbosulfan, dichlorvos, dimethoate, acetamiprid, imidacloprid, fenitrothion, cyhalothrin, bifenthrin and cyfluthrin were detected in canal, handpump, tubewell and weil waters, Concentrations of monocrotophos are higher in handpump than other water samples. Some synthetic pesticides like propergite, avetamiprid and imidacloprid have higher residues in water samples collected from Sindh than Punjab. It is clear from the data (Table 4.11) that samples of Sindh cotton-belt with positive detection of pesticides have more frequeney of pyrethroid residues as compared to the samples of Punjab cotton-belt. Some pesticides like heptachlor, p, p-DDT, p, p-DDE, methamidophos, dimethoate, dichlorvos and imidacloprid were found in high frequency in surface and ground waters of Sindh reservoirs as compared to Punjab. Rice fields are irrigated by canal and tubewell waters and the residents of this area drink handpump and well waters and use this water for other purposes like washing, cooking and bathing, Water samples taken from rice-belt were analyzed with gas chromatographic methods and the data is given in Table 4,12, From the data it is evident that only two chlorinated pesticides were detected in the surface and groundwater samples. Higher concentration of “heptachlor” was found in Sialkot and Sheikhupura districts as compared to Gujranwala and Faisalabad territories. Frequency of “carbofuran” and “cartap” residues was lower in Faisalabad but “chlorpyriphos”, “diazinon”, “triazophos” and “carbaryl” were higher as compared to other areas. There is a significant difference between the frequencies of residues in rice-belt samples (Fig. 4.10). The concentration of pyrethroids found in rice-belt a1Results and Discussion is in the range of O.0lygl to 0.28 ygL, “Alphamethrin” was found in higher concentrations in Sialkot area while all others pesticides belonging to this group (pyrethroid) have nearly same residues in surface and groundwaters, “Chlorothalonil”, the only fungicide, was detected in canal water (Gujranwala), in surface and ground water of Sialkot and also in canal and handpump waters of Sheikhupura, “Butachlor” was the only herbicide detected in rice-belt samples and this compound had dominant residue in Sheikhupura areas. 04: MGujranwala OSialkot Sheikhupura DFaisalabad Carboturan Cartap Chiorpyriphos —Melamidophos —‘Triazophos Fig. 4.11 Pesticide residues in canal water of rice area Water samples from vegetable growing areas were analyzed (Table 4.13), “Alpha- endosulfan”, “lindane” and “p, p-DDT" were found in all samples except tubewell water. “o-Endosulfan” has significant concentration in water samples following “lindane” and “p, p-DDT”. Most water samples were contaminated with fungicides and their concentrations in canal water were not same in all the provinces (Fig. 4.12). The concentration of “isoproturon” was higher in NWFP water samples as compared to Punjab and Sindh. “Methamidophos”, “monocrotophos” and “phosphamidon” residues were found higher inResults and Discussion canal water in Punjab, Sindh and NWFP (Fig, 4.13) but these compounds have lower residues in tubewell waters in these provinces. 0.05 0.045 Punjab 0.04 OSindn maNWrP > ° 8 & 0.03 0.025 0.02 Concentration (ugh) Benomy Tritoun Carbendazim Chiorothatonit Fig. 4.12 Fungicide residues in canal water of vegetable grown areas orResults and Discussion © Methamidophos CMonocrotophos WPhosphamidon ‘Concentration (ugh) e fe 8 e Fig, 4.13 Organophosphorus pesticide residues in canal waters in vegetable areas of three provinces Pyrethroids are used to protect vegetables from infestation like jassid and aphid, The residues of “cypermethrin”, “bifenthrin” and “cyhalothrin” were found in the present study. “Cyhalothrin” and “bifenthrin” had dominant residues in surface and ground water of the earlier mentioned locations (Punjab, Sindh and NWFP) whereas lower concentration of “cypermethrin” was found in canal and handpump of all provinces. A comparison of the pyrethroid residues in surface and ground waters is presented in Fig.4.14. From the data, it is evident that lower concentration of “lindane” (0.004 jig) was observed in tubewell waters in Punjab and Sindh while maximum residues of "methamidophos” was detected in canal and handpump waters near the treated fields or at the farm locations.Results and Discussion WBifenthrin Cyhatothrin WCypermethrin Concentration (ugh) Punjab Fig, 4.14 Pyrethroid residues (ygl') in surface and ground waters in vegetable grown areas of three provinces Discussion Pesticide pollution of water may be due to the routine use of pesticides in agriculture but also accidental spillage and non-agricultural uses. Pesticides reach water bodies through leaching, run-off or spray drift. Pesticide pollution in water bodies has become a major concem in the world as well as in Pakistan in recent years. In the present study, data showed that surface and ground waters