VOL. 2, NO. 2, March 2012 ISSN ARPN Journal of Science and Technology All rights reserved.

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1 Pesticide Residues in Bean Samples from Northeastern Nigeria 1 Obida M. Gwary, 2 Stephen S. Hati, 1 Goni A. Dimari and 1 Victor O. Ogugbuaja 1 Department of Chemistry, University of Maiduguri, Nigeria 2 Department of Chemistry, IBB University, Lapai, Nigeria stevehati@yahoo.com ABSTRACT A study was conducted to measure residues of pesticides in samples of Beans (Phaseolus vulgaris) collected in both field and storage facilities in six local area councils of Borno State, northeast Nigeria. Multi-residue pesticide analysis technique consisting of the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) sample preparation and GC-MS detection was employed. Method validation for this study maintained a RSD ±13% and percentage recoveries were in the range of %. Results show that out of the residues of eight analytes determined, three analytes (Dichlorvos, Endrin and DDT) were detected in both field samples (Pre Storage) and stored sample (post harvest). Another set of three analytes (Lindane, Diazinon and Aldrin) were only detected in the pre storage samples, while the remaining two analytes (Carbofuran and Dieldrin) were not detected at all. In general, the concentrations of these analytes in the bean samples are in the order: Endrin > DDT > Dichlorvos. The study observed that Dichlorvos was mainly utilized in storage of beans from insect attack and suggested that stringent monitoring of use of pesticides in agriculture and food storage in Nigeria is required. Keywords: phaseolus vulgaris, multi-residue, GC-MS, borno state. 1. INTRODUCTION Pesticide residue are substances or mixtures of substances in food for man or animals resulting from the use of pesticide including any specified derivatives, such as degradation and conversion products, metabolites, reaction products and impurities considered to be of toxicological significance[1, 2]. Several reports show that global pesticide usage has increased significantly during the last three decades consequent with changes in farming practices and the increasing intensive agriculture. This extensive use of pesticides for agricultural and nonagricultural purposes has resulted in the presence of their residues in various environmental matrices, especially food stuff [3, 4] proving the high risk of these chemicals to human health and environment. Pesticide residues most commonly found in food samples of vegetal origin are pesticides that are intentionally applied to the plants to attack invertebrate pests (insecticides, acaricides, etc.) and plant diseases (fungicides). Every cultivated crop may be attracted by insects from the day it is planted until harvest. Hence, the value of chemical pesticides in controlling mosquitoes, termites, cockroaches, weevil and bowers, snails, rats and multitude of others animals have been great, and it is difficult to envisage modern disease control and agricultural programmes without some form of chemical control [5, 6]. If modern agriculture attempted to operate without chemical pesticides of some sort, production would probably decline precipitously in many areas, food prices would soar far higher and food shortages would become more severe [3]. Borno State, in Northeastern Nigeria contributes significantly to national cereals and legumes production and has received international support for these agricultural activities [8]. High levels of pesticides residues arising from improper application and multiple sprays of sub lethal doses have been reported to be responsible for the poisoning and deaths of people in both rural and urban areas of Borno State and Nigeria in general. The following episodes have been reported concerning poisoning from bean foods in Nigeria: NAFDAC report revealed that the offensive beans were sourced from Taraba State (Northeast Nigeria). NAFDAC laboratory analysis reports of the moi-moi and beans from the homes of the victims and beans from the open market the State contained outrageously high organophosphates, carbamates, fenithrothion and chloropyrifos which are highly toxic pesticides. They must have resulted from either higher doses then recommended or wrong application. However, in April 2008 several National dailies reported 120 students of Government Girls Secondary School, Doma in Gombe metropolis (Northeast Nigeria) were rushed to the Gombe Specialist Hospital after consuming a meal of beans that was suspected to have been preserved with poisonous chemicals - 10 of these students were reported to be in Critical condition. Samples of the cooked beans consumed, samples of uncooked beans, palm oil, onions, maggi, salt and other condiments used in cooking the beans were also tested in NAFDAC Laboratories to ascertain the cause of the problem [6]. The result of the analysis showed that samples of the cooked beans and the uncooked beans contained outrageously high levels of lindane, an organ chlorinated pesticide that was banned under the 1989 Rotterdam convention [8]. Thus this study attempts to provide monitoring information on the levels of pesticides residues in bean samples cultivated and stored in Borno State, northeast Nigeria. 