SURVEY OF PERSISTENT ORGANOCHLORINE CONTAMINANTS (PCDD, PCDF, DL-PCB) IN BALTIC FISH AND FISH MEALS

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1 Bull Vet Inst Pulawy 53, , 29 SURVEY OF PERSISTENT ORGANOCHLORINE CONTAMINANTS (PCDD, PCDF, DL-PCB) IN BALTIC FISH AND FISH MEALS JADWIGA PISKORSKA-PLISZCZYŃSKA, RADOSŁAW LIZAK, SEBASTIAN MASZEWSKI, PAWEŁ MAŁAGOCKI, AND TADEUSZ WIJASZKA Department of Radiobiology, National Veterinary Research Institute, 24-1, Pulawy, Poland Received for publication August 27, 29 Abstract The concentration and congener-specific profile of polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), and dioxin-like polychlorinated biphenyls (dl-pcb), were determined in edible marine fish tissues of salmon, herring, and in sprat and fishmeals collected between 26 and 28. Average concentrations of the sum of the compounds ranged from 4.5 ±5.46 to 9.23 ±2.64 pg WHO-TEQ/g f.w. for the tested fish species. The highest concentration was found in salmon up to pg WHO-TEQ/g f.w. of the sum of PCDD, PCDF, and dl-pcb. Only seven out of 55 fish meals exceeded the maximum permitted level for PCDD/PCDF (over 1.25 ng WHO-TEQ/kg) and 4 pg/p for total TEQ. Dioxin-like PCB make up about 5% of the concentration. The PCDD, PCDF, and dl-pcb accumulation pattern found in the analysed samples showed typical distribution reported for marine samples. The following congeners dominated: 2,3,7,8-PCDF, 2,3,4,7,8-PeCDF, 1,2,3,7,8-PeCDF, and PCB 15,118, and 156. Key words: fish, fish meal, polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, dioxin-like polychlorinated biphenyls, Baltic Sea. Fish are important in a healthy diet. The nutritional benefits of fish consumption are well known. Low levels of saturated fats, high amounts of omega-3 polyunsaturated fatty acids, which seem to have protective effects in preventing coronary heart disease, and in imparting other health benefits, a high content of high-quality proteins, vitamins, and other essential nutrients. Some fish may contain dioxins or other harmful chemicals at sufficiently high levels to be a concern. Organochlorine compounds such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs), are widely distributed pollutants in the environment. Due to their lipophilic properties, they concentrate and biomagnify in the food chain, mainly related to fat. A number of toxicological studies over the last 4 years have concerned the biological effects of these compounds, ranging from mortality to chloracne. These effects are commonly referred to dioxin-like toxicity and are displayed not only by the dioxins but also by other closely-related chlorinated compounds - PCBs. This dioxin-like toxicity within PCBs is displayed primarily by non-ortho-chloro-substituted and some mono-ortho chloro-substituted PCBs (2). In response to this potential health risk, Community legislation has been put in place with the aim of reducing dioxin contamination and consumer exposure to these chemicals (3). Regulation of these contaminants, and maximum permissible limits for a range of foods and feeds, have been established within the European Union (5-7, 16). The ingestion of contaminated foods contributes more than 9% of total human exposure, and fish and seafood are recognised as one of the main contributors (8, 12, 15, 17-19). Maximum levels have been set for the sum of dioxins and dioxin-like PCBs expressed in the World Health Organisation (WHO) toxic equivalents, using the WHO-TEFs, as this is the most appropriate approach from a toxicological point of view. Regulation 1881/26 and Directive 22/32 establish maximum (Community) levels for dioxins in foodstuffs, including fish and fish products. This paper presents an overview of the data from our study concerning the concentration of PCDD/Fs and dl-pcb in edible marine species and fish meal from the Baltic Sea. The study covers the period from 26 to 28. Official controls of Baltic fish and fish meal were done on a regular basis from and according to Commission Recommendations 24/75 and 26/794/EC. A national control survey of dioxins and related compounds in fish involved seven

