Polycyclic aromatic hydrocarbons and risk elements in honey from the South Moravian region ( Czech Republic )

The aim of this study was to determine the actual content of some exogenous contaminants in the honey from the South Moravian region of the Czech Republic. The content of polycyclic aromatic hydrocarbons (PAHs) and risk elements (Mercury Hg, Cadmium Cd, Lead Pb, and Arsenic As) in multifloral blossom and honey dew samples of honey were determined by high-performance liquid chromatography and atomic absorption spectroscopy methods. The samples were collected form beekeepers and from retail stores located in South Moravia in the Czech Republic. Concentrations of individual PAHs in honey samples ranged between 0.02 μg·kg-1–1.93 μg·kg-1. The limit of benzo[a] pyrene for infant formula (1.0 μg·kg-1) was not exceeded and fluoranthene was not quantified in any of the samples. Concentrations of Hg, Cd, and Pb were in the range of 3.24 μg·kg-1–11.31 μg·kg-1, 0.95 μg·kg-1–32.35 μg·kg-1, and 22.80 μg·kg-1–177.85 μg·kg-1, respectively. Concentration of As exceeded the detection limit only in three samples, ranging from 3.51 μg·kg-1 to 4.35 μg·kg-1. Acceptable limits for trace elements in infant formula were met. Results of this study complete present knowledge of the contaminant content in Czech honey and confirmed high quality and safety of honey from the South Moravian region. Persistent organic pollutants, toxic elements, HPLC, atomic absorption spectroscopy Honey contains a number of nutritionally valuable compounds and is appreciated for its healing and prophylactic properties. These result from its composition and are reflected by physicochemical and chemical indicators (Lachman et al. 2010). According to some authors, honey is considered an environmental marker due to its ability to contain harmful substances coming from polluted environment and beekeeping practices, e.g. trace elements (Conti and Botre 2001), pesticides (Blasco et al. 2003), antibiotic residues (Hammel et al. 2008), and polycyclic aromatic hydrocarbons (PAHs) (Dobrinas et al. 2008). On the other hand, honeybees use their own detoxification mechanisms to decrease the content of harmful substances (Ferreira et al. 2010; Niu et al. 2011). The PAHs belong to the family of persistent organic pollutants with properties negatively impacting the human organism such as carcinogenicity, mutagenicity, etc. (Ramesh et al. 2004). Inhaled air and foodstuffs represent the main exposure source of PAHs for most of the population (Alexander et al. 2008). Data on the PAHs content in honey are very rare, nevertheless, some authors reported high concentrations of PAHs in honey (Dobrinas et al. 2008). Trace concentration of metals in honey and their high variability are dependent very strongly on the botanical and geographical origin, and less on the climatic and seasonal criteria (Čelechovská and Vorlová 2001). Remarkably elevated values of trace elements in honey were reported in samples from industrial and otherwise polluted areas (Bogdanov et al. 2007). The influence of environmental anthropogenic contaminations on the risk element content in honey is still discussed (Claudianos et al. 2006; Fredes and Montenegro 2006; Tuzen et al. 2006) because of evident low metal bioaccumulation (Conti and Botre 2001). On the other hand, according to previous studies the presence of toxic metals in honey is ACTA VET. BRNO 2012, 81: 169–174; doi:10.2754/avb201281020169 Address for correspondence: Ing. Petra Batelková Department of Milk Hygiene and Technology Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Palackého tř. 1/3, 612 42 Brno, Czech Republic Phone: +420 777 887 933 Fax: +420 541 562 711 E-mail: batelkova@email.cz http://actavet.vfu.cz/ significantly influenced by the environmental pollution (Toporčák et al. 1992; Pisany et al. 2008). The aim of our study was to determine the actual content of exogenous contaminants, i.e. PAHs and toxic elements (Hg, Cd, Pb, and As) in honey from the South Moravian region of the Czech Republic, to compare our results with known data obtained from previous studies, and to confirm the quality and safety of the honey tested. Materials and Methods Sample characterization Samples of honey were obtained from beekeepers located around the city of Brno (honey no. 1–10) and from Brno retail stores (honey no. 11–20). The country of origin of all honey samples was declared as the Czech Republic. Samples no. 1 and 2 were honey dew honeys and the remaining samples were multifloral blossom honeys. PAHs analysis We monitored the content of 15 PAHs pollutants in honey according to the US EPA (EPA/630/R-98/002, 1986), namely acenaphtene (Ace), anthracene (Ant), benzo[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[g,h,i] perylen (BghiP), benzo[b]fluoranthen (BbF), benzo[k]-fluoranthene (BkF), chrysene (Chr), dibenzo[a,h] anthracene (DahA), fluorene (Fl), fluoranthene (Fu), indeno[1,2,3-c,d] pyrene (IPy), naphthalene (Na), phenanthrene (Phe), and pyrene (Py). The honey sample (10 g) was mixed with anhydrous sodium sulphate, and 40 ml of dichloromethane were added and mixture was extracted by means of ultrasonic bath and Ultra Turrax. After filtration the solvent was evaporated to dryness (Büchi, Flawil, Switzerland), redissolved in 1 ml of acetonitrile, filtered (0.45 μm nylon membrane filter) and analysed by HPLC – Waters 2695 Alliance chromatographic system equipped with Empower 2 software, and the Waters 2475 Fluorescence detector (Waters, Milford, USA) using an excitation and emission wavelength program. The PAH C18 column (250 mm × 4.6 mm I.D. 5 mm – Waters, Milford, USA) was used. Column temperature was 30 °C, mobile phase A (water) and B (acetonitrile). The gradient elution was programmed as follows: 50% B (0–5 min), 50% B to 100% B linearly (5–20 min), 100% B (20–28 min), 100% B to 50% B linearly (28–32 min). The flow rate was set at 1.4 ml·min-1. Quantification was carried out by the external standard method. Risk element analysis Risk elements Cd and Pb were determined by electrothermal atomization HR-CS AAS (High Resolution Continuum Source Atomic Absorption Spectrometry) on the ContrAA 700 apparatus (Analytik, Jena, Germany) after mineralization of the sample with concentrated nitric acid and hydrogen peroxide in a laboratory autoclave with microwave heating (Ethos Sel, Milestone, Sorisole, Italy). For determination of As, the sample (after mineralization in autoclave) was ashed with the addition of magnesium nitrate in a muffle oven at 450 °C. The ash was dissolved in hydrochloric acid and AsV was reduced to AsIII. Arsenic was determined by hydride technique in graphite atomizer, HS 60 (Analytik). The apparatus was set to the conditions recommended by the producer and the temperature program was optimized for the individual elements (see Table 1). The measurement of Hg was done by HR-CS AAS on an Advanced Mercury Analyzer AMA – 254 (ALTEC, Dvůr Králové nad Labem, Czech Republic). Statistics Basic statistical characteristics (mean, standard deviation, minimum, maximum) for the exogenous elements were calculated using Microsoft Excel 2003. Statistical assessment of the data was carried out using the statistical and graphic software STAT Plus (Matoušková et al. 1992).

