Heavy Metal Content in PolyfloralHoney and Potential Health Risk. A Case Study of Copșa Mică, Romania

Honey is both a complex food and medicine as well as a healthy alternative to refined sugar. Besides a complex mixture of carbohydrates, honey contains other minor substances which may threaten human health in excess concentrations. Several environmental conditions can affect the quality of honey. This research paper aims to measure the degree of heavy metals (Lead (Pb), Cadmium (Cd), Zinc (Zn), and Copper (Cu)) in some polyfloral honey from an industrial area of Romania, considered to be one of the most polluted regions in Eastern Europe. The samples were collected from six stationary apiaries and analysed using the atomic absorption spectrometry method. The content of Pb was higher in the sampling areas exposed directly to the polluted air masses. Cd concentration decreases exponentially while Cu concentration increases as the distance from the source of pollution increases. The checking of the quality of polyfloral honey from local producers is imperative because this product is intended to be consumed by the beekeeper’s family or the local community without being sold to an authorised processor. The results of the study can help to set a threshold for the concentration of Pb and Cd in honey marketed in the European Union.


Introduction
Food safety has become an essential food quality attribute, not only because of the major role played by foodstuffs within a human healthy diet, but also the issue of public concern [1]. The presence of heavy metals in foodstuffs is becoming increasingly more obvious and food consumption represents the main way of access to the human body [1,2] with potentially negative effects on human health, especially because heavy metals de-regulate the immune system [3] and lead to severe diseases, including cancer, cardiovascular diseases, and neurological disorders [4][5][6]. Heavy metals persist and biomagnify in trophic chains, and when in concentrations above the maximum permissible limits, they become toxic [7]. High concentrations of heavy metals pose a risk to consumer health, while prolonged consumption of honey containing Cu and Fe causes gastrointestinal disorders [8]. Heavy metals can metal pollutants. Due to an increase in the anthropological footprint of ecosystems and the degree of pollution thereof, the quality of honey is an obvious and stringent issue which concerns both honey producers and processors, where appropriate, and especially consumers who consume unprocessed honey directly from producers.
The purpose of this research is to determine heavy metal content (Pb, Cd, Cu, and Zn) in honey from private apiaries located in the most well-known historical polluted area in Romania. The analytical values obtained can provide information on the regional dynamics of pollution and allow for the adoption of acceptable thresholds in terms of the heavy metal content of natural honey, which are currently not in place.

Sample Collection Area
The polyfloral honey samples were collected directly from local beekeepers at six stationary apiaries (four samples from each apiary) from the town of Cops , a Mică, Romania and the neighboring villages of Micăsasa, Târnava, Valea Viilor, andŞeica Mică (Table 1, Figure 1).

Method of Analysis
The collected samples were stored in hermetically sealed plastic containers and kept in a cool and dark space at temperatures ranging between 4-5°C until analytical determinations were carried out. Before analysing the polyfloral honey samples without visible granules, the samples were This paper aims to identify the presence of potentially toxic heavy metals in the honey collected from an environment polluted for over 60 years by industrial activity with non-ferrous metals on the industrial platform of Cops , a Mică. The dispersion of pollutants emitted by the main polluter of the Cops , a Mică area is governed by the local climate. The wind regime of the area is influenced by the land orography, especially the presence of the Târnava Mare valley, the main air masses being channeled into the corridor of the Târnava Mare river (northeastern and southwestern air circulation) and the corridor of the Visa river (South-North air circulation).
The source of the pollution whose effects are analyzed in this article is an industrial park in the town of Cops , a Mică (46 • 06 59.10" N and 24 • 13 15.43" E) in the center of Romania.

