Polychlorinated Naphthalene Levels, Distribution, and

Polychlorinated Naphthalene Levels,. Distribution, and Biomagnification in a Benthic Food Chain in the Baltic. Sea. KJELL LUNDGREN* AND MATS TYSKLIND...
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Environ. Sci. Technol. 2002, 36, 5005-5013

Polychlorinated Naphthalene Levels, Distribution, and Biomagnification in a Benthic Food Chain in the Baltic Sea KJELL LUNDGREN* AND MATS TYSKLIND Environmental Chemistry, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden RASHA ISHAQ AND DAG BROMAN Institute of Applied Environmental Research, Stockholm University, SE-106 91 Stockholm, Sweden BERT VAN BAVEL MTM Research Centre, O ¨ rebro University, SE-701 82, O ¨ rebro, Sweden

The scientific literature contains little information regarding bioaccumulation and biomagnification of polychlorinated naphthalenes (PCNs) in food webs. Here we present new information on the food chain transfer of PCNs within a food chain in a subarctic environment. PCNs (tetra- to heptachloro congeners) were measured in surface sediments and in a marine benthic food chain, comprising amphipods, isopods, and fourhorned sculpins. Samples were collected from five locations in the Gulf of Bothnia, northern Baltic Sea. PCN concentrations in the sediments were similar to background levels determined previously in sediments from the northern hemisphere. Measurement of the carbon content of the sediments allowed the calculation of biota to sediment accumulation factors (BSAFs). Tetra- and penta-CNs exhibited BSAF values greater than one, while BSAFs for the more chlorinated PCNs were less than one. This suggests more efficient assimilation, by amphipods, of the less chlorinated PCNs. A decrease in ΣPCN concentrations from the lowest to the highest trophic level was demonstrated (amphipods: 10-69 ng/g lw; isopods: 3.9-16 ng/g lw; fourhorned sculpins: 0.54-1.5 ng/g lw). Biomagnification factors (BMFs) were calculated based on the concentrations of the congeners. These indicated that a few congeners biomagnified significantly: the highest BMFs (0.09-1.4) were found for 2,3,6,7-substituted congeners and those lacking adjacent hydrogen-substituted carbon atoms.

Introduction Polychlorinated naphthalenes (PCNs) are ubiquitous environmental contaminants that originate, like polychlorinated biphenyls (PCBs), in technical mixtures (e.g. Halowaxes, Nibren waxes, Seekay waxes, Clonacire waxes, and Cerifal material) used in a variety of industrial applications. The production of technical PCN mixtures has ceased in many countries, but they are still found, for example, in electrical * Corresponding author phone: +46 - (0)90 - 786 93 24; fax: +46 - (0)90 - 12 81 33; e-mail: [email protected]. 10.1021/es0201146 CCC: $22.00 Published on Web 11/02/2002

 2002 American Chemical Society

equipment (1). In addition, PCNs are formed and released into the environment via processes such as incineration (2, 3) and chlor-alkali production (4, 5). This group of compounds has also been found as a byproduct in technical PCB mixtures (6, 7). The PCN molecule consists of two fused aromatic rings with 1-8 chlorine atoms substituted to the naphthalene molecule skeleton, so, theoretically, there are 75 possible congeners. Among the 48 tetra- to hepta-CN congeners, 28 PCN congeners (18 TeCNs, 8 PeCNs, and 2 HxCNs) have two or more adjacent carbon atoms substituted with hydrogen, and 6 PCN congeners have chlorine atoms substituted in all the 2,3,6,7-positions (PCN 48, 54, 66, 67, 70, and 73). The PCN congener numbering throughout this paper follows ref 8. PCNs tend to accumulate in biota and persist in the environment as a result of their physicochemical properties. Consequently, PCNs have been detected in a wide variety of organisms and systems, including marine mammals (9, 10), fish (11-15), sediments (13, 14, 16), and air (17-19). Although PCNs are ubiquitous pollutants, reports of levels of PCNs in the environment are scarce in comparison to PCBs, for example. The molecular structures of PCNs are similar to those of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and PCBs. The few studies of the toxicity of PCNs have shown that the more chlorinated PCN congeners elicit similar biochemical responses as 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). These responses include the induction of hepatic drug-metabolizing activity, such as aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin-O-deethylase (EROD) activities (20-24). In toxicological tests, inducing Ah-receptor responses, the more chlorinated PCNs, especially 2,3,6,7-chlorine substituted PCNs (24), have a relative potency similar to the mono-ortho PCBs, i.e., 3-4 orders of magnitude less potent than TCDD. Recently, in a feeding experiment involving PCN contaminated food, juvenile Baltic salmon suffered hepatotoxicity, dose-dependent induction of EROD activity, negative effects on ovaries, and delayed development (25). The fact that PCNs are found in the environment at levels comparable to, or higher than the non-ortho PCBs, suggests the need to include this group of compounds in environmental monitoring studies. Only a few studies of biomagnification of PCNs in food chains have been reported (26-30). The following have been studied: a pelagic food chain (plankton, herring, and harbor porpoise) (26), black cormorant in relation to fish (27), fish in relation to mussel (28), and salmon in relation to food (29). A large number of the biomagnification factors (BMFs) for the individual PCN congeners in these food chains showed that many of the members of this group of compounds were less assimilated by the higher trophic level species. There were only a few PCNs that biomagnified to a greater extent. The food chains studied in the literature were in the southern part of the Baltic Sea. In this study, it was also possible to calculate biota (amphipod) to sediment accumulation factors (BSAF values). To our knowledge, this is the first time, in the scientific literature, that BSAF values from Europe have been reported for PCNs. In this paper the concentrations of tetra- to hepta-CNs are presented for a benthic food chain (surface sediment amphipod - isopod - fourhorned sculpin) from five coastal locations in the northern part of the Baltic Sea (Gulf of Bothnia: Bothnian Bay and Bothnian Sea). The PCN concentration data obtained allowed the calculation of BSAF VOL. 36, NO. 23, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Sampling locations in the Baltic Sea. and BMF values for individual PCNs present in the food chain studied. In addition, the change of PCN homologue profiles and PCN congener patterns were investigated.

