INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 5, Copyright by the authors - Licensee IPA- Under Creative Commons license 3.

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1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 5, 2013 Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0 Research article ISSN Studies on residues of insecticide used to spray pineapples grown at Samsam in the Amasaman District (Ghana) Tordzagla Nestor, Adosraku K. Reimmel and Okine N.A. Nathaniel Department of pharmaceutical chemistry, Faculty of Pharmacy and Pharmaceutical sciences, Kwame Nkrumah University of Science and Technology, Ghana tordzaglanestor@yahoo.com doi: /ijes ABSTRACT The residues resulting from the misuse of pesticides on fruits are a major concern in many countries as well as in Ghana.On the other, the use of pesticides is one of the important measures of modern agricultural practices in protecting the crops from different pests. However, the hazards to health can be minimized to a great extent if these residues are kept below their prescribed safe level. A field survey was conducted at Samsam in Amasaman District regarding the type of insecticide used by local farmers on pineapples.in order to assess the residue level of insecticide in fresh fruits; samples were collected at harvest from the fields of farmers at two sites. The analysis of insecticide was limited to the treatment history obtained from the fruit growers. The blended epicarps and mesocarps were extracted with ethyl acetate. The extracts were cleaned up (purified) by using florisil adsorption column chromatographic method. The purified extracts from epicarps and mesocarps were concentrated and analysed separately by a reversed phase HPLC isocratic method with 60% methanol and 40% water mixture as the mobile phase. Only two out of 20 samples (10 epicarps and 10 mesocarps) were contaminated with certain levels of chlorpyrifos and the residues levels were above European Commision (E.C.) Maximum Residue limit (MRL) set (2006) for pineapple. The MRL set by European Commission (2006) is 0.05mg/kg but the residue levels detected were 0.18mg/kg and 0.13mg/kg representing 10% of sampled pineapples analysed. Keywords: HPLC, Chlorpyrifos, pineapple, insecticide residues, Ghana. 1. Introduction The use of insecticides has increased over time in Ghana and is particularly elevated in the production of high value cash crops and vegetables (Gerken et al. 2001). Pineapple (Ananas cosmosus) is a rosette plant with spiky leaves, about a metre in height. The fruit is rich in vitamins A and C. Pineapple production is increasing in Ghana and it is currently one of the top five major non- traditional export crops in Ghana. About 71,804 tonnes of pineapple was exported in 2004, but this declined to 46,694 tonnes in 2005 (Ofori, 2007). The major pineapple producing areas in Ghana include the Eastern, Central and Greater Accra regions.mealybug wilt of pineapple caused by microsplama and transmitted by mealybugs is a major constraint to pineapple production. A nematode complex of several species of rootknot nematodes often limit fruit production and may require soil fumigation before planting. A few fruit-boring caterpillars and maggots also sometimes attack the fruits (Ofori, 2007). The pests can be controlled by the use of pesticides (insecticides). Commercial neem insecticides have shown to be effective against pineapple mealybugs in Ghana. Moreover, Received on January 2013 Published on April

