Lead Content in Multifloral Honey from Central Croatia over a Three-Year Period
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- Bilandžić, N., Sedak, M., Đokić, M. et al. Bull Environ Contam Toxicol (2012) 88: 985. doi:10.1007/s00128-012-0585-z
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Lead concentrations were analysed by atomic absorption spectrometry in multifloral honeys collected in central Croatia (Zagreb County) during a three-year period from 2009 to 2011. The mean levels of elements (μg/kg) in honey samples measured were: 90.8 in 2009, 189 in 2010 and 360 in 2011. Significant differences were observed, and Pb levels determined in 2009 were significantly lower than those in 2010 and 2011 (p < 0.05, both). In 2009 there was no concentration found above 300 μg/kg. However, in 2010 and 2011 levels exceeding 300 μg/kg were found in 28.6 % and 25 % of samples. Trace element levels of Pb determined in multifloral honey were generally higher than concentrations obtained from other geographical origins and neighbouring countries. These high concentrations of Pb may be related to the fact that the central region is becoming increasingly urban and the network of motorways is growing. Accordingly, the risk of positioning hives near zones of busy highways and railways is increasing. This underlines that particular attention should be paid to toxic Pb levels, due to their gradual increased during the study period.
Rapid developments and increases in mining and industrial activities have gradually redistributed many of the toxic metals from the earth’s crust to the soil, atmosphere and waters, foods and plants. Metals accumulate in the topsoil, thus endangering crops, vegetables and microflora and causing their incorporation into the food chain, which can entail a wide variety of adverse effects on animals and humans due to contaminant accumulation. Soils contaminated by metals, especially Pb, are an environmental issue and a priority.
Honey is a natural, sweet product and its chemical composition gives it many nourishing, prophylactic and healing properties. It is also used as an ingredient for sweetness, colour, flavour, caramelisation and viscosity in many type of food products (Rashed and Soltan 2004). The mineral content in honey ranges from about 0.04 % in pale honeys to 0.2 % in dark honeys (Anklam 1998). Investigations of environmental, geographical and botanical aspects of the mineral content in honey have shown that botanical factors have the greatest influence on trace element content with regard to soil and climate characteristics (Bogdanov et al. 2007). Elevated toxic element levels in honey, such as heavy metals (As, Cd, Hg, Pb), may be the result of elevated metal contents in plant nectar. High heavy element contents have been determined in honey from industrial areas with heavy industrial activities or near busy highways (Toporcák et al. 1992).
Widespread environmental contamination with Pb during the past 50 years has been caused by the use of Pb in batteries, bearing metals, cable coverings, gasoline additives, explosives and ammunition and in the manufacture of pesticides, antifouling paints and analytical reagents (Johnson 1998). It is a ubiquitous though biologically non-essential element, and its toxicity is caused by the ability of Pb2+ to interfere with several enzymes. Lead may cause a large variety of toxic effects, including gastrointestinal, muscular, reproductive, neurological or behavioural effects, including sleeplessness, fatigue, hearing and weight loss, and genetic malfunctions (Johnson 1998). Exposure to Pb may manifest as peripheral nerve degeneration with detrimental effects on intelligence in the case of prolonged ingestion of small amounts of metal. It may also cause poisoning in domestic animals, with elevated blood Pb levels detected in cattle from industrial areas (Swarup et al. 2005). Lead is one of the least soluble metals with a very long retention time, and its contamination is mainly restricted to surface soil in boreal forests that are rich in humic material with a podsolic stratification. Honeybees forage on plants growing over a relatively large area (more than 7 km2) and move from flower to flower, and are thus in constant contact with air, water and soil, branches and leaves. Therefore, honey is the result of a bio-accumulative process that is useful for collecting information about the environment, and thus may be considered a bioindicator of environmental pollution (Conti and Botre 2001; Pisani et al. 2008).
Multifloral honey is one of the most common honey types in Croatia. The aim of the present study is to examine the concentrations of Pb in multifloral honey collected over 3 years in the same geographical region, i.e. in Zagreb County in central Croatia.