Materials and Methods Sampling Locations, Sampling Methodology, and Samples. Between autumn 1991 and autumn 1993 surface sediments, amphipods (Monoporeia affinis), isopods (Saduria entomon), and fourhorned sculpins (Oncocottus quadricornis) were collected at five different coastal locations in the Gulf of Bothnia. All came from sea-floor areas characterized by sediment accumulation (Figure 1). The sampling locations, from north to south, were as follows: Harufja¨rden (HF), Umeå (UM), Hornslandet (HL), Ga¨vlebukten (GB), and Simpna¨s (SN). The samples were taken from the second accumulation depression from the coastline in order to establish background PCN levels. The benthic food chain studied consists of species living in or close to accumulation areas. Amphipods and isopods are sediment dwelling crustaceans. Amphipods (maximum size 8 mm) feed on material from the sediment and serve as a food source for isopods (maximum size 8.6 cm), which are also carrion feeders. Fourhorned sculpins are bottom dwelling fish that feed on both isopods and amphipods. The sculpins are sedentary and are, therefore, good indicators of environmental pollution within the region they inhabit. A detailed description of the sampling methods used is given elsewhere (31). Briefly, amphipods were extracted from the sediments by sieving; isopods were collected in cages placed on the bottom; and fourhorned sculpins were caught in fishing-nets by local fishermen. The surface sediment samples (0-1 cm) were collected with a modified Ponar grab sampler (32), which allows free passage of water through the sampler during descent and sediment penetration. Great care was taken to minimize disturbance of the samples (33). In total, 16 surface sediment samples, 14 pooled whole-body amphipod, 18 isopod, and 14 fourhorned sculpin samples were analyzed. The number of replicates analyzed at each station was between one and five. All samples from the locations HF, UM, HL, and GB were collected during the autumn, but the samples from location 5006

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FIGURE 2. The principal analytical flow diagram, showing the cleanup and analysis of PCNs. SPM, semipermeable membrane; MACs, monocyclic aromatic compounds; DACs, dicyclic aromatic compounds; PACs, polycyclic aromatic compounds. SN were collected during both the autumn, SN(a), and the spring SN(s). Extraction and Cleanup. A multiresidue nondestructive analytical procedure was applied to all the samples. This allowed the analysis of other contaminants in addition to the PCNs analyzed in this investigation (31). A detailed flow diagram of the analytical procedure is presented in Figure 2. All samples were homogenized and stored at -20 °C until extraction. The biological material was first homogenized and pooled and then subsampled into replicates. The homogenate was placed in pre-extracted cellulose thimbles and extracted wet in a Soxhlet apparatus, equipped with a Dean Stark trap for the collection of water. The homogenate was extracted with 300 mL of toluene for 24 h followed by a 300 mL of an azeotropic mixture of acetone-n-hexane (59:41, v/v), for another 24 h. The two extracts were combined and after evaporation of solvents the total lipid weight (organic phase) for each sample was determined gravimetrically. The total organic carbon content (TOC) in the sediment samples was determined for a subsample using a hightemperature combustion elemental analyzer (Carlo Erba EA 1108), following standard procedures. Prior to extraction, a 13C-labeled non-ortho PCB (PCB 126) was added to the sample as an internal standard. The lipid phase derived from the extraction was dissolved in cyclopentane and transferred into a bottom sealed semipermeable membrane (SPM, 500 mm length, 26 mm wide, and 80 µm thick) mounted inside a dialysis funnel (500 × 45 mm i.d.). Cleanup was achieved by dialysis through the membrane, using cyclopentane, to reduce the bulk of the lipids (34). Dialysis through the polymeric film was accomplished by changing the outer cyclopentane (dialysate) after 16, 40 and 64 h. The dialysate fractions, containing about 1-10% of the original lipids, were combined and carefully concentrated to a few milliliters using a rotary evaporator. Prior to the silica column stage, the sample extract was split into two parts: 90% was used for the analysis of PCNs

