Heavy Metals in Honey Collected from Contaminated Locations: A Case of Lithuania

Abstract

Honey, as a bioindicator, can be used to determine the level of pollution in the environment with selected pollutants, including heavy metals. Twelve locations were selected for experimental studies near the main sources of pollution: industrial sites, landfills, railways, and highways. The honey samples were burned to ash, and the heavy metals in ashes were determined using aqua regia digestion in the microwave digestion system. The concentration of heavy metals (Cd, Cr, Cu, Pb, and Ni) was determined using a Buck Scientific model 210 VGP atomic absorption spectrophotometer with a graphite furnace atomizer and an acetylene-air flame. These median amounts of heavy metals were found in the analyzed honey samples: 0.0030 mg/kg for Cd, 0.0179 mg/kg for Pb, 0.0317 mg/kg for Cr, 0.0999 mg/kg for Cu, and 0.0332 mg/kg for Ni. The obtained results were compared with honey samples research conducted in other countries. It is difficult to compare the level of heavy metal results found in honey from different countries, as the type of honey, soil composition, rainfall, air temperature, the plants from which it was harvested, its vegetation and flowering duration, and the degree of anthropogenic pollution in the area differ. The heavy metal content tested in honey was found to be low, except for the Pb content in one sample of honey, and did not pose a risk to human health. A statistical analysis including average, median, standard deviation, confidence intervals, and Spearman coefficients was performed for the evaluation of the relationships between the heavy metal quantities and the determination of the impact of pollution sources (transport and industry). The correlation analysis showed a strong negative correlation coefficient between heavy metals and distance (r = −0.593 to −0.204).

1. Introduction

In recent years, a healthy environment has become the main goal for European countries. As stated in the EEA Report, a significant part of the burden of disease in Europe is attributed to manmade environmental pollution [1]. Industrial and agricultural activities, transport, and other anthropogenic activities release large quantities of heavy metals into the soil, atmosphere, and water. As a result, concentrations of heavy metals in the environment increase significantly and affect the ecological balance and human health [2]. Heavy metals in the atmosphere pose a serious threat to health and the environment, including food safety, human health, and ecosystems, as these heavy metal particles can be deposited on water and land surfaces and readily bioaccumulate in food chains [3]. Heavy metals are potentially harmful compounds that enter foodstuffs from the environment, enter the human body through food, and are quickly absorbed into the biological cycle. Food safety has become an essential quality feature of food [4,5].

Honey is a natural, highly nutritious bioproduct with bactericidal and stimulating properties [6]. Its value depends on the species, location, environment, collection time, and climatic conditions [4]. The valuable properties of honey as a natural organic product depend on its variety and origin. The quantitative and qualitative ratio of chemical elements is characteristic of each flower of the plant from each region of the country, so the total amount of minerals depends on the location [7].

Heavy metals and other contaminants in honey are the subject of research in a number of countries, since honey is a good indicator of the level of pollution: in Europe (France [8], Greece [9], Italy [10], Lithuania [7], Poland [11,12,13], Romania [4,11,14], and Slovakia [15]) and other continents (Australia [16], Bangladesh [17], China [5], Iran [18], and Turkey [6,19,20]).

The main concentrations of metals in honey come from the soil. They are transported through the root system to the honey plants, enter the nectar, and then the honey produced by the bees [21]. Pesticides, heavy metals, and radioactive substances enter plants; air; water; and, finally, enter nectar, pollen, and honey [22]. Honey can be contaminated by two sources: the environment where bees collect honey and the beekeeping method. The foraging area of bees covers about 7 km² and includes various environments, plants, and foods. When foraging for nectar, honeydew, and pollen in such a large area, bees come into contact with plants, air, water, and soil [21]. In the case of environmental pollution, bees also become contaminated and carry the pollutants from the environment into the hive itself or the collected raw materials (Figure 1).

The content of honey might be an indicator of pollution in the environment or the location of its genesis [16]. The total amount of minerals in honey is largely dependent on geography, because each region has its unique qualitative and quantitative ratio of chemical components.

The mobility and accumulation of heavy metals in the environment, as well as their consequences in the ecosystems and humans, require as much data as possible. The amount of heavy metals in plants varies depending on their location, the type and pollution of the soil, the distance from the transport routes, and the industrial locations that release heavy metals [23]. In the air, heavy metals settle on the surface of the soil and migrate to deeper layers [24]. The highest concentration is in the topsoil layer [25]. It is noticeable that, despite the protective function of roots, heavy metals still enter the upper parts of the plant. The amount of heavy metals entering the stems and leaves increases with the increasing concentration of heavy metals in the soil. In soils contaminated with heavy metals, relatively high concentrations of these metals are found in the aboveground parts of plants [26].

