Polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls in bivalve molluscs. Risk to Polish consumers?

Altogether 32 samples of six species of molluscs were collected, comprising five dog cockles (Glycymeris glycymeris), five Manila clams (Ruditapes philippinarum), five Atlantic jackknife clams (Ensis directus), ten blue mussels (Mytilus edulis), five Pacific oysters (Crassostrea gigas) and two common cockles (Cardium edule). Frozen samples were taken randomly from retail markets and immediately shipped to the National Reference Laboratory for Halogenated Compounds at the National Veterinary Research Institute. The molluscs were caught in the North Sea, Atlantic Ocean and Mediterranean Sea.

The following analytes were investigated: seven 2,3,7,8-substituted PCDDs (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentachloro-dibenzo-p-dioxin (PeCDD), 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) and octachlorodibenzo-p-dioxin (OCDD)), ten 2,3,7,8-substituted PCDFs (2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-hexachlorodibenzofuran (HxCDF), 1,2,3,6,7,8-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF,1,2,3,4,6,7,8-heptachlorodibenzofuran (HpCDF), 1,2,3,4,7,8,9-HpCDF and octachlorodibenzofuran (OCDF), and twelve DL-PCBs (PCB 77, PCB 81, PCB 126, PCB 169, PCB 105, PCB 114, PCB 118, PCB 123, PCB 156, PCB 157, PCB 167 and PCB 189).

The concentrations of ¹³C12-labelled internal standards were 25 pg mL⁻¹ and 400 pg mL⁻¹ for PCDD/F and DL-PCB congeners, respectively. The recovery control ¹³C12-labelled standards concentration was 25 pg mL⁻¹ for 1,2,3,4-TCDD (PCDD/F fraction) and 400 pg mL⁻¹ for PCB 111 (PCB fraction). As a reference material T-0645–fish oil was used (FAPAS, Fera Science, Sand Hutton, UK).

Dichloromethane, toluene, n-hexane and n-nonane were supplied by LGC Standards (Wesel, Germany). Sodium sulphate and 98% ACS grade sulphuric acid were purchased from Merck (Darmstadt, Germany), and diatomaceous earth from Restek (Bellefonte, PA, USA). Helium (purity 99.9999%) and nitrogen (99.999%) were sourced from Messer (Gumpoldskirchen, Austria). All of the organic solvents were of suitable purity for the residue analysis. All standards were purchased from the Cambridge Isotope Laboratory (Andover, MA, USA) or Wellington Laboratories Inc. (Ontario, Canada). Ready-made columns for sample purification (silica PCB-HCDS-ACD-TFC, silica PCBS-ABN-STD, alumina PCBA-BAS-011 and carbon PCBC-CCE-034) were supplied from Fluid Management Systems (Billerica, MA, USA).

After homogenisation, samples were freeze dried. Before pressurised liquid extraction (Fluid Management Systems) samples were spiked with ¹³C12-internally labelled standards. A mixture of dichloromethane/hexane (50/50, v/v) was used for extraction under high pressure (10 bar) at 120°C. The next step was purification carried out using an automated Power Prep sample preparation system (Fluid Management Systems). After fat removal with silica gel in the first two columns, the extract was subjected to fractionation in the activated alumina and carbon columns. Two fractions were collected. The first (including the 8 DL-PCB congeners 105, 114, 118, 123, 156, 157, 167 and 189) was eluted from the alumina using hexane/dichloromethane (98:2, v/v) and hexane/dichloromethane (1 : 1, v/v) and from the carbon with ethyl acetate/toluene (1 : 1, v/v) and hexane. To elute the second fraction from the carbon column (comprising all 2,3,7,8-PCDD/F and the 4 DL-PCB congeners 77, 81, 126 and 169), toluene was used. Before instrumental analysis, internal recovery standards were added to the fractions.

High-resolution gas chromatography coupled with high-resolution mass spectrometry was used for detection and the apparatus was an Ultra Trace GC gas chromatograph, TriPlus autosampler, and DFS dual-focusing mass spectrometer (Thermo Scientific, Bremen, Germany). Positive electron ionisation operating in selected-ion monitoring mode at a resolution of 10,000 was employed. Chromatographic separation was carried out in a DB-5 MS fused-silica capillary column (60 m × 0.25 mm × 0.1 mm). The limits of quantification (LOQs) were 0.01–0.12 pg g⁻¹ w.w. for PCDD/Fs and 0.09–1.16 pg g⁻¹ w.w for DL-PCBs. Estimation of the LOQs of individual congeners was performed in accordance with the European Commission Joint Research Centre’s Guidance Document on the Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food (11).

Blank samples and reference material were analysed in every series of samples. The trueness for the reference material analysis ranged between −20% and +20% and the recoveries of the internal standards ranged between 60% and 120% in all samples, which met the criteria set out in European Commission Regulation 2017/644/EU (10). The method performance was verified by successful participation in the proficiency testing (PT study) organised by the European Union Reference Laboratory for Halogenated Persistent Organic Pollutants in Feed and Food (Freiburg, Germany).