and other planar compounds, and 10% was used for the analysis of organochlorine pesticides and the bulk of PCBs. In addition to the cleanup steps described above the sediment samples were treated with pure copper (regenerated with hydrochloric acid) between the dialysis and the silica column stages to remove elemental sulfur from the sample matrix. The remaining lipids were removed on a 10% water deactivated silica column with 50 mL of n-hexane. After solvent reduction, the extract was fractionated on a high performance liquid chromatography (HPLC) aminopropylsilica column (300 × 7.8 mm, 10 µm) (35). The HPLC aminocolumn separated the solutes according to the number of aromatic rings and the resulting fractionation produced three fractions: one containing aliphatic compounds and monocyclic aromatic compounds (MACs); one containing dicyclic aromatic compounds (DACs, e.g. PCBs, PCNs, and PCDD/ Fs); and one containing polycyclic aromatic compounds (PACs, e.g. polycyclic aromatic hydrocarbons, PAHs). The fraction from the amino-column containing the DACs was then introduced onto an HPLC column (250 × 4.5 mm) containing 100 mg PX-21 activated carbon (particle size 2-10 µm) on 2.20 g Lichroprep RP-18 (15-25 µm). This produced a final separation of planar PCNs (the planar fraction, Figure 2) from less planar compounds (e.g. poly-ortho substituted PCBs and mono-ortho PCBs, the nonplanar and semiplanar fractions, Figure 2) and other interfering compounds. This was achieved by gradient elution with a mixture of dichloromethane (DCM, 1%) in n-hexane and toluene (0-10%) (36). PCNs were backflushed from the column with 80 mL of pure toluene. All solvents were degassed with argon and the solvent flow-rate was 4 mL/min. Prior to injection (150-300 µL), the column was washed and reconditioned with toluene (5 min, backflush) and n-hexane-DCM (5 min, backflush) followed by n-hexane-DCM for 10 min flowing forward. A tetradecane keeper (30 µL) was added to the planar compound fraction prior to evaporation, and a recovery standard (13C-labeled PCB 101) was added prior to the final analysis. Procedure blanks were processed concurrently with each batch of 10 samples. HRGC-HRMS Analysis. The extracts (3 µL) were injected in splitless mode on a Hewlett-Packard 5890 gas chromatograph system coupled to a VG Analytical 70-250S mass spectrometer (HRGC-HRMS). PCN separation was performed on an RTx-5 capillary column (60 m × 0.32 mm i.d., 0.25 µm film thickness) using the following temperature program: 180 °C (2 min), 20 °C/min, 200 °C, 4 °C/min, 300 °C (15 min). The carrier gas was helium and the column head pressure was 18 psi (∼120 kPa). Electron ionization (EI) was used at 35 eV and the HRMS operated at a mass resolution of >8000. The injector, interface, and ion source temperatures were 250, 280, and 250 °C, respectively. The PCNs were recorded using selected ion recording (SIR), monitoring two of the most abundant ions in the molecular ion chlorine distribution cluster for the tetra-, penta-, hexa-, and hepta-CN homologues (m/z 263.9067/ 265.9038, 297.8677/299.8648, 331.8288/333.8258, 365.7898/ 367.7868). For the 13C-labeled PCB 101 and 126 m/z 337.9207 was monitored. The detection limit (S/N > 3) was approximately 0.1 pg during the analyses. The HRGC-HRMS quantification of the PCNs was based on PCN and 13C-PCB chromatographic peak areas. When the ratio between the relative abundances of the detected chlorine cluster molecular ions was within 10% of the theoretical value, relative response factors (RRFs) of the available native HxCN (PCN 66/67 and 71) and HpCN (PCN 73) standards together with the internal standard (13C-PCB 126) were used for the quantification of the PCNs. Standards of individual native TeCNs and PeCNs and their 13C-labeled analogues were not available. The molar response factors of

TABLE 1. Total Average ΣPCN Levels in Samples from the Baltic Sea ΣPCN levelsa

sampling site Harufja¨ rden (HF) Umeå (UM) Hornslandet (HL) Ga¨ vlebukten (GB) Simpna¨ s (SN, autumn) Simpna¨ s (SN, spring)

sediment amphipod isopod (ng/g dw) (ng/g lw) (ng/g lw) 0.33 0.088 0.72 0.48 1.9 1.1

69 12 23 17 32 10

16 8.5 3.9 b 7.5 9.3

fourhorned sculpin (ng/g lw) 0.54 1.3 0.90 1.4 1.2 1.5

a ΣPCN level ) sum of the concentrations of 4-7 chlorine substituted PCNs. b Not sampled.

hexa-CNs were, therefore, used to quantify the tetra- and penta-CNs without correction for the differences in the ionization cross-sections (Q). These differences were 33.7, 36.9, and 40.1 × 10-16 cm2 for the tetra-, penta-, and hexaCNs, respectively (8). The identification of PCNs was based on retention data from the literature (37) and using a Halowax 1014 mixture.

