IJFS#1765_bozza


	

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PAPER 
 
 
 
 
 

TREND OF POLYCHLORINATED 
DIBENZO-P-DIOXINS AND DIBENZOFURANS 

(PCDD/PCDFS) IN BEEHIVE MATRICES 
 
 
 
 
 
 
 

S.M.R. TULINIa*, R.M. SPECCHIAb , O.R. LAIb, C. MUCCIOLOc, M. AMORENAa 
 and G. CRESCENZOb  

aDepartment of Bioscience and Agro-Food and Environmental Technology, Teramo University, 
Località Piano d’Accio, 64100 Teramo, Italy 

bDepartment of Veterinary Medicine, University of Bari Aldo Moro, 70121 Bari, Italy 
cSalerno’s A.S.L., Department of Prevention - Food Hygiene Service, 84135 Salerno, Italy 

*Corresponding author: Tel. +390861266988, Fax: +390861266987 
Email address: stulini@unite.it 

 
 
 
 

ABSTRACT 
 
Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/PCDFs) 
are well-known persistent organic pollutants (POPs) with highly toxic potential. These 
compounds are released in the environment as a complex mixture of various congeners 
which shown significant physico-chemical differences, as well as different environmental 
fates. PCDD/PCDF mixtures change spatially and temporally in the environment and 
biota, complicating the risk assessment and regulatory control for human and animal 
exposure. Considering the well-known role of honeybees as bioindicators for pesticides, 
heavy metals and other chemicals, the present study has been developed to assess the use 
of honeybees and honeybee products in biomonitoring projects about PCDD/PCDFs. 
Three Dadant-Blatt type beehives, located since March 2017 in the headquarter of Ducati 
Motor Holding S.p.A. (Borgo Panigale, Bologna, Italy) have been used as monitoring 
stations. Honeybees, honey and beeswax have been sampled and analyzed for 
PCDD/PCDFs detection in June and in September of the same year. Among the analyzed 



	

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matrices, beeswax has shown the highest WHO-TEQ values, probably due to its lipidic 
nature capable of accumulating fat-soluble, non-volatile, persistent organic pollutants. 
Hexaclorodibenzo-p-dioxin (HxCDD), usually measured in vegetables and fruits, has been 
detected only in honey samples. Maximum levels of PCDD/PCDFs are settled by 
Commission Regulation (EC) No 1259/2011 of 2 December 2011, but only on animal-
derived products. Considering the role of dietary-model adopted by the consumers on 
toxic substances dietary intake and associated exposure risks, limits on botanical derived 
products are needed. But more controls about bee-products are advisable also in order to 
reduce the exposure risk for bees and for protecting biodiversity. 
 
 
 
 

Keywords: POPs, PCDD/PCDFs, honeybees, bio-indicators, environment, health 



	

