Major and trace elements levels in multifloral and unifloral honeys in Croatia

Journal of Food Composition and Analysis 33 (2014) 132–138

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Original Research Article

Major and trace elements levels in multifloral and unifloral honeys in Croatia Nina Bilandzˇic´ a,*, Milica Gacˇic´ b, Maja Ðokic´ a, Marija Sedak a, Ðurpica Ivanec Sˇipusˇic´ b, Ana Koncˇurat c, Ivana Tlak Gajger d a

Laboratory for Residue Control, Department for Veterinary Public Health, Croatian Veterinary Institute, 10000 Zagreb, Croatia Food Control Center, Faculty of Food and Biotechnology, University of Zagreb, 10000 Zagreb, Croatia Laboratory for Culture Media Preparation and Sterilisation, Veterinary Institute Krizˇevci, 48260 Krizˇevci, Croatia d Department for Biology and Pathology of Fish and Bees, Faculty of Veterinary Medicine University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 May 2012 Received in revised form 4 November 2013 Accepted 17 December 2013

Different honey types were collected in Croatia during 2010 and 2011: 7 multifloral orchard honeys, 7 multifloral meadow honeys, 19 black locust, 9 chestnut, 11 lime and 6 sage honeys. Elements were measured using graphite (As, Cu, Cd, Pb, Se) and flame atomic absorption spectrometer (Ca, Fe, K, Mg, Na, Zn) and by mercury analyser (Hg). Significant differences in Ca, Fe, K, Mg, Zn, As and Hg levels were observed between honey types. In chestnut honey were determined (K, Ca, Mg: mg kg 1; Hg, Ad, Cd: mg kg 1): the highest concentrations of K 2824.4, Ca 486.7, Mg 59.1 and Hg 2.52; the lowest of As 24.1 and Cd 2.52. Lime honey has been shown the highest content of Cu (20.6 mg kg 1), Zn (6.78 mg kg 1), Cd (2.14 mg kg 1) and Pb (810.3 mg kg 1). The lowest levels of following elements were determined in black locust honey (Fe, K, Mg: mg kg 1; Hg: mg kg 1): Fe 2.77, K 304.7, Mg 8.02 and Hg 0.82. Sage honey had the lowest Ca and Na content (173.9 and 31.8 mg kg 1). Among the multifloral honeys, the following was determined (Fe, Na, Cu: mg kg 1; As, Pb: mg kg 1): orchard honey – highest of Fe 5.17 and As 276.1, lowest Pb 301; meadow honey – highest Na 36.1, lowest Cu 4.38. The average Ca, Cu and Pb levels found in multifloral honey types were much higher than those reported in other European countries. Calcium levels in lime, chestnut and black locust were higher than those from other countries. ß 2014 Elsevier Inc. All rights reserved.

Keywords: Trace elements Food composition Unifloral honey Multifloral honey Black locust Chestnut Lime Sage Atomic absorption spectrometry Croatia

1. Introduction Honey is a natural, sweet product used as an ingredient for sweetness, colour, flavour, caramelisation and viscosity in many types of food products (Rashed and Soltan, 2004). Honey is composed mainly of fructose and glucose (65%), and water (18%), with low protein levels (Silva et al., 2009). The mineral content in honey is low and ranges from 0.04% in pale honeys to 0.2% in dark honeys (Anklam, 1998; Ferna´ndez-Torres et al., 2005). In investigations of the environmental, geographical and botanical aspects of the mineral content in honey, botanical factors have been shown to have the greatest influence on trace element content (Bogdanov et al., 2007). Honey may contain high levels of toxic elements, such as As, Cd, Hg and Pb, as a result of the elevated metal contents in plant nectar. High concentrations of these elements have been determined in honey from industrial areas with heavy industrial activities or near busy highways (Gajek et al., 1987; Toporca´k et al., 1992). Therefore, honey may be considered a

