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Environ. Sci. Technol. 2004, 38, 3505-3513

Toxicokinetics of PCDD, PCDF, and Coplanar PCB Congeners in Baikal Seals, Pusa sibirica: Age-Related Accumulation, Maternal Transfer, and Hepatic Sequestration H I S A T O I W A T A , * ,† M A F U M I W A T A N A B E , † YUKA OKAJIMA,† SHINSUKE TANABE,† MASAO AMANO,‡ NOBUYUKI MIYAZAKI,‡ AND EVGENY A. PETROV§ Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan, International Coastal Research Center, Ocean Research Institute, The University of Tokyo, 2-106-1 Akahama, Otsuchi, Iwate 028-1102, Japan, and Limnological Institute of the Siberian Division of the Academy of Science of Russia, 664033 Irkutsk, Uran-Batorskaya 3, Russia

To assess the toxicokinetic behavior and potential toxicity of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (PCBs) in Baikal seals, congener-specific levels and tissue distribution were evaluated in the liver and blubber, and the effects of biological factors including sex and growth were assessed. Total 2,3,7,8-TCDD toxic equivalents (TEQs) were in the range of 210-920 pgTEQ/g fat wt (180-800 pgTEQ/g wet wt) in the blubber and 2907800 pgTEQ/g fat wt (10-570 pgTEQ/wet wt) in the liver. Nonortho coplanar PCB126 was the most TEQ-contributed congener accounting for 37-59% of the total TEQs in the liver. From the unique congener profiles, weak metabolic properties of Baikal seals for 2,3,7,8-TCDF and 1,2,3,7,8-P5CDF are suggested. Concentrations of most congeners linearly increased with age in male seals, whereas in adult females the levels revealed an age-related decline. The increasing and declining rates were congener-specific. Maternal transfer rates of 5 representative congeners from adult female to pup through lactation, which was estimated from male-female differences in the body burden, was 1.1 ngTEQ/kg/day for the first pup and decreased with every lactational epoch. The liver-blubber distribution of 1,2,3,4,7,8H6CDD, 1,2,3,6,7,8-H6CDD, PCB81, PCB126, and PCB169 was dependent on the hepatic total TEQ, indicating hepatic sequestration by induced cytochrome P450 (CYP). These results indicate that congener profile in Baikal seals is governed by complex factors including sex, tissue concentration, binding to CYP, and rates of absorption and metabolism/excretion.

* Corresponding author phone/fax: +81-89-927-8171; e-mail: [email protected]. † Ehime University. ‡ The University of Tokyo. § Limnological Institute of the Siberian Division of the Academy of Science of Russia. 10.1021/es035461+ CCC: $27.50 Published on Web 05/22/2004

 2004 American Chemical Society

Introduction Lake Baikal is exposed to a considerable influx of anthropogenic pollutants. During the past decade, high levels of persistent contaminants such as polychlorinated biphenyls (PCBs) and DDTs have been measured in the lake ecosystem (1, 2). Baikal seals (Pusa sibirica), an endemic species, are particularly vulnerable to the exposure to persistent contaminants due to their high biomagnification through the food chain (2, 3). In 1987-1988, an outbreak of the morbillivirus infection resulted in mass mortality of Baikal seals. Immunosuppression elicited by chronic exposure to environmental contaminants has been speculated as a contributing factor for this epizootic, although the direct cause for this outbreak was the infectious disease (4). The chemical analyses of Baikal seal tissues demonstrated high contamination by PCBs and DDTs (2, 3). Highly toxic planar halogenated aromatic hydrocarbons (PHAHs) such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and coplanar PCBs are ubiquitous environmental contaminants. Due to the efficient transfer of PHAHs in the food web, lipophilic PHAHs are highly accumulated in a variety of aquatic species. PHAHs elicit a wide range of toxic and biological effects including reproductive failure, immune deficiency, and teratogenesis in certain laboratory animals (5, 6). Mechanisms through which aquatic mammal species exhibit these effects are unclear but are likely to involve the aryl hydrocarbon receptor (AHR) signaling pathway including cytochrome P450 (CYP) 1A and 1B induction (7, 8). In recent studies, full-length AHR sequences have been isolated from two aquatic mammals, the beluga (Delphinapterus leucas) and the harbor seal (Phoca vitulina) (7, 8). The dioxin-binding affinity of these AHRs was at least as high as that of the AHR from a dioxin-sensitive (C57/BL) strain of mice, suggesting that these aquatic species may be sensitive to PHAH effects. At present, our group has cloned and sequenced the distinct full-length cDNA of AHR from Baikal seals (9). Comparison of AHR amino acid sequences indicated a high degree of sequence conservation (98%) between Baikal and harbor seals. The high conservation of AHRs shows that AHR proteins of these seals are structurally closely related, suggesting that Baikal seals may also be sensitive to PHAH exposure as the harbor seal. This speculation raises concerns about the toxic effects of PHAHs on the health, hormonal circulation, and reproductive performance (10-13) of Baikal seals. However, the magnitude of the risk that PHAHs may pose to the health of Baikal seals is still uncertain, because only very little information is available on the residue levels of PHAHs in Baikal seals (14, 15). Toxicokinetics is also another factor that contributes to in vivo toxic potency of individual congeners in a given species. Available data indicate that there are marked species differences in the toxicokinetics of these compounds. Seal and other wildlife populations are exposed to a complex mixture of PHAHs. Thus, risk assessment of PHAHs on wildlife should also consider toxicokinetic data of individual congeners, which is influenced by various factors such as sex, age, tissue, and dose (16). Nevertheless, most of the toxicokinetic data available have been established with a single or repeated dose(s) of a particular congener via various administration routes in short-time experiments. Validity of such experimental designs is still unproven, and therefore the data obtained from these studies, which are controversial in some cases, may lead to false evaluation of toxicokinetic VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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properties of PHAH congeners in the wild populations, thus making it difficult to assess their toxicity with high accuracy. The objective of this study is to evaluate the levels of PHAHs in Baikal seals and to understand the toxicokinetics of individual PHAH congeners in which factors including sex, age, and tissue-distribution are involved. In addition, we also assessed the potential risk of offsprings exposed to PHAHs through maternal transfer.