The European Union is the second-largest honey producer in the world after China. Every year, some 600,000 beekeepers and 20 million hives produce about 218,000 tons of honey [27]. The consumption of honey varies greatly from country to country. The annual consumption of honey is increasing and is estimated at 0.3 to 0.4 kg per capita in Italy and France, 1.0–1.8 kg in Germany and Austria, and 0.07 kg in Brazil [28].

The results of the studies are evaluated by comparing them with the levels permitted in the main nutritional products, the presence of which may pose a risk of toxicity. The maximum levels of the chemical elements (MRL) are established following the recommendations of the World Health Organization (WHO), the Joint Food Code Commission (FAO), the Republic of Lithuania’s Food Law, and the actual situation in the country. In many countries, health authorities adopt recommended dietary guidelines for the population, which, in addition to basic food components, contain the necessary daily nutrient limits and limit them. Concern for food quality requires constant monitoring, analysis of the latest research methods, and evaluation of the heavy metal contents [15]. However, heavy metals in honey are not regulated by LT and EU legislation.

Currently, Lithuania is governed by Commission Regulation (EC) No. 1881/2006, which establishes limit levels for pollutants in specific commodities (OJ 2006 L 364, p. 5). It sets limit values for Pb, Cd, Hg, and Sn in foods of plant and animal origins. According to the directive 2001/110/EC of the Council relating to honey, “Honey is the natural sweet substance produced by Apis mellifera bees 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”. This term defines honey as of plant and animal origin, so the limit value can only be determined as a range: for Pb, 0.1–0.5 mg/kg, while the cadmium rate is 0.05–0.3 mg/kg, according to the Commission Regulation (EC) No. 1881/2006 of 19 December 2006.

Thus, the assessment of the heavy metal content in honey serves not only to evaluate the quality of these products but also to determine the general level of environmental pollution. The aim of the study was to use honey as an indicator for evaluating heavy metal contamination in the environment in potentially contaminated areas of Lithuania. The scientific hypothesis was checked during the research: the concentration of selected heavy metals (Cd, Cr, Cu, Pb, and Ni) in honey samples is reducing with increasing distance from potential sources of pollution.

2. Materials and Methods

2.1. Sampling Places

Twelve locations were selected for experimental studies (Figure 2), where specific “small hives”—the nucleus—were used for research. Čepkeliai Reserve (Lynežeriai Village) and Druskininkai in the village of Mizarai honey was sampled from hives. Bees can potentially collect honey up to 4 km from the hive, so the heavy metal contamination of honey samples reflects environmental pollution at this distance from the hive. The description of the sampling sites, GPS coordinates, and distance from the pollution source are provided in

Table 1. Description of the sampling sites. Pollution sources and distances from them.

No of the Site on the Map	Sampling Site, Location of Hives, GPS Coordinates of the Site (Latitude; Longitude)	The Main Source of Pollution	Distance from Pollution Source, km
1	Čepkeliai reserve (Lynežeriai village) (54.062486, 24.570274) 54°03′45.0″ N 24°34′13.0″ E	Background	unpolluted area
2	Klaipėda, Jakai settlement (55.697356, 21.219722) 55°41′50.5″ N 21°13′11.0″ E	Jakai road transport ring	1.4 km
3	Jonava (Paneriai st.) (55.074389, 24.290667) 55°04′27.8″ N 24°17′26.4″ E	SC Achema (produces nitrogen fertilizers)	2.7 km (to the West from the factory)
4	Kaunas, Domeikava (54.963519, 23.926490) 54°57′48.7″ N 23°55′35.4″ E	The Western Bypass Transport Hub	3.4 km
5	Kėdainiai (Taučiūnai village) (55.282074, 24.054986) 55°16′55.5″ N 24°03′18.0″ E	SC Lifosa (produces mineral fertilizers)	2.5 km (to the North-East from the factory)
6	Vilnius (Bajorai settlement) (54.754128, 25.235967) 54°45′14.9″ N 25°14′09.5″ E	Vilnius-Panevėžys motorway	1.5 km
7	Jonava (Jurkonys village) (55.081986, 24.417128) 55°04′55.2″ N 24°25′01.7″ E	SC Achema (produces nitrogen fertilizers)	3.8 km (to the East from the factory)
8	Mizarai village near Druskininkai (54.018321, 23.937369) 54°01′06.0″ N 23°56′14.5″ E	Background	unpolluted area
9	Sitkūnai (55.040385, 23.815676) 55°02′25.4″ N 23°48′56.4″ E	The road transport junction	0.6 km
10	Vievis (54.770950, 24.826148) 54°46′15.4″ N 24°49′34.1″ E	The railway line Vilnius-Klaipėda and the highway Vilnius-Kaunas.	0.3 km from the railway line and 0.1 km from the highway
11	Kėdainiai (Paobelys settlement) (55.264325, 23.964224) 55°15′51.6″ N 23°57′51.2″ E	The SC Lifosa (produces mineral fertilizers)	2 km (South-West from the factory)
12	Rykantai (54.716856; 24.985023) 54°43′00.7″ N 24°59′06.1″ E	The Kariotiškės landfill	1.4 km