Results and Discussion General Results. The analyses of the surface sediment samples show that the average concentrations of total PCNs were lowest in Bothnian Bay (stations HF and UM) and highest in the Bothnian Sea (stations HL, GB, and SN). The results were 0.088-0.33 and 0.48-1.9 ng/g dw, respectively (Table 1). These results indicate a higher level of deposition of PCNs in the southern part of the Gulf of Bothnia. Similar background surface sediment PCN levels (0.27 to 2.5 ng/g dw) have been determined previously near these locations (16). Sample data and average PCN levels for all the analyzed PCN congeners from all the sample locations are presented in Tables 2 and 3. The results of the analysis of replicate samples are presented in the form of relative standard deviations (RSDs). The RSDs ranged from 2 to 34%, which are acceptable figures for this type of environmental analysis. The average recovery of the internal standard was 61%, ranging from 39 to 93%. Samples with low recoveries were omitted from subsequent analysis. The carbon content of the sediments was similar (∼3%) at all stations except station UM (1.5%) located in the Bothnian Bay. The biological samples contained 40-62, 11-20, and 18-35% lipids for the amphipods, isopods, and fourhorned sculpins, respectively (Tables 2 and 3). The difference in PCN levels between the different stations was less pronounced in the biota. The total average concentration of PCNs in the lipids decreased from the bottom to the top of the benthic food chain (CΣPCNs,amphipod > CΣPCNs,isopod > CΣPCNs,fourhorned sculpin). The average levels in amphipods were between 10 and 69 ng/g lw and were 3.9-16 ng/g lw in the isopods. There appears to be a similar spatial variation in the ΣPCN levels between the amphipods and the sediments (Table 2) reflecting reduced metabolic capacity in the amphipods. The ΣPCN levels in the fourhorned sculpins (0.54-1.5 ng/g lw) were very similar in all the samples analyzed. For comparison, reported ΣPCN levels in flounder (Platychthis flesus) caught in the southern part of the Baltic Sea (Gulf of Gdan ˜ sk) were typically in the range of 36-83 ng/g lw (28) and 0.2-1.4 ng/g lw in tuna and swordfish (38) collected in the Mediterranean Sea near the Italian coast. Homologue Profiles. The PCN homologue compositions (%) in the food chain studied here were compared to different environmental matrices from other studies (Figure 3). In the surface sediment samples tetra- and penta-CNs were the VOL. 36, NO. 23, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Average PCN Levels in Surface Sediment (pg/g dw) and Amphipod (ng/g lw) Samplesa sediment

amphipod

type of sample locationb

HF

UM

HL

GB

SN(a)

SN(s)

HF

UM

HL

GB

SN(a)

SN(s)

42 33/34/37 44/47 36 28/43/45 27/29 30/32 35/39 38/40/48 46 31 41 TeCNs 52/60 58 61 50 51 54 57 62 53/55 59 49 56 PeCNs 66/67 64/68 69 71/72 63 65 70 HxCNs 73/74 HpCNs ΣPCNs no. of samples RSD (%) carbon content (%) organic phase or lipid content (%)

1.1 29 11 6.1 17 15 2.0 13 33 13 0.43 2.0 140 19 2.1 15 8.4 6.9 4.8 9.5 12 10 10 1.8 1.1 100 29 6.1 5.6 11 3.3 0.72 n.d. 55 27 27 330 3 18 3.1 2.5

0.14 10 4.7 3.4 7.4 5.1 1.2 4.6 14 3.9 0.3 1.3 56 2.2 0.3 3.3 2.4 2.1 1.0 2.8 3.1 2.7 2.1 0.3 0.3 23 1.4 1.1 1.2 0.9 0.5 0.2 n.d. 5.2 4.4 4.4 88 2 2 1.5 0.83

1.5 91 42 34 51 44 4.6 28 86 28 1.4 2.1 410 53 5.4 30 13 11 5.5 22 25 19 21 1.9 1.9 210 16 10 12 17 5.5 4.2 n.d. 65 31 31 720 3 7 3.3 2.7

2.8 75 27 19 34 19 2.9 16 49 15 0.88 2.8 260 36 3.3 22 11 10 5.3 16 17 15 12 1.4 1.4 150 17 7.9 8.6 5.0 4.0 1.9 n.d. 45 20 20 480 3 11 3.2 2.6

15 310 140 49 220 68 22 150 300 150 3.1 6.5 1400 120 12 67 30 25 12 35 50 51 49 7.0 2.6 460 27 9.2 11 5.2 5.2 1.8 n.d. 60 11 11 1900 2 32 3.1 2.5

3.5 180 69 39 95 37 9.4 52 140 50 1.8 8.2 690 69 7.4 43 20 18 9.2 28 32 27 27 2.7 2.3 290 21 9.3 11 7.6 5.3 2.6 n.d. 57 21 21 1100 3 10 3.2 2.4

0.60 8.2 3.9 1.4 7.6 5.0 0.76 5.6 12 6.3 0.10 0.32 52 3.3 0.37 2.1 1.1 0.80 0.37 1.0 1.6 1.7 1.9 0.30 0.089 15 1.1 0.33 0.33 0.39 0.17 0.070 n.d. 2.4 0.87 0.87 69 3 19

0.024 1.2 0.70 0.26 1.4 0.70 0.20 1.1 2.5 1.0 0.070 0.14 9.3 0.25 0.026 0.35 0.27 0.21 0.090 0.23 0.34 0.33 0.37 0.057 0.019 2.5 0.13 0.089 0.094 0.062 0.040 0.022 n.d. 0.44 0.13 0.13 12 2 4