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1. INTRODUCTION 
 
Various attributes make the honeybee (Apis mellifera) the “ideal bioindicator” (TONG et al., 
1975; STÖCKER, 1980; WALLWORK-BARBER, 1982; RAES et al., 1992; LEITA et al., 1996; 
ZHELYAZKOVA, 2012). 
Due to the intense forager activity and the high sensitivity of bees toward toxic substances, 
the hives can give informations about environmental pollution via health-status and high 
mortality of bees or via the residues detection in honey, pollen, propolis, beeswax, royal-
jelly, larvae and bees (CONTI and BOTRÈ, 2001; CRANE, 1984; BOGDANOV, 2006; 
CHAUZAT et al., 2011; PERUGINI et al., 2017).  
As far as the biomonitoring of environmental pollution is concerned, honeybees have been 
used in a lot of investigations to evaluate different type of contaminants (KIRKHAM and 
COREY, 1977; BROMENSHENK et al., 1985; TONELLI et al., 1990; FRANCO et al., 1997; 
FRANCO et al., 1998; CELLI and MACCAGNANI, 2003; BALAYIANNIS and 
BALAYIANNIS, 2008; PORRINI et al., 2014). However, only few studies have tried to 
evaluate the possible application of honeybees as bioindicators for dioxin and furan 
detection (PORRINI et al., 2014; ÖZKÖK et al., 2018). Polychlorinated dibenzo-p-dioxins 
and polychlorinated dibenzofurans (PCDD/PCDFs) are well-known persistent organic 
pollutants (POPs), with highly toxic potential (SEMANAINEN et al., 2002; Birnbaum et al., 
2003). These compounds are released in the environment as a complex mixture of various 
congeners, produced primarily as by-products of chemical manufacturing activities and 
during the combustion of municipal and chemical waste (HUTZINGER et al., 1985; 
MENESES, 2004). Atmospheric transport and deposition processes lead to the dispersion 
of these compounds into soils, plant surfaces, bodies of water and sediments (VAN DEN 
BERG et al., 1994; LOHMANN and JONES, 1998). Due to the significant differences in 
physico-chemical properties (solubilities, volatilities, rates of degradation/metabolism, 
exc.) of each congener, the complex mixtures of PCDD/PCDFs change spatially and 
temporally in the environment and in animal tissues (SCHRENK et al., 1991; WEGIEL et 
al., 2018; ZHENG et al., 2008). Due to their lipophilic properties, PCDD/PCDFs may 
concentrate in fatty tissues and bioaccumulate through the food chain (TRAAG et al., 
2006).  
Considering the well-known role of honeybees as bioindicators, the present study has 
been developed to evaluate the distribution of PCDD/PCDFs in the hive. Due to their 
wide use, honey and beeswax contamination could also represent an important safety 
concern. Nevertheless, this investigation has the main purpose of improving data about 
the possible application of honeybees as bioindicators for monitoring environmental 
pollution and human exposure risks.  
 
 
2. MATERIALS AND METHODS 
 
2.1. Beehives location 
 
Monitoring station in the headquarter of Ducati Motor Holding S.p.A. (Borgo Panigale, 
Bologna, Italy), was placed in an important Italian industrial area, at less than 7 Km from 
Bologna Central Station. Bologna city represents one of the most populated cities in Italy 
(2783 p/Km2) and its province extends for about 3702 Km2 representing in Italy, the most 
productive industrial area for metalworking and engine sector. The monitoring station, 
consisting of three beehives (BH1, BH2, BH3), Dadant-Blatt type with 12 frames have been 



	

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located in the headquarter of Ducati Motor Holding S.p.A. (Borgo Panigale, Bologna, Italy) 
at the end of March 2017. The hives, homogeneous in colony strength (colonies bring up 
on 10 frames) were placed into a wooden gazebo open frontally and laterally, with roof on 
top, at the distance of 40 cm between them. As suggested by PORRINI et al, (2014), all the 
beehives have been provided with cages for dead bees sampling (under-basket type) and 
were periodically monitored. 
 
2.2. Sampling 
 
Honeybees, honey and beeswax were sampled from each beehive in June and in 
September 2017.  
Bees have been collected alive in airtight container directly from the combs, taking care to 
not involve the queen. Honey and wax have been collected as two honeycomb centrifuged 
for honey extraction.  
Stored at −20°C for 24 hours, each sample (50g) was homogenized with liquid nitrogen by 
a crushing mill (IKA, Wilmington, NC), then analyzed for PCDD/PCDFs detection. The 
analyzed congeners have been reported in Table 1. 
 