Honey contains a number of nutritionally valuable compounds and is appreciated for its healing and prophylactic properties.These result from its composition and are reflected by physicochemical and chemical indicators (Lachman et al. 2010).According to some authors, honey is considered an environmental marker due to its ability to contain harmful substances coming from polluted environment and beekeeping practices, e.g.trace elements (Conti and Botre 2001), pesticides (Blasco et al. 2003), antibiotic residues (Hammel et al. 2008), and polycyclic aromatic hydrocarbons (PAHs) (Dobrinas et al. 2008).On the other hand, honeybees use their own detoxification mechanisms to decrease the content of harmful substances (Ferreira et al. 2010;Niu et al. 2011).The PAHs belong to the family of persistent organic pollutants with properties negatively impacting the human organism such as carcinogenicity, mutagenicity, etc. (Ramesh et al. 2004).Inhaled air and foodstuffs represent the main exposure source of PAHs for most of the population (Alexander et al. 2008).Data on the PAHs content in honey are very rare, nevertheless, some authors reported high concentrations of PAHs in honey (Dobrinas et al. 2008).Trace concentration of metals in honey and their high variability are dependent very strongly on the botanical and geographical origin, and less on the climatic and seasonal criteria (Čelechovská and Vorlová 2001).Remarkably elevated values of trace elements in honey were reported in samples from industrial and otherwise polluted areas (Bogdanov et al. 2007).The influence of environmental anthropogenic contaminations on the risk element content in honey is still discussed (Claudianos et al. 2006;Fredes and Montenegro 2006;Tuzen et al. 2006) because of evident low metal bioaccumulation (Conti and Botre 2001).On the other hand, according to previous studies the presence of toxic metals in honey is significantly influenced by the environmental pollution (Toporčák et al. 1992;Pisany et al. 2008).
The aim of our study was to determine the actual content of exogenous contaminants, i.e.PAHs and toxic elements (Hg, Cd, Pb, and As) in honey from the South Moravian region of the Czech Republic, to compare our results with known data obtained from previous studies, and to confirm the quality and safety of the honey tested.

