Study of Contamination Level of Pesticide Residues in Grapes (Vitis vinifera) in Maharas...
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1 Page 1 of 7 Study of Contamination Level of Pesticide Residues in Grapes (Vitis vinifera) in Maharashtra By Mukesh Kumar Raikwar*, Vikas Bhardvaj, Urmila Sawant & Virendra Vora September 2011 The authors are Specialists at the Department of Pesticide Residue, Reliable Analytical Laboratories Pvt Ltd, Bhiwandi, Thane, India *Corresponding Author Abstract Maharashtra is the major grape growing state in India with an estimated cultivation area of around 35,000 H.A. and with an annual production of approx. 9 lacs M.T. Increasing quantities are being exported from Maharashtra to European countries. To grow grapes economically and to get a high yield of grapes with a good quality, farmers are using lots of (toxic and nontoxic) pesticides. It is necessary to ensure that such residues should not be found in food or feed at levels presenting an unacceptable risk to humans. Maximum residue levels (MRLs) are therefore set by the European Commission to protect consumers from exposure to unacceptable levels of pesticides residues in food and feed. During the winter season of 2010 & 2011 contamination of a total of 171 pesticides in grapes were surveyed by using LCMS & GCMS instruments. For this study a total of 2656 samples were collected from farmers fields and from local market in Maharashtra and were analyzed by using validated extraction method. From the results it was concluded that 27 pesticides out of 171 pesticides can be found usually in grape samples which indicates that the stability of these pesticides is very high or they retain in the grape fruit for a long time after use of them. Some pesticides which failed most to meet the requirement of EC MRL were Chloramquate chloride (CCC), Chlorpyriphos, Azoxystrobin, Carbendazim and Kresoxym Methyl compared to other pesticides. This article can help farmer in spray of pesticides as few out of many pesticides are toxic and more stable so that avoidance of these pesticides can improve the quality (Low contamination of Pesticides) of grapes and also can help to prevent the financial loss of farmer and exporter too. Keywords: Grapes, Pesticides, Maharashtra, European Commission, LCMS and GCMS Introduction India is an agriculture land, due to versatility in nature of weather and soil thousands types of fruits and vegetables are grown in different parts of India. Every year million tonnes of fruits and vegetables are exported to all over the world and it has been seen a steep rise in the export of fruits. The total production of fruits and vegetables in the world is around 370 MT. India ranks first in the world with an annual output of 32 MT. it is become a large variety of fruits (Apple, Litchi, Papaya, Pineapple and Sapota and mango) are grown in diverse agro-climatic zones in India. A highest fruit producing state in India is Maharashtra & Grape contributes major percentage in fruit production and export. In Maharashtra, mainly grape is grown in Sangli, Solapur, Latur, Pune and Nasik area. The major varieties of grapes grown in India are Thomson Seedless, Sonaka, Anab-e-Shahi, Perlette, Banglore blue, Pusa seedless, Beauty seedless etc. Production of grape in Maharashtra is around 0.68 MT which followed by Karnataka seating at second position. Other grapes growing states are Punjab, Andhra Pradesh and Tamil Nadu.