Method of Analysis
The collected samples were stored in hermetically sealed plastic containers and kept in a cool and dark space at temperatures ranging between 4-5 • C until analytical determinations were carried out. Before analysing the polyfloral honey samples without visible granules, the samples were shaken for homogenization purposes, and those containing crystallised sugar (four samples) were heated to 65 • C in a water bath for 30 minutes to homogenise and solubilise the crystals. From the homogenised samples, 1 g of honey was weighed into polypropylene tubes, which were dissolved in 100 ml of heated, deionised, ultra-pure water(18.2 MΩ-cm resistivity from the water purification system Direct Q3UV Smart, Milipore, SAS, Molsheim, France). The solutions thus obtained were subjected to wet mineralisation, using 0.5 ml of analytically pure HNO 3 (Merck, Darmstadt, Germany) concentrate (65% v/v) as an oxidising agent, and the Top Wave Analytic Jena AG (Germany) microwave system was also used, designed for pressure digestion at temperatures up to 230 • C and pressures up to a maximum of 100 bars (1450 psi). The digester could contain a maximum of 24 digestion vessels made of modified polytetrafluorethylene (TFM-PTFE). Vessels were cleaned with 50 ml HNO 3 before each mineralization. The glassware used was left overnight in a 10% HNO 3 solution and rinsed prior to use with purified water to prevent possible contamination with Pb, Cd, Cu, and Zn. The extract was then filtered, ransferred to 25 mL volumetric flasks, and made to volume with deionised water. Mineralization was carried out in four steps, at temperatures of 145, 170,190, and 100 • C ( Table 2). Digestions of polyfloral honey samples were carried out in triplicate. A blank digest was carried out.
For the quantitative analysis of Pb, Cd, Zn, and Cu in the obtained extracts, we used the atomic absorption spectrometry method (AAS) because the harvested honey originated from a medium historically affected with a significant load of metal pollutants [41]. Quantitative determination of Cu and Zn was realized by FAAS (flame atomic absorbtion spectrometry) [42] while the concentration of non-essential trace metals Pb and Cd was performed using GFAAS (graphite furnace atomic absorption spectrometry) [43]. All samples were analysed in triplicate, and mean values were obtained. We used the Shimadzu AA-6300 Spectrometer equipped with a graphite furnace and single-element hollow cathode lamps adapted to eachanalysed metal (Cu was measured using the multi-element hollow method lamp), and the D2 lamp for background correction (BGC-D2). In FAAS for determination of Zn and Cu, an air-acetylene flame was used (Acetylene purity 98%, flow rate 1.8-2.0 L/min.) and a slot burner head from titan which was 10 cm long. For graphite furnace determination, argon (Ar) was used (flow rate 0-1.5L/min). The instrument parameters were optimised following the manufacturer's recommendations (Table 3). Merck standard solutions (1000 mgL −1 ) of Pb, Cd, Zn, and Cu were used to prepare the working standards [44]  During the analyses, the argon flow rate through the graphite tube was 250 mL/min. Settings for graphite furnace atomic absorption spectrometry (GFAAS) are summarized in Table 4.

Results
All samples subjected to analytical determinations had detectable Pb, Cd, Cu, and Zn content. Concentration values show a significant dispersion; the differences between the apiaries were statistically less reliable for copper (Table 5). Statistical analyses was performed using STATISTICA 8.0 (Stat Soft, Tulsa, OK, USA). The order of the accumulation degree expressed in median concentration values mg/kg for the elements surveyed is Zn > Cu > Cd > Pb.
The differences in terms of frontal exposure versus secondary valley exposure are statistically significant only for the Pb content (Table 6), which is, on average, 1 mg/kg higher in the apiaries located on the main valley, which is the driver for the pollutants of the apiaries on the side valleys.

