J Arthropod-Borne Dis, June 2019, 13(2): 185–190 F Bagheri et al.: Detection of Diazinon … 185 http://jad.tums.ac.ir Published Online: June 24, 2019 Original Article Detection of Diazinon Residue in Honey and Honey Bee (Apis mellifera) in Bandar-Abbas and Meshkinshahr, Iran Fatemeh Bagheri1; *Hassan Vatandoost1,2; Mansoureh Shayeghi1; Yavar Rassi1; Ahmad Ali Hanafi-Bojd1,2; Abbas Rahimi-Foroushani3; Alireza Razavi4; Fatemeh Nikpour-Alkaran2 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Environmental Chemical Pollutants and Pesticides, Institute of Environmental Research, Tehran University of Medical Silences, Tehran, Iran 3Department of Epidemiology and Biostatistics School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 4Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 24 Sep 2018; accepted 15 Dec 2018) Abstract Background: The excessive use of pesticides for crops by farmers, their destructive effects on beneficial organisms, such as bees, have become a big problem today. This study was designed to find out if the honey bee (Apis mellifera) and the honey be affected by diazinon. Methods: Six hives were purchased, 3 hives in Bandar-Abbas and remaining were considered for Meshkinshahr. Plants around the hive were sprayed with diazinon at a concentration of 2/1000. Sampling took place 15d after spray- ing, and diazinon residue was measured by the HP TLC. The study was conducted in 2017–2018. Results: The amount of diazinon residue in honey and honey bee was measured and compared with existing studies. The amount of diazinon residue in honey bee was found to be 0.017mg/kg in Bandar Abbas, and 0.005mg/kg in Meshkinshahr hives. There was nothing in honey. Conclusion: Honey is safe for consumers. Keywords: Honey; Apis mellifera; Diazinon reside; HP TLC Introduction Honey is a sweet, sticky and thick liquid produced by bees from nectarine flowers and contains a significant amount of minerals, vit- amins, and enzymes (1). Honey is composed of main fructose (about 38.5%) and glucose (about 31.0%) (2). It is known as a nutrient and much healthier than sugar (3, 4). Honey is widely used for nutritional and medicinal purposes and can be used to treat wound in- fections and cancers (5). It can be used to treat cough and sore throat, stomach ulcer, earache, measles, and eye diseases (6). Honey is widely used worldwide as a food or medicine. Feeding babies with honey help to improve the memory and growth of children, as well as reduce anxi- ety and increase the growth performance dur- ing the life of individuals (7). Honey is used in cosmetics as well as a natural sweetener in food production. While nutritional value and different aspects of the quality of honey are im- portant, ensuring its chemical safety is also im- portant for consumer acceptance. Benefits of health and nutrition Honey if it is contaminat- ed with toxic chemicals, such as contamination of honey with residues of pesticides or other environmental pollutants (8). It can be severely exposed to various types of contaminations in the environment. Even *Corresponding author: Prof Hassan Vatan- doost, E-mail: hvatandoost1@yahoo.com mailto:hvatandoost1@yahoo.com J Arthropod-Borne Dis, June 2019, 13(2): 185–190 F Bagheri et al.: Detection of Diazinon … 186 http://jad.tums.ac.ir Published Online: June 24, 2019 small amounts of contaminants in the environ- ment, such as residues of pesticides, can enter the honeybee and enter honey during pro- cessing. Since pesticides are widely used in ag- ricultural fields and gardens to control pests and plant diseases, and hives are often found along- side the same fields and gardens, the potential for contamination of bees and consequently honey is very high. While the use of synthet- ic pyrethroids and carbamates as insecticides, herbicides, and fungicides are increasing, but the use of organochlorines (mainly as insecti- cides in food products) has been severely re- duced since their ban in 1999 (9). However, organochlorine pesticide residues are still recognized in the environment and in various types of foods because of their re- sistance to staying in the environment and il- legal use. Even the newer generation of pes- ticides (artificial pyrethroids, organophosphates, and carbamates) is not as stable as the first gen- eration organochloride, but they have very high acute toxicity. Organophosphate pesticides have been found in some areas even in fruits and vegetables that enter the market (10). All pes- ticides are poisonous and many of them have high potential for carcinogenesis and may even cause severe chromosomal aberrations (11). Pesticides can also interfere with endocrine dis- ruptions, reproductive