2 826 congeners of 2,3,7,8-polychlorinated dibenzo-p-dioxins (PCDD), 1 congeners of 2,3,7,8-polychlorinated dibenzofurans (PCDF), and 12 dioxin-like PCBs. The concentration and composition profiles of 29 toxic compounds were analysed. Material and Methods Fish and fish meal sampling. The sampling methods and methods of analysis for the official control of dioxins and the determination of dioxin-like PCBs in foodstuffs were laid down in Commission Directive 22/69/EC, 24/44 and present Commission Recommendation 1883/26 (7). Fish samples (Baltic herring - Clupea harengus membras, salmon - Salmo salar, and sprat - Sprattus sprattus balticus) and fish meal samples were collected by veterinary inspection each year and tested by the National Veterinary Research Institute in Pulawy. Solvents and standards. The solvents and Florisil used were obtained from LGC Promochem (Wesel, Germany). Carbopack C and silica were from Sigma-Aldrich (Poznan, Poland), and sodium sulphate and sulphuric acid were from Merck (Darmstadt, Germany). All standards ( 13 C labeled and unlabeled) were obtained from Cambridge Isotope Laboratories (Andover, USA). Fish sample preparation. One sample comprised three to 12 individuals for herring, and 3 to 6 individuals for sprat. Salmon samples were tested individually. All the samples were homogenised by blending and freeze-dried prior to extraction and the water content was determined gravimetrically. Extraction and purification. Each sample (5 g) was spiked with 13 C labeled PCDD/Fs and dl-pcbs and extracted with dichloromethane/n-hexane (5/5, v/v) by accelerated solvent extraction (ASE 3). Classical column chromatography was used for cleanup. The samples were defatted on acidic silica columns and then purified and fractionated on Florisil and Carbopack C. Instrumental analysis. Dioxins and PCBs were determined by high resolution gas chromatography coupled to high resolution mass spectrometry (HRGC- HRMS). MAT 95XP (Thermo Scientific, Germany) coupled with an Ultra Trace GC (Thermo Scientific, Italy) with GC PAL and with auto sampler (CTC Analytics AG, Switzerland). Chromatographic separation was achieved by split less injection of 1 µl on a DB-5MS (6 m, id.25 mm,.1 µm, J&W Scientific, Folsom, USA). The MS was operated in SIM mode at a resolution equal or superior to 1,. The two most intense ions were monitored for native and labeled compounds. Calculations. Toxic equivalents (TEQ) for PCDD/Fs and dl-pcbs were calculated according to toxic equivalency factors (TEF) adopted by the WHO. The concentrations below LODs were equated to the LOD (upperbound concept). For congener pattern determination, standardisation was performed on complete congener specific data, normalised to the sum of the congeners (individual congener concentration divided by sum of PCDD/Fs and the sum of dl-pcbs, respectively) to eliminate differences in concentration levels and other sample-specific variations. All data are expressed as pg WHO-TEQ per g of fresh weight (f.w.). Quality assurance/quality control. All PCDD/PCDFs and PCBs data were assessed for compliance with published acceptance criteria and the method performance criteria guidelines laid down in EU Commission Regulation 1883/26. The GC MS analytical run for each set of analyses was preceded by a reference standard solution used to check system performance and calibration validity prior to continuation of the run. The reference standard solution was also analysed during and at the end of the analytical run. All integrated chromatograms were examined to assess chromatographic peak shape, resolution, and signal-to-noise; and for high-resolution mass spectrometry. Isotope ratios for signal peaks were assessed for agreement with theoretical abundances and the variation in response factors for reference standard solutions within a run was limited to 15%. The method was validated and uncertainty of measurement was estimated (16.4% for PCDD/Fs and 18.8% for dl-pcbs). The limits of detection (LODs) for PCDD/F and dl-pcb congeners were isomer dependent and varied between.2 and.5 pg/g f.w. The recoveries of the internal standards ranged between 65% and 12%. Bioassay. Fish meal samples were analysed by biological screening procedure with XDS CALUX bioassay and HRGC/HRMS analysis was used for confirmation of suspected samples. The method was validated, expanded uncertainty was estimated, and the requirements of dioxin and dl-pcb analysis laid down in the Commission Directive 22/74 and Commission Regulation 1883/26 were fulfilled. Results Baltic fish. The overview of the actual contamination levels of dioxins in tested fish species, the number of samples analysed during and number noncompliant with regulation 1881/26 samples are summarised in Table 1. Individual fish samples, which exceeded the permitted level of PCDD/PCDFs and the sum of PCDD/PCDF/dl-PCBs are introduced in Table 2. Dioxin data are shown on the fresh weight basis (f.w.). Percentage of dioxins and dl- PCBs in sum of tested compounds are shown in Table 2. The marine fish species listed in Tables 1 and 2 were caught at ICES subdivision 27. Fourteen from 32 salmon samples exceeded permitted levels and half of concentration was created by dl-pcbs. Only one herring and one sprat samples were over the acceptable level 4. pg WHO-TEQ/g f.w. (1881/26). Dl-PCBs concentration was 62%, 52%, and 58 % of the tested compounds in salmon, herring, and sprat, respectively. The congener profiles showed the similarity of the toxicity pattern of dioxins and furans in herring and other tested species (Fig. 1). Generally, the toxicity