2.3. Chemical analysis 
 
Determination of PCDD/Fs were performed following analytical methods based on 
international norms for dioxin analysis, such as EPA 1613 (EPA, 1994), and following the 
requirements of European Directives related to this subject.  
Extraction of the fat fraction, including the compounds of interest, was performed in 
Soxhlet apparatus with solvents (hexane/dichlormethane or hexane/diethyl ether). 
Beeswax samples were directly dissolved in 20 ml of hexane. Sample extracts were 
purified in a 4 cm diameter multilayer column, containing (top to bottom) Na2SO4 , 44% 
H2SO4/silica, 22 %H2SO4/silica, NaOH/silica and AgNO3/silica. PCDD/Fs were eluted 
with hexane. The purified extracts were fractionated in SPE pre-packed carbon tubes 
(Supelclean Envi-Carb), from SUPELCO (Bellefonte, PA, USA). The obtained PCDD/F 
fractions were evaporated to 15 µl under nitrogen stream and corresponding PCDD/F 13C 
syringe standards (1,2,3,4-TeCDD and 1,2,3,7,8,9-HxCDD) were added.  
Samples were analysed in a 6890N gas chromatograph (Agilent, Santa Clara, CA, USA), 
coupled to an Autospec Ultima high resolution mass spectrometer (Micromass, 
Manchester, UK), operating in electronic impact ionization mode and at 10,000 resolving 
power. For the PCDD/F analysis, samples were injected (2 µl) on splitless mode (1 min) 
into the injector at 280 ºC. The chromatograph was fitted with a RTX-5MS column (60 m x 
0.25 mm i.d., 0.25 µm) from Restek (Bellefonte, PA, USA). Carrier gas was helium at 250 
kPa constant pressure mode. The temperature program was 150 ºC (held for 1 min), 
increased at 30 min-1 to 200 ºC, increased at 3 ºC min-1 to 235 ºC (held for 10 min) and 
increased at 6 min-1 to 300 ºC (held 17 min). Monitored masses were those proposed by 
EPA 1613 method (EPA, 1994). Samples were quantified according to the isotopic dilution 
method, with the use of 13C12-labelled PCDD/F as internal standards. Among 200 
PCDDs and 70 PCDFs, 17 congeners considered dangerous from a toxicological point of 
view (Council Regulation (EU) 1259/2011), have been evaluated for the present 
investigation (Table 1). 
 
 
 



	

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Table 1. LOQ, LOD and WHO-TEFs (Van den Berg et al., 2006) of researched PCDD/PCDF congeners. 
 
 LOQ (pg/g) LOD (pg/g) WHO 2005 TEFs 
2, 3, 7, 8 - Tetrachlorodibenzo-p-dioxin (TCDD) 0.04 0.02             1 
1, 2, 3, 7, 8 - Pentachlorodibenzo-p-dioxin (PeCDD) 0.05   0.025             1 
1, 2, 3, 4, 7, 8 - Hexachlorodibenzo-p-dioxin (HxCDD) 0.10 0.05             0.1 
1, 2, 3, 6, 7, 8 - Hexachlorodibenzo-p-dioxin (HxCDD) 0.10 0.05             0.1 
1, 2, 3, 7, 8, 9 - Hexachlorodibenzo-p-dioxin (HxCDD) 0.10 0.05             0.1 
1, 2, 3, 4, 6, 7, 8 - Heptachlorodibenzo-p-dioxin (HpCDD) 0.25 0.13 0.01 
1, 2, 3, 4, 6, 7, 8, 9 - Octachlorodibenzo-p-dioxin (OCDD) 0.50 0.25     0.0003 
2, 3, 7, 8 - Tetrachlorodibenzofuran (TCDF) 0.04 0.02             0.1 
1, 2, 3, 7, 8 - Pentachlorodibenzofuran (PeCDF) 0.05   0.025 0.03 
2, 3, 4, 7, 8 - Pentachlorodibenzofuran (PeCDF) 0.05   0.025             0.3 
1, 2, 3, 4, 7, 8 - Hexachlorodibenzofuran (HxCDF) 0.10 0.05             0.1 
1, 2, 3, 6, 7, 8 - Hexachlorodibenzofuran (HxCDF) 0.10 0.05             0.1 
2, 3, 4, 6, 7, 8 - Hexachlorodibenzofuran (HxCDF) 0.10 0.05             0.1 
1, 2, 3, 7, 8, 9 - Hexachlorodibenzofuran (HxCDF) 0.10 0.05             0.1 
1, 2, 3, 4, 6, 7, 8 - Heptachlorodibenzofuran (HpCDF) 0.25 0.13 0.01 
1, 2, 3, 4, 7, 8, 9 - Heptachlorodibenzofuran (HpCDF) 0.25 0.13 0.01 
1, 2, 3, 4, 6, 7, 8, 9 - Octachlorodibenzofuran (OCDF) 0.50 0.25             0.0003 
 