2 Page 2 of 7 Pesticide are substance or mixture of substance designed for preventing, destroying, repelling or lessening the damage of a pestwhich help directly or indirectly in growth of plant and crop. They have been used in agricultural crops, residential and commercial buildings, ornamental gardens and plants and also to control Mosquitoes & insects. Pesticides use has no doubt increased the agricultural production economically but long term stability and carcinogenic side effect of residues has made limitation in use. Continuous use of pesticide in large quantity has leaded accumulation of it in the Agriculture crop (Andersen JH & Dogehim SM). The problem of PR (pesticide residue) accumulation needs more attention in vegetables and fruits because most of time these are consumed either raw or without much storage time. Due to carcinogenic effect of these pesticides EC has tighten the rules and regulation for import of grapes. Million Tonnes of Grapes exported every year in winter season from different region of Maharashtra to Europe country for liquor production and for other use. European commission put MRL (Annexure ) for every PR as per potency and stability of pesticide as they have carcinogenic effects. Decided MRL of PR is at ppb level so that GCMS & LCMS can be used (Anna S et. all) to detect the residue of PR in Grape. In this project we need to fully validate the method to detect and quantify the each PR level. To get relevant conclusion, need to perform this project on large number of samples obtained from different region of Maharashtra and to make it more perfect two years samples need to be collected. This project can guide the farmer in use of pesticides on grape fruit. By finding the higher possible PRs contamination in grape, precaution can be taken in advance to decrease the level of pesticides contamination in grape and so that failing of samples will be reduced and more export of Fruits can be seen which means more benefit to the Farmer, exporter and so that to the country. 2. Materials and Methods Chemicals Methanol & Water (HPLC Grade) of J T Backer (as a Mobile phase). Chilled Distilled water (to maintain the temp. of matrix as increase in temp cause degradation of residues like, captan, captafol and dicofol) and Ethyl Acetate (to prevent degradation of some PRs in Extraction), PSA (Primary secondary amine for cleaning of matrix interference in sample), Activated Sodium sulphate (to remove water traces), Formic Acid (to acidify extraction solvent), Ammonium formate (as a buffer for mobile phase), all these were purchased from Merk. 10% DEG (Di ethylene glycol) in methanol (as a analytes keeper & protector in MS). Acetic acid (to acidify final volume) and PTFE (Polytetrafluoroethylene) Filter of 0.22 µm (used to filter final injection volume before going to fill vial). Pesticide Standard The Pesticides standards were purchased from Dr. Ehrenstorfer GmbH, Merck or Riedel-deHaen and Accu standard. The purity of all pesticide standards were greater than 95%. All standards were tuned to obtain optimized MRM and qualifier (for confirmation of PR) for analysis. Preparation of Stock Solutions of Standard Pesticides: The stock solutions for pesticides were prepared separately 1mg/ml by dissolving pure pesticide standards in Methanol. Stock solutions of Organochlorine and Synthetic pyrethroids were prepared with Ethyl acetate. Theses individual std stock solutions were mixed appropriately to obtain desire concentration of Pesticides and then stock solution of std mix was serially diluted with methanol to 1 µg/ ml to spike samples for recovery and method validation. Internal standard C 13 was used for CCC to compensate instrument error and manual error during analysis as contamination of it is very considered in Europe. Samples for analysis: The samples of grapes were collected from the Maharashtra region, from the farmer and Grape Wholesale market. Different types (Black & Green) of Grape samples were received in fresh condition and stored at -21 C.