Discussion
The polluters on the industrial platform were present mainly in the valley of the Târnava Mare river being scattered to the West (village of Micăsasa and city of Blaj), the East (city of Mediaş and town of Dumbrăveni), or South in the direction of the Șomârd-Şoala villages [45]. The preferential circulation of the air masses guided by the orography of the land resulted in the accumulation of significant amounts of Pb and Cd in the honey sampled from the localities of Târnava (located east of the main polluter) and Valea Viilor (southeast of Copşa Mică). The analytical values of Zn and Cu are above those determined in the honey sampled in the town of Copşa Mică, which supports the model of the remote dispersion of pollutants and the importance of the role of orography.
The accumulation of Pb in the environment is mainly due to mining, the melting and production of metals, and the battery industry, as Pb is a non-essential metal that has no physiological role in the metabolism of plants or animals. The exposure to Pb occurs through contact with soil, air, water, and contaminated food [46], and in time it causes anemia, neurological effects,

Discussion
The polluters on the industrial platform were present mainly in the valley of the Târnava Mare river being scattered to the West (village of Micăsasa and city of Blaj), the East (city of Mediaş and town of Dumbrăveni), or South in the direction of the S , omârd-Şoala villages [45]. The preferential circulation of the air masses guided by the orography of the land resulted in the accumulation of significant amounts of Pb and Cd in the honey sampled from the localities of Târnava (located east of the main polluter) and Valea Viilor (southeast of Copşa Mică). The analytical values of Zn and Cu are above those determined in the honey sampled in the town of Copşa Mică, which supports the model of the remote dispersion of pollutants and the importance of the role of orography.
The accumulation of Pb in the environment is mainly due to mining, the melting and production of metals, and the battery industry, as Pb is a non-essential metal that has no physiological role in the metabolism of plants or animals. The exposure to Pb occurs through contact with soil, air, water, and contaminated food [46], and in time it causes anemia, neurological effects, nephropathy, renal tubular dysfunction, as well as impairment of reproductive function in both genders [47].
Although it is easily surpassed by Cd in terms of average and maximum levels, the honey sampled contained significant amounts of concentrated Pb (Table 2). Golob et al. [48] determined a maximum Pb concentration of 79.1 mg/kg in honey sampled in Slovenia, and Yilmaz and Yavuz [49], discovered high values of 4.2-6.3 mg/kg Pb in honey sampled in the Southeast Anatolia Region, Turkey. Concentrations of up to 80.37 mg/kg were detected in honey sampled in the polluted areas of Italy by Dambrosio and Marchesini [50]. Frias et al. [51] determined high Pb concentrations of 31.50 mg/kg in the honey sampled in Tenerife, Spain. The maximum Pb concentrations are lower than those obtained between 2005-2011 by Berinde and Michnea [52], who also determined Pbconcentration values ranging between 0.18-20.34 mg/kg in the area of the city of Baia Mare, which is also a historical polluted area of Romania. Bogdanov [30] reviewed the results presented in various papers in the literature and noted concentrations of Pb in honey ranging between 0.01-1.8 mg/kg and concentrations of Cd ranging between 0.03-2.1 mg/kg.
As far as the Pb content in Polish honey goes, Pb concentration values above 1 mg/kg were also obtained by Roman [27] from the honey sampled in the Legnica and Glogow industrial regions (Pb median values ranging between 0.465-1.097 mg/kg) and between 0.17-1.90 mg/kg in the Wroclaw region (Lower Silesia), [53]. Oddi and Bertani [54], Conti et al. [55], and Porrini et al. [28] determined maximum Pb concentration values ranging between 1.10-1.74 mg/kg in polyfloral honey sampled in Italy. Singh et al. [56], reported Pb concentration values ranging between 0.2-4.2 mg/kg in the case of 13 samples of monofloral and polyfloral honey collected from different regions of Karnataka, India. An analysis of some honey batches collected on the Czech market in 1999 from different honey producers indicated Pb concentration values ranging between 0.0184-1.0003 mg/kg [57].