 
2.4. WHO-TEF and WHO-TEQ 
 
The concepts of toxic equivalency factor (TEF) and total toxic equivalent (TEQ) have been 
developed and introduced by the World Health Organization (WHO) to facilitate risk 
assessment and regulatory control of exposure to PCDD/PCDFs mixtures. The WHO-TEF 
estimates the toxic potential of each congener comparing its affinity for a cytosolic 
receptor protein (aryl hydrocarbon receptor – AhR) with the highest affinity associated to 
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Table 1).  
The WHO-TEQ is operationally defined by the sum of each compound concentration 
multiplied by its TEF value and represents an evaluation of the total 2,3,7,8-TCDD–like 
activity of the PCDD/PCDFs mixture, as well as of their total potential toxicity (VAN 
DEN BERG et al., 1998, VAN DEN BERG et al., 2006; VAN DEN BERG et al., 2013).  
Two different methods can be used for WHO-TEQ evaluation. Usually, it is calculated as 
lower-bound for environmental matrices, considering the undetectable concentrations equal 
to zero. Instead, for high-lipid-content food products it is calculated with the upper-bound 
method, considering the undetectable concentrations equal to the detection limit of each 
congener (LOD) (Commission Regulation (EU) No 589/2014). For the present study the 
WHO-TEQs were calculated on honeybees, honey and beeswax PCDD/PCDFs 
concentrations with both lower-bound and upper-bound methods (Table 2). 
 
 
3. RESULTS AND DISCUSSION 
 
Ingestion of contaminated food is the principal way of human exposure to PCDD/PCDFs, 
accounting for 90% if compared to other ways such as inhalation and dermal contact 
(SWEETMAN et al., 2000). This concern about the human health impact and continuous 



	

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encouragement from scientific committees to monitor food samples across Europe, have 
led to numerous international and local studies on the concentration of dioxins in 
particular food items or on the estimation of the daily intake from food (European 
Commission, Brusseles, June 2002; FOCANT et al. 2002; KARL et al., 2002). Seafood 
represents the most contaminated foodstuff and the congeners most frequently detected in 
all type of analyzed foodstuff were OCDD and HpCDD, as well as PeCDD. Regarding 
dietary intake evaluation on human health it was carry out combining data on 
consumption habits with the different concentrations of PCDD/PCDFs expressed in 
WHO-TEQ found in food samples (BORDAJANDI et al., 2004).  
Honey and other bee-products are included in nutritional habits of a lot of country and 
currently are widely used also as dietary supplements for health purposes. However these 
product were never been taking into account for human dietary exposure calculation and 
then never analysed for PCDD/PCDFs evaluation. This study has been performed for 
improving this lack o data profile on food PCDD/PCDFs concentration and to evaluate a 
possible application of honeybee as bio-indicators for PCDD/PCDFs monitoring in the 
environment. Results are showed in Tables 2 and 3. 
Octachlorodibenzo-p-dioxin (OCDD), as well as being reported in other studies regarding 
PCDD/PCDFs, is the congener most frequently detected during the present investigation 
(BORDAJANDI et al., 2004; DOMINGO et al., 1999). It has been quantified in all the 
analysed matrices (Figure 1 and Figure 2). Aside for one honeybee sample collected in 
September (BH1), that reported 0.07 pg/g of 2, 3, 7, 8 – tetraclorodibenzofuran (TCDF), 
OCDD was the only congener detected in honeybee samples. This trend is confirmed also 
for honey samples. TCDF has been quantified only in one sample of honey collected in 
September from BH1 (0.07 pg/g), while interesting concentrations of OCDD have been 
detected in all honey samples collected in June and in September (Figs. 1 and 2). 
Detectable concentrations of 1, 2, 3, 4, 7, 8 - hexaclorodibenzo-p-dioxin and 1, 2, 3, 6, 7, 8 - 
hexaclorodibenzo-p-dioxin (HxCDD), usually measured in vegetables and fruits 
(DOMINGO et al., 1999), have been detected, in June and in September, only in honey 
samples (Figs. 1 and 2). Honey is a natural product that honeybees make from blossom’s 
nectar or from secretions coming from living parts of plants (ÖZKÖK et al., 2017), but 
ÖZKÖK et al. (2018) monitoring PCDD/PCDFs in honeybee pollen (honey component) 
had encountered different results. 2, 3, 7, 8 – TCDF, 1, 2, 3, 7, 8 – PeCDD, 2, 3, 4, 7, 8 – 
PeCDF, showed the higher concentrations with both analytical methods employed for the 
study. However, mentioned studies confirm with present data, that maximum levels 
should be established also for cereals, vegetables and bee-products, in which not 
negligible concentrations have been reported (ÖZKÖK et al., 2018). Moreover, vegetables 
represent the most frequent consumed food for a healthy diet and dietary-model adopted 
by the consumers should be considered important for assessing daily pollutants intake for 
humans (DOMINGO et al., 1999; SCHECTER et al., 2006). 
The most toxic PCDD/PCDF congeners are 2,3,7,8-substituted tetra-, penta-, and 
hexachloro compounds that, in addition to OCDD, have the greatest tendency to 
bioaccumulate (COHEN et al., 2002; BOCIO and DOMINGO, 2005). Nevertheless, the 
highest concentrations registered during the present investigation and associated to 
heptaclorodibenzo-p-dioxin (HpCDD) and octachlorodibenzo-p-dioxin (OCDD), have 
been detected in beeswax samples collected in June (Fig. 1). 
 