2. METHODOLOGY Study Area Borno State is located between latitude 10 N and 13 N and longitude 12 and 15. It is in the north-easterly region of Nigeria, with an area of 70, 898 km 2. Borno State is divided into 27 local government areas (LGAs) with a total population of 4, 171, 104 [9]. The three seasons observed are: the cool dry (harmattan) season (October- March), hot dry season (April-June) and rainy season 79

2 (July-September). Temperatures are generally high all the year round, with hot season temperatures ranging between 39 C and 40 C under the shade. Average annual rainfall is between 500mm and 800mm. The variability of rainfall is over 100 per cent. The relative humidity is generally low throughout the state, ranging from as low as 13% in the driest months of February and March to the highest values of 78% in the rainy season months of July and August [10]. Sampling Samples of Beans (Phaseolus vulgaris) were collected in both field and storage facilities in six Local council areas (LGA): Bama Biu, Damboa, Gubio, Maiduguri Metropolitan, and Monguno, in Borno State. Sampling was conducted in two phases. Firstly, during the harvest periods (November - December 2008) from eighteen different farms (three each at the six LGAs) and secondly, six months post harvest period at storage and preservation facilities. The post harvest samples consists of samples not limited to the last farm harvests. Ten kilograms composites of each sample were collected in sterile poly bags on location basis. Determination The determination of pesticide residue followed the multiresidue pesticide analysis technique consisting of the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) sample preparation method by AOAC Method [11] with slight modifications for number of analytes spiking was adopted in the work. clean-up tube, capped tightly and vortexed for 30 seconds. After centrifuging for 5 min at 3,000 rpm, 200 μl of the clear extract were transferred to an auto-sampler vial with a small glass insert for analysis on the GC-ion trap MS. The individual calibration levels were spiked into each extract for the calibration curve in matrix before the final clean-up step. GC/MS Injection, Separation and Detection The SHIMADZU GC/MS (GC-17A), equipped with fluorescence detector was used for the chromatographic separation and was achieved by using a 35% diphenyl/65% dimethyl polysiloxane column. The oven was programmed as follows: initial temperature 40 C, 1.5 min, 25 C/min to 150 C, 0.0 min, 5 C/min to 200 C, 7.5 min, 25 C/min to 290 C with a final hold time of 12 min and a constant column flow rate of 1 ml/min. The detection of the pesticides was performed using the GCion trap MS with optional MSn mode. This scanning mode offered enhanced selectivity over either full scan or selected ion monitoring (SIM). The GC-ion trap MS was operated in the MSn mode and performed tandem MS functions by injecting ions into the ion trap and destabilizing matrix ions, isolating only the pesticide ions. The retention time, peak area and peak heights of the sample were compared with those of the standards for quantization. Data Analysis Results obtained were analysed on Microsoft Excel 2007 spreadsheet. Extraction A thoroughly homogenized 15 g sample of beans was weighed into an extraction tube. Then, 15 ml of 1% acetic acid-mecn extraction solvent was poured into the tube on top of the sample. The surrogate and the pesticide solutions were spiked into this MeCN layer for the method validation (MVD) and method detection limit (MDL) samples. The tube was capped and vortexed for 30 seconds. The cap was removed and the powder reagents were poured slowly into the MeCN layer. The cap was tightened securely on the 50-mL extraction tube and was vortexed for 30 seconds until all of the powder reagents were mixed with the liquid layers. The tube was placed on a mechanical shaker for 5 min and then centrifuged for 5 min at 3, 000 rpm. Following this, 11 ml of the top MeCN layer were removed and transferred to a 15-mL clean-up tube. This tube was capped and vortexed for 30 seconds and centrifuged for 5 min at 3,000 rpm. A 5-mL aliquot of the top layer was transferred into a clean test tube for solvent exchange. Solvent Exchange The 5-mL aliquot of the cleaned-up extract was blown down to dryness with a gentle stream of nitrogen at 40 C in approximately 1 hour. Care was taken to remove the tube immediately when dried. A 900-μL aliquot of hexane/acetone (9:1) was added