0.13 2.6 1.5 0.49 2.6 1.6 0.24 1.7 3.8 1.6 0.034 0.044 16 1.4 0.16 0.97 0.40 0.31 0.12 0.35 0.56 0.59 0.60 0.080 0.023 5.6 0.40 0.18 0.19 0.14 0.077 0.061 n.d. 1.1 0.20 0.20 23 2 19

0.10 2.3 1.3 0.42 2.0 0.71 0.21 1.0 2.4 0.84 0.028 0.038 11 1.3 0.13 0.73 0.33 0.28 0.11 0.29 0.43 0.35 0.37 0.051 0.023 4.3 0.62 0.13 0.13 0.26 0.054 0.025 n.d. 1.2 0.40 0.40 17 2 10

0.20 4.0 2.2 0.57 3.7 1.0 0.36 2.5 4.7 2.4 0.043 0.055 22 1.8 0.19 1.0 0.42 0.36 0.17 0.41 0.66 0.80 0.70 0.11 0.035 6.7 1.2 0.093 0.13 0.68 0.062 0.024 n.d. 2.2 0.93 0.93 32 3 16

0.10 1.9 0.81 0.25 1.2 0.34 0.14 0.75 1.5 0.67 0.011 0.042 7.7 0.53 0.056 0.34 0.15 0.13 0.054 0.15 0.23 0.24 0.23 0.033 0.013 2.1 0.10 0.040 0.043 0.022 0.020 0.0098 n.d. 0.24 0.032 0.032 10 2 31

52

48

53

43

40

62

a

n.d., not detected; RSD, relative standard deviation. (autumn), SN(s) ) Simpna¨ s (spring).

b

HF ) Harufja¨ rden, UM ) Umeå, HL ) Hornslandet, GB ) Ga¨ vlebukten, SN(a) ) Simpna¨ s

dominant homologues comprising 65 and 27%, respectively of the total PCNs. Ja¨rnberg et al. (39) found similar profiles (tetra- and penta-CN dominance) in surface sediments from two background locations, one in the Baltic proper and the other in Lake Storvindeln. Generally, naphthalenes with low levels of chlorination were found in the food chain base and more chlorinated homologues accumulated toward the top of the chain. The amphipod samples showed no major deviation in homologue distribution from the sediments in which they lived. This may be due to the limited capacity of these organisms to excrete or eliminate/metabolize such compounds. However, when moving up the food chain the profiles became dominated by more chlorinated homologues. This trend is especially obvious in the fourhorned sculpins where the HxCNs comprised 42% of the ΣPCNs analyzed. This may indicate less assimilation in the isopods and the fourhorned sculpins of the less chlorinated congeners, in comparison to the amphipods. The homologue profiles in flounder from the southern part of the Baltic proper differ from the fourhorned sculpin profiles in that they were dominated by tetra- and penta-CNs, accounting for approximately 30% and 60%, respectively (28). The PCN homologue composition of an air sample collected in the northern part of Sweden (18) resembles the 5008

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composition in the sediments, being dominated by the TeCNs. The relatively high vapor pressures of TeCNs (∼0.048 Pa) favor their mobility and long-range transportation in the atmosphere when compared with more chlorinated congeners (estimated vapor pressures for PeCNs through OCN are 0.0043 Pa to 0.00013 Pa) (40). These findings indicate that a major fraction of the PCNs deposited in the sediments originates from the air. The homologue composition of all samples was different to the technical mixture Halowax 1014. Congener Pattern. PCN congener patterns (TeCNs HxCNs) for the benthic food chain studied, together with those for air (18) and Halowax 1014 (37) presented in the literature, are shown in Figure 4. In general, the PCN pattern shifts from a sediment-like pattern (sediment, amphipod) to a nonsediment-like pattern (isopod, sculpin) when moving from the bottom to the top of the food chain. The differences in patterns might be a reflection of congener-specific rapid excretion, intestinal absorption, and metabolic transformations in the species studied. When comparing air and amphipod samples with sediment samples, similarities are observed for the less chlorinated congeners. However, differences in the composition of HxCNs are observed. Congeners 66/67 and 71/72 contribute more to the total HxCNs in the amphipods than in the sediments. In the air sample PCN 71/72 and 69 contribute

TABLE 3. Average PCN Levels (ng/g lw) in Isopod and Fourhorned Sculpin Samplesa type of sample locationb

HF

UM

isopod HL

SN(a)

SN(s)

HF

UM

42 33/34/37 44/47 36 28/43/45 27/29 30/32 35/39 38/40/48 46 31 41 TeCNs 52/60 58 61 50 51 54 57 62 53/55 59 49 56 PeCNs 66/67 64/68 69 71/72 63 65 70 HxCNs 73/74 HpCNs ΣPCNs no. of samples RSD (%) lipid content (%)