were contaminated with organochlorines, organophosphates, pyrethroids and fungicides. Some compounds like p, p-DDT, p, p-DDE, dimethoate, parathion-methyl, imidacloprid, acetamiprid and pyrethroids were detected below the permissible limits (0.1 pel) in the water samples of canal, handpump, well and tubewell at all sites of Punjab but as a whole the residues were above the tolerance level (0.5-0.1 igh"), Jimenez er al. (1997) 08Results and Discussion reported 18 pesticides in smali water loughs used to irrigate surrounding crops. They determined the residues in water samples by using gas chromatography with electron capture and nitrogen phosphorus detectors and also confirmed with high performance. liquid chromatography with diode-array detection. Most of the compounds detected in water samples were above the maximum residue limits. “Cypermethrin”, “dimethoate”, “isoproturon”, “captan”, “chlortoluron” and “carbaryl” were the compounds detected in higher frequencies. Some organophosphates sprayed in Sindh colton-area had greater residues (Table 4,11) than the water samples of colton-area in Punjab (Table 4.10). Residues of methamidophos in Punjab aquifer (0.009-0.025 pL”) were lower than that of Sindh (0.007-0.035 pgL"'), Some other authors reported that intensive spray of insecticides in cotton-area might contribute water quality problem due to run-off or leaching and pose health risk to the ultimate users. (Crutchfield ef al., 1992 and Tian et al., 1994), Pesticide may reach surface water bodies in either dissolved or particulate form, which may impair the water quality and jeopardize the aquatic species, Many pesticides were found in canal and well waters located in the cultivated area of cotton, rice and vegetable which is a continuous threat to animals and human beings who have been drinking this polluted water. “o-Fndosulfan", “heptachlor”, “lindane” and metabolite of DDT were detected in most aquifers from farming areas, “Atrazine” and “2, 4-D" herbicides have been reported individually at concentration upto 2 jigL’' in potable and public water supplies in arable areas (Croll, 1985 and Foster, 1976). Water resources are highly susceptible to contamination by agronomic practices because the contamination of yround water resources originates us excess rainfall and excess irrigation infiltration to this land, Rice-belt in Pakistan is over flooded all the time and average rainfall in this area (200-450 mm) is very high as compared to cotton-belt. The data present (Table 4.13) show a very high concentration of residues in numerous aquifers. Significant concentrations of “carbofuran”, “cartap”, “chlorpyriphos”, “‘methamidophos", “monocrotophos” and “quinalphos” were detected in the water samples after clean up and concentrate by solid-phase extraction. 96Results and Discussion Field monitoring of surface and ground water showed that the chlorinated pesticides like “lindane”, “p, p'-DDT”, “p, p-DDE” had lower residues level than the recommended ones (0.1 pgL or 0.5 pgL"'), The range of these pesticides was |-12 ngL" in Punjab aquifers and in Sindh cotton areas. Bailey and White (1970) reported that there are a number 1-16 ngi of compounds specific factors that influence the environmental behavior of pesticides in water and soil. The factors which are responsible for the leaching of pesticide from the treated fields are chemical character (molecular shape and configuration), acidity or alkalinity, water solubility, charge distribution on the organic cations, polarity and molecular size. Soil organic matter also has influence on the mobility of chemicals through ion- exchange, hydrogen bonding, Vander Waals forces, acid coordination, attached metal ion and ligand exchange (Stevenson, 1985). Krapac and others (1995) found DDT residues in soil in Illinois, USA, Mean DDT residues were 40 pkg and maximum concentration of DDT was 1200 jigkg"" after five years application. Solubility of lindane and DDT is 10 mg L” and 5.5 ngl" respectively (WHO, 1989; Chiou ef a/., 1986). Organic matter can change the solubility of DDT in water and also the adsorption and desorption of DDT in soil, which can minimize the leaching of DDT and its metabolite from treated fields. Another study showed that pH, calcium concentration, zinc contents and concentration of organic matter influence the binding of DDT to humic materials (Carter and Suflet, 1982), Haarstad (2002) mentioned that the concentration of DDT in river water was 0-70 gL"! and in ground water 0-40 pgL™ and also found that pesticide partition happened due to organic carbon and binding of non- polar compounds increased with high content of humic substances that can increase the risk for the mobilization of organochlorine compounds. There had been wide sprayed distribution of DDT and its metabolite as residues in milk, water and adipose tissue due to their lipophilic nature, Chlorinated pesticides have been found by Parveen and Masud (1988) in sample of cattle drinking water from Karachi Cattle Colony. Residues of DDT and its degradation products in human milk and adipose tissue samples in Faisalabad have also been reported by Hussain vr al. (1993). From the results of the previous and the present studies, it is evident that these persistent organi¢ pollutants still have a big threat to our wildlife as well as to buman beings in Pakistan. 