IJFS#1765_bozza Ital. J. Food Sci., vol. 32, 2020 - 858 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 Ital. J. Food Sci., vol. 32, 2020 - 859 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 Ital. J. Food Sci., vol. 32, 2020 - 860 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 Ital. J. Food Sci., vol. 32, 2020 - 861 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). Ital. J. Food Sci., vol. 32, 2020 - 862 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 Ital. J. Food Sci., vol. 32, 2020 - 863 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). Ital. J. Food Sci., vol. 32, 2020 - 864 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)