* Corresponding author. Tel.: +385 1 612 3601; fax: +385 1 612 3636. E-mail address: [email protected] (N. Bilandzˇic´). 0889-1575/$ – see front matter ß 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jfca.2013.12.002

bioindicator of environmental pollution (Conti and Botre, 2001; Pisani et al., 2008). Essential metals such as copper, iron, manganese and zinc play an important role in a number of biochemical processes, but can also have toxicological implications for humans when ingested in ˜ ez, 2005; Fraga, 2005). In the excessive doses (Arredondo and Nu´n past decade, trace metal contents have been determined in different honey types in European countries: France (Devillers et al., 2002), Italy (Conti, 2000; Buldini et al., 2001; Pisani et al., 2008), Poland (Przybyłowski and Wilczyn´ska, 2001), Slovenia (Golob et al., 2005; Kropf et al., 2010), Czech Republic (Lachman et al., 2007), Romania (Bratu and Beorgescu, 2005), Spain (Terrab et al., 2004; Herna´ndez et al., 2005; Garcia et al., 2006) and Turkey (Tuzen, 2002; Tuzen and Soylak, 2005; Tuzen et al., 2007; Silici et al., 2008). Most studies have only tested the differences in element composition among regions related to the geographical origin of honey, and therefore neglected the botanical influence on honey composition. Among the many studies, most of which pertain to multifloral honeys, few studies have also been conducted on unifloral honey types. Studies conducted on different unifloral honeys demonstrated great differences in elemental composition (Ferna´ndez-Torres et al., 2005; Golob et al., 2005;

N. Bilandzˇic´ et al. / Journal of Food Composition and Analysis 33 (2014) 132–138

Lachman et al., 2007; Pisani et al., 2008). In Croatia, in addition to the common production of multifloral honeys, the most common unifloral honey types are black locust (Robinia pseudoacacia L.), chestnut (Castanea sativa Mill.) and lime (Tilia spp.). On the other hand, unifloral honeys originating from Lavandula stoechas and Salvia officinalis are rare and are indicative of the southern geographical origin in the country. In the present study, the element composition of honey samples of different botanical and geographical origins in Croatia were studied. Differences in trace element contents between multifloral and unifloral honey types were examined. 2. Materials and methods 2.1. Honey samples

133

Ultra pure grade carrier argon (Ar, 99.9995% pure) and acetylene (99.9995% pure) were supplied by UTP d.o.o. (Pula, Croatia). 2.3. Sample preparation 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, then ramped for 1 min and hold for 4 min. The second step began at a power of 1000 W ramped for 5 min and hold for 5 min. The third step began at a power of 1400 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.

Fifty-nine honey types of different botanical origin were collected during 2010 and 2011: multifloral orchard honeys, multifloral meadow honeys, black locust (R. pseudoacacia L.), chestnut (C. sativa Mill.), lime (Tilia spp.) and sage (S. officinalis) honeys. Honey samples were collected in five continental regions of Croatia: Centre (Zagreb and Karlovac Counties), North (Varazˇdin County), Northeast (Bjelovar-Bilogora and Krizˇevci-Koprivnica Counties), Northwest (Krapina-Zagorje County) and South (Zadar and Split-Dalmatia Counties) (Table 1). The majority of samples were collected from the primarily agricultural Bjelovar-Bilogora and Krizˇevci-Koprivnica Counties, with cultivated fields, vineyards and orchards. The region has no considerable industrial activities and vehicular traffic is rather low in comparison with European standards and the more heavily populated, urbanised and industrialised Centre region around the capital city of Zagreb. Only sage honey was collected on island locations far from urbanisation and traffic, in southern Zadar County and southwest Primorje-Gorski Kotar County. All the honey samples were taken directly from containers used by beekeepers. Upon collection, all honey samples (500 g) were placed into clean glass bottles, labelled and transferred to the laboratory and kept at 4–8 8C until analysis.

2.4. Apparatus The analyses of As, Cd, Cu and Pb were conducted by graphite furnace-atomic absorption spectroscopy (GFAAS) using an AAnalyst 800 atomic absorption spectrometer (Perkin Elmer, USA) equipped with an AS 800 autosampler (Perkin Elmer, USA). For graphite furnace measurements, argon was used as the inert gas. Pyrolytic-coated graphite tubes with a platform were used. The analyses of Zn, Fe, Ca, Mg, Na and K were conducted by flame atomic absorption spectroscopy (FAAS) using a SpectrAA 220 atomic absorption spectrometer (Varian, Australia). Gases used for flame atomic absorption spectroscopy were acetylene, air and nitrous oxide. The atomic absorption signal for GFAAS and FAAS were measured in peak area and integration measurement mode against a calibration curve. Mercury levels in honey samples were quantified without acid digestion using the AMA-254 (Advanced Mercury Analyzer, Leco, Poland), which employs direct combustion of the sample in an oxygen-rich atmosphere. The instrumental settings and optimising temperature programs of the graphite and flame spectrometer and mercury analyser are summarised in Table 2.