97Results and Discussion Many pesticides like ‘carbofuran”, “cartap”, “chlorpyriphos”, “o-Endosulfan’, “diazinon”, “methamidophos”, “alphamethrin” and “decamethrin” were detected in reasonable concentrations in the water reservoirs located in rice-belt. From the results it is clear that all aquifers located in rice-belt areas were contaminated with the extensive use of pesticides on this crop by leaching or run-off processes. The high residue levels may be due to water solubility or partition coefficient (Koc) of the applied pesticides in this area. “Carbofuran” and “imidacloprid” have very high water solubility (320-610 mgL"'). . Many authors have detected different pesticides in aqueous samples, including ultrapure water, environmental waters (surface and ground waters) and drinking water using solid phase microextraction (SPME) around the European limit of 100 ngL" for individual pesticides (Moder er al., 1999; Boyd-Boland and Pawliszyn, 1995). Aquatic resources (ponds, lakes, rivers and streams) are valuable natural assets, which are being enjoyed by thousand peoples, birds and animals. The U.S. Environmental Protection Agency (U.S. EPA) estimated that pollution affects about two percent of ground water in U.S. and an increasing amount of surface water is at risk [rom contamination. Petroff (2002) mentioned that persistence, volatility, soil adsorption and water solubility are the main factors responsible for the contamination of aquifers. Some pesticides like carbaryt and methamidophos has high Koc and tends to mobile in the soil bed whereas diazinon, malathion and parathion-methy! have low Koc values that’s why they have relatively high leaching potential in the treated areas. Water solubility and adsorption to soil particles are inversely related for most compounds. if Koc value is low (<300-500), the water solubility is high. Pesticides with solubility greater than 30 mgkg'' and Koc: values less than {00 are considered a concem in sandy soil. Likewise pesticides with high Koc values are more likely to run-off with soil than leached independently. ‘Those pesticides, which have high volatility, can pollute the water resources by leaching or run-off processes. Soil erosion by wind and water can pollute the surface and ground waters. The pollution of surface and ground water may be due to point or non-point sources. Point sources are small while non-point sources have wide range and are not easy to locate, Pesticides were found to have the potential to accumulate in ORResults and Discussion sediment and aquatic biota if they had a octanol-water partition coefficient (Kow) greater than 1000 and a soil half-life greater than 30 days (Mayewski and Capel, 1995). Pesticide residues were also monitored in natural water aquifers from vegetable grown areas, which are very much close to urban territory/localities, Peoples or farm owners also used sewage water side by side with ground and canal waters to irrigate their fields containing lot of burden of microorganic pollutants. ‘The data of pesticide residues in canal, handpump and tubewell waters taken from Punjab, Sindh and NWFP is presented in Table 4,10 ~ 4.13. Significantly high concentrations of “a-Endosulfan”, “lindane”, ‘p, p-DDT", ‘methamidophos”, “monocrotophos”, “phosphamidon” and “dichlorvos” were found in canal water collected from three provinces and lower concentration of the above mentioned compounds alongwith many others was in tubewell water. The contamination of canal water may be due to wind or water, which erode soil that contain pesticide residues. Even comparatively insoluble pesticides and pesticides with high adsorption properties can move with eroding soil, Washing of tractor and spray machines near the canal may also be responsible for the pesticide residues in surface waters. Fifteen pesticide compounds were detected from water samples collected from all vegetable grown areas. The most frequently detected compounds in water samples belonging to vegetable grown area were “a- Endosulfan’, “lindane”, “methamidophos”, “monocrotophos” and “cypermethrin”. Detection frequencies were highest in the water samples at vegetable growing areas. ‘Three pesticides (a-endosulfin, p, p-DDT and lindane) were detected most frequently at urban sites. Carabias-Martinez ef al, (2000) also detected triazine herbicides in natural water by solid- phase extraction and non-aqueous capillary zone electrophoresis. The detection limits of micropollutants in