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Issues in Biological Sciences and Pharmaceutical Research Vol.3(9),pp.100-106,September 2015 Available online at http://www.journalissues.org/IBSPR/ http://dx.doi.org/10.15739/ibspr.019 Copyright © 2015 Author(s) retain the copyright of this article ISSN 2350-1588

Original Research Article

Evaluation of residue in rice, wheat and pulses of Bidar district Karnataka,

Received 10 August, 2015, Revised 26 August, 2015 Accepted 30 August, 2015 Published 11 September, 2015

1Jagadish G.K., Pesticide residues in rice, wheat and pulses of Bidar district of north 2Jaylakshmi S.K Karnataka were evaluated in this study. (OP), Organochlorines (OC), Synthetic (SP) and residues and were monitored. A total of 250 samples from different markets of Bidar 1 *Sreeramulu K district, Karnataka were analysed by gas chromatography (GC) and high performance liquid chromatography (HPLC). Recovery studies carried out 1 Department of Biochemistry, for all 4 groups of were within the acceptable range of 70-120% gulbarga University, gulbarga- before the analysis of field samples. Sample extraction and partitioning was 585106,India carried out by multi residue method. Among the samples analysed, 80 2 College of Agriculture samples were found to be contaminated with different groups of pesticides. (University of Agricultural Pesticide residues were found to be above the Maximum Residue Limit Sciences-Raichur), Aland Road, (MRL) in 22 samples and below MRL in 58 samples. More numbers of Gulbarga-585101,India. pesticides were detected in wheat followed by rice and pulses. In pulses, red

*Corresponding Author gram was the major commodity contaminated with multiple pesticide Email: [email protected] residues followed by black gram and green gram. OP pesticides were dominated OC and SP in all the studied commodities. Most commonly Tel.: +91-8472-263289, detected OPs were , , , and triazofos. Fax : +91-8472-245632 Key words: Pesticides, GC, HPLC, pulses, rice and wheat

INTRODUCTION

Karnataka is one among the major states in production of pesticide residues causes resistance of pests to the pulses, rice, wheat, sorghum, fruits and vegetables. The chemicals indicating 50-70% of contamination with north regions of Karnataka (Bidar) have semi-arid dry residues. Public concern towards pesticide conditions with temperatures ranging from 26-40 oC in residues has risen over the few decades to identify the summer and the agriculture is mainly dependent on rain. point where it has become a significant food safety issue The major crops grown in these areas are red gram, green (Column et al., 1999). The determination of pesticide gram, black gram; wheat and rice; fruits like mango and residues in food has become an essential requirement for banana. India is an agriculture based country with second consumer, producers and authorities responsible for largest producer of vegetables after china, and access for food quality control (Aguilear et al., 2003). Environmental 13.4% of the world production. pollution is one of the serious predicaments of the modern The growing demand for food and feed is due to world (Hela et al., 2005). During the last decade, significant consistent increase in population and the use of chemical increase in environmental pollutants and lack of pesticides has been increased to obtain more yield and precautionary measures or observance of the protection of crop from pests and diseases. Through which environmental regulations has become a global problem the crop protection has been increased to 100% but the (Bondarenko et al., 2004; Hela et al., 2005; Abdel-Halim et cropping area has increased marginally 20%. But the al., 2006; Wilsont and Foos, 2006). indiscriminate usage of pesticide leads to accumulation of Pesticides applied to food crops in the field can leave pesticide residues in food chain, aquatic system and soil potentially harmful residues; OC in particular is persistent (Jayashree and Vasudevan, 2007). The non-degradability of in foodstuffs for longer periods. If crops are sprayed on to Issues Biol. Sci. Pharma. Res. 101