and 100 μl of the internal standard (d10-parathion) was spiked into the organic solution. The tube was capped and vortexed for 15 seconds. Next, 1 ml of extract was transferred to a 2-mL 3. RESULTS An overall summary descriptive result of pesticides residues determined in the bean samples is presented in APPENDIX 1. The result shows that residues of eight analytes were determined. Three analytes (Dichlorvos, Endrin and DDT) were detected in both pre storage harvest samples and post storage harvest samples analysed. Another set of three analytes (Lindane, Diazinon and Aldrin) were only detected in the pre storage samples, while the remaining two analytes (Carbofuran and Dieldrin) were not detected at all. Figure-1 show the average DDT concentrations in the PRE (1.47 ± 1.08 µg/g) samples at the different location as generally higher than the POST (1.13 ± 0.83 µg/g). This trend in concentration variations across location was regular and consistently show that Damboa LGA was lowest in both PRE (0.93 ± 0.79 µg/g) and POST (0.83 ± 0.70 µg/g) DDT concentrations. The highest concentration of DDT was recorded in Gubio LGA in both PRE (2.63 ± 0.49 µg/g) and POST (1.98 ± 0.37 µg/g). The average concentration of Endrin in PRE and POST samples are 2.46 ± 1.85 µg/g and 2.15 ± 1.65 µg/g respectively. The variations of Endrin concentrations across locations are similar to DDT (Figure-2). The lowest concentrations of Endrin are recorded in Damboa, PRE (0.49 ± 0.18 µg/g) and POST (0.36 ± 0.16 µg/g), while the highest concentrations was recorded in Maiduguri LGA in both PRE (5.54 ± 1.23 µg/g) and POST (4.83 ± 1.11 µg/g). Converse to DDT and Endrin, the average concentration of 80

3 Dichlorvos in POST (0.262 ± µg/g) is much higher than concentrations in PRE (0.008 ± µg/g) samples. Figure-3. Pre and Post Storage Residue Concentration of DICHLORVOS in Bean Samples. Figure-1. Pre and Post Storage Residue Concentration of DDT in Bean Samples. Figure-4 shows the overall percentage difference of pesticide residues concentrations between the PRE and POST samples of beans. Dichlorvos increased by about 97% between PRE and POST samples analysed, while Endrin and DDT decreased by about 15% and 30% respectively. In general the order of concentration for these analytes in the beans samples is Endrin > DDT > Dichlorvos. Method validation for this study consisted of sample runs for spiked analytes quality control, which was maintained at ± 13% deviation. The percentage recoveries were in the range of %. Based on a confidence level of 95%, the uncertainty of measurement is within the acceptance criteria. Figure-2. Pre and Post Storage Residue Concentration of ENDRIN in Bean Samples. Figure-3 shows the variations of Endrin concentrations at the different locations. The lowest concentrations of Dichlorvos were consistently recorded in Biu LGA, PRE (0.003 ± µg/g) and POST (0.009 ± µg/g), while the highest concentrations were recorded in different locations, Gubio LGA for PRE (0.013 ± µg/g) and Maiduguri for POST (0.51 ± 0.30 µg/g). Figure-4. Percentage Variations of Pesticide Residues Concentrations between the PRE and POST samples. 4. DISCUSSIONS Organochlorines, organophosphates, carbamates and pyrethroids pesticides are the common and major classes of pesticides applied to food crops especially beans [12] in the field and at storage [13]. The eight pesticides, mainly as insecticides, determined in this study covered these 81

4 common and major classes of pesticides. However not all the insecticides of organochlorines (Aldrin, Endrin, DDT, Dieldrin and Lindane), organophosphates (Diazinon and Dichlorvos) and carbamates (Carbofuran) determined was detected in the samples. A number of factors such as non availability to farmers and or non application of these insecticides during the period of study, and also from low concentration levels below the limits of quantitation [14] may be responsible of the non detection of Carbofuran and Dieldrin in both the pre storage and post harvest beans samples analysed. However, Lindane, Diazinon and Aldrin were detected only in the pre storage bean samples, while DDT, Dichlorvos, and Endrin were present in both pre storage and in storage samples. Pesticides residues in crops at harvest may result from uptake by the plant of soil-applied pesticides or otherwise, translocation of pesticides applied to the plant before the edible part of the plant is formed. Residues at harvest from these circumstances are usually low and often below the limit of determination, but the majority of significant residues at harvest result from applications when the edible part of the plant is already present [4, 15]. DDT, Dichlorvos, and Endrin are obviously heavily utilized in this region due mainly to their easy availability and dependability. This is also reflected in the almost similar trend in their concentrations