2.2. Standard preparation

2.5. Quality parameters

Analytical grade reagents (HNO3 (65%, v/v), H2O2 (30%, v/v) K2Cr2O7 and HCl) were purchased from Kemika (Zagreb, Croatia). All solutions were prepared and diluted with ultra-pure water (18.2 MV cm resistivity at 25 8C) obtained by the purification system NIRO VV UV UF 20 (Nirosta d.o.o., Water Technologies, Osijek, Croatia). Plastic and glassware were cleaned by soaking in diluted HNO3 (1/9, v/v) and by subsequent rinsing with ultra-pure water and drying prior to use. Standard stock solutions containing 1000 mg L 1 of As (1001  3 mg L 1), Cd (999  3 mg L 1), Cu (1001  3 mg L 1), Hg (1000  3 mg L 1), Se (1004  3 mg L 1), Pb (1001  3 mg L 1) were purchased from Perkin Elmer (Waltham, Massachusetts, USA). Also, standards containing 1000 mg L 1 of Zn (1001  2 mg L 1), Fe (1000  2 mg L 1), Ca (999  2 mg L 1), Mg (1000  2 mg L 1), Na (1001  5 mg L 1) and K (1002  5 mg L 1) were purchased from Merck (Darmstadt, Germany). The stock solution and working standards were diluted in 0.2% (v/v) HNO3. Four calibration points were prepared for calibration curve for each element. In the preparation of Hg working standards, 1 mL of HNO3 conc., 0.1 mL 10% K2Cr2O7, and 0.1 mL HCl conc. were added to all working standards and prepared in brown glass volumetric flasks. The following were used as matrix modifiers (all, Perkin Elmer, USA) in each atomisation: for Cd, Cu and Se 0.005 mg Pd(NO3)2 and 0.003 mg Mg(NO3)2, for Pb 0.050 mg NH4H2PO4 and 0.003 mg Mg (NO3)2 and for As 0.01 mg Pd(NO3)2 and 0.005 mg Mg(NO3)2.

The limits of detection (LODs) were determined as the concentration corresponding to three times the standard deviation of twenty blanks. All specimens were run in batches that included blanks, a standard calibration curve, two spiked specimens, and one duplicate. To calculate the recovery percentage, ten honey samples spiked with known amounts of element analytical standards were processed. The limits of detection, recovery and linear range of the calibration curve for elements are presented in Table 3. The quality of data showed good accuracy, with recovery rates from 93.0% to 99.2%. For Zn determined at levels lower than the LOD value (0.01 mg kg 1), values were expressed as 0.01. Also, for Cd level lower than LOD (1 mg kg 1), values were expressed as 1. 2.6. Data analysis All calculations and statistical analysis were performed using the Statistica 6.1 software package (StatSoft1 Inc., Tulsa, USA). One-way analysis of variance was used to test for differences in honey metal concentrations. Data were log-transformed to improve normality prior to analysis to meet the underlying assumptions of the analysis of variance. The differences in metal concentrations between different honey types were analysed using the t-test. A probability level of p  0.05 was considered statistically significant.

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Table 1 Classification and geographical origin of honey samples. Sample

Honey type

S S S S S S

Sage Sage Sage Sage Sage Sage

1 2 3 4 5 6

BL BL BL BL BL BL BL BL BL BL BL BL BL BL BL BL BL BL BL

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(Salvia (Salvia (Salvia (Salvia (Salvia (Salvia

Black Black Black Black Black Black Black Black Black Black Black Black Black Black Black Black Black Black Black

locust locust locust locust locust locust locust locust locust locust locust locust locust locust locust locust locust locust locust

C C C C C C C C C

1 2 3 4 5 6 7 8 9

Chestnut Chestnut Chestnut Chestnut Chestnut Chestnut Chestnut Chestnut Chestnut

L L L L L L L L L L L

1 2 3 4 5 6 7 8 9 10 11

Lime Lime Lime Lime Lime Lime Lime Lime Lime Lime Lime

MF-0 MF-0 MF-0 MF-0 MF-0 MF-0 MF-0

1 2 3 4 5 6 7

MF-M MF-M MF-M MF-M MF-M MF-M MF-M

1 2 3 4 5 6 7

officinalis) officinalis) officinalis) officinalis) officinalis) officinalis) (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia (Robinia

