Contamination levels of organophosphorus and synthetic pyrethroid pesticides in cocoa beans from Ghana

Food Control xxx (2016) 1e8

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Contamination levels of organophosphorus and synthetic pyrethroid pesticides in cocoa beans from Ghana Elvis D. Okoffo, Benedicta Y. Fosu-Mensah*, Christopher Gordon Institute for Environment and Sanitation Studies (IESS), University of Ghana, P.O. Box 209, Legon, Accra, Ghana

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 August 2016 Received in revised form 12 October 2016 Accepted 1 November 2016 Available online xxx

The concentrations of pesticide residues in fermented dried cocoa beans were assessed from sixteen (16) selected cocoa farms in the Dormaa West District in the Brong Ahafo Region of Ghana. The samples were extracted and analysed for 13 organophosphorus and 9 synthetic pyrethroid pesticide residues using a partially modified multi-residue method for agricultural chemicals by a Varian CP-3800 Gas Chromatograph equipped with a pulse flame photometric detector (PFPD) and 63Ni electron capture detector (ECD), respectively. The study revealed the presence of three organophosphorus pesticide residues namely; chlorpyrifos (0.04 mg/kg), pirimiphos-methyl (0.03 mg/kg) and diazinon (0.01 mg/kg), with chlorpyrifos occurring most frequently, and six synthetic pyrethroid pesticide residues namely; allethrin (0.01 mg/kg), lambda-cyhalothrin (0.03 mg/kg), deltamethrin (0.03 mg/kg), cypermethrin (0.04 mg/kg), bifenthrin (0.02 mg/kg) and permethrin (0.02 mg/kg), with cypermethrin occurring most frequently. None of the detected pesticides’ average residues did exceed their European Union Maximum Residue Limits for cocoa beans. Considering levels of pesticide residues in the fermented dried cocoa beans analysed against the European (EU) commissions’ regulations on pesticides residues, cocoa beans from the study area will not pose any significant threat to the cocoa industry in Ghana as far as shipment to Europe is concern. The presence of organophosphorus and synthetic pyrethroid pesticide residues in the cocoa bean samples analysed could be attributed to their usage by cocoa farmers in the study area. The routine monitoring of pesticide residues in the study area is necessary for the prevention, control and reduction of environmental pollution, so as to minimize health risks. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Cocoa beans Contamination levels Pesticide residues Organophosphorus Synthetic pyrethroids Ghana Health risks

1. Introduction Agriculture is the mainstay of Ghana’s economy, providing the major source of employment and income. Tree crops such as cocoa, coffee, oil palm, cashew and rubber dominates Ghana’s agricultural sector (Institute of Statistical Social and Economic Research (ISSER), 2012; Danso-Abbeam, Addai, & Ehiakpor, 2012). However, the most dominant cash crop in the agricultural sector has been cocoa, (Theobroma cacao). Over 70% of the world’s cocoa is produced in West Africa, with Ghana producing about 21% (Afrane & Ntiamoah, 2011). Cocoa production is an important source of livelihood for many smallholder producers in Ghana. For instance, the cocoa sector in Ghana employs over eight hundred thousand (800,000) smallholder farm families and contributes about 70e100% of their

* Corresponding author. E-mail addresses: [email protected] (E.D. Okoffo), [email protected]. edu.gh (B.Y. Fosu-Mensah), [email protected] (C. Gordon).

annual household income (Anim-Kwapong & Frimpong, 2004; Appiah, 2004, p. 32; Danso-Abbeam, Setsoafia, Gershon, & Ansah, 2014). In spite of the significant contribution of cocoa production to the economy of Ghana, the crop has been confronted with a lot of challenges like pests and diseases infestation over the years, which have contributed to Ghana losing its position as the leading cocoa producer in the world (Anim-Kwapong & Frimpong, 2004). Cocoa is easily attacked by insect pest and disease such as cocoa mirids (capsids) locally known in Ghana as ‘akate’, the Phytophthora pod rot, commonly called ‘black pod’ and the swollen shoot virus, also known locally as ‘cocoa sasabro’ (Afrane & Ntiamoah, 2011; Ayenor, €ling, 2007; Dormon, Huis, & Leeuwis, Huis, Obeng-Ofori, Padi, & Ro 2007). Duguma, Gockowski, and Bakala (1998), Lass (2004) and Dormon et al. (2007) estimates losses by insect pests and diseases to be 30% of global yields of cocoa annually, whereas site-specific losses can range from 10 to 80% annually. In order to reduce the incidence of insect pests and diseases and to improve cocoa yields, the applications of synthetic pesticides