 
 



	

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Table 2. Concentrations detected in June 2017. 
 

 Bees Honey WAX 
Analyzed 

congeners 
BH1* 
(pg/g) 

BH2* 
(pg/g) 

BH3* 
(pg/g) 

Average* 
(pg/g) SD 

BH1* 
(pg/g) 

BH2* 
(pg/g) 

BH3* 
(pg/g) 

Average* 
(pg/g) SD 

BH1* 
(pg/g) 

BH2* 
(pg/g) 

BH3* 
(pg/g) 

Average* 
(pg/g) SD 

2,3,7,8 - 
Tetraclorodibenzo-
p-diossina (TCDD) 

<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,7,8 - 
Pentaclorodiben 

zo-p-diossina 
(PeCDD) 

<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,4,7,8 - 
Esaclorodibenzo-p-
diossina (ExCDD) 

<LOD <LOD <LOD ND ND 0,11 0,11 0,15 0,12 0,02 <LOD <LOD <LOD ND ND 

1,2,3,6,7,8 - 
Esaclorodibenzo-p-
diossina (ExCDD) 

<LOD <LOD <LOD ND ND <LOD 0,12 0,12 0,08 0,07 <LOD <LOD <LOD ND ND 

1,2,3,7,8,9 - 
Esaclorodibenzo-p-
diossina (ExCDD) 

<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,4,6,7,8 - 
Eptaclorodibenzo-p-
diossina (EpCDD) 

<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 2,01 1,39 1,73 1,71 0,31 

Octaclorodibenzo-p-
diossina (OCDD) 0,52 0,56 <LOD 0,36 0,31 0,54 0,52 0,51 0,52 0,02 12,05 9,08 9,97   10,37 1,52 

2,3,7,8 - 
Tetraclorodibenzofu

rano (TCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 0,09 0,07 0,08 0,08 0,01 

1,2,3,7,8 - 
Pentaclorodiben 

zofurano (PeCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 0,07 0,07 0,08 0,07 0,01 

2,3,4,7,8 - 
Pentaclorodiben 

zofurano (PeCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,4,7,8 - 
Esaclorodibenzo 
furano (ExCDF) 

<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 



	

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1,2,3,6,7,8 - 
Esaclorodibenzo 
furano (ExCDF) 