harvest without an appropriate waiting period, even OP can Bhalki, Aurad, Basvakalyan, Humnabad and Bidar. From persist in food (Bull, 1992). Pesticide residues in foods are a each taluk, 10 samples of rice, wheat, red gram, black gram growing source of concern for the general population and green gram were collected in air tight polyethylene zip (Torres et al. 1996). A substantial body of laboratory and cover bags and stored in refrigerator until complete epidemiological evidence suggests that certain pesticides analysis. Approximately 1-2 kg of each sample was are associated with carcinogenesis, immunotoxicity, collected. Sample extraction and purification was , behavioural impairment, reproductive completed within 24 hours of collection. dysfunction, endocrine disruption, developmental disabilities and respiratory diseases, such as asthama Preparation of standard solutions (Solomon et al., 2000). Cereals and pulses form a large proportion of the global diet and control of pests during Stock solutions of OP mixture, OC mixture and SP mixture storage is a bigger problem. Storage pests can be controlled (500 µg mL-1) were prepared in hexane: (9:1). by the use of as residual protectants. This grain Stock solution of (500 µg mL-1) was prepared in treatment method can be easily adaptable to all types and HPLC grade acetonitrile. OP mixture contains , sizes of storage with minimum expense. This post harvest dichlorovos, methyl parathion, , , application of pesticides leads to the persistence of profenofos, , chlorpyrifos, quinalfos, ethion, and pesticides in food grains and may cause health hazards triazofos. Mixture of OC contains alpha-HCH, gamma-HCH, (Webley, 1994). The short as well as long term impacts of alpha-, endosulfan sulphate, and p.p’-DDT. the use of pesticides on biological systems are being SP mixture contains , , - evaluated continuously in an effort to minimize the lambda, and . Working standard hazards. A wide spread use pesticides, their toxic residues solutions were prepared by further dilutions of the stock have been reported in various environmental matrices by solutions. the researchers (Kumari et al., 2002; Kumari et al., 2003; Singh et al.,2004). Extraction and Purification The presence of pesticide residues in rice, wheat and pulses can be a significant route to human exposure and Cereal and pulse samples (1-2 kg) were homogenized in a most of (OC) pesticides have been banned because they are warring blender. To the flask containing 25 g of a highly persistent insecticides, and their residues still powdered sample, 100 mL mixture of and acetone appear as pollutants in food and environment (European (35:65 v/v) was added and kept for shaking for about 30 Community, 1990). Organochlorine pesticides are minutes in a mechanical shaker and filtered under vacuum characterized by high lipid solubility and high persistence through a Buchner funnel. The extract was transferred to a and hence they tend to accumulate in fatty tissues. To flat bottom flask and concentrated to near dryness in a ensure the safety of food for consumers, numerous rotary evaporator. The aqueous extract was partitioned legislations such as the European Council (EC) directives using 50 mL mixture of hexane: (1:1). The have established MRLs for pesticides in food. No reports process was repeated twice. The organic fraction obtained have yet been published about OC, OP, SP and carbamate after the partitioning was transferred to a 250 mL flat pesticides in pulses, wheat and rice from North Karnataka. bottomed flask filtered through sodium sulphate to absorb In the present study, the monitoring of OC, OP & SP in the residual moisture. The organic portion was cereals and pulses, which are undesirable substances in concentrated up to dryness in a rotary evaporator and re- food according to the Indian Community directives, have dissolved using 10 mL n-hexane: acetone (1: 1). Final been investigated. volume of 10 mL sample was taken in a 25 mL centrifuge tube and 250 mg primary secondary (PSA) and 1 g magnesium sulphate was added to it. The contents of the MATERIAL AND METHODS tubes were mixed in a vortex mixer and centrifuged at 10000 rpm for 10 mins. 3 mL of this sample was Primary secondary amine (PSA) was procured from Agilent evaporated and re-dissolved in HPLC grade acetonitrile for Technologies, Pvt. Ltd, India. Reference standards of OC, OP, analysis in HPLC. The samples were transferred to 1.5 mL SP and Carbamates were procured from Dr. Ehrenstorfer glass vials for analysis by GC and HPLC. (Augsburg, Germany). All other chemicals used were of analytical grade. Chromatographic analysis