.

Honey samples were collected during the 2020 summer season. The following locations were identified as potential sources of air, precipitation, and soil pollution. Sampling sites (1) and (8) were selected as a couple of the least polluted areas of Lithuania (background). The hives were built far from the sources of pollution, so this area was chosen as the control (background).

2.2. Reagents and Equipment

All chemicals and reagents that were used in the experiments were of high purity (Sigma Aldrich, Darmstadt, Germany). Aqueous solutions were prepared by mixing standards with high-purity deionized water. Metal standards manufactured by VWR Chemicals (Lutterworth, UK) were used to calibrate the graphite furnace atomic absorption spectrometer (GFAAS) and the flame atomic absorption spectrometer (FAAS). All flasks used in the experiments were drained for 24 h with 5M HNO₃ and then carefully washed with deionized water 3 to 4 times. High-precision analytical balance Radwag AS 60/220.R2 was used to weigh the honey samples.

2.3. Sample Preparation for Metal Analysis

As it is assumed that the heavy metal concentration could be very low and difficult to measure, the honey samples were concentrated by burning them; after which, the ash samples were microwave-digested. Each honey sample (100 g) was dried in a furnace until it reached a constant mass at 100 ± 5 °C. After that, in a muffle furnace, the temperature was gradually raised for 2 h to 500 ± 20.0 °C from 100 °C/1 h. The ash that resulted was allowed to cool to an ambient temperature. The moisture in the ashes gradually evaporated during the cooling to room temperature. The total recoverable heavy metals in the ashes were determined using the aqua regia (3:1, v/v, HCl to HNO₃) digestion technique. The honey ash samples (approximately 1.0 g) were digested in Teflon containers for around 50 min using the Milestone Ethos Touch Control microwave digestion system (Milestone SRL, Sorisole, Italy). The solution was filtered through a 0.45-m PTFE membrane filter, quantitatively transferred to a 50-mL volumetric flask, and supplemented with deionized water to the mark after cooling to room temperature. After that, the concentration of heavy metals was determined using a Buck Scientific model 210 VGP atomic absorption spectrophotometer with a Buck Scientific model 220-GF graphite furnace atomizer and an acetylene–air flame. The measurement wavelengths for different heavy metals were Cd (228.8 nm), Cr (357.9 nm), Cu (324.8 nm), Pb (283.3 nm), and Ni (232.0 nm). A slit width of 0.7 nm was used for Cu, Pb, Cr, and Cd, while a slit width of 0.2 nm was used for Ni.

The amount of heavy metals in honey ash was calculated by taking into account the measured concentration of the analyte in solution, the volume of the sample, the dilution factor, and the amount of honey ash taken for the test. The quantity of heavy metals in the honey samples was calculated by multiplying the quantity of the analyte in the ash by the weight of the ash (in grams) obtained by burning 100 g of honey in a muffle furnace and dividing by 100 (the initial weight of the honey before burning in grams).

2.4. Quality Control

The concentrations of all the metals in the standard solutions used were the same and equal to 1000 mg/L in 2% HNO₃. Working aqueous standard solutions containing Cu, Pb, Cd, Cr, and Ni were prepared by serial dilution of the certified reference metal standard solution for atomic absorption spectroscopy with deionized water. Calibration curves were constructed for each chemical element using appropriate standard solutions by diluting stock solutions of 1000 mg/L of each chemical element supplied by VWR Chemicals (England). Free samples of honey were taken from each hive. All experiments were conducted in triplicate, and the mean of the three was presented. Two blanks (without metal ions) were used for each determination.