Materials and Methods
Zagreb County covers an area of about 3,078 km2 with 9 urban centres having a total population of 96,928 and 25 municipalities, together with more than 300,000 inhabitants. This is the most populated, urbanized and industrialized region in Croatia. The capital city of Zagreb has more than 800,000 inhabitants and pronounced industrial activities: production of electronic devices, chemical and pharmaceutical industries, textile industry and food production. Also, small scale industry is situated near the small settlements. The capital and neighbouring cities are crossroads of European transport routes, with a network of busy highways and railways. Airport facilities are also located in the southerneastern part of the capital.
Upon collection, all honey samples (500 g) were placed into clean glass bottles, labelled and transferred to the laboratory and kept at 4–8°C until analysis.
The acids used, HNO3 and HCl, were of analytical reagent grade (Kemika, Croatia). Ultra high purity water processed through a purification system NIRO VV UV UF 20 (Nirosta d.o.o. Water Technologies, Osijek, Croatia) was used for all dilutions. Calibrations were prepared with element standard solutions of 1 g L−1 of Pb (Perkin Elmer, USA). Stock solution was diluted in HNO3 (0.2 %). As matrix modifiers in each atomization for Pb, 0.005 mg NH4H2PO4 (Perkin Elmer, USA) and 0.003 mg Mg(NO3)2 (Perkin Elmer, USA) were used.
Plastic and glassware were cleaned by soaking in diluted HNO3 (1/9; v/v) and by subsequent rinsing with high purity water and drying prior to use.
Honey samples (0.5 g) were digested with 4 mL HNO3 (65 % v/v) and 2 mL H2O2 (30 % v/v) with a Multiwave 3000 microwave closed system (Anton Paar, Germany). A blank digest was carried out in the same way. The digestion programme began at a power of 500 W, ramped for 1 min and hold for 4 min. The second step began at a power of 1,000 W ramped for 5 min and hold for 5 min. The third step began at a power of 1,400 W, ramped for 5 min and hold for 10 min. Digested samples were diluted to a final volume of 50 mL with double deionised water.
Detection limits for Pb were determined as the concentration corresponding to three times the standard deviation of twenty blanks. All samples were run in batches that included blanks, a standard calibration curve, two spiked honey samples, and one duplicate. To calculate the recovery percentage, ten honey samples were spiked with known amounts of Pb.
The analyses of Pb were performed by graphite furnace-atomic absorption spectroscopy with an AAnalyst 800 atomic absorption spectrometer (Perkin Elmer, USA), equipped with an AS 800 autosampler (Perkin Elmer, USA) set at 283.3 nm. Argon was used for graphite furnace measurements. Pyrolytic-coated graphite tubes with a platform were used.
All calculations and statistical analysis were performed using the software package Statistica 6.1 (StatSoft® Inc., USA). Data were log-transformed to improve normality prior to analysis to meet the underlying assumptions of the analysis of variance. One-way analysis of variance was used to test for differences in honey metal concentrations. The differences between the metal concentrations within different years were analyzed using the t test. A probability level of p ≤ 0.05 was considered statistically significant.
Results and Discussion
In addition to the environmental concern regarding honey element composition, quality control of honey is also important given the increasing global trends in total honey production and the fact that the European Union is the world’s largest consumer of honey (Vanhanen et al. 2011). Usually, the heavy metal content of honey is sufficiently small that even 100 g daily intake of honey would not contribute appreciably to dietary requirements. A provisional tolerable weekly intake of 25 μg/kg b.w. has been established as an acceptable level of Pb that may accumulate in the body and can be ingested on a weekly basis (WHO 2000).
On the other hand, when the environment consists of one or more metals, the content in honey may be enhanced and thereby influence health. Toxic trace metal contents have been presented in different honey types, mainly multifloral, from European countries during the past 10 years (Przybyłowski and Wilczyńska 2001; Bratu and Georgescu 2005; Golob et al. 2005; Tuzen et al. 2007; Pisani et al. 2008; Silici et al. 2008).