natural water varied between 0.0] and 0.05 gL"! depending on the type of matrix analyzed. The continuous use of pesticides in agriculture has great potential to reach groundwater by leaching, nioff or percolation. “Carbofuran”, “cartap”, “carbaryl” and “carbosulfan” were also found in appreciable concentration in water samples (canal and hand pump) and this may he attributed to potential leaching and high water solubility of carbamate insecticides (Gustafson, 1998). Concentration of “isoproturon” has been found to be 5.5% in 99Results and Discussion water by Hartmann and others (2000). Rao and Homsby (2002) also reported the behaviour of pesticides in soil and water. From the study, they concluded that those pesticides with greater half-life (Ty) and Koc values increased the persistence in sub-soils and ground water because they remain on the application site for long time. Our results are in line with the findings of Potter er al. (2000). They determined many cotton defoliant, herbicide and insecticide residues in water by solid-phase extraction and GC-NPD, GC-MS and HPLC- diode array detections. Concluding Remarks About 97% of the world's fresh water is groundwater, while streams, rivers and lakes hold only about 3%, Ground water provides a critical source of water for agricultural ierigation and industries. Surface and ground waters in cotton-belt, rive-belt and vegetable areas were found (o be contaminated with insecticides, herbicides and fungicides. Persistent pesticides like a-Endosulfan, heptachlor, lindane, DDT and its main metabolite DDE were found in cotton-belt aquifers. Alpha endosulfan concentration was higher in well water as compared to other aquifers, Carbosulfan concentration was higher in canal water whereas least was found in tubewell waters, Dichlorves, acetamiprid, imidacloprid, fenitrothion and ethion were significantly in high concentrations in canal and well waters. ‘The concentration of pyrethroids e.g, fenvalerate, cyhalothrin deltamethrin and cytluthrin were found in higher concentrations than the tolerance limits in fresh and ground waters, No fungicide residues were detected in cotton-belt water samples. Water samples in rice-belt were contaminated with chlorinated, organophosphate, carbamate and pyrethroid pesticides. Higher concentrations of pesticides were found in Sialkot and Sheikhupura aquifers. Organophosphate residues were higher in rice-belt water samples and residues of fungicide were not detected in vegetable grown areas, Endosulfan (a-) residue was higher in canal water and lower residue of p, p/DDT was found in surface and ground waters. highest concentration in Methamidophos, monocrotophos and phosphamidon were found i canal and handpump water samples. Cyhalothrin was the only pyrethroid that had higher 100,Results and Discussion concentrations in surface and ground waters. The natural water resources in Pakistan found to be contaminated with synthetic pesticides and had high concentration than the recommended EU maximum limits of 0.1 web’! for any individuat pesticides and 0.5 pel” for the total concentration of all pesticides. . 101Results and Discussion 4.2.2 Pesticide residues in water samples analyzed by liquid chromatography Water samples of cotton belt and vegetable areas were analyzed by high performance liquid chromatographic methods using fix wavelength after solid-phase extraction and results are presented in Table 4.14. High residues of carbofuran, chlortohuron, isoproturon, cypermethrin and cyhalothrin were detected in canal water samples of cotton and vegetable areas (Fig. 4.15) whereas handpump water samples of both cotton and vegetable areas have lower residues of p,f-DDT, carbendazim and imidacloprid. Tabled.14 Pesticide residue (sgl!) in water samples collected from cotton-belt and vegetable area Cotton-belt Vegetable area Pesticide Canal water | Handpump | Canal water | Handpump Carbofuran 0.150 (3) O180(7) | 0.210 (5) 0.790 (a) Shydroxy carbofuran | 0.014 (7) 0.01298) | 0.0167) 0.009 (3) Chlortoluron 0.250 (3) 0.1806) | 0.1909) 0.012 30 Tsoproturon 0.280 (6) 0.150(7) | 0.210(5) 0.025 (8) Cypermethrin 0.350 (4) 0.048(9) | 0.320 (4) 0.160 (9) Cyhalothrin 9.160 (5) 0.052(7) | 0.270 (3) 0.012 (10) Imidacloprid 0.190 (8) 0.034 (8) | 0.120 (7) 0.011 (7) p.p-DDT 0.024 (9) 0.009 (10) [0.018 (12) [0.007 9) Carbendazim: 0.075 (5) 0.003 (9) | 0.078 (7) 0.076) Triforia 0.035 (3) 011) 10.085 (5) 0.180 (5) Data ure mean of 5 replicates; values in parenthesis indicate relative standard deviationResults and Discussion A metabolite of carbofuran (3-hydroxy carbofuran) was also found in canal and handpump water samples. High residue of this metabolite was detected in canal water as compared to aandpump samples but less than the maximum residue limits of BU (0.1 pigl") established oy European Economic Community (Fielding ef af. 1992). Residue content of carbofuran in water samples belonging to