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Determination of Physicochemical Properties, Heavy Metals and Pesticide Residues of Honey Samples Collected from Walmara, Ethiopia

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Determination of Physicochemical Properties, Heavy Metals and Pesticide Residues of Honey Samples Collected from Walmara, Ethiopia International Journal of Advanced Research in Chemical Science (IJARCS) Volume 6, Issue 7, 2019, PP 23-33 ISSN No. (Online) 2349-0403 DOI: http://dx.doi.org/10.20431/2349-0403.0607004 www.arcjournals.org Determination of Physicochemical Properties, Heavy Metals and Pesticide Residues of Honey Samples Collected From Walmara, Ethiopia Deressa Kebebe1*, Alemayehu Paulos2, Ermias Haile3 Department of Chemistry, Hawassa University, Hawassa, Ethiopia *Corresponding Author: Deressa Kebebe, Department of Chemistry, Hawassa University, Hawassa, Ethiopia Abstract: This study was conducted to investigate pesticide residues, heavy metals and physicochemical parameters levels in honey samples. In this study, the results of moisture content, electrical conductivity, pH and ash were 15.15 –21.75%, 0.45–1.55 mScm-1, 3.50–4.50, and 0.18–0.80% and that of Fe, Zn, Cu, Pb, Cd, Ni and Cr were analyzed by using flame atomic absorption spectrophotometer and the concentrations ranged from 4.87-11.79 µg/g, 1.41-6.94 µg/g, 0.22-1.22 µg/g, 0.37-0.90 µg/g, 0.04-0.70 µg/g, 0.26-0.60 µg/g and 0.16-0.50µg/g with mean concentration ranges, respectively. All metals were determined by using atomic absorption spectrophotometer except Fe that was determined by using UV-visible spectrophotometer. The percentage recovery for metal analyses was from 85% to 104%. Cd, Cu, Cr and Pb concentrations in honey samples from all sites were not significantly different but Fe, Zn and Ni levels were significantly different at (p<0.05). Pesticide residues in the honey samples were determined by using gas chromatography coupled with mass spectrometer technique. The residues in all samples were found to be below detection limits. The detection limits range was from 0.001 to 0.017 ng/g for the residues analyzed while the recovery percentages were range from 75-105 %. Most trace metals and all residues have been found to be within the acceptable range set by national and international standards except Pb and Cd contents in some samples which need further study in the future. Keywords: Flame Atomic Absorption Spectrophotometry, Gas Chromatography-Mass Spectrometry, Heavy Metals, Honey, Pesticide Residue. 1. INTRODUCTION Bee honey is syrupy and viscous substance that produced by honeybees from the nectar of flowers or from the secretion of living parts of plants, in which honeybees transform through enzymatic activity and store it in wax structures called honeycombs until maturation (Adenekan, 2010, Awad and Elgornazi, 2016) should be safe for all consumers and pollinators. It has been used by humans since ancient times in various foodstuffs and drinks as a sweetener and flavoring (Kowsalya, 2012, Omoya et al, 2014).It has also medicinal and therapeutic effects (Eyobel et al., 2017). Since the forage area of the hive is very large (more than 7 km2) and the bees come in contact not only with air but also with soil and water, the concentration of heavy metals in honey reflects their amount in the whole region. Therefore, honey has been recognized as a biological indicator of environmental pollution (Przybylowski, 2001). Determination of heavy metals in honey is of high interest mainly for quality control and nutritional aspect (Buldini, 2001). Chemical pesticides are conventionally synthetic materials that directly kill or inactivate the pest (Rama et al., 2011). Depending on their chemical properties they can enter the organism, bio accumulate in food chains and consequently influence also human health (PAN Europe, 2010). Pesticides use is usually accompanied with deleterious environmental and public health effects. Pesticides hold a unique position among environmental contaminants due to their high biological activity and toxicity (acute and chronic). Although some pesticides are described to be selective in their modes of action, their selectivity is only limited to test animals. Thus pesticides can be best described as biocides i.e., capable of harming all forms of life other than the target pest (Zacharia, 2011). International Journal of Advanced Research in Chemical Science (IJARCS) Page | 23 Determination of Physicochemical Properties, Heavy Metals and Pesticide Residues of Honey Samples Collected From Walmara, Ethiopia The study district is identified as one of the potential areas for beekeeping in the country and honey is an important source of income for farmers in the area. But it is exposed to pesticides that may be released from floricultural industries and other agricultural activities. In addition, there are floriculture industries and factories that may release trace metals to the area because their applications are highly increasing from time to time (Belay, 2015).Therefore, the objective of this study was to determine physicochemical properties, pesticides residues and heavy metals of honey and comparing their levels with international and national standards. 