(Castanea (Castanea (Castanea (Castanea (Castanea (Castanea (Castanea (Castanea (Castanea

(Tilia (Tilia (Tilia (Tilia (Tilia (Tilia (Tilia (Tilia (Tilia (Tilia (Tilia

pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia pseudoacacia

sativa sativa sativa sativa sativa sativa sativa sativa sativa

L.) L.) L.) L.) L.) L.) L.) L.) L.) L.) L.) L.) L.) L.) L.) L.) L.) L.) L.)

Mill.) Mill.) Mill.) Mill.) Mill.) Mill.) Mill.) Mill.) Mill.)

spp.) spp.) spp.) spp.) spp.) spp.) spp.) spp.) spp.) spp.) spp.)

Region

County

Pag – – Punat Rab –

Zadar Zadar Primorje-Gorski Primorje-Gorski Primorje-Gorski Primorje-Gorski

Mali Bukovac Krizˇevci Krizˇevci Virje Ðurpevac Gola Zˇabno Ðurpevac Ðurpevac Podgajec Molve Koprivnica Koprivnica Zagreb Zagreb Belec Konjsˇcˇina Konjsˇcˇina Sisak

Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Zagreb Zagreb Krapina-Zagorje Krapina-Zagorje Krapina-Zagorje Sisak

Garesˇnica Daruvar Ðurpevac Varazˇdin Varazˇdin Dugo Selo Glina Petrinja Petrinja

Bjelovar-Bilogora Bjelovar-Bilogora Koprivnica-Krizˇevci Varazˇdin Varazˇdin Zagreb Sisak-Moslavina Sisak-Moslavina Sisak-Moslavina

Karlovac Varazˇdin Virje Koprivnica Koprivnica Krizˇevci Ðurpevac Ðurpevac Ðurpevac Ðurpevac Ðurpevac

Karlovac Varazˇdin Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci

Kotar Kotar Kotar Kotar

Multifloral Multifloral Multifloral Multifloral Multifloral Multifloral Multifloral

– – – – – – –

orchard orchard orchard orchard orchard orchard orchard

flower flower flower flower flower flower flower

Novigrad Podravski Batinjani Ðurpevac Krizˇevci Koprivnica Orle Varazˇdin

Bjelovar-Bilogora Bjelovar-Bilogora Koprivnica-Krizˇevci Koprivnica-Krizˇevci Koprivnica-Krizˇevci Zagreb Varazˇdin

Multifloral Multifloral Multifloral Multifloral Multifloral Multifloral Multifloral

– – – – – – –

medow medow medow medow medow medow medow

flower flower flower flower flower flower flower

Ivanic´ Grad Jastrebarsko Zagreb Zagreb Zagreb Knin Josipdol

Zagreb Zagreb Zagreb Zagreb Zagreb Split Osijek-Baranja

3. Results and discussion In the present study, concentrations of twelve elements were determined in six different honey types in Croatia. Mean element contents are shown in Table 4. The results showed that the influence of botanical origin on the elemental composition of honey is evident for the toxic elements As and Hg and for the essential elements K, Ca, Mg, Fe and Zn. Statistical analyses by oneway ANOVA showed significant differences in the content of Ca, K,

Mg, Hg, As (p < 0.001, all) and of Fe and Zn (p < 0.05, both) between the tested honey types. The highest total mean concentrations were measured for K (1147.4 mg kg 1), followed by Ca (345.3 mg kg 1), Na (34.1 mg kg 1), Mg (23.9 mg kg 1) and Cu (14.4 mg kg 1). Iron contents were in the range of 3.57– 5.17 mg kg 1. Zinc concentrations differed between the lowest levels found in black locust (0.55 mg kg 1) and the highest measured in lime (6.78 mg kg 1). Mean Pb concentrations were between 301.0 and 560.2 mg kg 1. The mean Hg content ranged

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135

Table 2 Instrumental conditions for the determination of Pb, Cd, Cu and As by graphite furnace atomic absorption spectrometry (GFAAS), Zn, Fe, Ca, Mg, Na, K by flame atomic absorption spectrometry (FAAS) and Hg, using a mercury analyser. Conditions for graphite furnace atomic absorption spectrometry (GFAAS)