vegetable area was substantially higher than cotton-belt, however zotton-belt area water samples have more residues of chlortoluron (0.250 gL) than the avaler samples of vegetable areas (0.190 gL”). Imidacloprid residues were more in canal water samples of cotton belt and vegetable areas as Compared to hand pump samples, Carbendazim, DDT and triforin residues found in canal and hand pump water samples were below the EU maximum permissible levels (O.1pgb"). O48 coton ve Cana! Vegetable tet! Carat 03 {nlerogear/L) 2 ot Fig. 4.15 Pesticide residue concentration in canal waters collected. from cotton and vegetable areaResults and Discussion Concluding remarks High performance liquid chromatography (HPLC) proved an efficient technique for the analysis of labile and non-labile compounds. ‘The analyses showed good repeatability (RSD) <5 and linearity R? > 0.928. From the data obtained, it is clear that HPLC parameters used were good and showed high sensitivity for organophosphate, carbamate, pyrethroid and organochlorine pesticides. Successive and concentrated treatments to cotton and rice belt areas may contribute the residues in aquifers. High residues of pesticide (carbofuran, chlortoluron, isoproturon, cypermethrin, cyhalothrin) were found in canal waters belonging to cotton and vegetable growing areas. The residues were certainly higher than individual as well as total pesticide residues recommended by European Union Commission.Results and Discussion 43 Pesticide residue in fruits and vegetables Agriculture sector contributes 25% of total GDP in Pakistan. Pakistan produces about 1.8 million tones of a large variety of vegetables and fruits of tropical, subtropical and temperate groups on 150,000 hectares. These are very good source of vitamins, proteins, carbohydrates, fibre and mineral source to sustain and maintain the health of human beings, The residue of pesticides in fruits and vegetables are of great concern to every one. Because of the inherent toxicity and ubiquity, the residues of these chemicals have received a good deal of attention throughout the world. Consequently many countries have introduced legislation for protection of the consumer’s health from hazards of pesticides, Unfortunately no such regulations have been enforced in Pakistan which may arose the public awareness and hence protects the people and livestock. The possible reason seems to be the non- availability of sufficient data regarding the pesticide residues status in agricultural products in the country. Different types of pesticides have been used for the protection of vegetables from different diseases and invaders. No comprehensive studies have taken into account the determination of pesticide residues in vegetables, fruits and water in the country. Pesticide residues can be determined qualitatively and quantitatively by HPTLC, GC and HPLC methods, All the methods were tried to analyze the samples of vegetables, fruits. 4.3.1 Validation of thin layer chromatographic (TLC) methods Validation is a prerequisite of any reliable chromatographic analysis (Levison et al., 1995). Many chromatographic parameters have been proposed for inclusion in the validation process, such as linearity of the calibration curve, sensitivity and selectivity of solute detection, interday and interaday reproducibility, instrument precision, detection limit, quantitation limit, recovery and ruggedness (Gurley et al., 1995 and Lee er al., 1995), Before analyzing the actual samples of fruits, vegetables 105Results and Discussion and water samples, different TLC detection methods (Section 3.4.2.2) were validated and results are presented in Table 4.15. Table 4.15 Ryand MDQ values of standard pesticides by TLC methods Pesticide R MDQ, ng ab e die ff 30 40 a-Endosulfan B-Endosulfan Acephate Atrazine BHC Captan Carbendazim Carbofuran Carbosul fan Chlorfenvinphos Chlorbromuren Chlortoluron ichlofluanid Dichlorvos Dieldrin Dioxacarb Fenarimol Fenitrothion Fenthion Imidacloprid Tsoprotwron Lindane Linuron, Methamidophos Methomyl Metoxuron Parathion-methyl Prochloraz Quinalphos Thiobendazole 25 Thiophanate-methyl | 0.580} 50 Rrvalues are mean of 5 replicates a= O-toludine + KI method; b = Silver nitrate + UV exposition, ¢ = Hill reaction d= Fungi spore inhibition; e ~ Enzyme inhibition with cow liver extract and B-naphthyl acetate; f= Enzyme inhibition with horse blood serum and acetylthiacoline iodide substrate 30 20 0.5 25 20 30 100 C 90 60 | 24 78 50, 25 25 2 10 : 20 4L 2.2 20 196 ©Results and Discussion 4.3.2. Behaviour of marker compounds in TLC methods Different classes of pesticides were used as marker compounds in different detection methods, which gave good response (spot visibility) during the validation of TLC methods. Linear response was observed between quantity of pesticide and average spot diameter (horizontal and vertical diameter) in different TLC detection methods that are shown