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were introduced. However, along with the positive effects of the use of pesticides, some negative impacts on the environment and uniquely their replicating effect on human health have also been caused (Dankyi, Gordon, Carboo, & Fomsgaard, 2014; Essumang, Togoh, & Chokky, 2009). For instance, pesticides in the environment have the potential to cause toxicity to plants, their product and contaminate the food chain when taken up by plant roots from soils and nutrients solutions. In addition, pesticides used on farms can cause destruction to soil flora and fauna and contaminate sources of drinking water through leaching and surface runoffs. Human beings are exposed to the harmful effect of these chemical when contaminated food and drinking water are consumed (Bempah, Buah-Kwofie, Denutsui, Asomaning, & Osei-Tutu, 2011a; Bempah, Donkor, Yeboah, Dubey, & Osei-Fosu, 2011b; William et al., 2008). Pesticide residues exposure have been reported to affect thyroid function, cause low sperm count in males, birth defects, increase in testicular cancer, reproductive and immune malfunction, endocrine disruptions, cancers, immunotoxicity, neurobehavioral and developmental disorders (Cocco et al., 2013; Gill & ^ mois, & Seralini, 2010; Garg, 2014; Mesnage, Clair, de Vendo Tanner et al., 2011). Many cocoa consuming countries have expressed their concerns regarding the health risks associated with the use of pesticides in cocoa production. This has led to the prescription of tolerance Maximum Residue Limits (MRLs), and Acceptable Daily Intake (ADI) as well as No Observable Adverse Effect Level (NOAEL) for various pesticide residues in food (including cocoa beans) especially by the Codex Alimentarius Commission (CODEX), the World Health Organisation (WHO) (Sosan, Akingbohungbe, Ojo, & Durosinmi, 2008) and other cocoa importing countries and authorities (Bateman, 2009). In view of the strict enforcement of the MRLs harmonization, export of cocoa beans from Ghana in particular to its main partners in Europe and Asia are liable to possible rejections if prohibited substances (including pesticide residues) are found in the product or at levels above the MRLs. The rejection of cocoa beans as a result of high levels of prohibited substances will threaten the livelihood of smallholder farmers, thereby worsening unemployment and poverty as well as the foreign income earnings of the country. There is therefore the pressing need for the control and monitoring of pesticides use in cocoa production in Ghana. The Brong Ahafo region is one of the major cocoa producing regions in Ghana. Farmers in this region use pesticides extensively, particularly, organophosphorus and synthetic pyrethroids to control insect pests and diseases in order to increase cocoa yields (Denkyirah et al., 2016; Fosu-Mensah, Okoffo, & Mensah, 2016; Fosu-Mensah, Okoffo, Darko, & Gordon, 2016a,2016b; Okoffo, FosuMensah, & Gordon, 2016; Okoffo, Mensah, & Fosu-Mensah, 2016). Unfortunately, there is limited data and studies on organophosphorus and synthetic pyrethroid residue levels in quality cocoa beans produced from farms. The objective of this paper is to evaluate the residual concentrations of organophosphorus and synthetic pyrethroids in cocoa beans produced from the Dormaa West District (which is known to be one of the major cocoa producing Districts in the Brong Ahafo Region of Ghana), and to assess the levels of the detected pesticide residues against their respective European Union (EU) MRLs for cocoa beans. 2. Materials and methods 2.1. Study area The study was carried out in the Dormaa West District located at the western part of the Brong Ahafo Region of Ghana with slightly hilly terrain (240e300 m above sea level). It has boundaries in the north by the Dormaa Central Municipality, in the east by Asunafo