Wavelength (nm) Argon flow (mL min 1) Sample volume (mL) Modifier volume (mL)

As

Cd

Cu

Pb

Se

193.7 250 20 5

228.8 250 20 5

324.8 250 20 5

283.3 250 20 5

196.0 250 20 5

Heating program temperature 8C (ramp time (s), hold time (s)) Drying 1 Drying 2 Pre-ashing Cooling Ashing Atomisation Cleaning

110 (1, 30) 130 (15, 30)

110 (1, 30) 130 (15, 30)

110 (1, 30) 130 (15, 30)

110 (1, 30) 130 (15, 30)

1600 (10, 20) 2000 (0, 5) 2450 (1, 3)

700 (10, 20) 1550 (0, 5) 2450 (1, 3)

1200 (10, 20) 2000 (0, 5) 2450 (1, 3)

900 (10, 20) 1850 (0, 5) 2450 (1, 3)

110 130 600 20 1300 2100 2400

(5, 10) (15, 15) (20,40) (1,40) (10, 20) (0, 5) (1, 5)

Conditions for flame absorption spectrometry (FAAS)

Wavelength (nm) Lamp current (mA) Fuel Support Flame stoichiometry

Ca

Fe

K

Mg

Na

Zn

422.7 10 Acetylene Nitrous oxide Reducing

248.3 5 Acetylene Air Oxidising

766.5 5 Acetylene Air Oxidising

285.2; 202.6 4 Acetylene Air Oxidising

589.0; 589.6 5 Acetylene Air Oxidising

213.9 5 Acetylene Air Oxidising

Conditions for mercury analyser Hg Wavelength (nm) Drying time (s) Decomposition time (s) Wait time (s) Weight/volume of sample Working range

253.65 60 150 45 100 mg/100 mL 0.05–600 ng

between 0.47 and 2.52 mg kg 1 while Cd concentrations were in the range 0.47–2.14 mg kg 1. Only Se was negligible in all honey samples, with contents lower than the LOD value of 10 mg kg 1. Literature values for different types of honey originating from European countries are given in Table 5. It must be emphasised that there are far more studies on multifloral honeys than on unifloral studies. The literature results show significant differences in element concentrations between different honey types (Golob et al., 2005; Lachman et al., 2007; Pisani et al., 2008). The most abundant element K in the present study decreased in content in the following order: chestnut > lime > multifloralorchard flower > multifloral-meadow flower > sage > black locust. As previously stated, significant differences in K content were determined between the tested honey types. It was

1

previously reported that K is the most representative element among other abundant elements (K > P > Mg > Ca) in chestnut honey (Pereira-Lorenzo et al., 2006; Neri et al., 2010). A recent study of soil element content in Croatia showed that concentrations of K in soils of northern and central Croatian clearly differed from those in southern regions (Halamic´ and Miko, 2009). The soil of central and north Croatia is high in K, in concentrations in the range 1.71–2.26%, which is higher than K contents measures in the southern regions, i.e. in the range 0.18–1.25%. These is relevant as there are environmental influences on K content in honey, considering that all honey samples were collected in central Croatia, in contrast to the sage honey samples collected in the southern regions. In a previous study in Turkey, it was also concluded that differences in element concentrations depend on

Table 3 Major (Na, K, Ca, Mg) and trace element (Fe, Cu, Zn, Se, As, Cd, Pb, Hg) concentrations and recoveries in spike honey samples. Element

Linear range (mg L

As Cd Cu Hg Se Pb

0.5–20 0.2–5 1–40 1–5000 1–50 0.5–20

Element

Linear range (mg L

Ca Zn Fe Mg Na K

1–300 1–200 6–1500 0.3–100 0.2–100 0.3–200

1

)

LOD (mg kg

1

)

5.0 1.0 1.2 0.1 10 4.7 1

)

LOD (mg kg 1 1 6 0.3 0.2 0.3

Spiked value (mg kg

1

)

10 10 50 10 50 50 1

)

Spiked value (mg kg 1.0 1.0 10 10 10 10

Measured value (mg kg

1

)

9.59  0.27 9.67  0.33 49.4  5.23 9.92  0.14 47.9  0.14 48.9  6.45 1

)

Measured value (mg kg 0.81  0.12 0.93  0.09 9.67  0.33 9.43  0.51 9.78  0.19 9.53  0.22