<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

2,3,4,6,7,8 - 
Esaclorodibenzo 
furano (ExCDF) 

<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,7,8,9 - 
Esaclorodibenzo 
furano (ExCDF) 

<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,4,6,7,8 - 
Eptaclorodibenzo 
furano (EpCDF) 

<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 0,41 1,20 0,86 0,82 0,40 

1,2,3,4,7,8,9 - 
Eptaclorodibenzo 
furano (EpCDF) 

<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 0,25 <LOD <LOD 0,08 ND 

Octaclorodibenzofu 
rano (OCDF) <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 0,84 0,66 0,88 0,79 0,12 

 
 
Table 3. Concentrations detected in September 2017. 
 

 Bees Honey WAX 

Analyzed congeners BH1* (pg/g) 
BH2* 
(pg/g) 

BH3* 
(pg/g) 

Average* 
(pg/g) SD 

BH1* 
(pg/g) 

BH2* 
(pg/g) 

BH3* 
(pg/g) 

Average* 
(pg/g) SD 

BH1* 
(pg/g) 

BH2* 
(pg/g) 

BH3* 
(pg/g) 

Average* 
(pg/g) SD 

2,3,7,8 - Tetraclorodibenzo-p-
diossina (TCDD) <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,7,8 - Pentaclorodibenzo-p-
diossina (PeCDD) <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,4,7,8 – Esaclorodibenzo-
p-diossina (ExCDD) <LOD <LOD <LOD ND ND 0,11 0,16 0,15 0,14 0,03 <LOD <LOD <LOD ND ND 

1,2,3,6,7,8 – Esaclorodibenzo-
p-diossina (ExCDD) <LOD <LOD <LOD ND ND 0,11 0,18 0,12 0,14 0,04 <LOD <LOD <LOD ND ND 

1,2,3,7,8,9 - Esaclorodibenzo-p-
diossina (ExCDD) <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,4,6,7,8 - 
Eptaclorodibenzo-p-diossina 

(EpCDD) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 0,58 1,36 1,36 1,10 0,45 

Octaclorodibenzo-p-diossina 
(OCDD) 0,77 0,90 0,79 0,82 0,07 0,52 0,59 0,52 0,54 0,04 5,68 8,42 10,04 8,05 2,20 

2,3,7,8 - 
Tetraclorodibenzofurano 

(TCDF) 
<LOD <LOD 0,07 0,07 0,00 <LOD <LOD 0,07 0,07 0,00 0,14 0,15 0,16 0,15 0,01 



	

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1,2,3,7,8 - 
Pentaclorodibenzofurano 

(PeCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 0,05 0,08 0,05 0,06 0,02 

2,3,4,7,8 - 
Pentaclorodibenzofurano 

(PeCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 0,09 0,08 0,05 0,07 0,02 

1,2,3,4,7,8 - 
Esaclorodibenzofurano 

(ExCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 0,12 0,10 0,10 0,11 0,01 

1,2,3,6,7,8 - 
Esaclorodibenzofurano 

(ExCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

2,3,4,6,7,8 - 
Esaclorodibenzofurano 

(ExCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,7,8,9 - 
Esaclorodibenzofurano 

(ExCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,4,6,7,8 - 
Eptaclorodibenzofurano 

(EpCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

1,2,3,4,7,8,9 - 
Eptaclorodibenzofurano 

(EpCDF) 
<LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND 

Octaclorodibenzofurano 
(OCDF) <LOD <LOD <LOD ND ND <LOD <LOD <LOD ND ND <LOD <LOD 1,31 1,31 0,00 



	

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Figure 1. Average concentrations of PCDD/PCDFs detected in hive matrices in June 2017. 
 

 
 

Figure 2. Average concentrations of PCDD/PCDFs detected in hive matrices in September 2017. 
 