North Municipality, in the west by Cote d’Ivoire and in the south west by Bia East District (Fig. 1). It has a population of 47, 678, comprising of 24, 681 (51.8%) males and 22, 997 (48.2%) females (Ghana Statistical Service (GSS), 2014). It lies in the sub-humid zone (with annual total rainfall of 800 mme1200 mm) and has a bimodal rainfall regime. The highest mean temperature of the District is about 30
C which occurs between March and April and the lowest about 26.1
C in August. The climatic condition of the study area is suitable for the cultivation of various cash crops such as cocoa, coffee and food crops such as plantain, cocoyam and cassava. Farmers farm lands varied from small (0.5 ha) and spatially dispersed parcels to much greater plots (10 ha) due to land fragmentation of the arable land. The district is generally an agrarian economy which contributes immensely to the food basket of the country. Agriculture is the main source of employment (82%) in the district. The major economic activities in the district include; the cultivation of food and cash crops (including cocoa), poultry and livestock farming, oil palm extraction, cassava processing and sand winning (Ghana Statistical Service (GSS), 2014). Soils in the District belong to the Bekwai-Nzema compound Associations. The soil types within the study area support cultivation of both commercial and domestic food crops, which include cocoa, coffee, oil palm, citrus, cola-nuts, plantain, cassava and maize. The area is well drained as evidenced by the network of rivers spread out within the district. The rivers are mostly perennial due to the double maxima rainfall, which is experienced in the area. Notable among them are the Bia, Nkasapim and Pamu rivers. These rivers are mostly used as a source of water for the cultivation of vegetables such as tomatoes, pepper and okra during the dry season. There are however, traditional restrictions on the use of the rivers for fishing (Ghana Statistical Service (GSS), 2014). 2.2. Chemicals and reagents used Certified reference standards of methamidophos, ethoprophos, phorate, diazinon, fonofos, dimethoate, pirimiphos-methyl, chlorpyrifos, malathion, fenitrothion, parathion, chlorfenvinphos, profenofos, allethrin, bifenthrin, fenpropathrin, lambda-cyhalothrin, permethrin, cyfluthrin, cypermethrin, fenvalerate and deltamethrin which had a purity of 98e100% were purchased from Dr. Ehrenstorfer GmbH, (Augsburg, Germany). Pesticide grade acetonitrile was obtained from BDH, England. Analytical grade acetone, ethyl acetate, anhydrous sodium sulphate (Na2SO4) and sodium chloride (NaCl) were purchased from BDH, England. Envi-carb/LCNH2 (graphite carbon/aminopropylsilanized silica gel mini column) (500 mg/500 mg/6 ml) and Bond elute C-18 (octadecylsilanized silica gel mini column) (1000 mg/6 ml) Solid phase extraction (SPE) cartridges were purchased from Supelco Inc, USA and Phenomenex, USA, respectively. 2.3. Pesticide solution Pesticides stock solutions (1000 mg/ml) of each of the certified reference organophosphorus and synthetic pyrethroid pesticides standards were prepared by pipetting the appropriate aliquot or weight of the certified reference pesticide into a 25 ml volumetric flasks, and then dissolving and diluting to the mark with ethyl acetate with the aid of a vortex mixer (Thermolyne Maxi Mix-Plus). Stock solutions were stored in a freezer at e 18
C in ample bottles. Working solutions of the pesticides standards for use as fortification standards in the procedural recovery process, and as calibration standards in the instrument calibration were freshly prepared through the dilution of an appropriate aliquot of the stock solutions with ethyl acetate.

Please cite this article in press as: Okoffo, E. D., et al., Contamination levels of organophosphorus and synthetic pyrethroid pesticides in cocoa beans from Ghana, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.11.004

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Fig. 1. Map of Dormaa West District showing selected communities.

2.4. Sampling and preparation of cocoa beans This study was conducted during the months of December 2014 to February 2015. Four cocoa growing communities namely; Nkrankwanta, Diabaa, Krakrom and Kwakuanya as shown in Fig. 1 were randomly selected from the district (study area) and coded as sites S1, S2, S3 and S4, respectively. Within each selected community, four cocoa farms were identified and selected. A total of sixteen (16) cocoa farms were selected with the age of farm (farm not less than 8 years and not more than 20 years with a history of at least five years of pesticides application) and the density of cocoa production being the determining factors. Each selected cocoa farm was divided into two quadrats (80  80 m). For each quadrat, five (5) cocoa trees were randomly selected, where five (5) matured and ripped cocoa pods were randomly taken and kept in labelled bags. The pods were transported to the laboratory [Ecological Laboratory (ECOLAB), of University of Ghana, Legon] where they were broken with wooden sticks and the cocoa beans fermented for seven days. The fermented beans were then sun-dried for 21 days. The dried cocoa

beans from each quadrat of a farm were then bulked together to form a composite sample. The composite cocoa bean samples were well mixed and 2 kg sub-samples were taken into clean labelled polythene bags and transported to the Ghana Standards Authority Pesticides Residue Laboratory in Accra, Ghana for analysis. Two cocoa bean replicates were collected from each cocoa farm. These gave a total of 32 cocoa bean samples from the study area. Prior to analysis, each labelled fermented dried cocoa bean (2 kg) was thoroughly grounded into fine powder and collected into a new sample bag and stored in a freezer at 18
C until extraction. Approximately 10 g of the samples were used for pesticide residues analysis. 2.5. The analytical method The analysis (extraction and clean-up of cocoa bean) was performed by following the Japanese multi - residue method for agricultural chemicals by GC/MS (agricultural products) released by the Department of Food Safety, Ministry of Health, Labour and Welfare (2006), (No. 0124001). This analytical method was