0.16 1.5 0.42 0.32 0.77 1.0 0.089 0.79 1.4 0.59 0.0016 0.025 7.1 1.5 0.26 2.7 0.47 0.34 0.49 0.27 0.59 0.26 0.20 0.032 0.024 7.2 0.98 0.18 0.19 0.11 0.073 0.010 n.d. 1.6 0.10 0.10 16 5 30 11

0.037 0.693 0.276 0.198 0.516 0.318 0.055 0.495 0.920 0.304 0.016 0.085 3.9 0.18 0.18 0.34 0.20 0.18 0.16 0.49 0.59 0.45 0.42 0.018 0.005 3.2 0.040 0.11 0.30 0.42 0.12 0.15 n.d. 1.1 0.19 0.19 8.5 1 14

0.014 0.19 0.058 0.049 0.086 0.11 0.024 0.11 0.18 0.059 n.d. 0.0063 0.88 0.26 0.035 0.63 0.14 0.10 0.13 0.073 0.11 0.049 0.025 n.d. n.d. 1.5 0.67 0.082 0.075 0.27 0.025 0.0040 n.d. 1.1 0.39 0.39 3.9 2 20 20

0.34 0.98 0.22 0.21 0.28 0.19 0.14 0.21 0.36 0.15 n.d. 0.0093 3.1 0.77 0.15 1.9 0.14 0.20 0.35 0.090 0.18 0.057 0.043 0.010 0.013 3.9 0.34 0.031 0.047 0.015 0.012 0.0007 n.d. 0.45 0.0029 0.0029 7.5 5 12 13

0.29 1.3 0.35 0.23 0.57 0.26 0.081 0.49 0.69 0.30 n.d. 0.011 4.5 0.85 0.18 1.9 0.18 0.23 0.29 0.12 0.28 0.12 0.088 0.013 0.014 4.3 0.33 0.042 0.066 0.032 0.023 0.0076 n.d. 0.50 n.d. n.d. 9.3 5 19 14

0.0019 0.036 0.020 0.0038 0.042 0.014 0.0058 0.040 0.056 0.024 n.d. 0.0020 0.25 0.019 0.012 0.035 0.0046 0.0025 0.036 0.0062 0.0077 0.0068 0.010 0.0015 n.d. 0.14 0.017 0.0065 0.056 0.055 0.0019 0.0019 n.d. 0.14 0.014 0.014 0.54 5 13 18

0.016 0.057 0.069 0.006 0.092 0.031 0.023 0.14 0.086 0.046 n.d. 0.001 0.57 0.026 0.017 0.046 0.006 0.006 0.074 0.022 0.021 0.024 0.049 0.006 n.d. 0.30 0.026 0.012 0.20 0.137 0.003 0.002 n.d. 0.38 0.049 0.05 1.3 1 18

a n.d., not detected; RSD, relative standard deviation. (autumn), SN(s) ) Simpna¨ s (spring).

b

fourhorned sculpin HL GB 0.0012 0.050 0.041 0.0053 0.044 0.021 0.014 0.059 0.047 0.025 n.d. 0.0015 0.31 0.025 0.0071 0.047 0.0035 0.0053 0.074 0.011 0.017 0.010 0.025 0.0021 n.d. 0.23 0.018 0.018 0.12 0.11 0.0035 0.0053 n.d. 0.27 0.10 0.10 0.90 1 35

0.021 0.14 0.055 0.0053 0.030 0.023 0.013 0.053 0.12 0.034 n.d. 0.0029 0.49 0.057 0.017 0.072 0.010 0.0082 0.024 0.042 0.047 0.039 0.056 0.0046 0.0014 0.38 0.059 0.026 0.19 0.18 0.0082 0.020 n.d. 0.49 0.083 0.083 1.4 2 28 26

SN(a)

SN(s)

0.0029 0.043 0.025 0.0029 0.025 0.0054 0.010 0.033 0.027 0.016 0.0008 0.0007 0.19 0.32 0.0098 0.055 0.0036 0.0029 0.045 0.0060 0.010 0.0083 0.012 0.0015 n.d. 0.48 0.36 0.019 0.11 0.027 n.d. n.d. n.d. 0.52 0.043 0.043 1.2 2 34 33

0.0028 0.041 0.026 0.0041 0.030 0.0093 0.0078 0.035 0.042 0.023 0.0007 n.d. 0.22 0.18 0.0066 0.057 0.0039 0.0084 0.036 0.0099 0.016 0.012 0.014 0.0013 n.d. 0.35 0.63 0.019 0.13 0.042 n.d. n.d. n.d. 0.82 0.085 0.085 1.5 3 32 20

HF ) Harufja¨ rden, UM ) Umeå, HL ) Hornslandet, GB ) Ga¨ vlebukten, SN(a) ) Simpna¨ s

FIGURE 3. PCN homologue composition in surface sediment, amphipod, isopod, fourhorned sculpin, Halowax 1014 (37), air (18), flounder (28), and human adipose tissue (45). Levels of HpCNs were not detected in human adipose tissue and air samples. much more to the total HxCNs than PCN 66/67, which dominate the HxCN patterns in the sediment and amphipod samples.