Sample collection Analysis of OP, OC and SP residues was performed using a Bruker gas chromatograph (GC), Model 450-GC. Electron A total of 250 samples of cereals namely rice, wheat and capture detector (ECD) was used for the analysis of OC and pulses (red gram, black gram and green gram) cultivated SP and nitrogen phosphorous detector (NPD) was used for during the kharif season of the year 2014 were collected the analysis of OP. A capillary column DB-5MS (30m × from different farmers’ fields, whole sale market and retail 0.25mm, 0.25µm film thickness; stationary phase, 5% markets of Bidar district, which includes 5 taluk’s namely phenyl / 95% dimethylarylene siloxane) was used for the Jagadish et al. 102

Figure 1: GC chromatogram of selected OP mix 1ppm 1.Dichlorovos, 2.Phorate, 3.Dimethoate, 4.Methylparathion, 5.Fenitrothion, 6.Malathion, 7.Chloropyriphos, 8.Quinolphos, 9.Ethion, 10.Triazophos

analysis. The injector was kept at split less mode. Column extracts of rice, wheat and pulses. Linearity curve was oven was initially maintained at 80 °C with a hold time of 1 drawn by analysing standards in the range of 0.01-1 µg mL- .to 150 °C with a hold 1 1־min, then increased at 15 °C min to 250 °C and 1־time of 5 min and then increased at 4 °C min to 280 °C with a hold time 1־further increased at 10 °C min of 15 min. The temperatures of the injector and detector RESULTS AND DISCUSSION were maintained at 280 °C and 300 °C, respectively. One microliter of samples was injected with the auto-sampler. The method used was efficient to extract and analyse all the Ultra high pure nitrogen was used as the carrier gas at a 4 groups of pesticides. The recoveries obtained were within At the above mentioned conditions the acceptable range of 70-120%. The LOD of OC and SP .1־flow rate of 1 mL min the analysis of pesticide residues was performed. was 0.01 µg mL-1; LOD of OP was 0.02 µg mL-1. Carbamates Analysis of carbamate residues in cereals and pulses were determined at the LOD of 0.1 µg mL-1 (Figure 3). The were carried out by HPLC (Shimadzu, LC 2010 CHT) with a LOQ of OC, SP and OP in cereals and pulses was 0.05 mg kg- PDA detector. The column used was Phenomenex Luna C- 1; LOQ of carbamates in cereals and pulses was 0.5 mg kg-1. 18, 100A, 250×4, 60mm. The mobile phase composition The method was linear for all the pesticides with a used was acetonitrile: water (50:50, v/v) at a flow rate of correlation coefficient >0.99. The GC chromatogram of OP 1.0 mL min-1 and an injection volume of 20µL was used. standard mixture is given in Figure 1.

Method validation Pesticide residues in cereals

The multi residue method (AOAC-2000) used for the Rabbi and kharif are two major cultivation patterns in Bidar analysis of all 4 groups of pesticides (OC, SP, OP and mainly depend on rain. Major crops in this area are pulses, carbamates) was performed according to SANCO guidelines wheat, and nuts; while minor are sugarcane, oil seeds and (SANCO, 2013). The recovery experiments were carried out vegetables. Wheat is the optimum crop for the farmer in to determine the accuracy of the method by using the above this area. Out of 50 wheat samples analysed from different mentioned procedures. The limit of detection (LOD), the sources, 24 samples were contaminated with pesticide lowest amount of analyte that can be detected with a signal residue in which 8 are above MRL and 16 samples are to noise ratio of ≥ 3 was determined by analysing lower below MRL (Table 2). In wheat and rice, the major groups concentrations of pesticides. Limit of quantification (LOQ), of pesticides found were OP dominated most than SP the lowest amount of analyte that can be detected at a followed by OC; carbamates were not found in any of the signal to noise ratio of ≥10 was determined by analyzing samples analyzed (Table 1). Among OP, chloropyrifos is the the lower concentration standards prepared in the blank most abundant pesticide residue in wheat and rice followed Issues Biol. Sci. Pharma. Res. 103

Table I. Detection of pesticides in different commodities collected from different places of Bidar District of Karnataka state, India