 

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,	8
	-	
TC
D
F	

1,
	2
,	3
,	7
,	8
	-	
Pe
CD
F	

2,
	3
,	4
,	7
,	8
	-	
Pe
CD
F	

1,
	2
,	3
,	4
,	7
,	8
	-	
H
xC
D
F	

1,
	2
,	3
,	6
,	7
,	8
	-	
H
xC
D
F	

2,
	3
,	4
,	6
,	7
,	8
	-	
H
xC
D
F	

1,
	2
,	3
,	7
,	8
,	9
	-	
H
xC
D
F	

1,
	2
,	3
,	4
,	6
,	7
,	8
	-	
H
pC
D
F	

1,
	2
,	3
,	4
,	7
,	8
,	9
	-	
H
pC
D
F	

1,
	2
,	3
,	4
,	6
,	7
,	8
,	9
	-	
O
CD
F	

Co
n
ce
n
tr
at
io
n
	p
g/
g	

Bees		

Honey		

Beeswax		

0	

1	

2	

3	

4	

5	

6	

7	

8	

9	

2,
	3
,	7
,	8
	-	
TC
D
D
	

1,
	2
,	3
,	7
,	8
	-	
Pe
CD
D
	

1,
	2
,	3
,	4
,	7
,	8
	-	
H
xC
D
D
	

1,
	2
,	3
,	6
,	7
,	8
	-	
H
xC
D
D
	

1,
	2
,	3
,	7
,	8
,	9
	-	
H
xC
D
D
	

1,
	2
,	3
,	4
,	6
,	7
,	8
	-	
H
pC
D
D
	

1,
	2
,	3
,	4
,	6
,	7
,	8
,	9
	-	
O
CD
D
	

2,
	3
,	7
,	8
	-	
TC
D
F	

1,
	2
,	3
,	7
,	8
	-	
Pe
CD
F	

2,
	3
,	4
,	7
,	8
	-	
Pe
CD
F	

1,
	2
,	3
,	4
,	7
,	8
	-	
H
xC
D
F	

1,
	2
,	3
,	6
,	7
,	8
	-	
H
xC
D
F	

2,
	3
,	4
,	6
,	7
,	8
	-	
H
xC
D
F	

1,
	2
,	3
,	7
,	8
,	9
	-	
H
xC
D
F	

1,
	2
,	3
,	4
,	6
,	7
,	8
	-	
H
pC
D
F	

1,
	2
,	3
,	4
,	7
,	8
,	9
	-	
H
pC
D
F	

1,
	2
,	3
,	4
,	6
,	7
,	8
,	9
	-	
O
CD
F	

Co
n
ce
n
tr
at
io
n
	p
g/
g	

Bees		

Honey		

Beeswax		



	

Ital. J. Food Sci., vol. 32, 2020 - 868 

 

Among the analysed matrices, beeswax has shown the highest number of detectable 
congeners. HpCDD, OCDD, TCDF, pentachlorodibenzofuran (PeCDF), 
heptachlorodibenzofuran (HpCDF) and octachlorodibenzofuran (OCDF) have all been 
detected in beeswax (Figs. 1 and 2). A larger number of congeners have been quantified in 
beeswax samples collected in September as compared to those collected in June (Fig. 2) 
and indeed, the highest WHO-TEQ values calculated for the present investigation have 
been associated to them (Table 4). 
 
 
Table 4. Average WHO-TEQ lower- and upper-bound values, calculated on the average PCDD/PCDF 
concentrations measured in honeybee, honey. 
 