3 Page 3 of 7 Preparation of Grapes matrix: Initially grapes were plucked and collected in washed & dried 1000 ml Beaker. Then sample was crushed in Grinder and stirred for 15 minutes to homogenize the sample matrix thoroughly. Sample Extraction Methods: Fully validated methods were used for different PR groups like PGR and PR (Zweig G et. all, Kaphalia BS et. all, K Banerjee et. all). Which are detailed in Fig 1&2. Spiking of std PR mix was done on the same method for validation. Fig 1: PGR extraction method for Grape Fig 2: PR extraction Method for Grape 10 ± 0.1 gm Grapes matrix in 50 ml Poly propylene tube 20 ml 0.1 % Formic Acid in Methanol Vortex for 5 min Centrifuge for 10 min at 5000 rpm Filter supernatant through 0.22 µm PTFE filter into the vial for LCMS 10 ± 0.1 gm Grapes matrix in 50 ml Poly propylene tube 5 ml Chilled Water and vortex for 5 min 10 ml Ethyl acetate and vortex for 5 min 10 gm Activated Sodium sulphate Centrifuge for 5 min at rpm 1 ml supernatant for GCMS in eppendort tube + 25 mg PSA Centrifuge for 5 min at rpm Use supernatant for GCMS injection 5 ml supernatant for LCMS in eppendort tube + 25 mg PSA Centrifuge for 5 min at 5000 rpm Transfer 4 ml supernatant in Bowl µl DEG for Drying Reconstitute with 1 ml Methanol + 1 ml 1% Acetic acid in water Filter through 0.22 µm PTFE and transfer into the vial for LCMS Instrument: For Analysis of Multi residue in grapes were performed by LCMSMS and GCMSMS (Triple Quadruple). The condition of each instrument is given below. GC/MS Conditions and Parameters The volatile PRs were analyzed on Agilent 7890A GC/MS, parameters were: Source Temp 230 C
4 Page 4 of 7 MS1 Quad 150 C Column Gas Flow 1.2 ml/min (Carrier Gas - Nitrogen) Collision Gas Flow 1.5 ml/min (Collision Gas - Helium) Quench Flow 2.25 ml/min LC/MS Conditions and Parameters The PGR and PR were analyzed and validated on Triple Quadrapole LCMS (Agilent 6460 & Bio MDS Sciex 2000). Table 1 Monthly Agilent LC-MS Parameters used for the analysis of pesticides Gas temp 330 C Gas flow 6 l/min Sheath gas flow 8 l/min Sheath gas temp 310 C Neb Pressure 30 psi Capillary ve ve Nozzele Voltage 500 +ve 500 -ve Collision Gas Nitrogen Ionization ESI(+) & (-) Mobile phase: Same Mobile phase were used for both PR & PGR are A: Water: Methanol in 8:2 ratio with 5 mm ammonium formate (Buffer to ionize molecules in MS). B: Methanol: Water in 9:1 ratio with 5 mm ammonium formate buffer Eclips XDB C18 column of 5 µ particle size with 4.6 mm diameter and of 150 mm length was used for LC/MS. For CCC Atlantis Waters HILIC column of 5µ particles size with 4.6 mm diameter & 150 mm long column was used. Validation of Method Highly sensitive GCMS and LCMS instruments were used for the analysis of PR in grapes. Before analysis the efficiency of the methods were evaluated as recovery experiment by spiking samples with std pesticides at 3 concentrations. To spike well homogenized sample was used. Three replicates at 3 different concentrations were extracted with the solvent and cleaned on PSA and then were analyzed on GCMS and LCMS. Blank extracting solvent with no added pesticide was also processed in a similar manner. Fig-3: Six Points linearity of Iprovelicarb plotted from 10 to 200 mg/kg in matrix Fig-4: Two qualifiers and 1 quantifier with MRM of Iprovelicarb Fig-5: Chromatogram of all PR in Grape spiking at 20 ppb on LCMS Fig-6: Chromatogram of volatile PR in Grape spiking at 20 ppb on GCMS 3. Results and Discussion Before analysis of grape samples, the method of extraction and Mass parameter were validated twice So that there is very less chances of error and we can also be sure that obtained data are more accurate and precise as theses data going to tell the level of contamination of pesticides in soil & environment and so that in fruit. For Validation following parameters were used and optimum response was obtained and maintained. Linearity range was from 0.01 mg/kg to 0.2 mg/kg. Total 3
5 Page 5 of 7 product ions (Daughter ion) (fig-4) of precursor were used so that presence of PR can be confirmed with more proof. Nº Name of Pesticides Table 2 Parameters included in method validation with obtained maximum and minimum results range for every pesticide Parameters Precision (CV) > 98% Linearity coefficient > 0.99 Result/Response Recovery 80 to 105 % Selectivity Ruggedness Specificity (20 blank runs) 2 Qualifier ions < 2 % RSD between 2 days No Peak Uncertainty ± 20 % LOD/LOQ LOD Limit of detection LOQ limit of quantification CV Coefficient variation LOD (mg/kg) Total Samples Range (mg/kg) 0.01mg/kg Table 3 Contamination results of Multi residues in Analyzed grape samples in year 2010 & 2011 Found samples Cont.* % Cont. EU MRL (mg/kg) Samples > MRL 1 Carbendazim Thiophenate methyl CCC Fipronil Metalaxyal CPPU Flusiazole Captan Lamda Chlorpyriphos Methomyl BA Myclobutanil Spinosad Fenamidone Acetamiprid Famaoxadone Kresoxim methyl Azoxystorbin Triadminol Iprovalicarb Abmectin Pyroclostorbin Thiomethoxam Ethion