In the present study, Pb concentrations were determined in multifloral honey collected over 3 years from the same geographical origin. There are no mining activities in Zagreb County or in the territory of Croatia. However, industrial production is centred around large cities, primarily in the central Zagreb County region.
Lead concentrations (mean and range; (μg/kg, w.w.) in multifloral honey over a three-year period from central region of Croatia
No. of samples
The mean lead content in all honey samples investigated was 229 μg/kg. The highest mean Pb concentration was 1,521 μg/kg, measured in 2011. The results obtained in 2010 and 2011 were higher than previously determined values in the same region of Croatia (Bilandžic et al. 2011).
Frequency distribution of Pb concentrations in multifloral honey over a 3-year period in central region of Croatia
No. of samples
Number of samples in range (μg/kg)
Measured Pb concentrations in the present study were similar to Pb levels measured in honeys in Hungary, i.e. ranging from 18 to 163 μg/kg in floral honey, and from 17 to 144 μg/kg in accacia honey (Ajtony et al. 2007). However, the Pb contents in the present study were higher than levels determined in Turkey, a country known for its large honey production: 17.6–32.1 μg/kg (Tuzen and Soylak 2005), 8.4–106 μg/kg (Tuzen et al. 2007), 3.2–36.7 μg/kg (Silici et al. 2008). The observed concentrations were also higher than those found in honey (25–70 μg/kg) from Poland (Przybyłowski and Wilczyńska 2001) and in blossom multifloral honey (0.042 mg/kg) and rape blossom honey (0.096 mg/kg) from Czech Republic (Vorlová and Čelechovská 2002). Lead concentrations ranging from 0.0001 to 0.200 mg/kg were determined in Romanian honey (Antonescu and Mateescu 2001). Very low Pb contents were measured in different honey types from Romania (mg/kg): mixed flowers 0.07, Linden tree 0.09, rape 0.03, acacia tree 0.089 and dark honey 0.18 (Bratu and Georgescu 2005).
Lead content in multifloral honey measured in Italy in the past decade were (μg/kg): <50 in Lizzano and 620 in Bologna (Buldini et al. 2001), <2.0–63.0 in the City of Rome and Rome County (Conti and Botre 2001), 28.2–304 in Siena County (Pisani et al. 2008).
High Pb concentrations of 1.86–4.3 mg/kg of Pb were determined in acacia honey from Croatia’s neighbouring country, Slovenia (Golob et al. 2005). Also, high Pb levels of 6.3, 5.7 and 4.2 mg/kg were determined in Egyptian sesame, orange and clover honeys, respectively (Rashed and Soltan 2004). Furthermore, high Pb levels were measured in mixed flower honey of 920 μg/kg from Kashmir and 695 μg/kg from Ashok originating from India (Buldini et al. 2001).
In a recent study in New Zealand, Pb was not detected in five of ten honey types analysed, and the mean Pb content was 0.017 mg/kg (Vanhanen et al. 2011). It is suggested that New Zealand soils are classified as the lowest international Pb decile with a declining national median Pb level.
Lead is a widespread metal pollutant that can reach humans and animals through air, water and food. The fact that Pb is detected in honey is already evidence of pollution of the environment with this metal. The results of the present study show that the trace element levels of Pb determined in multifloral honey were generally higher than concentrations obtained from other geographical origins in Europe. These high concentrations of Pb in multifloral honey samples in the central region may be related to the fact that this region has become more urban and that the network of motorways is growing every year. Accordingly, the risk of positioning hives near to zones of busy highways and railways is increased. This underline that particular attention should be paid to toxic Pb levels regarding the fact that the Pb levels gradually increased during the study period.
We thank Marijana Fluka and Mirjana Hren for assistance in sample preparation. This study was supported with a grant from the Croatian Veterinary Institute, Zagreb, Croatia.