2 dipping the planting material such as the slips in insecticide solutions and stacking them vertically for 24 hours to allow the insecticide to accumulate at the leaf bases before planting helps to prevent pineapple from being attacked by insects or pests. In severe infestation, the base of the plant should be sprayed with any appropriate organophosphorus insecticide such as chlorpyrifos, diazinon, parathion, dimethoate and bromophos (Ofori, 2007). Agricultural insecticides can be classified as organosphosphates, carbamates, pyrethroids and organochlorine. However, organochlorine insecticides have been banned because of their toxicity persistence and bioaccumulation in the environment (Molto et al., 1991; Heitefusset al., 1989). The organophosphate insecticides have considerable advantages such as good efficiency against sucking insects, rapid penetrative potential into plants (fruits) and systemic action (Heitefusset al., 1989). Some of the organophosphate insecticides are chlorpyrifos, parathion, diazinon, bromophos, malathion, chlorfenvinphos, trichlorphon. The insecticide use in Ghana is well below the level of the developed countries, but due to ineffective legislation, little awareness of harmful effects and technical know-how among farming communities, insecticide usage is not being properly regulated. The injudicious and indiscriminate application of insecticides to crops result in residues in food and food commodities with consequential hazards. The extent of hazard depends on the amount of insecticide residue on crops and their toxicity. Since most insecticides are toxic in nature, their continuous ingestion by man even in trace amounts, can result in accumulation in body tissues with serious adverse effects on health (Handa et al., 1999).Considering the food losses caused by insect pests, it is not feasible to completely dispense with the application of insecticides. However, the use of insecticides can be regulated to ensure minimum residues on food, which can be considered safe for human consumption and for the environment, as well. Every insecticide has a withholding period, waiting period, lapse period or pre-harvest interval which is defined as the number of days required to lapse between the date of final insecticide application and harvest, for residues to fall below the tolerance level established for that crop or for a similar food type. The pre-harvest period differs from insecticide to insecticide and from crop to crop. Food products become safe for consumption only after withholding period has lapsed. By this time the insecticide residues get dissipated. However, the extent and rate of dissipation depends on the nature of the insecticide, crop, cultural practices and various environmental conditions under which the crop is grown or a treated commodity is stored (Handaet al., 1999). Various chemical techniques can be used to determine pesticide residues but the most popular methods for this type of analysis are gas chromatography (G.C.) and liquid chromatography (LC) combined with UV or florescence detector. Also, mass spectrometry (MS) has been used as one of the methods of analysis in multiresidue determinations. However, thin layer chromatography and spectrometric methods had been widely used in the 1960s and 1970s for pesticide residue analysis, but have been used only to limited extent since gas-liquid chromatography(glc) and high performance liquid chromatography (HPLC) became readily available (Sherma, 1997; 1999; and 2000) and (Horwitz and Latimer, 2005). A diet rich in fruits or vegetables is thought to reduce the risk of some types of cancer and heart diseases. However, commercially grown produce often contains high levels of insecticide residues that can lead to serious health problems when consumed. This could be attributed to local farming practices concerning application of insecticides and subsequent harvest of treated crops as well as the behaviour of insecticides in different environments. The detection and identification of pesticides in our environment is a problem of increasing public interest (Agnihotri et al; 1974; Burns et al; 1974). Hence there is a need to investigate insecticide residues in some fruits. 1578

3 2. Materials and methodology 2.1 Materials: Instrumentation Shimadzu LC-6A Liquid chromatograph, Applied 783A Programmable Absorbance, SGE 100µl Biosystems, UV Shimadzu CR 501 Chromatopac, Reversed-phase Column (ODS- 1317), 140mm 4.6mm internal diameter Quartz cuvette, Buchi Rotavapour R-114,Buchi Vacuum Pump V-700, Buchi water bath B-480, 10ml, 25ml, 50ml, 100ml, 200ml volumetric flasks,flat-bottom flasks,round-bottom flasks,beakers, Electrothermal Melting Point Apparatus,Libror EB-430Hw Shamzu Capacity g, Pipettes 0.1ml, 1ml, 2ml, 5ml, 10ml, Measuring Cylinders: 50ml. 100ml, 500ml, Conical flasks, funnels, sinta glass, Column for absorption column chromatography,ph-meter: Eutech instruments ph510, High speed blender, knife,chopping board,mechanical shaker. 2.2 Materials: Reagents and chemicals Ethyl acetate (BDH Laboratory Reagents), Methanol (Fisons Scientific Equipment),florisil (ALDRICH Chemicals), Activated charcoal (ALDRICH Chemicals), Anhydrous sodium sulphate (ALDRICH Chemicals),Chlorpyrifos, Hexane (BDH Laboratory Reagents), Chloroform (BDH Laboratory Reagents), Dichloromethane (BDH Laboratory Reagents), Acetone (BDH Laboratory Reagents). 3. Methodology Figure 1: Chemical structure of chlorpyrifos 3.1 Field Survey to identify insecticides used to spray pineapples The field survey included collection of information from farmers through interviews. Samsam in Amasaman district was selected for the sampling. Only farmers actively involved in pineapple farming were interviewed. Total of 62 pineapple farmers were interviewed. Questionnaires were developed and administered to the fruit growers. On the basis of answers obtained from the questionnaires, some farmers were interviewed and the following details were obtained. These include the types of insecticides used, the frequency and amounts of insecticide application. Others are the time of insecticide application, harvest time, time of sale of treated crops and also the contacts they make with insecticide dealers, researchers, extension workers, in order to make decisions regarding the use of insecticides. Before conducting an interview, the objective of the study was briefly explained to the respondents highlighting the need and importance. 3.2 Sampling of fruits for pesticide residues 1579