Results are presented as a TEQ, which is calculated using the following equation: TEQ=∑​i=17(PCDDi×TEFi)+∑​j=110(PCDFj×TEFj)+∑​k=112(PCBk×TEFk)

World Health Organization toxic equivalency factors (TEF) were established by Van den Berg (41). World Health Organization toxicity equivalents were expressed as upper-bound concentrations (all values of the congeners below LOQ were equal to their LOQ). In accordance with European Commission Regulation 1259/2011/EU results are expressed on a wet weight basis (9).

Since there are no data regarding average consumption of bivalve molluscs in Poland, three weekly intake levels were assumed: 25 g, 50 g and 100 g. The calculations were performed for an adult of 70 kg body weight and 3–10-year-old children of average 23.1 kg body weight (13). To characterise the potential health risk associated with the intake of dioxins and DL-PCBs, the doses ingested with molluscs were expressed as a percentage of the TWI established by the EFSA (2 pg WHO-TEQ kg⁻¹ b.w.).

The content of PCDD/Fs and PCBs in bivalve molluscs is summarised in Table 1. All samples were compliant with Commission Regulation 1259/2011/EU, which sets the maximum levels for PCDD/Fs and DL-PCBs in foodstuffs at 3.5 pg WHO-TEQ g⁻¹ w.w. for PCDD/Fs and 6.5 pg WHO-TEQ g⁻¹ w.w. for PCDD/Fs/DL-PCBs (9). The highest mean concentrations of all three groups of analysed compounds were found to be in the Pacific oyster. The mean concentration of PCDD/Fs of 0.30 pg WHO-TEQ g^–1 w.w. was almost twelve times lower than the maximum level but twice as high as in blue mussels and around three times higher than in the remaining species. The mean level of DL-PCBs in Pacific oysters of 0.19 pg WHO-TEQ g^–1 w.w. was also considerably higher than those in other species. The sum contents of PCDD/Fs and DL-PCBs were severalfold lower than the limit in all samples (Table 1).

Three different profiles of PCDD/Fs were identified (Fig. 1). In the first type of profile which was observed in dog cockles and Manila clams, OCDD and 2,3,7,8-TCDF dominated, but concentrations were very low at a respective 0.16 pg g⁻¹ w.w and 0.07 pg g⁻¹ w.w. in dog cockles and 0.23 pg g⁻¹⁻¹ w.w and 0.16 pg g⁻¹ w.w. in Manila clams. The second congener profile was observed in Atlantic jackknife clams, blue mussels and common cockles. In these species, apart from the dominant congeners (OCDD and 2,3,7,8-TCDF), 1,2,3,4,7,8,9-HpCDF and 1,2,3,4,6,7,8-HpCDD were also present. These congeners were at concentrations many times higher than in dog cockles and Manila clams. Because OCDD, 1,2,3,4,7,8,9-HpCDF and 1,2,3,4,6,7,8-HpCDD have lower TEF values (0.0003, 0.01 and 0.01, respectively) they are less important from a toxicological point of view. The 2,3,7,8-TCDF (TEF 0.1) and 2,3,4,7,8-PeCDF (TEF 0.3) congeners are more important, and these were the dominant congeners in Pacific oysters. The mean level of 2,3,4,7,8-PeCDF in Pacific oysters of 0.28 pg g⁻¹ w.w was 2–5-fold higher than in other species except dog cockles, in which it was not detected. The most toxic congeners, with TEF = 1 (2,3,7,8-TCDD and 1,2,3,7,8-PeCDD), were not detected in any samples of dog cockles or Manila clams. The following PCDD/Fs congeners were not found in any of the 32 analysed samples: 1,2,3,4,7,8-HxCDD, 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF and 1,2,3,4,7,8,9-HpCDF.

Fig. 1.

The profiles of DL-PCBs were alike in all species, and PCB 118 were found to be most abundant (Fig. 1). Its concentrations ranged from 7.6 pg g⁻¹ w.w. in dog cockles to 664.5 pg g⁻¹⁻¹ w.w. in blue mussels. This compound has very low toxic potential and its TEF is 0.00003. The congener with the highest TEF of all DL-PCBs, PCB 126 with 0.1, was detected in all Atlantic jackknife clam, blue mussel, Pacific oyster and common cockle samples. The highest mean concentration in Pacific oysters was 1.68 pg g⁻¹ w.w. Polychlorinated biphenyl 169 was not present in any sample.

Dietary intake of PCDD/Fs and DL-PCBs via one assumed weekly serving for adults was from 0.05 to 0.7 pg WHO-TEQ kg⁻¹ b.w. Consumption of the 25 g serving led to intake below 4% of the TWI and eating the 100 g serving exposed the individual to below 16% of the TWI (Fig. 2). The maximum PCDD/F and DL-PCB amounts were ingested via Pacific oysters.

Fig. 2.

Higher exposure was noticed for children: from 0.14 to 2.12 pg WHO-TEQ kg⁻¹ b.w. This corresponds to intake of 3–48% of the TWI. The highest exposure was from consumption of Pacific oysters; nevertheless, it did not result in the TWI being exceeded. It should be noted that this calculation does not take into account other consumed food commodities which may also contain PCDD/Fs and DL-PCBs, such as fish, eggs, milk or meat (22, 24, 26, 28, 29, 43). However, bearing in mind the low consumption of bivalve molluscs in Poland, especially by children, they should not be of concern as sources of PCDD/F and DL-PCB exposure.