Detection of heavy metals in honey samples using Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

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1 Research Article Open Access Received: November 16, 2017 Accepted: December 22, 2017 Published: December 28, 2017 Detection of heavy metals in honey samples using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Ishana Aggarwal* Modern School, Barakhamba Road, India *Correspondence: Ishana Aggarwal, Modern School, Barakhamba Road, New delhi, India Abstract Honey is a composite mixture of various carbohydrates, enzymes, flavonoids and organic acids. It is used for a wide variety of purposes and is a known antimicrobial agent. Heavy metals may be present in trace quantities in honey and their detection is important for the quality control of honey and it also serves as an indicator of environmental pollution. For this study, eight samples of honey were collected from New Delhi. Using inductively coupled mass spectrometry (ICP-MS), the honey samples were tested for the presence of seven elements - Copper, Zinc, Arsenic, Cadmium, Mercury, Tin, and Lead. Copyright: 2017 Ishana Aggarwal. PRAS is licensed to the public under a Creative Commons Attribution 4.0 license. Your use of this site is governed by the Terms of Use Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

2 INTRODUCTION According to the Codex Alimentarius, honey is defined as the the natural sweet substance, produced by honeybees from the nectar of plants or from secretions of living parts of plants, or excretions of plant-sucking insects on the living parts of plants, which the bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in honeycombs to ripen and mature" (1). Honey is a composite mixture of carbohydrates such as fructose, glucose, sucrose, and maltose; enzymes such as invertase and amylase; vitamins; minerals; flavonoids and organic acids (2). Due to its highly unique and complex chemical nature, honey finds a host of applications as a sweetener, a potent antimicrobial agent and it is also used extensively for the treatment of burns, wounds, and skin ulcers (3). To fulfill all these roles, it is important for honey to be free from contamination. One source of contamination, examined by this paper, is the presence of heavy metals such lead, cadmium, arsenic, and mercury. Detection of heavy metals in honey serves a necessary purpose in quality control and in monitoring of bee environments. Heavy metals, if present in significant amounts, adversely impact human health and signal contamination of bee environments. Hairy bodies of bees may gather heavy metals from the atmosphere and bring them back to the hive with pollen. In addition, the heavy metals may be absorbed by the bees together with the nectar of the flowers or through water and honeydew. Polluted soil may lend heavy metals to the flower nectar as these may be absorbed by the roots of plants from contaminated soils. Consequently, the presence of heavy metals in honey can be seen as a valuable indicator for environmental contamination (4). The use of trace elements such as copper, cadmium, lead, mercury, and zinc is prevalent in the human economy. Due to expansive industrial growth, heavy metals have become common environmental pollutants. In honey, particularly, these metals may originate from external sources such as industrial smelter pollution, industrial emissions and unsuitable procedures during the different stages of honey production. In addition, the origin of metals in honey may be agrochemicals such as organic mercury, cadmium containing fertilizers and arsenic-based pesticides (5). Elements such zinc, iron, and copper are vital micronutrients for humans and are needed in adequate amounts to maintain normal growth and development. Their deficiency is linked to impairment in cognitive performance, lowered work capacity, lowered immunity to infections, and pregnancy complications. However, elements such as lead and arsenic have little to no known requirements in the human diet. In fact, exposure to these elements is toxic as these trace elements damage human metabolism and has known cytotoxic and carcinogenic effects. (6). The maximum permissible limit of heavy metals in food in India is prescribed by the Prevention of Food Adulteration (PFA) Act of Rule 57 states the limits of contaminants under the category Foods Not Specified, which includes honey. Samples were collected from New Delhi and were tested for the presence of the metals Copper, Zinc, Arsenic, Cadmium, Mercury, Tin, and Lead using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The ICP-MS method for testing the presence of trace elements is suitable as multi-elemental analysis with high sensitivity is possible.

3 Table (1): Maximum permissible limits of heavy metals set by PFA. Element Maximum permissible concentration () Lead 2.5 Copper 30 Arsenic 1.1 Tin 250 Zinc 50 Cadmium 1.5 Mercury 1 Methyl Mercury 0.25 EXPERIMENTAL METHOD Sample Collection: Eight samples of honey were collected from the local markets of New Delhi. The samples were collected in airtight glass containers and stored in room temperature in a lightless place till analysis. Sample Preparation: 1.0 g of each sample of honey was digested in a microwave digester with 5.0 ml concentrated nitric acid (65% HNO3 suprapure) and 1 ml hydrogen peroxide (30% H2O2 pure). The instrumental parameters and settings for the microwave digester were 120 C and 700 Psi for the first ten minutes and 130 C and 800 Psi for the next fifteen minutes. The sample was then filtered using a filter paper with a pore size of 0.45 microns and diluted up to 20 ml with deionized ultrapure water. Apparatus: Agilent 7500ce Inductively Coupled Plasma Mass Spectrometer with an Octopole Reaction System and standard sample introduction system (Nickel cones, glass concentric nebulizer, a quartz Peltier cooled spray chamber and quartz torch) was used for elemental analysis. Table (2): Operating conditions of the ICP-MS. RF Generator (W) 1550 Plasma Gas Flow Rate (L/Min) 15 Auxiliary Gas Flow Rate (L/Min) 0.9 Carrier Gas Flow Rate (L/Min) 1 Sample Introduction Flow Rate (L/Min) 1.1 Nebulizer Pump (rps) 0.1 Elemental analysis was carried out by inductively coupled plasma mass spectrometry (ICP-MS) after microwave-assisted acid digestion. All glassware used for the analysis was cleaned with %10 (v/v) HNO3 solution and rinsed with ultrapure water. The concentrations of seven elements (Cu, Zn, As, Cd, Hg, Sn, Pb) were determined in honey samples. All samples were measured in triplicate by the ICP-MS.