4 During the field survey, fresh samples of fruits and vegetables were collected at harvest from farmers fields to assess the residue levels of insecticides on these crops before their release into market. Fresh samples of fruits were collected from each grower s field. A total of 15 fruits (samples) were collected. The fruits (samples) were transported to the laboratory immediately after collection and analysed. Samples were prepared as described by Codex Alimentarius (FAO/WHO, 1993). Analysis of insecticide residues was mostly limited to the treatment history obtained from farmers in the survey. 3.3 HPLC conditions and systems Mobile Phase: A mixture of 60% Methanol and 40% Water, Wavelength of detection = 254nm ph of mobile phase = 7.13 Flow rate =1.5ml/min Injection volume = 20µl Column: Reversed-phase (C18) Temperature: ambient temperature Chart recorder speed: 5ml/min Sensitivity: Attenuation: Calibration curve for pure Chlorpyrifos Concentrations of g/l, g/l g/l, g/l, g/l, g/l were prepared. The resulting solutions were filtered and some of the filtrates were taken from each and were injected into the column one after another and their retention times recorded. The Chlorpyrifos sample was analysed at wavelength of 254nm. 3.5 Extraction procedure and clean-up The fruits were peeled and the epicarps were separated from the mesocarps. Each portion of the epicarp or mesocarp was chopped with a sharp knife and mixed thoroughly. The chopped sample was transferred in a high-speed blender; and was thoroughly blended to obtain a homogeneous representative sample for weighing. 100g of sample was weighed into a 250ml flat-bottom flask for extraction, 150ml ethyl acetate and 50g anhydrous sodium sulphate were added to the content of the flask and shaken for 1hour at 60cycles/min in a horizontal shaker. The ethyl acetate extract was filtered. In order to achieve the sensitivity required for the analysis, the extract of insecticide residues was cleaned up or purified to remove any interfering substances co-extracted with insecticide residues. Column adsorption chromatography technique using florisil was employed. 29cm x 34 mm chromatographic column was used for the clean-up. The column was filled with florisil (15g) that had been activated at a temperature of 100 o C for 12 hours, 2g of activated charcoal and 5g of anhydrous sodium sulphate were added one after the other. Prior to loading the extract on the adsorbents, the column containing adsorbents was washed with ethyl acetate to make the adsorbents compact. After preparation of the column, the extract (sample) was transferred quantitatively to the column. The eluate was collected and filtered. After filtering, the ethyl acetate extract was poured into 250ml round flask and the rotary evaporator was set at 40 0 C and used to evaporate and concentrate the extract to dryness. The dried extract was redissolved in the mobile phase,volume made up to up 400µl and chlorpyrifos residue then quantified using HPLC technique. 3.6 HPLC analysis of extracts HPLC analysis was performed in isocratic system using LC-6A shimadze liquid chromatograph pump, syringe loading sample injector fitted with external 20ul loop. Applied biosystem 783A programmable absorbance UV detector, and integrator shimadzu CR

5 chromatopac. The stationary phase used was HPLC column containing silica bonded to octadecylsilyl (ODS) groups (ODS-1317, length 140mm x 4.6 mm internal diameters). A mixture of methanol 60% and water 40% was used as a mobile phase. Prior to use, the mobile phase as well as the extract was filtered. The mobile phase was first loaded into sample injector, and then later the extracts were also loaded. The response was reproducible and coextracts in the samples showed less interfering peaks under UV detection. Before analyzing the sampled pineapples, the HPLC method was validated using chromatographic parameters including recovery, repeatability, reproducibility, and linearity, limit of detection (LOD) and limit of quantitation (LOQ).The two parameters (LOD and LOQ) were calculated by the use of standard deviation of the calibration line by determining the slope and standard deviation (SD) of the response. Thus LOD = (3.3σ) / S and LOQ = (10σ) / S Where σ = standard deviation of the response. S = Slope of the calibration graph. For recovery experiments, untreated pineapples were spiked with a known concentration of chlorpyrifos. For each fortification level, five replicates were analyzed by HPLC using UV detector. The results were found to be satisfactory. 4. Results and discussion 4.1 Field survey (Insecticides used to Spray Pineapples) The field survey that was done at Samsam in Amasaman District to identify insecticides used to spray pineapple indicates that Chlorpyrifos is the most commonly used insecticide representing 80.6%, thus out of 62 pineapple farmers that were interviewed 50 farmers used Chlorpyrifos, 7 used Diazinon, 3 used Dimethoate and 2 used Lambda-cyhalothrin. According to the farmers who use chlorpyrifos, it is a broad-spectrum organophosphate insecticide that could be used to spray other crops apart from pineapples. It has good efficiency in killing sucking insects and mites, rapid penetrative potential into the plant but rapid breakdown of the organic compounds in it, thereby reducing residual effects. 4.2 Recovery studies by using HPLC The percentage recoveries of five pineapples each of which was fortified with g/l of pure chlorpyrifos for five different durations gave the following results. The extracts from Epicarps showed higher percentage recoveries than their corresponding extracts from Mesocarps. The higher recoveries of extracts from epicarps are due to many factors. The large amount of insecticide that was retained by epicarps could be attributed to the thickness of the epicarps, also the epicarps were in direct contact with the insecticide during its application. Also, day 1 recovery of chlorpyrifos in extract from epicarp was 84.8% which is less than 100% even though the recovery was good, it could have been higher than this but the probability of the insecticide being oxidized due to solvent evaporation direct to air also exists and this could be a factor. According to E.C. Maximum Residue Limits (MRLs) established (2006) for chlorpyrifos in pineapple is 0.05mg/kg, but in the control analysis which lasted for 14 days, the last day gave MRL for extract from epicarp to be high than its corresponding mesocarp.the residue from the mesocarp was calculated to be 0.04mg/kg.This indicates that on the day 14 the chlorpyrifos residue in mesocarp was less than E.C. Maximum Residue Limit of 0.05mg/kg, hence the waiting or withholding period of 14 days as stated by the manufacturer is appropriate for pineapples, hence it is safe for consumption provided required dosage of insecticide is applied. Moreover, out of 10 other 1581