Recovery (%) 95.9 96.7 98.8 99.2 95.8 97.8

1

)

Recovery (%) 81.0 93.0 96.7 94.3 97.8 95.3

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Table 4 Statistics of the mayor and trace elements contents in different types of honey from different geografical origin in Croatia. Honey type

N

Statistics

mg kg Ca

Sage (S)

1

a

mg kg Cu

c

Mg

4.17 3.45

769.5 255.1

11.6 4.56

31.8 4.87

0.94 1.67

2.77 1.51

304.7 69.7

8.02 1.89

33.9 9.83

0.55 0.63

3.57 1.81

2824.4 734.4

59.1 44.5

35.8 5.03

0.89 1.08

Fe

K

d

Na

e

f

Zn

1

Cd

Hgg

Pb

64.7 53.7

1.65 1.21

0.96 0.47

512.0 547.4

127.9 234.9

1.97 1.95

0.82 0.52

560.2 732.3

1.31 0.47

2.52 1.79

302.9 369.3

As

6

Mean SD

173.9 88.3

19

Mean SD

349.3 251.7

9

Mean SD

486.7 131.1

11

Mean SD

387.8 82.1

20.6 37.9

4.02 1.41

1574.8 444.2

25.5 7.32

31.9 4.32

6.78 14.5

133.2 166.4

2.14 1.38

2.04 1.89

810.3 801.1

Multifloral – orchard flower (MF-O)

7

Mean SD

389.6 185.3

19.6 40.6

5.17 3.25

1191.0 564.7

27.1 8.11

35.6 7.81

2.95 5.13

276.1 183.6

1.86 1.21

1.40 0.61

301.0 209.1

Multifloral – medow flower (MF-M)

7

Mean SD Mean SD

188.9 125.7 345.3 196.1

4.38 5.05 14.4 29.7

3.29 2.92 3.61 2.26

890.0 330.7 1147.4 941.5

26.9 8.05 23.9 24.3

36.1 13.7 34.1 8.19

1.22 0.89 1.17 6.73

266.6 313.4 140.7 206.7

1.86 1.22 1.84 1.42

0.82 0.49 1.39 1.29

544.0 641.2 530.0 631.3

Black locust (BL)

Chestnut (C)

Lime (L)

All samples

59

7.85 9.91

b

18.6 35.8 6.19 9.03

24.1 7.88

Statistically significant differences in element content between different types of honey. a S-C p < 0.001; S-L p < 0.001; S-MO p < 0.05; C-MM p < 0.001; L-MM p < 0.001; MO-MM p < 0.01. b B-L p < 0.05; B-MO p < 0.05. c S-B p < 0.001; S-C p < 0.001; S-L p < 0.01; B-L p < 0.001; B-MO p < 0.001; B-MM p < 0.001; B-C p < 0.001; C-L p < 0.001; C-MO p < 0.001; C-MM p < 0.001; L-MM p < 0.01. d S-B p < 0.01; S-C p < 0.001; S-L p < 0.001; S-MO p < 0.001; S-MM p < 0.001; B-L p < 0.001; B-C p < 0.001; B-MO p < 0.001; B-MM p < 0.001; C-L p < 0.001; C-MO p < 0.01; CMM p < 0.01. e B-MM p < 0.05. f S-MO p < 0.01; B-MO p < 0.01; C-MO p < 0.001; L-MO p < 0.05. g B-C p < 0.01; B-L p < 0.01; C-MM p < 0.05.

Table 5 Overview of the element contents in different honey types from different geographical areas. Country

Element

Honey type Czech Republica

Italyb MF = mixed floral C = chestnut

64.9

–

254

MF 890j C < 50 MF 1.265 C 1.75 –

647j

Ca (mg kg

1

Cu (mg kg

1

)

0.42

Fe (mg kg

1

)

–

K (mg kg

)

1

)

Mg (mg kg 1) Na (mg kg 1) Zn (mg kg 1)

–

50.1 – 1.69

As (mg kg 1) Cd (mg kg 1)

– –

Hg (mg kg 1) Pb (mg kg 1)

– –

Se (mg kg a b c d e f g h i j

1

)

–

Lachman et al. (2007). Buldini et al. (2001). Pisani et al. (2008). Silva et al. (2009). Golob et al. (2005). Herna´ndez et al. (2005). Ferna´ndez-Torres et al. (2005). Tuzen et al. (2007). Silici et al. (2008). mg kg 1.