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validated and documented at the Pesticides Residue Laboratory at the Ghana Standard Authority. Ten grams (10 g) of the homogenous cocoa bean sub-samples were weighed into 250 ml Nalgene jar and labelled accordingly. A 20 ml of distilled water was then added to each sample, stirred to form a homogeneous mixture and left to stand for 15 min. A 40 ml acetonitrile was added and then homogenized using the Ultra Turrax T25 basic homogeniser (Staufen, Germany) for 2 min. They were then centrifuged (Jouan CR3i multifunction) at a speed of 3000 rpm for 3 min and decanted through filter papers into labelled 100 ml volumetric flasks. A 20 ml acetonitrile was added to the residues and further homogenized for 2 min, and 5 ml acetonitrile was used to rinse the dispersing element into the jars. The suspensions were then centrifuged (Jouan CR3i multifunction) at 3000 rpm for 3 min and filtered again into each corresponding labelled 100 ml volumetric flasks. A 15 ml acetonitrile was used to rinse the jars and residues filtered, and all filtrates adjusted to the 100 ml mark with acetonitrile. An aliquots of 20 ml were pipetted into labelled 250 ml separating flasks, and 10 g of NaCl and 20 ml of 0.5 mol/l phosphate buffer (pH 7.0) were added. The separating flasks were corked and shaken for 20 min using the horizontal shaker (Ika-Werke HS 501 Digital) and left to stand for 10 min until the solutions were clearly separated into layers. The NaCl and lower aqueous layers in each separating flasks were carefully removed and the organic layers (acetonitrile layers) transferred into labelled 50 ml beakers for clean-ups. Two solid phase extraction (SPE) cartridge clean-ups were employed; the first by bond elute C-18 (1000 mg/6 ml) and then followed an Envi-carb/LC-NH2 (500 mg/ 500 mg/6 ml) cartridge. For the first clean up, the bond C-18 cartridges were conditioned using 10 ml each of acetonitrile and labelled 30 ml pear shape flasks were placed under the cartridges to collect the elutes. The extracts from the extraction stage were loaded onto each corresponding cartridge, and eluted with 2 ml acetonitrile. After, a 5 g of anhydrous Na2SO4 were placed on filter papers in funnels and the extracts from the tubes dried over them. The receiving flasks were rinsed with acetonitrile and passed over the Na2SO4. Each filtrate was collected into labelled 100 ml round bottom flasks and concentrated at 40
C to dryness using the rotary evaporator (Bibby RE 200 and Buchi Ratovapor R-210). The residues were re-dissolved with 2 ml of acetonitrile in a ratio of 1:3 prior to the second cleanup step. For the second clean up, the Envi-carb/LC-NH2 cartridges were conditioned using 10 ml acetonitrile. Labelled 50 ml pear shape flasks were placed under the cartridges and the extracts from the previous clean-up step loaded onto the corresponding cartridges. The extracts were allowed to filter and the eluates collected. The cartridges were then eluted with 20 ml of the acetonitrile in four portions with intermittent vacuum use. All filtrates were then transferred into 100 ml round bottomed flasks and concentrated at 40
C to approximately 1 ml using the rotary evaporator (Bibby RE 200 and Buchi Ratovapor R-210). A 10 ml of acetone was added to each concentrated solution and further evaporated to dryness. The extracts were re-dissolved in 1 ml ethyl acetate by pipetting, and transferred into labelled 15 ml screw capped tubes, closed and placed in freezer at e 18
C for about 30 min. The extracts were removed and immediately centrifuged (Jouan CR3i multifunction) at 3000 rpm for 5 min, and the top layers carefully transferred into labelled 2 ml Gas Chromatography (GC) standard opening vials prior to quantification by Gas Chromatograph (GC) equipped with pulse flame photometric detector (PFPD) and Ni electron capture detector (ECD) for organophosphorus and synthetic pyrethroid pesticide residues, respectively. All extracts were kept frozen until quantifications were achieved.