in Figures 4.16 - 4.21. + mM Atrazine oe & + @- Diuron a Dioxacard Average spot diameter (mm) a0 80 Amount (119) Fig. 4.16 Response of marker compounds in o-toludine + KI method os = a Fs Bos 3 Bes + © Triforin gs ~m- Endosulfan i AS —*—Alphamethrin ° 50 100 130 200 250 Amount (ng) Fig. 4.17 Response of marker compounds in silver nitrate + UV exposition method 107Results and Discussion 6 € @ Atrazine = 884 —iChilortoluron 3 te Metaxuron Es = Bas £ 4 < 35 3 oO 25 5s 1S 10 Amount (og) Fig. 4.18 Response of marker compounds in Hill reaction method a“ @ Captan + Procloraz Average spot diameter (mm) Aumoont (ug) Fig. 4.19 Response of marker compounds in fangi spore inhibition method 108Results and Discussion Average spot diameter (mam) 2 = 2 e Be boo Fo & Amount (ng) Fig. 4.20 Response of pesticides in enzyme inhibition with cow liver extract and B-naphthyl acetate method + Qainaintor 2 ® not detected Table 4.22 Pesticide residues in brinjal by HPTLC methods Pesticide residues in brinjal_ (mg kg") Peaticile __ Sampling sites q 2 3 q 5 6 7 g Ri | MD@ a imidacloprid 008d [0.125 | 0065 | o.1b0 | O07S | G06 | 0.075 | ND. [0.226 [50 Benomyt oa [OUTS [Tas [UIs Ts PND. | 002s fToIs posi [I Chiorpyriphos [0.015 TN.D. | 0025 | 0.035 [0.045 [0.05 (ND. [0016 | 0.609 [05 o-Endosulfan ND. [0.075 [ND [0155 [0200 [0.08 [0145 | ND. [0.670 [50 Methamidophos | 0.025 [0.035 | 0.065 [ND- [0.035 [ND. | 0.025 0.045 [o.t10 [10 Cypermethrin 0125 TND. [6.200 [0.135] 0.145 [0.215 | ND. ]O.1s5 [0.673 | 100 Monocrotophes [0.135 } 0,168 [ND. {0.095 [o120 [N.D. [0130 | ND. [008 [as B-cyhalothein 0.125 [0150 fo2s0 PONS |ND. [ND. [0.135 [0.150 | oss0 [100 Dichlorvos ND. [0.025 [0022 [O03 |ND. [o0s [ND. [011s [0.504 | 10 * Values are mean of 23 samples from cach location and 1-8 are sampling sites (see in materials and methods) N.D- not detected 13Results and Discussion Table 4.23 Pesticide residues in pumpkin by HPTLC methods Pesticide residues in pumpkin (ing kg")* Pesticide ‘Sampling sites i 2 3 4 5 6 T 8 RE MDQ ng Imidacloprid 0.088 {N.D. [0.085 | 0.100 [0095 [006 | ND. [ND. {0.226 | 50 Benomyt ND. [0.025 [ND | 0045 [ND PND. [0.065 | 0.105 7 0.311 | 10 Chlompyriphos [0.015 [0.045 [0.025 | ND. [0.085 [0.06 |N.D. | 0.065 | 0.669 | 0.5 a-Endosulfan 0.100 [0.135 | 6.060 | 0075 [0.250 FO.15 [ND. | 0.075 [0.670 | 50 ‘Cypermethrin 0.175 |ND. [0150 [0145 [0.125 7 ND. [0.120 | ND, | 0.673 | 100 Monocrotophos 0.095 [0.125 [0.100 |N.D. [ND. [O14 [ND [0.155 | 0.08 | 88 B-cyhalotirin ND. [0125 [N.D. fotos [0165 1 ND. [0.145 | 0.150 | 0.550 | 100 Bifenthrin 0.200 [0.160 [0185 | ND. [0210 {0170 | ND. [0.185 [0520 [150 * Values ure mean of 23 samples from each location and 1-8 are sampling sites (sec in materials and methods) N.D.~ not detected Table 4.24 Pesticide residues in green peas by HPTLC methods ‘Green peas (mg kg)* Pesticide — ‘Sampling sites Y 2 7 a 5 6 7 a Rt | MDq L— bh ng Imidacloprid ND. [0.225 [0075 [0.150 | ND. [ND [0.075 [0.100 [0226 | 50 Benomyl 0.018 |ND. | 0015 [0035 [0075 [0.055 [0.035 | ND. | 0311 | 10 Chlorpyriphos | 0.015 [0.045 [0.103 | ND. [0.045 | 0.000 [N.D. [0.055 [0.669 | 05 ‘a-Endosulfan ND. | 0.135 [NO [0.055 [0.250 | 0.080 [0.145 [0.155 | 0.670 | 50 ‘Cypermethrin 0.250 |N.D. [0.145 [ND. | 0.150 [0.250 [ ND. | 0.185 | 0.673 [100 Heptachlor ND. [0.165 |ND. ToO17s [ ND. | 0.140 10.130 [ND. 10.790 [100 Cyhalothrin 0145 PND. [ND [0250 [0.130 [0.180 [ ND. [0.250 [0.540 | 120 Bifenthrin 0.150 [0.165 [O%65 |ND. [0220 ND. | 0.150 [0.205 [0520 | 150 + Values are mean of 33 saniples from each location and |-8 are sampling sites (see in materials and methods) N.D= not detected 14Results and Discussion Table 4.25 Pesticide residues in cucumber by HPTLC methods Pesticide residues in cucumber (mg kg™)* Pesticide Sampling sites 1 z 3 4 3 6 7 8 RF MDg ng_| Imidacloprid 0.070 | 0.125 | 0.085 [0150 [ND. [0.120 [ND | 0.350 [0.226 | 50 Benomyl ND. | 0.035 | 0.050 [0.035 [075 | N.D. [0.250 [0.150 [O31T [10 Chiorpyriphos | 0.050 | ND. [0.075 [0.015 | 0.065 [0.060 [N.D. | 0.025 | 0.669 | 0.5 a-Endosulfan 0.100 [ ND. PND. [0.065 ND. | 0.080 [0.145 | ND. | 0.670 | so Dichlorvos ND. | 0.075 [0.085 |N.D. [0.095 | 0.065 | 0.095 | 0.020 | 0.504 | 10 ‘Cypermethrin ND. [0.145 [0.205 [0.120 [0.145 [0.150 | ND. [0.160 | 0.673 | 100 Fenitrothion 0.150 [0.165 [N.D. | ND. [0.172 |ND. [0.130 [ ND. | 0.540 | 120 Bifenthrin 0.200 | ND. [0.175 10.205 |ND. | 0.186 [0.215 [0.200 [0.520 [150 Methamidophos | N.D. [0085 [0.050 [ND. [0.065 | 0.030 [0.125 [0.050 [o.110 | 10 Values are mean of 23 samples from cach location and 1-8 are sampling sites (see in materials and methods) N.D.