– – MF 3.205 C 2.64 – MF 305 C < 50 – MF 620 C < 50 –

Italyc Multifloral

2.49 1093

52.3 94.9 1.85 6.59 4.25

Portugald Multifloral + monofloral 59.88

Sloveniae L = lime C = chestnut F = floral L 43 C 159 F 49

Spainf Multifloral + monofloral 74.8

–

0.37

–

4.85

1150.1

35.57 261.43 –

L 780 C 3500 F 2000

1088

41.0 70.0 1.57

Spaing Rosemary

121–206.7

<0.531–0.693 – 483–886.5

Turkeyh Multifloral

Turkeyi Multifloral

–

8.04–197

0.23–2.41

9.97–29.5j

1.8–10.2

1.61–2.82

-

350–7030

19.7–37.82 16.17–36.8 1.441–4.496

– – 1.1–12.7

21.9–65.8 – 0.55–6.27

– –

– –

– –

– 0.9–17.9

– 0.57–2.03

– 76.4

– –

– –

– –

– 8.4–106

– 1.54–36.7

–

–

–

–

38–113

94.2–159

N. Bilandzˇic´ et al. / Journal of Food Composition and Analysis 33 (2014) 132–138

botanical origin, i.e. the type of flowers used by bees (Rashed and Soltan, 2004). Potassium contents in both multifloral honey types in this study corresponded with the literature data related to multifloral honey from Italy and Spain (Herna´ndez et al., 2005; Pisani et al., 2008; Silva et al., 2009). The highest level 7030 mg kg 1 was reported in Turkey (Silici et al., 2008). Sage K content (769.5 mg kg 1) in the present study was similar to levels determined in rosemary honey also related to the Mediterranean climate (Ferna´ndez-Torres et al., 2005). Furthermore, K levels determined in black locust, chestnut and lime honey corresponded with contents determined in the same honey type from Slovenia (Kropf et al., 2010). In the present study, Ca content decreased in the following order: chestnut > multifloral-orchard flower > lime > black locust > multifloral-meadow flower > sage. The significant differences in Ca concentrations between honey types are presented in Table 4. The Ca concentrations measured in the present study were higher than those reported in other European countries (Table 5). The levels obtained were 3–23 times higher than those reported in lime, chestnut and black locust honey types from Slovenia (Golob et al., 2005; Kropf et al., 2010). Also Ca content measured in multifloral honeys were higher than those from other countries (Herna´ndez et al., 2005; Lachman et al., 2007; Silici et al., 2008; Silva et al., 2009). In this study, the highest mean Mg contents were measured in chestnut honey and the lowest in sage honey. Compared with the results of other studies, the multifloral honeys analysed in this study contained Mg levels similar to those reported in the literature (Ferna´ndez-Torres et al., 2005; Pisani et al., 2008; Silici et al., 2008; Silva et al., 2009). Mean Na contents observed were similar for all honey types. The sodium content of multifloral honeys was lower than that reported in Czech Republic and Italy (Lachman et al., 2007; Pisani et al., 2008), but similar to contents measured in Spain and Portugal (Herna´ndez et al., 2005; Silva et al., 2009). In the present study, Cu mean concentrations ranged from the lowest concentration of 4.38 mg kg 1 found in multifloral meadow honey to the highest 20.6 mg kg 1 in lime honey. However, no significant differences were observed between honey types. Mean Cu levels found in both multifloral honey types were 10–55 times higher than those found in the literature (Conti, 2000; Golob et al., 2005; Herna´ndez et al., 2005; Lachman et al., 2007; Tuzen et al., 2007; Pisani et al., 2008) and more than 3 times higher than levels previously reported in Croatia (Bilandzˇic´ et al., 2011). On the other hand, results obtained were similar to those from the Black Sea region in Turkey (Silici et al., 2008). Copper levels determined in chestnut honey in this study were more than 40 times higher than those found in chestnut honey in Anatolia, Turkey (Kucuk et al., 2007). Mean Fe levels ranged from 2.77 mg kg 1 in black locust to 5.17 mg kg 1 in multifloral orchard honey. Iron concentrations determined in multifloral honey correspond to concentrations found in Spain, Turkey and Italy (Buldini et al., 2001; Herna´ndez et al., 2005; Tuzen et al., 2007; Lachman et al., 2007; Pisani et al., 2008; Silici et al., 2008). The mean Zn contents decreased in the order: lime > multifloral orchard > multifloral meadow > sage > chestnut > black locust. Zinc levels found in multifloral honeys were similar to the values found in other recent studies. In the present study, no significant differences were observed in Cd and Pb content among honey types. Cadmium content measured in multifloral honeys was similar to the content obtained in honeys from different geographical regions of Turkey (Silici et al., 2008), Macedonia (Stankovska et al., 2006/2007), Italy (Pisani et al., 2008) and to a previously report from Croatia (Bilandzˇic´ et al., 2011). A very high Cd content (305 mg kg 1) was determined in mixed-flower honey from Bologna, Italy (Buldini et al., 2001).