3 827 pattern of salmon, herring, and sprat is dominated by 2,3,7,8-TCDF, 2,3,4,7,8-PeCDF, 1,2,3,7,8-PeCDF, and to less extent of 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD. These five congeners contribute to more than 9% of the total WHO-PCDD/F-TEQs. But most sample toxicity (in WHO-TEQ) came from 1,2,3,7,8-pentachlorofuran. Dioxin-like PCBs represent mainly PCB 15, PCB 118, and PCB 156. Including WHO TEF dominating PCB congener was PCB 126 in all three tested species because of the very high value of TEF (.1). Fish meal. Relatively low dioxin concentrations were measured in the fish meal (Table 3) but seven samples (13%) from 55 tested passed permissible limit (Table 4). The concentration ranged from 3.29 to 6.9 ng WHO-PCDD/ F/dl-PCB-TEQ/kg. The results of noncompliant fish meal samples are presented in Table 4. Fish species Salm on, n=32 Herri ng, n=29 Sprat, n=29 Table 1 Contamination level of PCDD/PCDFs and dl-pcb, and sum of PCDD/F/dl-PCB in Baltic fish samples in pg WHO-TEQ/g f.w. Year PCDD/Fs dl-pcbs Sum of PCDD/F/dl- PCB ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±5.46 Sample tested/noncompliant (% noncompliant) 32/14 (43.8%) 29/1 (3.4%) 29/1 (3.4%) Fish specie Salmon n= 14 Table 2 Fish samples exceeding permitted level: PCDD/PCDFs 4 pg WHO-TEQ/g and 8 pg WHO-TEQ/g for sum of PCDD/PCDF/dl-PCBs PCDD/Fs dl-pcbs Sum of PCDD/F/dl-PCB Cod pg WHO- TEQ/g pg WHO- TEQ/g pg WHO-TEQ/g % % % number f.w. f.w. f.w. 7/ / O7/ // / / / / / / / / / / x ± SD 3.84 ± ± ± ± ± 1.88 Herring, n= Sprat, n=

4 828 Year Table 3 Fish meal. Average concentration of dioxins, furans, and dl-pcbs (ng WHO-TEQ/kg) Number of samples tested/ noncompliant 2 / 3 18 / 2 17 / 2 Total 55 / 7 % 1 / 13 PCDD/PCDF X ± SD range.94 ± ± ± dl-pcb X ± SD range.35 ± ± Sum of PCDD/F/dl- PCB X ± SD range 1.67 ± ± pg/g f.w herring salmon sprat 2378-TCDD PeCDD HxCDD HxCDD HxCDD HpCDD OCDD 2378-TCDF PeCDF PeCDF HxCDF HxCDF HxCDF HxCDF HpCDF HpCDF OCDF pg/g f.w herring salmon sprat PCB 77 PCB 81 PCB 126 PCB 169 PCB 15 PCB 114 PCB 118 PCB 123 PCB 156 PCB 157 Fig. 1. PCDD/PCDFs (upper) and dl-pcbs (lower) congener profiles in tested fish samples. PCB 167 PCB 189

5 pg/g product fish meal TCDD PeCDD HxCDD HxCDD HxCDD HpCDD OCDD 2378-TCDF PeCDF PeCDF HxCDF HxCDF HxCDF HxCDF HpCDF HpCDF OCDF pg/g product fish meal 1 PCB 77 PCB 81 PCB 126 PCB 169 PCB 15 PCB 114 PCB 118 PCB 123 PCB 156 PCB 157 PCB 167 Fig. 2. Typical congener profiles in fish meal. PCB 189 Table 4 Noncompliant fish meal samples. Maximum permit level of PCDD/PCDFs and sum of PCDD/F/dl-PCB is 1.25 ng WHO-TEQ/kg and 4.5 ng WHO-TEQ/kg, respectively Sample code PCDD/PCDF dl-pcb Sum of PCDD/F/dl-PCB 6/16/MD 6/118/MD 6/122/MD 7/8/MD 7/81/MD 8/51/MD 8/83/ MD