2.6. Gas chromatographic (GC) conditions for analysis of pesticide residues The organophosphorus pesticide residues were analysed by a Gas Chromatography (GC) - Varian CP-3800 (Varian Association Inc. USA) equipped with pulse flame photometric detector (PFPD) and Combi PAL auto sampler. The GC conditions used for the analysis were: capillary column (fused silica capillary) coated with VF1701 ms (30 m, 0.25 mm internal diameter, 0.25 mm film thickness). Nitrogen was used as carrier gas at a constant flow rate of 2.0 ml/ min and detector make-up gas of 17.0, 14.0 and 10.0 ml/min for Air 1, hydrogen (H2) and Air 2, respectively. The injector and detectorPFPD temperatures were held at 270
C and 280
C, respectively. The column oven temperature was programmed as follows: 70
C held for 2 min, ramp at 25
C min1 to 200
C, held for 1 min, and finally ramp at 25
C min1 to 250
C. The injection volume of the GC was 2.0 mL in a splitless mode. The total run time for a sample was 14 min. The synthetic pyrethroid pesticide residues were analysed by a Gas Chromatography (GC) - Varian CP-3800 (Varian Association Inc. USA) equipped with 63Ni electron capture detector (GC-ECD) and Combi PAL auto sampler. The GC conditions used for the analysis were: capillary column (fused silica capillary) coated with VF-5 ms (30 m þ 10 m EZ Guard, 0.25 mm internal diameter, 0.25 mm film thickness). Nitrogen was used as carrier gas at a constant flow rate of 1.0 ml min1 and detector make-up gas of 29 ml min1. The injector and detector-ECD temperatures were held at 270
C and 300
C, respectively. The column oven temperature was programmed as follows: 70
C held for 2 min, ramp at 25
C min1 to 180
C, held for 1 min, and finally ramp at 5
C min1 to 300
C. The injection volume of the GC was 1.0 mL. The total run time for a sample was 31.4 min. 2.7. Quantification and limit of detection The residue levels of organophosphorus and synthetic pyrethroid pesticides were quantitatively determined by the external standard method using peak area. Measurement was carried out within the linear range of the detector. A standard mixture of known concentration of organophosphorus and synthetic pyrethroid pesticides was run and the response of the detector for each compound ascertained. The peak areas whose retention times coincided with the standards were extrapolated on their corresponding calibration curves to obtain the concentration. All analyses were carried out in triplicates and the mean concentrations computed accordingly. The limit of detection of the pesticides was based on the extract of the fortified samples that were serially diluted by factor of two to give different concentrations. One out of each concentration that gave a response three times the standard deviation of the least fortified sample was noted. This was used to estimate the statistical significance of differences between low level analyte responses and the combined uncertainties in both the analyte and the background measurement. The limit of detection (LOD) for organophosphorus and synthetic pyrethroid residues in the cocoa bean samples were 0.010 mg/kg and 0.010 mg/kg, respectively. 2.8. Quality control and quality assurance All glassware used for analysis (extraction and clean ups) were rigorously washed with detergent and tap water. They were then rinsed with distilled water and thoroughly rinsed with analytical grade acetone and dried overnight in an oven at 150
C. The glass ware was then removed from the oven and allowed to cool down and stored in dust free cabinets. The quality of pesticide residues

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analysis was assured through the analysis of solvent blanks, and procedural matrix blanks. All analyses were carried out in triplicates and the mean concentrations were calculated based on the number of samples that tested positive to each residue. All reagents and solvents used during the analysis followed the same extraction procedures and subsequently run to check for interfering substances. In the blank for each extraction procedure, no pesticides (neither organophosphorus nor synthetic pyrethroid pesticides) were detected. The method was optimized and validated by spiking the cocoa bean samples with standard mixture before analysis to evaluate the recovery of compounds. Recalibration curves were run with each batch of samples to check that the correlation coefficient was kept above r2 > 0.99. The efficiency of the analytical methods (the extraction and clean-up methods) was determined by recoveries of an internal standard. The recoveries of internal standards ranged between 70% and 94% for organophosphorus and 73e100% for synthetic pyrethroid pesticide residues which shows that the method used was reproducible. 2.9. Data analysis Statistical Package for Social Sciences (SPSS) software version 20.0 was used to generate the means and the standard deviation for the various pesticide residues detected in the cocoa bean samples. One-way Analysis of variance (ANOVA) was used to test for the significant differences and similarities between the pesticide residues detected in cocoa beans from the various sampled sites. Differences were considered significant at p < 0.05. 3. Results and discussion In all twenty-two (22) pesticides comprising of thirteen (13) organophosphorus (methamidophos, ethoprophos, phorate, diazinon, fonofos, dimethoate, pirimiphos-methyl, chlorpyrifos, malathion, fenitrothion, parathion, chlorfenvinphos, profenofos) and nine (9) synthetic pyrethroids (allethrin, bifenthrin, fenpropathrin, lambda-cyhalothrin, permethrin, cyfluthrin, cypermethrin, fenvalerate, deltamethrin) were analysed in thirty-two (32) cocoa bean samples from the study area. However, the results revealed the presence of nine different pesticide residues, which is made up of three organophosphorus (diazinon, chlorpyrifos and pirimiphosmethyl) and six synthetic pyrethroids (allethrin, lambdacyhalothrin, cypermethrin, deltamethrin, permethrin and bifenthrin) in the cocoa bean samples analysed. All the samples analysed had at least one detectable pesticide residue which is an indication of how frequent organophosphorus and pyrethroids are used in cocoa production in Ghana. Among the pesticide residues detected, chlorpyrifos (organophosphorus) and cypermethrin (synthetic pyrethroid) occurred most frequently, occurring in 75% and 87.5% of the samples analysed, respectively. The results (minimum, maximum, average and % positive detection) of organophosphorus and synthetic pyrethroid residues detected in the cocoa bean samples analysed are shown in Tables 1 and 2, respectively. Diazinon was detected in 56.3% of the cocoa bean samples analysed. The measured diazinon concentrations ranged from <0.01 mg/kg at S3 to 0.01 mg/kg at S1, S2 and S4 with a mean value of 0.01 mg/kg. This mean value was below the EU MRL of 0.02 mg/ kg for diazinon in cocoa beans. In addition, the mean concentrations of diazinon recorded at the various sampled sites were below the EU MRL of 0.02 mg/kg for cocoa beans. There was no statistically significant site difference (p > 0.05) in mean diazinon recorded in the cocoa bean samples. Although diazinon as insecticide is not approved and recommended by Ghana Cocoa Board (COCOBOD) for use on cocoa farms, it is listed in the active ingredients