across the six sampling locations of the present study. However, the basic processes acting on pesticide residues in the field can continue to operate after crops are harvested. These include: volatilization, hydrolysis, penetration, metabolism, enzymatic transformation, oxidation and photodegradation, which generally ceases or is greatly reduced once a crop is removed from the field situation [16]. These were generally observed in trends of DDT and Endrin, but the higher accumulation of Endrin in beans samples may be due not only the volume of applications but also to the solubilities of Endrin [17] than DDT [18]. Residues of post harvest insecticide treatment on stored staples foodstuff generally decline only rather slowly [16] as the rate of reductions was rather low. The Organochlorines being chemically stable and persistent in the environment have been banned in most countries of the world but the less persistent classes are widely in use. Organophosphates are highly potent compounds used mainly as insecticides especially in the control of storage insects in food crops. They are very toxic and more often involved in acute poisoning than other classes of pesticides [19, 20]. The proliferation of Dichlorvos in this region has been well documented by [21], as a major component of otapiapia, a locally formulated insecticide for the preservation of foods stuffs. This may be responsible for the marked increases in Dichlorvos concentrations in the post storage samples. Foodstuff such as beans, corn and sorghum are frequently stored long term (3-36 months) at ambient temperatures in bulk silos where insecticides may be applied post-harvest to reduce losses from storage pests therefore have the potential to be a major source of residues in the diet for these insecticides [22]. Residues of pesticides in food are influenced by the storage, handling and processing that occurs between harvesting of raw agricultural commodities and consumption of prepared foodstuffs [16]. Residues of the more lipophilic materials tend to remain on the seed coat although a proportion can migrate through to the bran and germ which contain high levels of triglyceride. Also, storage fungi may assist in the degradation of insecticides [23]. The regulations for all controls on pesticide residues in crops are generally based on Maximum Residue Limits (MRL's) which are set using field trial data for a particular pesticide to arrive at the highest residue levels expected under use according to Good Agricultural Practice (GAP) [16, 24]. The organochlorines (Aldrin, Endrin, DDT, Dieldrin and Lindane) are banned products. However the allowable range for residues before then was from 0 to 0.1 ppm depending on the type of food product. For the organophosphates (Diazinon and Dichlorvos) the U.S. EPA has established maximum permissible levels of dichlorvos in various food products ranging from 0.02 to 2 parts per million (ppm) [17, 18, 25]. The average result of organophosphates in this study are within the MRL s and lower than the mean concentration range from 19.4 to 455.9μg/kg recorded in Lagos State [12]. This result also corresponds with the study reported in Brazil [26]. Beans as victuals are commonly consumed in Nigeria and around the world. The sensitization of populace to wash thoroughly before other forms of processing to reduce the pesticide residue content at the point of consumption residues [12] would greatly reduce the health hazards associated with pesticide residue levels in such foodstuffs, although, these insecticides were never completely eliminated by washing [12, 27]. Eradication of all streets hawking of locally adulterated, unregistered, unlabelled, repackaged, uncertified and expired chemical pesticides in the form such as Otapiapia among others [21] as well as the need for more stringent monitoring of importation and use of these pesticides in agriculture and food storage in Nigeria are required. 5. CONCLUSIONS a) This study revealed that DDT, Dichlorvos, and Endrin are heavily utilized in this region due mainly to their easy availability and dependability. b) Dichlorvos was observed to mainly be utilized in storage of beans from insect attack. c) Stringent monitoring of use of pesticides in agriculture and food storage in Nigeria is required ACKNOWLEDGEMENT The authors acknowledge the support of the Central Laboratory of the National Food Drug Administration and Control (NAFDAC), Lagos for supporting the analysis of the bean samples. REFERENCES [1] IUPAC Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled from (Glossary for chemists of terms used in toxicology (IUPAC Recommendations 1993) on Blackwell Scientific Publications, Oxford. p