= not detected Spinach samples (28) were analyzed with HPTLC methods. All the spinach samples were found contaminated with different pesticide residues and the concentration of each pesticide was varied from location to location, The pesticide residues of “decamethrin” in spinach were found at all sites (Section 3.2) in the range of 0.105- 0.200 mg kg". The residue of “chlorpyriphos” in spinach at site 3 (Hyderabad, Sindh) was higher (0.250 mg ky!) as compared to other sites. o-Endosulfan residues were found at sites I, 5, 6, 7 and 8 but higher concentrations were detected at sites 1 (Farrf field, Faisalabad) and 7 (Green market, Faisalabad) as compared to other sites. Heptachlor and lindane were. also detected in spinach samples. The endosulfan residues in vegetable samples are illustrated in Figure 4.22, 11SResults and Discussion efe§ fa f 8 Cone. (mg kg") 02 ous at vos Q ef ° Ss ee mo s Fig, 4:22 a-Endosulfan residues (mg kg") in vegetables. Spinach and cauliflower had higher residues of endosulfan as compared to other vegetable samples (Figure 4.22), Heptachlor residues were found in spinach and green peas whereas lindane residue was only detected in spinach samples. High concentration of “imidacloprid” residue was found in tomato and cauliflower whereas lower concentration of “chlorpyriphos” and “methamidophos” was detected in brinjal (eggplant) samples (Figure 4.23). Fenitrothion was found only in cucumber. Most of the vegetables in the present study were contaminated with “benomy!” but higher contents of this fungicide was found in cucumber (0.250 mg ky”) and lower one was found in green peas, tomato and brinjal (0.015 mg kg"). Dichlorvos residues were detected only in cucumber and brinjal as compared to other vegelables, Cauliflower, which is a fibrous vegetable, and tomato had higher levels of “carbendazim” (0.315 mg ky") and its residues were not detected in spinach, 116Results and Discussion Jadyfinger, bittergourd, brinjal, pumpkin, green peas and cucumber (Figure 4,24), Benomyl residues were found in most vegetables but higher concentrations were detected in cucumber (0.250 mg kg") at sites 7 (Green market, Faisalabad) and 4 (Hasilpur).. o4 0.35 ilmidactoprid on OChiorpyriphos @Methamidophos Cone. (mg kg-1) Fig. 4.23 Organophosphonus residues (mg kg") in vegetables Pyrethroids were also detected in vegetable samples. Residues of cypermethrin and bifenthrin were in maximum samples but decamethrin, cyhalothrin and B-cyhalothrin were found in spinach, tomato, potato, ladyfinger, brinjal, pumpkin and green peas. “Cypermethrin” concentration was higher in green peas (0.250mg kg") and tomato (0.245 mg kg") whereas high concentrations of “cyhalothrin” residues were also found in green peas and potato (Figure 4.25). 7Results and Discussion 0.45 os 0.38 _ 03 "2 mBenomy! g 025 OCarbendazim £ 02 8 015 on 0.05 ° Tat Cale Tue am Lage Dap ah fan Gen me Fig, 4.24 Fungicide residues (mg ky") in vegetables 03 MCypermethrin GQ Bitenthrin 02s BCyhalothrin Beta cyhalothrin _ 02 ¢ E ois & aos a Fig, 4.25 Pyrethroid residues (mg kg") in vegetables 118Results and Discussion Different fruits were collected from different gardens/orchards and also purchased from different locations/sites of Pakistan, The samples were blended, extracted in ethyl acetate, cleaned-up and analyzed by different HPTLC detection methods using silica gel plates. The results are given in Table 4.26- 4.35. Table 4.26 Pesticide residues in apple by HPTLC methods Pesticide residues in apple (mg kg")* Pesticide ‘Sampling sites 1 2 3 q 5 6 7 8 R | MDQ | ng Dimethoate N.D. 0.135 | 0.145 | 0.165 | N.D. 0.120 | 0.200 | 0.105 | 0.275 | 100 Benomy! ND. | 0.035 | 0.050 | 0.035 [0.073 | ND. [0.050 | ND. [0311 [10 Chlorpyriphos 0.050 |N.D. | 0.015 [o010 |N.D. [0.002 [ND | 0.025 [0.669 [0.5 a-Endosulfan 0.060 | 0.150 | ND. 0.065 | 0.075 | N.D. | 0.055 N.D. 0.670 | 50 Monocrotophos N.D. | 0,090 | 0.120 | 0.105 | 0.090 | N.D. 0.100 | N.D. | 0.08 88 Cypermethrin: ND. [0.105 } 0.110 | N.D. | Oits [0102 PND. 0.120 | 0.673 | 100 Parathion-methy! | 0,005 | N.D. | 0.005 | 0.050 | N.D. N.D, | 0,006 | 0.045 | 0.669 | 1 Bifenthrin 0.200 | N.D. | 0.175 | 0.155 | ND. 0.186 | N.D. 0,150 | 0,520 | 150 Mcthamidophos | N.D. 0,025 | 0.050 | N.D. 0.065 | 0.030 | 0.025 0.050 | 0.110 | !0 Diazinon 0.007 | 0.008 | 0.008 | N_D. N.D. | 0.012 | 0.030 | 0.060 | 0.660 | 0.2 + Values are mean of 23 samples from each location and 4-8 are sampling sites (see in materials and methods) N.D.- not detected 119Table 4.27 Pesticide residues in mango by HPTLC methods Results and Discussion Pesticide residues in mango (mg ke™)* —_] Pesticide ‘Sampling sites 7 2 3 4 3 6 7 8 R MDQ ug_| Dimethoate ND. [0.105 [6145 [ND [0.120 [0.140 [0.115 0.150 [0.275 | 100 Benomy! 0.020 | 0.035 [ND. | 0025 [ooIs |ND. [0.050 [ND. fos [10 aEndosuifan | ND. [0100 [ND. [ND [olds [ND [014s [ND. [0.670 [50 Monocrotophos | 0.135 | 0.100 | 0.120 |0.095 | ND. [0.099 [0110 [ND. [0.08 [88 Parathion-methyl | 0. oT ND. [0.010 [0.050 [ ND. [0.003 /0.020 |0.005"| 0.669 [7 Dichlorvos 0.004 | 0.005 | 0.020 | 0.005 [0.012 [0.015 | ND. | 0.008 | 0.505 [2 Methamidophos | 0,020 [0.015 {ND. | 0.050 | 0025 [0.070 | 0025 |ND. [0.110 | 10 Cyhalothrin 0.120 [ND. [0.130 } 0.135 [014s [0.130 | ND. | 0.120 [0.540 120 B-oyfluthrin 0120 [0.125 [0.145 | ND. ons | 0.130 [ND | ND. | 0.669 | 100 =” Values are mean of 23 samples from each location and 1-8 ave sampling sites (see in materials and methods) N.D.