Sample characterization
Samples of honey were obtained from beekeepers located around the city of Brno (honey no.1-10) and from Brno retail stores (honey no. 11-20).The country of origin of all honey samples was declared as the Czech Republic.Samples no. 1 and 2 were honey dew honeys and the remaining samples were multifloral blossom honeys.

Risk element analysis
Risk elements Cd and Pb were determined by electrothermal atomization HR-CS AAS (High Resolution Continuum Source Atomic Absorption Spectrometry) on the ContrAA 700 apparatus (Analytik, Jena, Germany) after mineralization of the sample with concentrated nitric acid and hydrogen peroxide in a laboratory autoclave with microwave heating (Ethos Sel, Milestone, Sorisole, Italy).For determination of As, the sample (after mineralization in autoclave) was ashed with the addition of magnesium nitrate in a muffle oven at 450 °C.The ash was dissolved in hydrochloric acid and As V was reduced to As III .Arsenic was determined by hydride technique in graphite atomizer, HS 60 (Analytik).The apparatus was set to the conditions recommended by the producer and the temperature program was optimized for the individual elements (see Table 1).The measurement of Hg was done by HR-CS AAS on an Advanced Mercury Analyzer AMA -254 (ALTEC, Dvůr Králové nad Labem, Czech Republic).

Statistics
Basic statistical characteristics (mean, standard deviation, minimum, maximum) for the exogenous elements were calculated using Microsoft Excel 2003.Statistical assessment of the data was carried out using the statistical and graphic software STAT Plus (Matoušková et al. 1992).

Risk elements
The method AAS showed the following detection limits (defined as the concentration equivalent to three times the standard deviation of the analytical blank signal, n = 10) 0.25 μg•kg -1 , 4.5 μg•kg -1 and 0.9 μg•kg -1 for Cd, Pb and As, respectively.The recovery test ranged from 93.5% to 104.1% for individual elements.Detection limit for mercury was 0.01 ng•kg -1 .Concentrations of Hg, Cd, and Pb were found in the range of 3.24 μg•kg -1 to 11.31 μg•kg -1 , 0.95 μg•kg -1 to 32.35 μg•kg -1 , and 22.8 μg•kg -1 to 177.85 μg•kg -1 , respectively.Concentration of arsenic exceeded the detection limit only in three samples, ranging from 3.51 μg•kg -1 to 4.35 μg•kg -1 (Table 4 and 5).Table 6 presents comparisons of toxicology limits of provisional tolerable weekly intake (PTWI), and the calculated amount of honey that would meet the toxicology limit (NTL -weight of honey sample PTWI -provisional tolerable weekly intake (JECFA/72/SC 2010), WHO -World Health Organization, bw -body weight, c -maximum concentration, D -tolerable weekly amount of pollutant expressed in micrograms per person; D = PTWI × W, W -average personal weight (70 kg), NTL -weight of honey sample for filling toxicology limits expressed in kilograms per week; NTL = D/c, a -value for inorganic Hg (other than fish and shellfish), b -value for inorganic As for meeting toxicology limits).Calculation was carried out for maximal concentration of target analytes in honey.

Table 1 .
Additional information about the experimental conditions of risk elements analysis in honey.

Table 5 .
Concentration of trace elements (μg•kg -1 ) in honey collected from beekeepers and from retail.

Table 6 .
Comparison of maximum concentration of pollutants in honey with toxicology limits of the World Health Organization.