5 [2] EC: European Communities Reproduction is authorized, except for commercial purposes, provided the source is acknowledged. Directorate-General for Health and Consumers European Commission B Brussels ISBN DOI / m. [3] Uygun U., Koksel H. and Atli A Residue levels of malathion and its metabolites and fenitrothion in post-harvest treated wheat during storage, milling and baking. Food Chem. 92, 643. [4] Tadeo J.L, Sánchez-Brunete C. and González L Analysis of pesticides in food and environmental samples, CRC Press Taylor and Francis Group. pp mites. Journal of Stored Products Research. 42: [14] CLSI Clinical and Laboratory Standards Institute Protocols for Determination of Limits of Detection and Limits of Quantitation, Approved Guidline, Wayne PA USA; CLSI Document EP17. [15] Bates J.A.R The Prediction of Pesticide Residues in Crops by the Optimum Use of Existing Data IUPAC Reports on Pesticides (26) Pure and Appl. Chem. 62(2): [16] Holland P. T., Hamilton D., Ohlin B. and Skidmore M. W Effects of Storage and Processing on Pesticide Residues in Plant Products, IUPAC Reports on Pesticides (31) Pure and Appl. Chem. 66(2): [5] NAFDAC: National Agency for Food and Drug Administration and Control Consumer Safety Bulletin, Banned/Restricted Chemicals in drugs, Agrochemicals, Industrial Chemicals and Household products. 2(3): [6] NAFDAC: National Agency for Food and Drug Administration and Control Consumer Safety Bulletin: NAFDAC Regulated Products. 6(1): 9. [7] IITA: International Institute of Tropical Agriculture Legume and cereal seed production for improved crop yields in Nigeria Proceedings of the Training Workshop on Production of Legume and Cereal Seeds. Edited by H.A. Ajeigbe, T. Abdoulaye, and D. Chikoye. [8] Awofadeji S Nigeria: Food Poisoning: How Many More Will Have to Die? This day online newspaper. Access date: February 23, [9] NPC: National Population Commission Federal Republic of Nigeria, Census Report Official Gazette. 94(4): B47-53, [10] Onlinenigria Borno State, Physical Setting. Community Portal of Nigeria. Online Nigeria, Accessed 10/12/2010. [11] Lehotay S AOAC Official Method Pesticide residues in foods by acetonitrile extraction and partitioning with magnesium sulfate. Journal of AOAC International. 90: [12] Ogah C.O., Coker H.B, Adepoju-Bello A.A Organophosphate and Carbamate Pesticide Residues in Beans from Markets in Lagos State, Nigeria. J. Innov. Res. Eng. Sci. 2(1): [13] Collins D. A A review of alternatives to organophosphorus compounds for the control storage [17] ATSDR: Agency for Toxic Substances and Disease Registry Public Health Toxicological Profile for Edrin U.S. Department of Health and Human Services Public Health Service. Division of Toxicology/Toxicology Information Branch 1600 Clifton Road NE, E-29 Atlanta, Georgia [18] ATSDR: Agency for Toxic Substances and Disease Registry Public Health Toxicological Profile for DDT, DDE, and DDD. U.S. Department of Health and Human Services Public Health Service. Division of Toxicology/Toxicology Information Branch 1600 Clifton Road NE, E-29 Atlanta, Georgia [19] Noble R.A., Hamilton D.J. and Osborne W.J Stability of pyre-throids on wheat in storage. Pestic. Sci. 11, [20] Mansour S.A Pesticide exposure - Egyptian scene. Toxicology. 198: [21] Musa U., Hati S. S., Mustapha A. and Magaji G Dichlorvos concentrations in locally formulated pesticide (Ota-piapia) utilized in northeastern Nigeria Scientific Research and Essay. 5(1): [22] Snelson J.T Grain Protectants. Australian Centre for International Research, Canberra. [23] Anderegg B.N. and Madisen L.J Degradation of 14C-Malathion in stored corn and wheat inoculated with Aspergilius glaucus, J. Econ. Entompl. 76: [24] Bates J.A.R. and Gorbach S Recommended approach to the appraisal of the risks to consumers from pesticides residues in crops and food commodities. Pure and Apl. C hem. 2, [25] ATSDR: Agency for Toxic Substances and Disease Registry Public Health Toxicological Profile for Dichlorvos U.S. Department of Health and Human Services Public Health Service. Division of 83

6 Toxicology/Toxicology Information Branch 1600 Clifton Road NE, E-29 Atlanta, Georgia [26] Ciscato C.H.P., Gebara A.B., Monteiro S.H., Souza G.S Pesticide Residues in Bean and Rice, a Basic Diet for Brazilian Consumers Pesticide Residues Laboratory (LRP) Biological Institute (IB), Sao Paulo, SP, Brazil. [27] Bhuiyan M.N.H., H.R. Bhuiyan, K. Ahmed, K.M. Dawlatana, F. Haque, M. Rahim and M.N.I. Bhuiyan Organochlorine insecticides (DDT and Heptachlor) in dry Fish: traditional washing and cooking effect on dietary intake. Bangladesh J. Pharmacol. 4: APPENDIX 1. Summary descriptive statistics for overall concentrations of pesticide residues in beans samples. Statistics Pesticides residue (µg/g) Dichlorvos-Pre Dichlorvos-Post Carbofuran Lindane -Pre Mean Standard Deviation Standard Error Median Sample Variance Kurtosis Skewness Range Minimum Maximum Diazinon -Pre Aldrin-Pre Dieldrin Endrin-Pre Endrin-Post DDT-Pre DDT-Post 84

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