5

registered in Ghana by Ghana EPA for the control of capsids and insect pests in cocoa (Environmental Protection Agency (EPA), 2013). Hence, the detection of diazinon in the samples suggests the use of diazinon as a pesticide in the control of insect pests by cocoa farmers in the study area. The mean value of diazinon recorded in this study was relatively lower compared to the mean value of 0.12 mg/kg reported by Aikpokpodion et al. (2012), but comparatively higher than the mean value of 0.005 mg/kg reported by Frimpong, Yeboah, Fletcher, Pwamang, and Adomako (2012b) in cocoa bean samples analysed from three ecological zones in Nigeria and in cocoa bean ready for export in Ghana, respectively. Among the organophosphorus residues detected, chlorpyrifos recorded the widest spread concentrations among the sampled sites (0.03 mg/kg to 0.05 mg/kg), occurring in 75% of the samples analysed. The average sum of chlorpyrifos in the samples analysed was 0.04 ± 0.01 mg/kg and ranged from 0.03 mg/kg at S3 to 0.05 mg/kg at S4. There was no significant difference (p > 0.05) in mean chlorpyrifos recorded among the sampled sites. The average concentrations of chlorpyrifos recorded in this study were below the EU MRL of 0.10 mg/kg for cocoa beans. The high values of chlorpyrifos recorded in the samples analysed suggests the extensive use of pesticides containing chlorpyrifos as its active ingredient in cocoa production in the study area. However, chlorpyrifos as insecticide is not approved and recommended by Ghana COCOBOD for use in cocoa production (EPA, 2013). The mean value of chlorpyrifos recorded in this study was higher than the mean value of 0.02 mg/kg reported by Daanu (2011) in cocoa bean samples from Asukese and its environs in the Tano North District of Ghana. On the other hand, the mean value of chlorpyrifos recorded in this study was lower compared to the mean values of 10.6 mg/kg and 0.05 mg/ kg recorded by Boakye (2012) and Frimpong et al. (2012b) in cocoa bean samples ready for export, and analysed from the Brong Ahafo and Ashanti Regions of Ghana, respectively. Pirimiphos-methyl, one of the three organophosphorus pesticides screened routinely for shipment to Japan and Europe (Ghana Standards Authority (GSA), 2011), was detected in 62.5% of the cocoa bean samples analysed. The measured mean concentrations ranged from 0.02 mg/kg at S2 to 0.03 mg/kg at S1 and S3 with a mean value of 0.03 ± 0.00 mg/kg. There was no significant difference (p > 0.05) in mean values of pirimiphos-methyl recorded among the sampled sites. The mean value of pirimiphos-methyl recorded in this study was below the EU MRL of 0.05 mg/kg for cocoa beans. In addition, the concentrations of pirimiphos-methyl recorded at the various study sites were below the EU MRL of 0.05 mg/kg for cocoa beans. Pirimiphos-methyl is among the approved and recommended insecticides for cocoa production in Ghana by Ghana COCOBOD (Denkyirah et al., 2016), hence its detection in the cocoa bean samples analysed suggest its use by cocoa farmers in the study area. The mean value of pirimiphosmethyl recorded in this study was similar to the mean value of 0.03 mg/kg reported by Frimpong et al. (2012b) in cocoa bean samples ready for export in Ghana. The mean concentrations of allethrin recorded in the cocoa bean samples analysed ranged from <0.01 mg/kg at S1 and S3 to 0.01 at S2 and S4 with a mean value of 0.01 mg/kg. This was detected in 50% of the cocoa bean samples analysed. There was no significant difference (p > 0.05) in mean values of allethrin recorded among the sampled sites. The mean concentrations of allethrin recorded in this study were below the EU MRL of 0.01 mg/kg for cocoa beans. Similarly, the detection of allethrin in the cocoa bean samples analysed suggest its use by cocoa farmers in the study area. However, the use of allethrin as insecticide in cocoa production in Ghana has not been approved and recommended by Ghana COCOBOD. The mean value of allethrin recorded in this study was similar to the mean value of 0.01 mg/kg reported by Frimpong, Yeboah, Fletcher,

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Table 1 Average concentration of organophosphorus residues (mg/kg) in cocoa bean samples from the study area. Sites

Nkrankwanta (S1)

Diabaa (S2)

Krakrom (S3)

Kwakuanya (S4)