 June 2017 September 2017 

 Honeybees Honey Beeswax Honeybees Honey Beeswax 

WHO-TEQ 
lower bound 

0.0001 
pg/WHO-

TEQ/g 

0.0088 
pg/WHO-

TEQ/g 

0.0382 
pg/WHO-

TEQ/g 

0.0025 
pg/WHO-

TEQ/g 

0.0164 
pg/WHO-

TEQ/g 

0.0630 
pg/WHO-

TEQ/g 

WHO-TEQ 
upper bound 

0.1884 
pg/WHO-

TEQ/g 

0.1913 
pg/WHO-

TEQ/g 

0.2159 
pg/WHO-

TEQ/g 

0.1894 
pg/WHO-

TEQ/g 

0.1971 
pg/WHO-

TEQ/g 

0.2181 
pg/WHO-

TEQ/g 
 
 
Higher values of WHO-TEQ lower- and upper-bound have been registered in September 
then in June for all the analyzed matrices (Table 2). The highest amounts of WHO-TEQ 
lower- and upper-bound have been associated to the beeswax samples in June and in 
September (Table 2). Beeswax composition, consisting in a mixture of fatty acids, fatty 
alcohols, paraffinic hydrocarbons and free fatty acids, is capable of accumulating of fat 
soluble, non-volatile and persistent pollutants (TULLOCH, 1980; JOHNSON et al., 2010; 
SERRA-BONVEHÍ and ORANTES-BERMEJO, 2010; RAVOET et al., 2015; PERUGINI et al., 
2017). However, the mechanisms of beeswax contamination have not been well 
investigated yet. Beeswax is made by young worker bees who have never been out of the 
hive and its contamination could be the result of chemicals transmigration between 
different matrices, as well as the result of degradation/metabolism processes allowed by 
the bees consuming contaminated pollen and nectar. Currently, regarding PCDD/PCDFs, 
a possible bioaccumulation phenomenon in the hive cannot be excluded. Similarly to 
animal’s fat-tissues, beeswax is the main reservoir for PCDD/PCDFs mixtures that change 
in the “hive tissues” during the exposure time according to specific 
degradation/metabolism processes. 
Although further studies would be advisable, honeybees, honey and beeswax data suggest 
the possible use of them as indicators for PCDD/PCDFs distribution in the environment 
(bees), in vegetable foodstuffs (honey) and in animal fat-tissues (beeswax).  
 
 
4. CONCLUSIONS 
 
“Honeybees monitoring stations” have been confirmed as an effective and inexpensive 
method for controlling the levels of PCDD/PCDFs, as well as other pollutants, in the 
environment. 
Nevertheless, honey and beeswax contamination also represents an important concern for 
beekeeping practices and for honeybee products consumer health.  



	

Ital. J. Food Sci., vol. 32, 2020 - 869 

 

Honey is an important food product in many countries and beeswax finds important 
applications in food, cosmetic and pharmaceutical industries, representing possible 
sources of exposure for humans (PERUGINI et al., 2017). Considering the beekeeping 
common practice to recycle not controlled beeswax for wax foundation sheets production, 
it can become a source of subsequent recirculation of pollutants in the hive, with serious 
risks for honeybee health and for biodiversity protection (MULLIN et al., 2010; WU et al., 
2011; WU et al., 2012).  
Based on WHO-TEF and WHO-TEQ concepts, the Commission Regulation (EC) No 
1259/2011 of 2 December 2011 set PCDD/PCDFs maximum levels modifying those 
established by the Commission Regulation (EC) No 1881/2006 of 19 December 2006. These 
limits are settled mainly for animal-derived products and expressed as pg WHO-
PCDD/F-TEQ/g, but currently, no maximum levels are applied to cereals, fruit and 
vegetables, or to honey and other honeybee products.  
According to many studies this lack should be revised in order to guarantee risk 
assessment and regulatory control of exposure to PCDD/PCDFs mixtures, taking into 
account real nutritional habit in different countries. Cereals, vegetables and bee-products 
have long been considered with a “low-impact” for humans daily intake, but recent 
studies (ÖZKÖK et al., 2018) have demonstrated that interesting concentrations can also be 
found in honeybee pollen (honey component) as well in cereals and vegetables 
(BORDAJANDI et al., 2004; DOMINGO et al., 1999; FOCANT et al., 2002; KARL et al., 2002; 
SCHECTER et al., 2006).  
 
 
ACKNOWLEDGEMENTS 
 
This work was made possible thanks to the commitment of Ducati Motor Holding S.p.A. (Borgo Panigale, Bologna, 
Italy). 
 
 
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Paper Received January 12, 2020 Accepted June 26, 2020