Commodity Sample collection Samples collected from Samples collected from Samples collected from point farmers field wholesale market retailers shop Sample Pesticide Sample Pesticide Sample Pesticide analysed residue analysed residue analysed residue detected detected detected Rice Bidar,Bhalki, Aurad, 20 1 20 2 10 1 Humanabad and Basvakalyan Wheat Bidar,Bhalki, Aurad, 20 1 20 3 10 2 Humanabad and Basvakalyan Red gram Bidar,Bhalki, Aurad, 20 1 20 2 10 1 Humanabad and Basvakalyan Black gram Bidar,Bhalki, Aurad, 20 0 20 2 10 1 Humanabad and Basvakalyan

Green gram Bidar,Bhalki, Aurad, 20 2 20 1 10 1 Humanabad and Basvakalyan

Figure 2: Number of Pesticide contamination in different commodities and type of pesticides detected.

by profenofos , malathion, whereas, triazofos concentration Pesticide residues in pulses is very negligible. Synthetic pyrithroids (cypermethrin and cyhalothrin-Lambda) are found in the wheat. However, out Major cultivation crops in pulses are red gram, black gram of 50 rice samples 18 are found to be contaminated as and green gram. Red gram is found to be contaminated with shown in Figure 2, in which 4 samples are above the MRL more number of pesticides followed by black gram and and rest are below MRL. Similar results in cereals were also green gram. Table 2 shows out of 50 samples of each pulses, reported in Tomer (2013). 17 red gram, 9 black gram and 12 green gram are Jagadish et al. 104

Table 2. Pesticide contaminated more than MRL and MRL of detected pesticide

Commodity No. of samples No. of samples with Pesticides detected MRL of pesticide contaminated with pesticides more (EU MRL) (mg/kg) pesticide residues than MRL Rice 18 04 Chloripyriphos 0.05 Ethion 0.01 Triazofos 0.02 Bifenthri 0.01 Fenpropathrin Not available Profenofos 0.01 Malathion 0.02 aldrin 0.01 Wheat 24 06 Chlorpyrifos 0.05 Triazofos 0.02 Profenofos 0.01 Malathion Not available Aldrin 0.01 Cypermethrin 2 Dichlorovos Not available Cyhalothrin-L 0.05 Red gram 17 04 Chlorpyrifos 0.05 Ethion 0.01 Triazofos 0.01 Profenofos 0.01 Malathion Not available Cypermethrin 0.05 Black gram 09 03 Chlorpyrifos 0.05 Ethion 0.01 Triazofos 0.01 Profenofos 0.01 Cypermethrin 0.05 Green gram 12 04 Chloripyriphos 0.05 Ethion 0.01 Triazofos 0.01 Profenofos 0.01 Malathion Not available Cypermethrin 0.05

contaminated with pesticide residues. The pesticides found food grains. Study of pesticide residue in holoestic pattern above MRL in 4 red gram, 3 black gram and 4 green gram is prime importance to implement the act in this region. samples. Multiple pesticides are detected in cereals and Table 2 depicts out of 50 samples of each commodity 20 pulses may be due to cocktails of various pesticides to samples from whole sale 20 from farmers field and 10 from increase the potency of the compounds as reported by retail shop was collected. Most of the wholesale and retail Danso et al. (2002) and Ntow et al. (2006). The most samples are contaminated with the OP, OC and SP pesticide commonly detected pesticide in cereals and pulses is residues followed by farmer field’s samples, there was no chloropyrifos which has effect on health and safety of detection of carbamates in any of the pulses samples. Study mammals has been assessed in numerous studies by concludes that food grains and pulses are cross Johnson et al. (1998); Clegg et al. (1999). with contaminated in storage may be by post-harvest application this compound can affect the central nervous system, of pesticide. Detection of these pesticide residues shows cardiovascular and respiratory system (Nolan et al., 1984). that the serious concern for the food grains and human Tomer (2013) elucidated the percent contamination of health. Measures to be taken for the monitoring and pesticides in the pulses. evaluation of pesticide residue at consumer level. An attempt has been made to study the monitoring and evaluation of pesticide residues in grown food grains of Acknowledgement Bidar area. This study suggests that minimize the indiscriminate, non-recommended usage of pesticides on One of the author Jagadish G.K. would like to thanks Issues Biol. Sci. Pharma. Res. 105

Figure 3: HPLC chromatogram of (a) and mixture of carboryl and (b)

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