6 83 The toxicity patterns of PCDDs, PCDFs, and dl-pcbs congeners was very similar to that of herrings (Fig. 2) but contain also a large amount of OCDD and a small amount of other dioxins. The PeCDF congeners contribute more than 5% to the PCDD/PCDFs toxicity. Dioxin-like PCB make about 5% of total PCDD/PCDFs and dl-pcbs. The four low chlorinated tetra- and penta-furans and tetra- and penta-dioxins contribute more than 8% to the total toxicity. About 5% of sum of total PCDD/PCDF/dl-PCB was from dl- PCDDs. Discussion The Baltic Sea is recognised as one of the most polluted seas in the world and persistent organic compound contamination of the sea has been known for years. Chloroorganic compounds enter the sea via a number of waterways or as air fallout and are stored in sediments, where they accumulate and also biomagnify in biota over decades due to lipophilic properties. The levels of these contaminants in Baltic fish are a major concern for the EU and Baltic States, which has led to numerous national surveys aimed at determining the dioxin and dioxin-like compound levels (1, 2, 8, 9, 11, 13, 14). Scientific and monitoring data show that the degree of dioxin contamination in fish varies geographically, from year to year, and between seasons. Dioxin contamination also varies with the fat content and increases with age of the fish. Thus in sea, fatty fish, such as herring, salmon, and sprat, contain the highest levels of dioxins, calculated by fresh weight (11, 17, 18). For herring, which is the most studied species, fish in the northern and eastern parts of the Baltic Sea have been shown to have higher dioxin levels (1). Maximum levels must not be exceeded in foodstuffs placed on the market in the EU. In addition, the Regulation states also that it is prohibited to mix products complying with the maximum levels with products exceeding these levels or to use products which do not comply with the maximum levels as an ingredient in the manufacture of other foodstuffs. Herring, salmon, and sprat became the major fish species examined in our study due to the highest rates of consumption, as well as the high levels of fat. The results of the presented study indicated that salmon was the most contaminated Baltic fish and very often dioxins and related compounds exceeded permitted level (44% samples tested). Herring and sprat very often go beyond action level (2 pg/g WHO-PCDD/F/dl-PCB- TEQ). WHO-TEQs were greater for the species at the high level in the trophic chain and fat content. The results were similar to obtained by us earlier (12, 14). Isosaari (8) has found PCDD/PCDF level up to 17.7 pg WHO-TEQ/g f.w. The EU maximum permitted level was exceeded in salmon and Baltic herrings (6). Karl et al. (9) have analysed the PCDD/PCDF content of 2 marine fish from German market. The highest dioxin level in edible tissues was found in Baltic herring. Fishery products from the Belgian market consist about 2.53 pg CALUX TEQ/ g f.w. (2). The review of international surveys shows that the comparison of results is rather complicated. This is not only because of the great variety of tested congeners, different ages of fish, fat content, and geographic origin but also differences in the expression of the results. The studies show that Baltic fish and fish meal may contain significant levels of polychlorinated pollutants. Herring and sprat from the Baltic Sea, used for human consumption in some Member States, may contain dioxin levels exceeding the Community action level (2. pg/g WHO-PCDD/F/dl-PCB-TEQ). The dietary intake of PCDDs, PCDFs, and dl-pcbs from Baltic Sea fish could be an important contribution to the total dietary toxic pollutants intakes of some subpopulations. The Domingo and Bocio (4) paper provides an overview of available information concerning PCDD/PCDFs and PCBs in marine species and the intake of these compounds through consumption. Fish are important in a healthy diet. They are a lean, low-caloric source of protein. However, some fish may contain dioxins or other harmful chemicals at sufficiently high levels to be a concern. Federal and local governments should issue fish consumption advisories when the fish are unsafe to eat. The advisories may suggest that people should avoid eating certain kinds or certain amounts of fish. Dioxins and other persistent organic pollutants (POPs) are also subject to the Stockholm Convention. The treaty obliges signatories to take measures to eliminate where possible, and minimise where not possible to eliminate, all sources of dioxins. Acknowledgments: The authors thank the General Veterinary Inspectorate and Veterinary Inspection of Poland for collecting samples. The authors are also grateful to Malgorzata Warenik-Bany, Magdalena Gembal, Joanna Cebulska, Halina Dudek, and Iwona Grochowska for their technical assistance in chemical and biological analysis. References 1. Aune M., Bjerselius R., Atuma S., Larsson L., Bergh A., Darnerud P., Andersson A., Arrhenius F., Bergek S., Tysklind M., Glynn A.: Large differences in dioxin and PCB levels in herring and salmon depending on tissue analyzed. Organohalogen Comp 23, 64, Bjerselius R., Aune M., Darnerud P.A., Atuma S., Tysklind M., Bergek S., Lundstedt-Enkel K., Karlsson L., Appelberg M., Glynn A.: PCDD/PCDF contribute with half of the total TEQ found in fatty fish from the Baltic Sea. Organohalogen Comp 22, 57, Community strategy on dioxins, furans and PCBs (COM (21) 593 final, EC 21). 4. Domingo J.L., Bocio A.: Levels of PCDD/PCDFs and PCBs in edible marine species and human intake: A literature review. Environ Int 27, 33, EC, 26. Commission Recommendation (EC) No. 26/88 of 6 February 26 on the reduction of the

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