Low concentrations of Pb which are below the values presented in our research were discovered in honey samples studied by Tuzen and Soylak [26] in Central Anatolia, with values ranging between 0.0176-0.0321 mg/kg; the same goes for Matusevicius et al. [58]  With regard to Cd concentrations, similar or lower levels in comparison with those determined in our research were reported by Dzugan et al. [60] (0.03 ppm) in Podcapacia, Southeast Poland; Derebasi et al. [59] (0.07 mg/kg) in Turkey; Tuzen and Soylak [26] (between 0.010-0.022 mg/kg) in Central Anatolia-Turkey; and Celechovska and Vorlova [57] (between 0.0005-0.0774 mg/kg) and Singh et al. [56] (0.005-0.76 mg/kg) in Karnataka India. Matusevicius et al. [58] reported maximum Cd concentrations (between 0.0039-0.0165 mg/kg) in different regions of Lithuania. Cd concentration values in polyfloral honey in the sample plot of Târnava, Micăsasa II, CopşaMică, and ValeaViilor surpassed the ones obtained by Bratu and Georgescu [62] in CopşaMică at a distance of 8-25 km from the main source of pollution. Polyfloral honey from the sample plot ofŞeicaMică and Micăsasa I was found to have a lower Cd content, as against the one analysed by Bratua nd Georgescu [62] (0.015-0.032 mg/kg).
The maximum concentration of Cd of 3.809 mg/kg that was determined from the polyfloral honey sampled from Târnava apiary was below the values observed by Frias et al. [51] (46.32 mg/kg Cd) in Tenerife, Spain. Simedru et al. [61] showed that the level of Cd in honey samples in Cluj County was below the detection limit of the used equipment. Devillers et al. [63] studied 150 samples of acacia honey from polluted areas (50%) and "virtually unpolluted" areas in the territory of France, and noticed the absence of Cd and Pb from the analysed samples, although other metals such as Ag, Zn, and Cr were detected as consequences of anthropogenic pollution.
Although zinc is an essential element, excessive bio-accumulated concentrations are the effect of anthropogenic intake. Acute poisoning with Zn occurs through inhalation at the workplace with symptoms such as nausea, vomiting, diarrhea, lethargy, and fever [64].
The honey examined by us contains the essential Zn and Cu trace elements in concentrations ranging between 15.00-36.40 mg/kg in the case of Zn, the median value being 20.400 mg/kg, and between 2.00-33.00 mg/kg in the case of Cu with a median value of 3.70 mg/kg. The maximum values close to the Zn values determined by us are those reported by Celechovska and Vorlova [57], as well as by Tuzen and Soylak [26] (Table 7). The level of heavy metals in honey can often be increased by poor harvesting and storage conditions, and contamination during the fumigation, extraction, and storage of honey which are non-compliant with hygiene standards recommended or required by law. This is why honey monitoring is required at both the producer and processor levels. The high values in terms of non-essential heavy metals concentration measured in sampled honey are a warning for the local population, as in terms of the provisional tolerable weekly intake (PTWI) for Pb and Cd, other food coming from a polluted environment also participates, and the potentially toxic heavy metal concentration in the human body subsequently increases. According

Conclusions
Historical pollution in the surveyed area, totaling over six decades of permanent contact with pollutants, marked the main bee product-honey. The results of this study offers valuable information regarding the effects of the environment on the quality of polyfloral honey produced in the Cops , a Mică area. Analytical determinations of the content of potentially toxic metal pollutants in the polyfloral honey collected from the local bee producers with stationary hives in the closing year of industrial production activity indicated high concentrations of accumulated heavy metals, posing a risk to the health of consumers. The apiaries located on the valley that channel the pollutants from the industrial platform are prone to higher Pb bioaccumulations than the apiaries situated on the side valleys. Concentrations of Cd were exponentially diminishing while moving away from the source of pollution, while Cu concentrations increased linearly.
The results are useful for improving the quality of the honey value chain. An integrated program on quality assurance should be conducted. Beekeepers should pay attention to the location of the apiaries since there still are high accumulations of heavy metals in the area. Because of the interrelation between environmental pollution and food, honey and bee products need to be carefully monitored to eliminate even suspected contamination, especially in a historically polluted area from appointed source pollution, such as the town of Cops , a Mică and its neighborhood. This study also has several limitations related to the limited number of analyzed apiaries and research areas. For future studies, the research area should be extended, and comparative analysis should be conducted.