= not detected Table 4,28 Pesticide residues in guava by HPTLC methods Pesticide residues in guava (mg kg") Pesticide ‘Sampling sites 7 2 3 4 3 6 7 8 Rr DO ng Benomyl ND. | 0.035 [0.050 [0.035 / 0075 |ND. [0.050 [oois [ott [10 Chlorpyriphos | 0.005 [N-D. | 0.007 | 0.008 [0.006 | 8.060 | ND. [0.025 [0.669 [05 a-Endosulfan | 0.100 [0.050 [ ND. | 0.065 [0.055 [0.080 [0.110 | ND. [0.670 | 50 Dichlorvos ND. [0.025 [0.035 [ND. [0.045 [0.065 0.095] 0.020 | 0.504 [10 Cypermethrin. | ND. [0.105 [0115 [0.120 | 0.125 [0104 | ND. [0.120 [0.673 | 100 Bifenthrin 0.208 (ND. | 0155 [O1GS |ND. [0.186 | 0.205] 0.156 | 0.520 [150 Methamidophos | ND. | 0.025 10.050 | ND. | 0.065 [0.030 [0.015 | 0.050 [0.110 | 10 Parathion-rethy! | 0.005 | 0.006 |N.D. | 0.002 [0.025 ND. [0.005 | 0.008 [0.669 [7 Monocrotophos | 0.089 { 0.090 | 0095 [0.100 [ND. [0.120 |ND. [0.120 | 0.080 [88 Values are mean of 23 samples from each location and 1-8 are sampling sites (see in materials and methods) N.D.= not detected 120Results and Discussion Table 4.29 Pesticide residues in grapes by HPTLC methods Pesticide residues in grapes (mg kg")* Pesticide ‘Sampling sites i 2 3 7 3 6 7 8 R MDQ Methamidophos | ND. | 0075 [0.015 [ND. | 0.020 [0.016] 0.045 [ND [0.110 To Benomyl ND. | 0.035 | ND. [001s | 0025 | ND. [0.050 | 0.020 0311 | 10 Carbaryl 0050 [ND. | 0015 [0025 [0.035 [0.060 | ND. | 0.025 | 0.598 ] 10 ‘@Endosulfan | 0.100 [0.035 | ND. 006s [0.055 [0.080 | 0.105 | ND. [0.670 | 50 Dimethoate ND. [0025 [008s PND. [oo1s [002s [oss |ao20 | 0508 | 10 Cypermethrin. | ND. [O11 [0125 [0105 [0105 [0.150 ND. | 0.160 [0.673 | 100 Bifenthrin 0170 [ND. [0200 [ ND. |[WD. f 0210 [O185 |ND. | 0.520 [1s0 Carbendazim N.D. [0.025 | 0.030 | 0.035 | 0.040 | ND. | N.D. | 0.060 | 0.299 | 20 Parathion-methyl | 0.605 | 0.006 | 0.025 | 0.036 | 0.007 [0.010 [0.009 [0.012 [0.669 | 1 * Vatues are mean of 23 samples from euch location and 1-8 ure sampling sites (sce in materials and methods) N.D not detected Table 4.30 Pesticide residues in melon by HPTLC methods Pesticide residues in melon (mg kg™)* Pesticide Sampling sites 7 z 3 4 3 6 7 e Rr MDQ ng Monocrotophos | 0.090 | ND. | 0.095 js 10 [0.120 [0.095 [0.100 | 0.096 | 0.080 | 88 ‘a-Endosulfan 0.100 [0.055 TND. | 0.065 | 0.150 [0.080 [0.145 | N.D. | 0.670 [50 Dichlorvos ND. [0.015 [0.028 |ND. [0.035 [0.015 [0.025 | 0.020 | 0.504 | 10 Cypermethrin ND. [Oras [0165 [0.120 [0.145 [0.150 | ND. [0.160 ; 0.673 [200 Bitenthrin 0225 ND. | 0175 [0.255 1ND. | 0.186 [0.245 [0.250 [0.520 | 150 Methamidophos | ND. | 0.015 | 0030 [Np [0025 [0.030 [0.025 | O050 [110 | 10 Carbendazim 0.025 | 0.030 [0.035 | 0045 ) 0025 | 0.030 [ND. [0.045 [0.299 | 20 Dimethoate 0110 [ ND. | 0.125 70.120 | 0.145 [0.107 PND. [0.120 | 0.275 | 100 Isoprowron 0.007 [0.010 | 0012 [ND. |ND. [0.012 [0.050 [0.045 | 0.386 [5 + Values are me: N.D.~ not deteeted ‘oF 23 samples from euch location and 1-8 ares 121 ng siles (see in maierials and methods)Results and Discussion Table 4.31 Pesticide residues in apricot by HPTLC methods Pesticide Pesticide residues in apricot (mg kg )* ‘Sampling sites T z 3 4 3 6 7 8 Rr MDQ Carbendazim | 0.070 [0.125 [0.655 [0085 [ND. [0.120 [ND | ND. [0.226 x Benomy! ND, [0.035 [0.050 [0.035 [0.045 | ND. [0.020 | 0.050 [Ott | 10 Cypermethrin | ND. [OTS [0.165 | 0.120 [0145 [O50 | ND. [ND. [0.673 [100 Bifenthrin 0.156 [ND. [0.175 [0185 [ND. [0.176 | 0205 [0.200 [0.520 [150 Meihamidophos | ND. | 0.025 [0.050 | ND. | 0.035 [0.030 [0.025 [0.050 [0.110 [10 a-Endosulfan | 0.065 [0.085 | 0.075 | 0055 10.096 | N.D. | 0.058 | 0.070 [0.670 | 50 Parathion-methy! | ND. [0.005 | 0.007 [ODDS {0.015 [ND. [0025 70.006 [0.669 [7 Monocrotophos | 0.125 [0.145 | 0.095 oan ND. [009% [0091 [ND | 0.080 | 88 Values afe mean oF23 samples from each Tocation and 1-8 ace sampling sites (See in materials and methods) N.D-= not detected Table 4.32 Pesticide residues in plum by HPTLC methods Pesticide residues in plum (mg kg)" Pesticide ‘Sampling sites r z 3 + 5 6 7 8 Rr MDQ og c-Endosulfan | 0.055 | 0.075 | ND. [0.105 [0.100 | N.D. [0.085 | 0.060 | 0.670 | 50 Benomyl ND. [0035 [2.020 [0.015 [0.035 |ND. [0.050 [0.020 Tosi | 10 Parathion-methyl | 0.00 | ND. [0.005 [003 | 0.065 [0010 [ND. | 0.025 | 0669 [1 Cypermethrin 10.105 [0.125 [ND [0122 [OI [ors fo1as |ND. [0673 [i100 Dimethoate ND. [0125 | 0.105 [ND. [O12 pons [0.120 | o.tas [ozs [100 Fenvalerate 0150 [9135 [ND foias for [Np. [0130 [ ND. [oss | 120 Bifenthrin 0.160 | N.D. | O.163 [0200 | ND. | 0.186 | 0.205 | 0.200 | 0.520 | 150 Chlorfenvinphos | ND. | 0.065 | N.p. ]0070 10.075 | 0.120 | 0.100 | 0.105 | 0547 | 50 * Values are mean of 23 samples trom each location and 1-8 are sampling sites (see in materials and methods) N.D.- not detected 122