4 RESULTS Table (3): Heavy metal concentrations (in ) in analyzed samples. Sample Lead S. No. (Pb) ± ± ± ± ± ± ± ± Mercury Copper (Hg) (Cu) ± ± ND ± ± ± ± ± ± ± ± ± ± ± ND ± Zinc (Zn) ± ± ± ± ± ± ± ± Arsenic (As) Cadmium (Cd) ± ± ± ± ND ± ± ND ± ± ± ± Tin (Sn) ± ± ± ± ± ± ± ± ± ± ND ± DISCUSSION In the present study, all the eight samples showed the presence of lead. Lead was found in quantities ranging from to Two samples were contaminated with high concentrations of lead, which exceeded the maximum permissible limit of 2.50 as prescribed by the PFA Rules. Lead, a highly toxic presence in foodstuff, has been linked to high blood pressure, heart disease, kidney disease, and reduced fertility. Overexposure to lead in infants can also lead to severe damage to brain development. High concentrations of lead in honey might be because of pollution from the vehicular traffic, heavy industries, and other anthropogenic activities (7). Mercury was detected, albeit in small quantities, in six out of the eight samples tested and ranged from to In one of the samples, the concentration of mercury was 1.124, which was beyond the permissible limit of 1.0. Excessive exposure to mercury is deleterious to human health. The brain remains the target organ for mercury, yet it can impair any organ and lead to malfunctioning of nerves, kidneys and muscles (8). In one of the samples, the concentration of arsenic detected was This was above the permissible limit of 1.1 as set by the PFA. Since arsenic is a known carcinogenic, strict attention must be given to regulate its presence in honey. The principal causes for presence of arsenic in honey include non-ferrous metallurgy, and agrochemicals such as fertilizers and

5 arsenic-based pesticides. Arsenic is also present in the soil, water, and air and hence may be absorbed by plants from these sources, consequently contaminating honey. The presence of arsenic beyond the permissible limits in honey samples is an indicator of micropollution and hence, the use of arsenic-based fertilizers should be checked to limit environmental pollution (9). Micronutrients zinc and copper were detected in all of the eight samples. Zinc and copper and essential for several functions of the body such as regulating the metabolism. Moreover, copper acts as an antioxidant whereas zinc is required for the proper functioning of the immune system. The presence of copper and zinc in honey were beneath the maximum permissible limits in all samples, indicating that there is scarce possibility of zinc or copper poisoning due to consumption of honey. Cadmium was detected in seven out of eight samples and its concentration ranged from to Tin was detected in all samples. Like cadmium, tin contamination did not exceed the permissible limits in any of the samples. Cadmium and tin are released into the environment through numerous industrial processes and enter the food chain through contaminated soil and water. Hence, cadmium and tin concentrations in different places depend on many factors, explaining the variability in the concentration of tin and cadmium tested in the honey samples. When compared with similar studies, one can conclude that the concentration of heavy metals is comparatively higher in urban areas than rural areas or areas with forest cover. The presence of heavy metals above permissible limits in some samples of local honey in New Delhi may be a result of pollution from vehicular traffic and heavy industries. The increasing amount of industries set up in Delhi, particularly in Wazirpur and Naraina, release disproportionately large amounts of contaminants into the atmosphere. This atmospheric fallout coupled with water polluted with chemical pesticides and fertilizers are the key sources of presence of heavy metals in plants. These trace elements eventually find their way in honey. CONCLUSION Eight samples of honey were collected from New Delhi and were tested for the presence of seven elements Copper, Zinc, Arsenic, Cadmium, Mercury, Tin, and Lead using ICP-MS. Two samples contained quantities of lead that exceeded the maximum permissible limit set by the Prevention of Food Adulteration Act of India. Arsenic was found exceeding the maximum limit in one sample and mercury in another. Copper, zinc, cadmium, and tin were present well below the limits. These results may be explained by the increasing urbanization in New Delhi, which results in air and water pollution that ultimately contaminate honey. The presence of heavy metals in honey is an indicator of environmental pollution and sufficient care must be taken to regulate the amount of heavy materials entering the environment as exposure to these elements can adversely affect human health REFERENCE 1. Codex Alimentarius Commission. Revised Codex Standard for Honey. Codex STAN , Rev. 1 (1987) 2: White JW Jr., Doner LW. Honey Composition and Properties. Beekeeping in the United States, Agricultural Handbook Number Bogdanov S, Jurendic T, Sieber R, et al. Honey for Nutrition and Health: a Review. Am J College of Nutrition (2008) 27: Singh C, Shubharani R, Sivaram V. Assessment of Heavy Metals in Honey by Atomic Absorption Spectrometer. World J Pharm and Pharmaceutical Sci 3.

6 5. Pisani A, Protano G, Riccobono F. Minor and trace elements in different honeytypes produced in Siena county (Italy). Food Chem (2008)107: Soetan KO, Olaiya CO, Oyewole OE. The importance of mineral elements for humans, domestic animals and plants: A review. African J Food Sci (2010) 4: Podgorski W, Kanoniuk D. Honey as marker of environmental contamination with heavy metals. Section EE Zootechnica (2004) 22: Jaishankar M, Tseten T, Anbalagan N, et al. Toxicity, mechanism and health effects of some heavy metals. 9. Aghamirlou HM.Heavy metals determination in honey samples using inductively coupled plasma-optical emission spectrometry.

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