6 pineapples collected from the farmers ' fields representing 20 samples (10 epicarps and 10 mesocarps) only in samples Ep2 and Ep4 that some levels of chlorpyrifos residues were detected. The levels detected were 0.18mg/kg and 0.13mg/kg for samples Ep2 and Ep4 respectively. This result showed that all the edible parts of pineapples analysed were safe for consumption. However, two epicarps(ep2 and Ep4) contained residues levels of 0.18mg/kg and 0.13mg/kg respectively. These residues were higher than MRL established by E.C. (2006). In all the samples analysed showed low percentage levels of residual insecticide in the fruits and this could be attributed to many factors. There is a probability of the insecticide being washed away by rainfall during the time the fruits were on the farm after they have been sprayed by chlorpyrifos. The probability of insecticide being oxidized due to direct interaction with air also exists. Finally, there is also a probability of part of the insecticide being broken down turning intodifferent by-products. This supports the finding that organophosphate insecticides have rapid break-down into some other compounds, making residual effects less (Heitefuss et al. 1989). Figure 2: Percentage Recovery of Chlorpyrifos from the Extracts of Epicarp Broken-down chlorpyrifos peaks Figure 3: Chromatogram of extract from fortified mesocarp with pure chlorpyrifos Table 1: Analysis of Pineapples Sprayed by Farmers using Chlorpyrifos (mesocarps) 1582

7 Number Chlorpyrifos peak height (cm) Peak area (cm 2 ) Amount of insecticide residue (mg/kg) M1 Nil Nil Nil M2 Nil Nil Nil M3 Nil Nil Nil M4 Nil Nil Nil M5 Nil Nil Nil M6 Nil Nil Nil M7 Nil Nil Nil M8 Nil Nil Nil M9 Nil Nil Nil M10 Nil Nil Nil Where Nil means no chlorpyrifos residue detected; M represents mesocarp Table 2: Results of Extracts from Epicarps Analysis E.C (2006) MRL=(0.0 5 mg/kg Number Chlorpyrifo s peak height (cm) Peak area (cm 2 ) Amount of insecticide residue (mg/kg) Ep1 Nil Nil Nil E.C (2006) MRL= (0.05mg/kg) Ep Ep3 Nil Nil Nil Ep Ep5 Nil Nil Nil Ep6 Nil Nil Nil Ep7 Nil Nil Nil Ep8 Nil Nil Nil Ep9 Nil Nil Nil Ep10 Nil Nil Nil Nil means no chlorpyrifos residue detected Ep represents Epicarp 4.3 Validation of method The method used for analysis was simple, fast, precise, accurate, reproducible and repeatable. The calibration curve of chlorpyrifos gave a linear correlation of the standard solution prepared. The aim of checking linearity is to derive a direct proportionally between the detector signal and concentration of the substance in the sample over a certain range. The regression coefficient of calibration curve of pure chlorpyrifos is r 2 = This is a good result in that a good regression coefficient must be This implies the value determined for regression coefficient met ICH standard. 1583