2.5. Statistical Procedures

Excel and Statistica were used to perform the statistical analysis of the data. The link between the heavy metal levels in honey samples was investigated using the Spearman’s rank correlation coefficient and cluster analysis. At a significance level of 0.05, the average, median, standard deviation, confidence intervals, and Spearman’s rank coefficients were calculated.

3. Results and Discussion

The following heavy metals were determined in honey: cadmium (Cd), lead (Pb), chromium (Cr), copper (Cu), and nickel (Ni). Basic statistical data such as the average value, standard deviation, and minimum and maximum values can be seen in Figure 3.

Comparison of the median of the heavy metals (Pb, Ni, Cu, Cr, and Cd) in honey shows that the median of the heavy metals decreases in the following order: Cu > Ni > Cr > Pb > Cd. The analyses of the data showed that the amounts of copper (Cu), lead (Pb), nickel (Ni), and chromium (Cr) were significantly higher than those of cadmium (Cd) (Figure 3).

The results obtained during this investigation were compared with those of other scientists. It was determined that the obtained results of Pb in the honey samples investigated during this research were (0.008–1.649 mg/kg) higher than those that were investigated in 2006 [7] in Lithuania; it should be noted that there were other sampling places. Seven trace elements were analyzed (Pb, Cd, Cu, Zn, Sr, Ba, and Rb). The concentration of Pb varied from 0.0029 to 0.0221 mg/kg. Possibly, the higher concentrations determined in our research could have been influenced by the increased number of cars in the streets and increased production. However, other scientists stated slightly, or much higher, concentrations compared in

Table 2. Comparison of the heavy metal contents in honey samples with results from different countries.

	Cd, mg/kg	Pb, mg/kg	Cr, mg/kg	Cu, mg/kg	Ni, mg/kg
Present study	0.002–0.013	0.008–1.649	0.019–0.051	0.024–0.262	0.012–0.087
Slovakia [15]	0.001–0.0524	0.007–0.085	0.027–0.043	0.045–2.015	0.023–1.359
Poland [11,12]	0.007–0.0021 [11]	0.05–0.098 [11]	0.037–0.055 [11]	0.05–1.38 [12]	0.184–1.241 [11]
Turkey [19]	0.001–0.0179	0.008–0.106	0.0025–0.0379	0.23–2.41	0.0053–0.0299
Greece [9]	-	0.26–0.41	-	0.41–1.68	0.10–1.63
Romania [4]	0.05–3.81	0.76–3.41	-	2.00–33.01	-
Italy [10]	0.001–0.04	0.01–1.39	0.02–2.04	-	-
Iran [18]	0.002–0.126	0.117–1.627	0.172–1.22	0.028–2.872	0.065–1.094
LIMIT EU *	0.05–0.3	0.1–0.5	-	-	-

* EU Commission Regulation (EC) No. 1881/2006.

. According to the data presented, honey picked in Greece, Romania, Italy, and Iran could potentially be harmful to human health according to the Pb level.

Meanwhile, the Cd concentration obtained in Lithuanian honey was lower (0.002–0.013 mg/kg) than in the samples gathered in Poland (0.007–0.0021 mg/kg) [11]. However, it was the same level as in another earlier research [7]. Staniškienė [7] referred to a very similar concentration of Cd 0.0041–0.0146 mg/kg. In other countries, different concentrations were reported (Table 2). According to the available data, honey from Slovakia, Romania, and Iran could potentially be harmful to health, depending on the Cd contents.

The lower Cu concentrations (0.024–0.262 mg/kg) in the samples measured in Lithuania than in the samples collected in Poland in 2016 (0.047–0.540 mg/kg) [12] were also detected during earlier research in Lithuania (Cu 0.1196–0.3429 mg/kg) [7].

In Lithuania and other countries, the Cr concentrations were found to be relatively comparable in Lithuania, Poland [11], Turkey [19], and Slovakia [15] (Table 2). Meanwhile, in Italy, there were much higher concentrations of Cr (0.02–2.04 mg/kg) [10]. The author suggested that these higher concentrations could have a connection with activities related to petroleum extraction. The Ni concentration was determined to be much lower in Lithuanian honey samples than in samples collected and measured in Poland [11].

There are no limit values set for Cu, Ni, or Cr, and it is difficult to evaluate the danger to human health according to these heavy metal contents.