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The World Health Organization (WHO) and the Food and Agriculture Organization (FAO) established provisional tolerable weekly intake (PTWI) guidelines, which propose the acceptable levels of major toxic elements that may accumulate in the body and can be ingested on a weekly basis (mg kg 1, b.w.): 25 for Pb, 7 for Cd, 15 for As and 5 for Hg (WHO, 1989, 2000; JECFA, 2005). Lead is one of the most widespread metal pollutants and has no beneficial role in human metabolism. It can cause progressive toxicity and health disorders such as fatigue, sleeplessness, hearing and weight loss. In the present study, Pb content decreased in the following order: lime > black locust > sage > multifloral meadow > chestnut > multifloral orchard. Concentrations measured in multifloral honey types were much higher than levels obtained in other countries: Italy (Buldini et al., 2001; Pisani et al., 2008), Poland (Przybyłowski and Wilczyn´ska, 2001), Turkey (Tuzen and Soylak, 2005; Tuzen et al., 2007; Silici et al., 2008) and Romania (Bratu and Beorgescu, 2005). The higher concentrations of Pb in honey samples may be due to the position of hives in zones near highways and railways, which is often the case. In this study, the highest As levels were measured in multifloral types of honey. In comparison with contents reported in the literature, As levels obtained were higher than levels found in Siena County, Italy (Caroli et al., 1999; Pisani et al., 2008) but less than half the levels (range 1.24–1.49 mg kg 1) reported in Slovenia (Golob et al., 2005). Mercury pollution in the environment is caused by mining and combustion, industrial and urban discharges and also by agricultural materials (Zhang and Wong, 2007). Mercury concentrations measured in this study were slightly more abundant in chestnut and lime honey than in sage, black locust and multifloral honeys. There are only a few literature reports for Hg concentrations in honey. Mercury levels ranged from 50 to 212 mg kg 1 in contaminated and from 1 to 3 mg kg 1 in uncontaminated areas in Slovakia (Toporca´k et al., 1992). In multifloral and other honey types examined in Siena County, Italy, Hg levels were lower than the quantification level of 2 mg kg 1 (Pisani et al., 2008). Previous studies suggested that the elemental content of honey can help to identify its geographical provenance, as this is consistent with environmental conditions (Przybyłowski and ˜ oz and Palmero, 2006; Baroni et al., Wilczyn´ska, 2001; Mun 2009). The present study showed differences in trace and mineral contents and confirmed the strong influence of botanical origin on the chemical composition of honeys. Also, the study indicated significant differences between sage honey from the south and all other honey types collected in the northern regions of Croatia, demonstrating that the mineral content and trace element in honey samples could give an indication of the geographical origin of honey. 4. Conclusions Fifty-nine honey samples from 6 different botanical origins collected in Croatia were analysed for 12 elements using the AAS technique. Significant differences in Ca, Fe, K, Mg, Zn, As and Hg levels were observed between honey types. Chestnut honey exhibited the highest concentrations of K, Ca, Mg and Hg and the lowest level of As and Cd. Lime honey showed the highest content of Cu, Zn, Cd and Pb. Black locust honey obtained the lowest levels of Fe, K, Mg and Hg. Sage honey had the lowest Ca and Na content. All Se values measured were below the limit of detection. Multifloral types of honey were defined by the highest level of Fe and As and the lowest Pb in multifloral orchard honey and the highest Na and the lowest Cu in multifloral meadow honey. Variation in the trace element content in different type of honeys confirmed differences due to botanical origin. Average Ca, Cu and Pb levels found in multifloral honeys were much higher than those

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