Pesticidese

Min

Max

Mean

Min

Max

Mean

Min

Max

Mean

Min

Max

Mean

Diazinon Chlorpyrifos Pirimphos-methyl

<0.01 0.02 0.01

0.01 0.06 0.04

0.01 0.04 0.03

<0.01 ND 0.01

0.01 ND 0.03

0.01 ND 0.02

<0.01 0.01 0.01

<0.01 0.05 0.04

<0.01 0.03 0.03

0.01 0.03 ND

0.01 0.06 ND

0.01 0.05 ND

Meana

SDb

EU MRLc

% Detectedd

0.01 0.04 0.03

0 0.01 0.00

0.02 0.10 0.05

56.3 75 62.5

b

EU MRLs

ND¼Non-detected. Limit of detection ¼ 0.010 mg/kg. a Mean of samples where residues were detected. b Standard deviation of samples where residues were detected. c European Union maximum residue limits for pesticides in cocoa beans. d Percent (%) of samples with positive pesticide detections. e Number of pesticides found in samples.

Table 2 Average concentration of synthetic pyrethroid residues (mg/kg) in cocoa beans samples from the study area. Sites Pesticides

e

Allethrin Lambda-cyhalothrin Cypermethrin Deltamethrin Permethrin Bifenthrin

Nkrankwanta (S1)

Diabaa (S2)

Krakrom (S3)

Kwakuanya (S4)

Min

Max

Mean

Min

Max

Mean

Min

Max

Mean

Min

Max

Mean

<0.01 0.01 0.02 0.01 0.01 0.01

<0.01 0.02 0.06 0.03 0.02 0.01

<0.01 0.02 0.04 0.02 0.02 0.01

<0.01 ND 0.01 0.02 0.01 0.01

0.01 ND 0.02 0.03 0.02 0.03

0.01 ND 0.02 0.03 0.02 0.03

<0.01 0.03 0.01 0.01 ND ND

<0.01 0.04 0.04 0.04 ND ND

<0.01 0.03 0.04 0.03 ND ND

0.01 ND 0.01 0.01 0.02 0.01

0.01 ND 0.06 0.03 0.03 0.01

0.01 ND 0.04 0.02 0.03 0.01

Mean

0.01 0.03 0.04 0.03 0.02 0.02

a

SD

0 0.01 0.01 0.01 0.01 0.01

0.01 0.05 0.10 0.05 0.10 0.10

c

% Detected

d

50 31.1 87.5 56.3 31.3 31.3

ND¼Non-detected. Limit of detection ¼ 0.010 mg/kg. a Mean of samples where residues were detected. b Standard deviation of samples where residues were detected. c European Union maximum residue limits for pesticides in cocoa beans. d Percent (%) of samples with positive pesticide detections. e Number of pesticides found in samples.

Pwamang, and Adomako (2012a) in cocoa bean samples ready for export in Ghana. Lambda-cyhalothrin concentration was detected only in cocoa bean samples analysed from S1 (0.02 mg/kg) and S3 (0.03 mg/kg) with mean a value of 0.03 ± 0.01 mg/kg. This was realised from 31.3% of the samples analysed. The mean values of lambdacyhalothrin recorded were below the EU MRL of 0.05 mg/kg for cocoa beans. The detection of lambda-cyhalothrin in the cocoa bean samples may be attributed to the current use of pesticides containing lambda-cyhalothrin (as active ingredient) by cocoa farmers in the study area. Lambda-cyhalothrin as insecticide is however not approved and recommended by Ghana COCOBOD for use on cocoa farms in Ghana. The measured mean concentration observed in this study was higher than the mean value of 0.01 mg/kg reported by Frimpong et al. (2012a) in cocoa bean samples ready for export in Ghana. Cypermethrin, a very popular synthetic pyrethroid in Ghana, but not approved and recommended for cocoa production by Ghana COCOBOD (EPA, 2013), was detected in 87.5% of the cocoa bean samples analysed. The measured means ranged from 0.02 mg/kg at S2 to 0.04 mg/kg at S1, S3 and S4 with a mean value of 0.04 ± 0.01 mg/kg. There was no significant difference (p > 0.05) in mean values of cypermethrin recorded among the sampled sites. The mean cypermethrin concentrations recorded at the various study sites were below the EU MRL of 0.10 mg/kg for cocoa beans and the findings of Boakye (2012). The use of cypermethrin in the control of cocoa insect pests by farmers in the study area might have accounted for its detection levels in the cocoa bean samples analysed. The mean cypermethrin concentration recorded in this study was higher than mean values of 0.02 mg/kg and 0.03 mg/kg reported by Daanu (2011) and Frimpong et al. (2012a) in cocoa bean samples analysed from Ghana, respectively.