The heavy metal quantities investigated in almost all honey samples were within the limits, indicating their purity. It is difficult to compare determined quantities of heavy metals in honey samples with results reported by other scientists due to differences in the type of honey, the composition of the soil, the amount of precipitation, air temperature, species of plant from which the honey was harvested, and the degree of pollution of that region. Continuous exposure to a small amount of Pb and Cd, even at low concentrations, can cause severe health problems, such as mental development disorders and irregularities in hematological function by limiting the formation of hemoglobin and shortening the lifespan of red blood cells [29].

The pollutant standardized mass concentration data were compared with the group sample locations using cluster analysis (CA). The distance between measurement locations was calculated using Euclidean distance. To determine the distances between groups, Ward’s hierarchical group method was used for the analysis of variance methodology (linkage) [13].

Figure 4 shows the dendrograms resulting from the application of CA to the concentrations of heavy metals from different sampling places. The results obtained showed that the sampling site can be grouped into three groups. It can be seen that sampling site No. 10 was clustered separately. This site was different from the others; the hive was located between the railway (0.3 km) and highway (0.1 km). The other two sampling places (No. 5 and 11) were located in Kėdainiai near the plant of fertilizer “Lifosa”. After the cluster analysis, they were divided into different clusters (Figure 4). These sampling places were located in different directions from the factory: site No. 5 was 2.5 km in the east direction, and site No. 11 was 2 km in the southwest direction. The prevailing wind in this area is south, southwest, and west (blowing from this direction). Higher concentrations of heavy metals were obtained in the sampling site located in the prevailing direction of the wind from the fertilizer plant.

The hypothesis was raised that the heavy metal quantities in honey are independent of each other. To test the hypothesis, it was conducted in the principal component analysis (PCA) and Spearman’s rank correlation analysis.

The PCA was used to analyze the data. The loadings on the first principal component were plotted against the loadings on the second principal component in a scatter plot (Figure 5). Two main components (PC) were identified as accounting for 69.04% of the variance in the dataset. PC1 accounted for 38.76% of the variability, whereas PC2 accounted for 30.28%.

The graph clearly distinguishes the variables that have a positive correlation between Cu and Cr, as well as Pb and Ni.

Table 3. Spearman’s rank correlation coefficients between different heavy metals in honey and distances (km) from the main sources.

	Cd	Pb	Cu	Cr	Ni
Cd	1
Pb	0.032	1
Cu	0.302	0.392	1
Cr	0.484	0.343	0.657	1.000
Ni	0.670	0.524	0.252	0.203	1
Distance	−0.319	−0.565	−0.204	−0.393	−0.593

shows the Spearman’s correlation relationship between the different heavy metals analyzed and the distance from the main pollution sources suggested near the sampling site. The strongest relationships were determined between Pb and Ni (r = 0.524), Cd and Ni (r = 0.670), and also Cu and Cr (r = 0.657). The correlation analysis revealed a negative relationship between these heavy metals and the distance from the main suggested pollution source. The strongest negative correlation coefficients were determined between Pb and Ni and the distance.

A scientific hypothesis was tested during the investigation: The concentration of selected heavy metals (Cd, Cr, Cu, Pb, and Ni) in honey samples decreases with increasing distance from the potential pollution sources. Spearman’s correlation analysis was used to test the established scientific hypothesis. According to the results obtained, this hypothesis is true. Spearman’s correlation analysis indicated a negative correlation coefficient between heavy metal concentrations and the distance from traffic roads. These results and conclusions based on these results were preliminary. Heavy metals released from transport can be assumed to fall near the source and are accumulated by plants or infiltrated into the soil.

4. Conclusions

In this research, honey collected from 12 different sampling sites in Lithuania was analyzed. The sampling sites represented potentially contaminated sites with different sources of pollution: two industrial sites (near the large fertilizer producers), landfills, railways, and highways and two unpolluted background locations. According to the cluster analysis, the sample locations could be divided into three groups. The sampling site located between highway and railway (No. 10) was selected in a different cluster; at this location, the highest concentration of Pb and Ni was determined. Honey samples collected near highways and industrial places showed a negative correlation between all the heavy metals (Pb, Cd, Cu, Cr, and Ni) and the distances from the roads (r = −0.204 to −0.593). It can be assumed that the heavy metals released from the source fall out near the source and accumulate in the plants. The locations of hives should be carefully selected by evaluating the sources of pollution around. Although the determined concentration of heavy metals was not very high, heavy metals in honey are not regulated by LT and EU legislation. However, the daily consumption of this honey can lead to an increase in the daily intake of toxic elements. Therefore, it is necessary to determine their permissible concentrations in honey.