organs, fertility and the nervous system (12-15). This study was designed to find out if the honeybee and honey are affected by diazinon. Materials and Methods This study was conducted in two cities of Bandar Abbas and Meshkinshahr in 2017– 2018. Meshkinshahr is located in the province of Ardebil in the northwest of Iran. This city is near the mountain and with relatively cold weather. Both plants that grow naturally in this area, as well as different herbaceous plants, will make beekeepers in the area more prev- alent. Bandar Abbas is located in Hormozgan Province in southern Iran. The climate of this city is warm and humid. The cold winters of the northern regions of Iran forced beekeep- ers to bring their hives to a warm tropical cli- mate, which in winter is pleasant spring weath- er like Bandar Abbas. Measurement of residues of diazinon in hon- ey For this purpose, 6 hives were purchased. Plants around the hive were sprayed up to radius of 200 meters in a hive with diazinon at a concentration of 2/1000, the same con- ventional concentration that farmers used to control the pests. Fifteen days after spraying, honey was harvested. The control beehive was placed at a point where no spraying is done and then honey is picked up. Honey harvest- ed from hives should be free of wax. To re- move wax from honey, wax honey was placed inside stainless steel and sub filters in a clean container that was washed with conventional detergents and placed under the sun. After 24 h, wax and honey were easily separated. Sub- sequently, samples of harvested honey were transferred to the laboratory. Steps to prepare honey samples Dissolve 50g of honey in a 50ml tube in 10ml of deionized water and place it in a blend- er for 1min, then acidify with acetonitrile with acidic (10ml) and 1g sodium acetate and 4g magnesium sulfate. Add water and shake for 1min. The samples were centrifuged for 4min at 2,000min. 6ml of high transparent liquid was added to 15ml polyethylene tubes con- taining 0.4g PSA and 0.6g magnesium sulfate without water and centrifuged at 4000 rounds per 2min. In this way, the solution was pre- pared to be placed on the chromatography de- vice (16). Stages of preparation of bee body samples The specimen from worker honey was transferred to disposable polyethylene bags. J Arthropod-Borne Dis, June 2019, 13(2): 185–190 F Bagheri et al.: Detection of Diazinon … 187 http://jad.tums.ac.ir Published Online: June 24, 2019 Honey bees were collected from honey from the honeycombs located in the furthest walls of the hive. These are typically older work- ing honeybees, which are likely to have the most residual pesticide residues, depending on their age. All samples were transported to a laboratory in a cool box and stored at 20 °C in the laboratory until freezing. Standard method for diazinon extraction was used (17). Subsequently, the extracted so- lution was measured by chromatography meth- od and the amount of pesticide residual was measured and compared with MRL and ADI. Detection and measurement of diazinon in honey samples To detect and determine the amount of pes- ticide, the first step is to apply a stain carried out using 20x20 plates covered with silica gel. A standard insecticide sample of diazinon is required to stain. Spotted plates are placed at a depth of 1.5cm inside the chromatography tank. To see the spots of the plate they were placed in a UV cabinet and observed at appro- priate wavelengths. Regarding standard spots and unknown spots, they can be compared with each other and determine the presence or ab- sence of pesticides in this way qualitatively. HP TLC Scanner Spot scan is done by the HP TLC. This de- vice is designed to monitor the donometry of chromatographic and electrophoretic purposes. After inserting the plates into the device chamber and adjusting the beam on the first spot, enter the necessary information to start the scan, such as the size of the gap, the speed of the scan, the wavelength, and the type of lamp in the corresponding section if the wavelength and the size of the correct gap are selected. Record spectrum The spectrum from the detected peaks is automatically used to determine the identifi- cation of the courier or the purity compari- son spectrum. Quantitative evaluation of results All plates are checked after chromatog- raphy and the proper wavelength detector de- termines this device, and the time required for this operation is very small. Using this device, the quantitative evaluation of the material at a higher speed and more precision is performed. Results The maximum residue of diazinon was in hive No. 2 in Bandar Abbas and the minimum s in Meshkinshahr No. 2 (Table 1). Table1. The number of diazinon residues in the hon- eybees' body in the examined hives Bandar-Abbas hives Diazinon residue Sample 1 (control) 0 Sample 2 0.025mg/kg Sample 3 0.009mg/kg Meshkinshahr hives Diazinon residue Sample 1 (control) 0 Sample 2 0.003mg/kg Sample 3 0.007mg/kg After analyzing the samples, the residues of dia- zinon in honey were not observed, and its value was considered zero. So the honey was com- pletely clean and free of insecticide. Discussion Diazinon residue in Bandar Abbas hive was 0.025 and 0.009mg/kg respectively in samples. The figures in Meshkinshar was 0.003 and 0.007mg/kg. In a study (16), 46 organochlorine, organophosphate, pyrethroid, and organonitro- gen pesticides residue were analyzed in hon- ey samples from 18 hives in 9 different loca- tions, followed the gas chromatography. The remaining pesticides tested in 55.6% of the samples collected, and most of them were de- tected more and more pesticides identified be- long to organochlorine and organophospho- rus groups. The highest amount of pesticide J Arthropod-Borne Dis, June 2019, 13(2): 185–190 F Bagheri et al.: Detection of Diazinon … 188 http://jad.tums.ac.ir Published Online: June 24, 2019 residues belonged to diclofen used in the hive, because of fighting the Varroa mites, and in 38.9% of honey samples and 81.8% of iden- tified pesticides exceeded the EU-approved honey limit, that is, MRL so the residue of di- azinon was set above the limit (16), while in our study, the amount of residual diazinon was less that MRL and honey was free of pesticide (16). In Ghana, the amount of residue of a large number of pesticides in honey was measured. Pesticides such as deltamethrin, permethrin, fenvalerate, chlorpyrophos, syphlotrin, diazinon and several other pesticides were studied. The residual of all measured pesticides is very low and below the maximum limit set by the Eu- ropean Union. So the analysis of residues of pesticides in honey samples shows that there are no health hazards for consumers (10). The results of this study are quite similar to the pre- sent study. In our study samples, the amount of residual diazinon was much less than de- tectable The pollen of the environment can be iden- tified with respect to the residual types of pes- ticides and heavy metals in honey and honey bee bodies. As one of the goals of our study was to measure the residue of the diazinon pesticide in honey and the honey bee, it is im- portant to ensure the integrity of this essen- tial food product and the impact of spraying the environment on the bee, which is a useful and strategic insect be aware. In our study, alt- hough some contamination was detected in the bee body, there was no contamination in hon- ey (18). Six villages were considered as research sites and by sampling bees and feces they meas- ured the remaining heavy metals by using the method of Atomic absorption spectrometry and showed that the bees changed to chemi- cal in the environment in which they live, es- pecially the increase in the number of heavy metals in soil, air and plants, and the residues of these substances in the body of the bee can represent the presence of these substances in nature. Although there may not be honey in the bee product, so these studies indicate that the bees are a reliable indicator of the clean- liness and lack of environment of any chem- ical (19). The l residue of different pesticides was investigated in local honey samples produced from different places in Jordan and from im- ported honey during 1995–1995. The remain- ing 50 pesticides were determined in 26 sam- ples of honey. The highest amount of pesti- cides in the samples was assigned to the chlo- rine group. Lower levels remained in some organophosphates and pyrothyroids. In the 11 samples examined, the remaining αHCH, βHCH, and lindane residues were detectable. In some of the samples, DDT, heptachlor, hep- tachlor oxide, dieldrin, and aldrin were also detected. The fluvalinate residue was found only in 4 honey samples. The amitraz residues, tetradifon and bromopropylate were not de- tectable in honey samples. The measurement of these pesticides in honey was carried out using the GC method. The similarity of this study with our finding is that the diazinon pesticides in honey samples were zero (20). Conclusion There was no pesticide residue in honey so it seems honey is safe for consumers. Acknowledgments The authors would like to highly appre- ciate the collaboration of the staff of De- partment of Medical Entomology and Vector Control School of Public Health, Tehran Uni- versity of Medical Sciences, Tehran, Iran. 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