2. METHODS AND MATERIALS 2.1. Study Area Description The study was under taken in Walmara district in central highland part of Ethiopia. The district total land area is 80,927hectares. This district is located at 25Km to the west of Finfinne (8.5°-9.5°N and 38.4°-39.2°E) with altitude of 2000-3380 m. It is bordered by Finfinne to the East; Ejere district to the West, Sululta district to the North, Sebeta Hawas district from the south and its weather condition is classified as 39% woinadega and 61% dega. The district has 1,853 traditional, 870 transitional, 843 modern beehives. The yields gained was 20Kg, 15 Kg and 5 Kg per hive per year from modern, transitional and traditional, respectively. 2.2. Sample Collections, Storages and Pretreatments Unprocessed representative honey subsamples, twenty in number were collected using purposive sampling technique from 20 selected beekeepers in clean honey sampling plastic jars. Honey samples of each weighing about 1 kg were collected directly from beekeepers during active season from January to May 2018 and then transferred to laboratory and preserved. Four composite samples (a sample from five apiaries) were mixed and homogenized to determine pesticides while 8 composited honey samples from similar sites were used to determine trace metals and physicochemical properties. The samples were placed in contaminant free polyethene plastic container labeled and stored in refrigerator at -20°C until analyses to determine pesticide residues in the four samples. 2.3. Equipments and Reagents 2.3.1. Equipments The laboratory apparatus that was used during the study include: measuring cylinders, funnel, filter papers, pipettes and micropipettes (Pyrex, USA) to dilute sample solutions and prepare standard solutions, round bottom flasks 250 mL fitted with reflux condenser were used in heating mantle (BI Barnstead Electro thermal, ԐC, UK) to digest honey samples, spiked honey samples and blank solutions as well as refrigerator to keep the collected samples and digested samples until analysis and digital Analytical Balance (Mettler Toledo, Model AG250, Switzerland with ± 0.0001 g) to weigh honey samples. An Agilent 7890B gas chromatograph connected to an Agilent 5977AMSD system equipped with an auto sampler G4513A, GC/MS (Agilent Technologies, USA) were used for detection of pesticide residues. 2.3.2. Reagents All Reagents and chemicals used in the analysis were of Analytical Grade. A combination of concentrated 69-72% HNO3 (Unichem) and 30 % H2O2 (UNI-Chem.), H2SO4 98% (Loba) were used in digestion of honey samples, blank and spiked solutions at optimum condition. Deionized water was used during the research for sample preparation, dilution and rinsing apparatus prior to analysis. Hydroquinone 99% (Kiran light Laboratory), 1,10-phenanthroline, sodium acetate trihydrate 99%, ferrous ammonium sulfate hex hydrate 98% were used for the determination of iron metal in the honey sample. Pesticide standards were used from JIJE Laboratory Services in Addis Ababa in Ethiopia with purity between 98.2 and 99.5%. Acetonitrile (99.99 %, Sigma Aldrich, Germany) high-performance liquid chromatography grade were used. Anhydrous magnesium sulfate, acetic acid and sodium acetate was obtained from Merck (China). Ultrapure water was prepared from a Milli-Q system (Millipore, Bedford, MA, USA). International Journal of Advanced Research in Chemical Science (IJARCS) Page | 24 Determination of Physicochemical Properties, Heavy Metals and Pesticide Residues of Honey Samples Collected From Walmara, Ethiopia 2.4. Sample Preparation for Pesticides Analysis 5 g of the honey samples were thoroughly homogenized with 10 ml ultrapure water and the homogenate was transferred into polypropylene centrifuge tube (50 ml). Then it was hand-shaken for 5 minutes. Thereafter, 15 ml of acetonitrile was added and mixed for 2 minutes, and the Quenchers salt kit was added. The samples were immediately hand shaken for 2 minutes and subsequently centrifuged at 3500 rpm for 5 minutes. Thereafter, 6 ml of supernatant was transferred in a 15 ml DSPE polypropylene tube. The tube was shaken for 30 seconds and subsequently centrifuged at 3500 rpm or 5 minutes. Finally, 0.5 ml of supernatant was taken into glass auto sampler vial (Kurtagićand Čopra-Janićijević, 2016). AOAC official method 2007.01 was used for analysis of pesticide in the honey samples. In this particular method, the Quenchers method were used for extraction of pesticide from honey samples using a single-step buffered acetonitrile extraction and salting out liquid–liquid partitioning from the water in the sample with MgSO4 (Lehotay, 2007). 2.5. Physicochemical Analysis of Honey Samples 2.5.1. Variation in Honey Color Honey color of the samples were measured using Pound color grader (AOAC, 1990). The scale used in the honey industry to describe the color of honey which consists of a wedge of amber-colored glass next to a wedge-shaped cell which is filled with honey. 2.5.2. Ash Content This variable was determined and calculated by adopting the 920, 181 method established by A.O.A.C and the ES (Belouali et al., 2008). Honey sample of 5 g was weighed accurately into a pre- weighed porcelain crucible and gently heated on a hot plate until the sample was turned into black and dry.
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