8 Figure 4: Chromatogram of pure chlorpyrifos: Retention time: 3.96 minutes Figure 5: Calibration Curve for Pure Chlorpyrifos Sample (peak area in cm 2 against concentration in g/ml) The limit of detection (LOD) and limit of quantitation (LOQ) were calculated statistically.lod and LOQ are based on the slope and standard deviation (SD) of the response LOD = (3.3 δ) / S where, S = slope of the calibration graph. δ = SD of the response= the residual standard deviation (Sres) of a regression line. LOD = (3.3 δ) / S = 0.122mg/l LOQ = (10 δ) / S = 0.368mg/l Repeatability (precision within- run) was calculated statistically. The values of standard deviation for concentrations g/l, g/l and g/l are , and respectively. Also, relative standard deviations RSD for above standard deviations are 1.69%, 1.40%, and 1.14% respectively. These values met Gomez, R. et al.,( 2009) standard. It states that relative standard deviation is determined using 5 injections if the requirement is 2.0%. Reproducibility (precision between-run) was calculated statistically. Two different concentrations were used and each concentration was run for three days by three different analysts. 1584

9 Table 3: Reproducibility of Method Results Concentration g/l g/l Day(s) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Mean ( ) Standard Deviation (SD) RSD (%) These results also met Gomez, R. et al., (2009) standard. It states that relative standard deviation (RSD) is determined using 5 injections if the requirement is 2%. 5. Conclusion The insecticides used to spray pineapples at Samsam were found to be Chlorpyrifos, diazinon, dimethoate and lambda-cyhalothrin. Most frequently used insecticide was chlorpyrifos representing 80.6% of the farmers; however lambda-cyhalothrin was the least frequently used insecticide representing 3.2% of the farmers. Analysis done by using HPLC gave good recoveries of the insecticide from various extracts analysed. The percentage recovery of 84.8% was observed for extract Ep1 used in control analysis. This result indicates good performance of extraction, clean-up and chromatographic parameters for the analysis of insecticide residues in the fruits. Out of total of 20 samples analysed only 10% of the samples were contaminated with chlorpyrifos residues. The residues level detected in them were above maximum residue limits established by European Commission (2006) for pineapples. These samples could pose health hazards to the consumers. Picking of fruits without taking into account their withholding periods may lead to residues higher than the tolerance limits. Fruits and other crops should be subjected to a random inspection in order to monitor and regulate the use of insecticides and other pesticides and products found to contain residue levels above the MRL should be discarded. In view of an increasing trend in insecticide use in Ghana, continuous monitoring for insecticides and other pesticides residues is needed in crops especially fruits in order to protect the end user from health hazards involved in the misuse of insecticides.. Acknowledgement We wish to extend thanks to both the academic and technical staff of the Department of Pharmaceutical Chemistry, K.N.U.S.T for their support throughout the research work. We are also thankful to the pineapple farmers at Samsam for their co-operation. 1585

10 6. References 1. Agnihotri N.P; Dewan R.S; Dixit A.K, (1974), Residue of insecticides in food commodities in food from Delhi, Vegetables, Indian J. Entromal, 36, pp Burns J.E, Miller F.M, Gomes E, Albert R, (1974), Hexachlorobenzene exposure from contaminated DCPA in Vegetables Spraymen, Arch Environ Health, 29, pp European Commission, amending Regulation (EC), (2006), No. 396/2005 of theeuropean Parliament and of the Council to establish Annex 1 listing the food and feed products to which maximum levels for pesticides residues apply. Off. J.E.U. 4. FAO/WHO, Codex Alimentarius, Codex, (1993), Committee on Pesticide Residues, Rome,Supplement 1, Volume Gerken, A. J. V; Suglo and M. Braun, (2001), Crop protection policy in Ghana.Pokuase Accra. Intergrated crop protection project, pp Gomez, R; Hiob, M.; Kunzle, J.; Schreiber, B. and Seyfarth, H, (2009), Laboratory and Analytical Controls, Excerpt from GMP Manual, pp Handa, S.K.; Agnihotri, N.P. and Kulshrestha, G, (1999), Pesticides residues; significance management and analysis, research periodicals and book publishing home; Texas, USA, Hardcover 226p. 8. Heitefuss, R.; Welch, J. and Francis, J, (1989), Crop and plant protection, The practical foundations, pp Horwitz, W. and Latimer, G. W, (2005) Official methods of analysis of AOAC International.18th Edition.Chapter Molto, J.C.; Pico, Y.; Font, G. and Manes, J, (1991), Determination of triazines and organophosphorus pesticides in water samples using solid phase extraction, Journal of Chromatography analysis, 55(5), pp Ofori, D.O, (2007), Major pests of food and selected fruits and industrial crops in West Africa, pp and Sherma, J, (1999), Recent advances in thin-layer chromatography and pesticides, Journal of AOAC International, 82, pp Sherma, J, (2000), Thin-layer chromatography in food and agricultural analysis, Journal of chromatography analysis.880, pp Sherma, J, (1997), Current status of pesticide residue analysis Journal of AOAC International, 80, pp