The HxCN pattern in the isopods resembles that in the surface sediments and the amphipods in contrast to the PeCN pattern, which is quite different between these samples. VOL. 36, NO. 23, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Tetra-, penta-, and hexa-CN congener patterns (% of total PCN homologue) found in the benthic food chain samples together with an air sample (18) and a Halowax 1014 mixture (37). Interestingly, in the isopod samples the PeCN congener 61 is more abundant than 52/60. The opposite is observed in the sediments and the amphipods. Furthermore, the relative 5010

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concentration of PCN 54 (1,2,3,6,7-PeCN) increases in the higher trophic levels. This increase suggests that PCN 54 might be more assimilated than other penta-chlorinated

TABLE 4. Calculated Average BSAF and BMF Values for PCNs in the Benthic Food Chainb PCN

BSAF (Camp/Csed)

BMF (Ciso/Camp)

BMF (Cscu/Ciso)

BMF (Cscu/Camp)

42 33/34/37 44/47 36 28/43/45 27/29 30/32 35/39 38/40/48 46 31 41 TeCNs 52/60 58 61 50 51 54 57 62 53/55 59 49 56 PeCNs 66/67 64/68 69 71/72 63 65 70 HxCNs 73/74 HpCNs ΣPCNs

4.1 2.2 2.8 1.7 3.5 2.6 3.2 3.6 2.9 4.0 2.3 1.4 2.9 1.6 1.6 1.4 1.4 1.3 0.94 1.0 1.3 1.6 1.9 1.8 0.87 1.4 1.0 0.74 0.76 1.4 0.69 1.1 a 0.88 0.90 0.90 2.0

1.3 0.34 0.21 0.48 0.21 0.33 0.30 0.28 0.22 0.19 0.047 0.26 0.27 0.69 2.3 2.1 0.61 0.81 2.3 0.72 0.76 0.44 0.35 0.19 0.41 0.92 1.3 0.73 1.2 2.1 1.0 1.6 a 1.3 0.69 0.69 0.45

0.11 0.090 0.24 0.037 0.18 0.072 0.25 0.22 0.11 0.16 a 0.082 0.13 0.18 0.089 0.057 0.023 0.028 0.28 0.073 0.065 0.11 0.33 0.12 a 0.092 0.73 0.29 1.4 0.88 0.039 0.31 a 0.69 0.22 0.22 0.15

0.15 0.027 0.036 0.012 0.022 0.021 0.056 0.048 0.022 0.024 0.014 0.023 0.040 0.12 0.17 0.087 0.017 0.025 0.45 0.059 0.049 0.045 0.069 0.046 0.010 0.10 1.1 0.19 1.4 0.96 0.049 0.17 a 0.65 0.23 0.23 0.18

aMissing data. b sed ) sediment; amp ) amphipod; iso ) isopod; scu ) fourhorned sculpin.

isomers in these organisms. The HxCN congeners 66/67 are dominant in the isopods and fourhorned sculpins (from SN) accounting for 50 and 74%, respectively, of the total HxCNs. It was possible to distinguish between two types of PCN patterns obtained in the fourhorned sculpins, one type from SN and the other from the remaining locations (Figure 4, top). The PCN pattern in the sculpins from SN is dominated by a few PCN congeners (PCN 52/60, 61, 54, 66/67, and 69) demonstrating a more congener-specific assimilation of PCNs compared to the other sculpins from the northern part of the Gulf of Bothnia. These sculpins may be suffering from stress due to the cold climate and the low salinity (3-6‰)

in the brackish waters of the north. It has been shown that PCN congeners without adjacent carbon atoms either on one or both of the aromatic rings unsubstituted with chlorine, bioaccumulate to a greater extent than other congeners (9, 41). Congeners 52, 60, 61, 66, and 67 fulfill this structure criterion. The TeCN patterns were similar in all samples, indicating nonisomer specific elimination of the TeCNs in the food chain. The PeCN and HxCN patterns but not the TeCN pattern of a Halowax 1014 mixture were quite different than the PCN patterns in the sediments and organisms investigated. Halowax 1014 does not seem to be responsible for the PCN pollution in the region studied. Biota to Sediment Accumulation Factors. The uptake of tetra- to hepta-CNs from sediments to amphipods was studied using biota to sediment accumulation factors (BSAF is calculated as the concentration in tissue, based on lipid weight, divided by the concentration in sediment based on the weight of organic carbon present, BSAF ) Camphipod, lw/ Csediment, org C). The results of these calculations, are shown in Table 4. The calculated average BSAF values, in decreasing order, were as follows: TeCNs 2.9 (1.4-4.1) > PeCNs 1.4 (0.87-1.9) > HxCNs 0.88 (0.69-1.4), and HpCNs 0.90. The BSAF values for the isomers within each homologue group were similar (narrow intervals) and in general the levels of BSAF were higher for less chlorinated PCN congeners than for more chlorinated congeners. The BSAF values reflect the total uptake of PCNs in the amphipods; this can take place in the gut, through the skin, and via the gills. The transport medium for all the uptake routes is water. PCN congeners that are more soluble in water (TeCNs between 4.0 and 8.3 µg/dm3; OCN 0.08 µg/dm3) (40) are therefore more readily available and seem to be associated with higher levels of accumulation. BSAFs for PCNs were in the range of 0.0012-0.00007 near a former chlor-alkali plant in Georgia U.S.A. (4). These figures are 3-4 orders of magnitude lower than the BSAFs determined in our study. This notably difference may be attributed to the habits of the species investigated. The amphipods studied here inhabit sediments and feed directly on them, unlike the Georgian species (blue crab, striped mullet, and boat tailed grackle). Biomagnification. The compositional patterns of PCNs for the species examined indicate that fourhorned sculpins and isopods are able to selectively excrete and/or eliminate/ metabolize many TeCNs and PeCNs. Excretion and/or elimination/metabolism of the more chlorinated HxCNs and HpCNs appears to be somewhat limited. Calculated BMFs for the predators (fourhorned sculpins, isopods), in relation to the their food are listed in Table 4. In general, low level BMFs (BMF ) Cfourhorned sculpin, lw/ Camphipod, lw) were found in the benthic food chain studied, again indicating that many congeners are excreted and/or eliminated/metabolized at the higher trophic levels. The