Deltamethrin was detected in 56.3% of the cocoa bean samples analysed with a mean value of 0.03 ± 0.01 mg/kg, and ranged from 0.02 mg/kg at S1 and S4 to 0.03 mg/kg at S2 and S3. There was no significant difference (p > 0.05) in mean values of deltamethrin recorded among the sampled sites. The mean concentrations of deltamethrin recorded in this study were below the EU MRL of 0.05 mg/kg for cocoa beans. The detection of deltamethrin in the sampled cocoa beans suggest the use of the pesticide by cocoa farmers in the study area, although it is not approved and recommended by Ghana COCOBOD for use on cocoa farms in Ghana. The mean residue concentration of deltamethrin recorded in this study was found to be higher than the mean value of 0.0003 mg/kg reported by Daanu (2011), but lower than the mean value of 0.04 mg/ kg reported by Frimpong et al. (2012a) in cocoa bean samples analysed from Ghana, respectively. The mean permethrin concentrations recorded in cocoa bean samples analysed ranged from 0.02 mg/kg at S1 and S2 to 0.03 mg/ kg at S4 with a mean value of 0.02 ± 0.00 mg/kg. This was realised from 31.3% of the samples analysed. There was however no significant difference (p > 0.05) in mean values of permethrin recorded among the sampled sites. The measured permethrin concentrations were below the EU MRL of 0.10 mg/kg for cocoa beans. The detection of permethrin suggests the use of pesticides with permethrin as its active ingredient in the study area. However, the use of permethrin as insecticide for cocoa production has not been approved and recommended by Ghana COCOBOD (EPA, 2013). The mean value of permethrin recorded in this study was lower compared to the mean value of 0.04 mg/kg recorded by Frimpong et al. (2012a), but higher than the mean value of 0.005 mg/kg recorded by Daanu (2011). Bifenthrin, an approved and recommended insecticide by Ghana COCOBOD for use in cocoa production in Ghana was

Please cite this article in press as: Okoffo, E. D., et al., Contamination levels of organophosphorus and synthetic pyrethroid pesticides in cocoa beans from Ghana, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.11.004

E.D. Okoffo et al. / Food Control xxx (2016) 1e8

detected in 31.3% of the samples analysed with a mean value of 0.02 ± 0.01 mg/kg. The measured means ranged from 0.01 mg/kg at S1 and S4 to 0.03 mg/kg at S2. There was no significant difference (p > 0.05) in mean values of bifenthrin recorded among the sampled sites. The mean bifenthrin recorded at the various study sites were below the EU MRL of 0.10 mg/kg for cocoa beans. The occurrence of bifenthrin in the cocoa bean samples suggests the current use of the pesticide in the study area since they are approved and recommended for use on cocoa in Ghana. The mean value of bifenthrin recorded in this study was higher than the mean value of 0.01 mg/kg reported by Frimpong et al. (2012a) in cocoa bean samples ready for export in Ghana, but lower than the value reported by Boakye (2012) in cocoa beans from the Brong Ahafo and Ashanti Regions of Ghana. Contamination of cocoa beans could have occurred directly by treating the crop with pesticides during the maturity (development) stages of the cocoa bean, where pesticide residues were adsorbed by the cocoa beans through the cocoa pod. 4. Conclusions and recommendations Pesticide residues were found in all the cocoa bean samples analysed from the study area. Three organophosphorus pesticides namely; diazinon, chlorpyrifos and pirimiphos-methyl and six synthetic pyrethroids namely; deltamethrin, cypermethrin, bifenthrin, permethrin, lambda-cyhalothrin and allethrin were detected in the cocoa bean samples analysed. The occurrence of the pesticides analysed indicates a higher preference for organophosphorus and synthetic pyrethroid pesticides among cocoa farmers in the study area, and that could be attributed to the fact that most of these pesticides are registered for use in Ghana for agricultural purposes (EPA, 2009; 2013). The measured means of the pesticide residues (organophosphorus and synthetic pyrethroids) recorded in the cocoa bean samples analysed from the study sites were below their respective EU MRL set for cocoa beans. Considering levels of pesticide residues in the fermented dried cocoa beans against the EU commission regulations on pesticide residues, cocoa beans analysed from the study area will not pose any significant threat to the cocoa industry in Ghana as far as shipment to Europe is concern. However, the application rate, nozzle size and the pre-harvest interval of pesticides application should be taken into consideration in order not to exceed their maximum limits for regulatory purposes. There should be education of farmers through extension services on the right kinds of pesticides, the right amounts and frequency of administration of pesticides in a farming season to reduce the amounts of pesticides in the environments. Competing interests All authors declare no competing interest. Authors’ contributions EDO, FMBY and CG designed the study and wrote the protocol, EDO collected data and conducted data analysis, FMBY and EDO, drafted the manuscript, and FMBY reviewed the manuscript. All authors read and approved the final manuscript. Acknowledgment The authors express their profound gratitude to the Pesticide Residue Laboratory of Ghana Standards Authority in Ghana for logistical assistance in the analytical work.

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