6 Page 6 of 7 26 Imidachlorpid Dimethomorph Cont. means contamination. Table 3 indicate the name of found pesticides in grape with number of samples, European commission MRL(Maximum residue limit), Detection limit, Failed sample and contamination range in ppm. For study 171 PR were monitored during 2 years (2010 to 2011) from which 27 PR were found in grape samples (see table 1). Total 2565 (1703 in 2010 & 862 in 2011) numbers of grape samples were analyzed in two year. Two year s obtained data (Tab-3) shows that the highest contamination in grape is of chlorpyriphos, 1120 samples (43.66 %) out of 2565 but only 8 samples exceeded the MRL value of 0.5 ppm given by European commission found values are from mg/kg range. The Highest MRL exceeded PR is captan in two years 35 samples were failed out of 2565 and in 2011 CCC (Plant growth regulator) was introduced in PR list for contamination testing and due to its high stability in soil and environment the level of contamination was highest in Grape fruits 206 out of 862. Due to high stability it is banned in so many countries too though contamination of it can be seen in Grape after many years. Contamination range of CCC was from mg/kg. Highest contain of PR is 2.5 ppm of Iprovelicarb but overall contamination is only 4.83 %. Out of found 27 pesticides Abmectin residue was found at lowest level 40 ppb and Ethion overall contamination was at lowest in samples 0.16 %. Azoxystribin contamination in grapes is at third highest % but exceeded samples are Zero due higher EU MRL at 2 ppm. Carbendazim contamination in 686 (26.74%) samples was observed. The degradation product Thiophenate methyl of carbendazin detected in 90 (3.51%) samples. Myclobutanil, Kresoxim methyl & triadminol contamination found in 561, 491 & 454 samples respectively. From this we can conclude that use of CCC will put sample in risk for long time even after stop in use of it so that complete avoidance of it needed. Other pesticides like Metlaxyl, Dimethomorph, Triadiminol and Azoxystrobin have higher MRL so that they are not that much concerned pesticides. Higher ratio in failing due to Thiophenate methyl, Fipronil, Captan, Flusilazole has also found higher so that use of such compound can also contaminate the sample higher than MRL. Acknowledgement We are very grateful to the Chairman and Managing Director of Reliable Analytical Laboratories Pvt Ltd, Mrs. Renu Kaushal for the essential and financial support. We are also thankful to the colleagues and lab technicians for giving moral support in this project. References Zweig G (1984). Analytical Methods for Pesticides and Plant Growth Regulator. In: Academic Press. New York. (Eds. Sharma J). pp Kaphalia BS (1990). Organochlorine pesticide residues in different Indian cereals, pulses, spices, vegetables, fruits, milk, butter, deshi ghee, and edible oils. In: Journal of AOAC Int. 73. (Eds. Takroo MS, Nigam U and Seth TD). pp Andersen JH (2001). Results from the monitoring of pesticide residues in fruit and vegetables in the Danish market In: Food Addit. Contam. 18 (Eds. Paulsen, ME). pp Dogehim SM (2001). Monitoring pesticide residues in Egyptian fruits and vegetables during In: Journal of AOAC Int. 84 (2). (Eds. Gad-Alla SA and Ei-Marsafy AM). pp Anna S (2004). Application of liquid chromatography with electrospray tandem mass spectrometry to the determination of a new generation of pesticides in processed fruits and vegetables. In: Journal of Chromatography A (Eds. Luciana B and Mirella B). pp K Banerjee (2007). Validation and uncertainty analysis of a multi-residue method for pesticides in grapes using ethyl acetate extraction and liquidchromatography-tandem mass spectrometry. In: Journal of Chromatography A (Eds. Oulkar DP, gupta S, Patil SB, Patil SH, Savant R, Pandurang G). pp Annexure 9 (2011) of Regulation of export of fresh table grapes to the European Union through control of residues of agrochemicals. ***
7 Page 7 of 7 0 Copyright 2011, ECO Services International
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