FIGURE 5. Average BMFs for tetra- to hepta-CN congeners in the benthic food chain studied. Fourhorned sculpins are related to amphipods. VOL. 36, NO. 23, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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average BMFs for PCN homologues were in the range of 0.040-0.65 (Table 4). HxCNs accumulate more than the other homologues. Five HxCN isomers (PCN 66/67, 69, and 71/72) dominate the BMF pattern (Figure 5). The BMF pattern obtained previously for a benthic food chain (flounder in relation to mussel) (28) resembled the pattern described here. Among all the PCN congeners analyzed, only PCN 66/67 (BMF ) 1.1) and PCN 69 (BMF ) 1.4) biomagnified. In the floundermussel study (28), the BMFs reported for the HxCNs was 1 order of magnitude higher for the HxCNs (BMFHxCNs, max ) Cflounder/Cmussel ) 16). In our study as well as in the floundermussel study the lowest BMF values among the HxCN isomers were observed for PCN 63 and 65. These isomers are the only HxCNs having two adjacent carbon atoms substituted with hydrogen. On the basis of BMF values, PeCN isomers (BMFs ) 0.0100.45) can be divided into three categories. One PeCN isomer, PCN 54, has the highest BMF value (BMF ) 0.45). This is the only 2,3,6,7-chlorine substituted PeCN isomer and is structurally similar to the 2,3,7,8-TCDD. PCN 52, 60, 58, and 61 are PeCN isomers with intermediate BMF values (BMFs ) 0.087-0.17). This group of PCN congeners lacks adjacent carbon atoms unsubstituted with chlorine. Low BMF values were found for the remaining PeCN isomers (BMFs ) 0.0100.069); these have adjacent carbon atoms substituted with hydrogen. PCN 55 belongs to the intermediate group but coelutes with PCN 53 on a nonpolar DB-5 type capillary GC column. The low BMF value may indicate that the eluting peak mainly consists of PCN 53. In addition, separation of these two isomers on an Rt-βDEXcst capillary GC column has shown that PCN 55 is not present in Halowax 1014 (42). The BMF values for most of the TeCNs were low (BMF e 0.056). Only PCN 42 has a relatively high BMF (0.15). PCN 44, 45, and 48 (three other isomers which lack adjacent carbon atoms substituted with hydrogen) unfortunately coelute with other TeCN isomers on the DB-5 type column making it impossible, here, to evaluate the biomagnification of these isomers. There is a need to separate all the PCN congeners on a single GC column or on a dual GC column system for this type of investigation. PCNs lacking, or with only a few chlorine atoms, are reportedly metabolized via arene oxides and, it has been suggested, form both hydroxylated and mercapturic acid pathway metabolites. Metabolism via arene oxides may be the reason PCN members with adjacent carbon atoms substituted with hydrogen are associated with the lowest BMF values (43). The PCN congeners with the highest BMFs (e.g. many of the 2,3,6,7-chlorine substituted PCNs) in our study were also the most potent in previous toxicological studies (21, 23, 24, 44). Unfortunately only 22 of all the 75 PCN congeners have been tested for dioxin-like toxicity. Quantitative structure-activity relationships (QSARs) have been established for the modeling of BMFs for the PCNs (29). In the QSAR study, several of the 16 PCN congeners with the highest biomagnification potentials were assigned to 2,3,6,7-substituted PCN congeners (PCN 48, 66, 67, 70, and 73).

Acknowledgments The financial support from the Kempe Foundation, the Swedish Forest Industries, the Swedish EPA, the Finnish National Board of Water and the Environment and the Boliden Mineral AB are gratefully acknowledged. Thanks must go to Orania Papakosta and Harald Pettersen (Institute of Applied Environmental Research, ITM, Stockholm University) and Bo Strandberg (Environmental Chemistry, Department of Chemistry, Umeå University) for their technical assistance. We also thank Eva Jakobsson (Stockholm University) for supplying the pure HxCN and HpCN standards. 5012

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Received for review June 12, 2002. Revised manuscript received September 25, 2002. Accepted October 1, 2002. ES0201146

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