Materials and Methods Sample Collection. The details of Baikal seal samples were reported previously (3). The same specimens examined for other organochlorines in ref 3 were used for the PHAH analysis in this study. Seals were collected from Lake Baikal in May-June in 1992 and immediately dissected on a board after measurements of biometry (body length, body weight, etc.). Livers from 13 male and 15 female seals were analyzed for age trend and sex difference of PHAH accumulation. Seven blubber tissues from male seals were also analyzed, and the data were used for understanding the age trend and tissue distribution in combination with data from livers. Total liver and blubber weights were measured in most of the specimens. Sampled animals appeared to be “normal”. There were only small variations in blubber thickness (male: 4.4 ( 0.46 cm; female: 4.8 ( 1.1 cm), and good correlations between body length and body weight (male: r2 ) 0.96; female: r2 ) 0.89). Ages of animals were determined from dentinal and cemental growth layers in a canine tooth. Subsamples of the tissues were stored in a freezer at -20 °C for chemical analysis. Chemical Analysis. Six grams of blubber and 10-20 g of liver was ground individually and extracted in a Soxhlet apparatus for more than 8 h with dichloromethane (DCM). The extract was concentrated, and an aliquot was used for lipid determination. 13C12-labeled PCDDs/DFs and coplanar PCBs (2,3,7,8-T4CDD/DF, 1,2,3,7,8-P5CDD/DF, 1,2,3,6,7,8H6CDD/DF, 1,2,3,7,8,9-H6CDF, 1,2,3,4,6,7,8-H7CDD/DF, OCDD/DF, PCB-77, -81, -118, -126, -156, -167, -169, and -189) were spiked into the remaining extract as internal standards. Lipid and biogenic materials in this solution were removed by gel permeation chromatography using a Bio-Bead S-X 3 packed glass column (50 cm × 2 cm i.d., Bio-Rad Laboratories). A mixture of 50% hexane in DCM was used as mobile phase at a flow rate of 5 mL/min. The first 120 mL of eluted solvent was discarded, and the following 100 mL fraction, which contained PCDDs/DFs and coplanar PCBs, was collected, concentrated, and passed through a 3 g activated silica gel packed glass column (Wako-gel S-1, Wako Pure Chemical Industries Ltd.). PCDDs/DFs and coplanar PCBs were eluted with 220 mL of hexane. After concentration, the extract was spiked into a 10 g activated alumina packed glass column (aluminum oxide 90, Brockman activity I, 70-230 mesh, Merck). The first fraction eluted with hexane contained most of the PCB isomers including most of the mono-ortho coplanar congeners, and the second fraction eluted with 50% of DCM in hexane contained the remaining mono-ortho coplanar PCBs, non-ortho coplanar PCBs, and PCDDs/DFs. The second fraction eluted from the activated alumina column was then passed through a 1 g activated carbondispersed silica gel packed glass column (1 cm i.d., Kanto Chemical Co. Inc). The first fraction was collected with 25% of DCM in hexane for mono-ortho coplanar PCBs and combined with the first fraction obtained from the alumina column. The second fraction eluted with 220 mL of toluene contained non-ortho coplanar PCBs and PCDDs/DFs. Both fractions were concentrated near to dryness, and 13C12-labeled CB-105, CB-157, and CB-180 prepared in decane were added to the combined first fraction, and 13C12-labeled 1,2,3,4-T4CDD and 1,2,3,7,8,9-H6CDD to the second fraction. The identification and quantification of mono-ortho coplanar PCBs were performed using a gas chromatograph (GC) 3506

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(Agilent 6890 series, Agilent Technology) with a benchtop double-focusing mass selective detector (MSD) (JEOL GCMate II, JEOL Ltd.). The GC column used was a DB-1 fused silica capillary (60 m length, 0.25 mm i.d., 0.25 µm film thickness, J&W Scientific Inc.). The identification and quantification of non-ortho coplanar PCBs and PCDDs/DFs were performed using a GC (Agilent 6890 series) with a highresolution MSD (JEOL JMS-700D). Separation was accomplished by a DB-5ms fused silica capillary column (60 m length, 0.25 mm i.d., 0.25 µm film thickness, J &W Scientific Inc.) for non-ortho coplanar PCBs and H7-O8CDDs/DFs and by a capillary column coated with CP-Sil 88 for dioxins (60 m length, 0.25 mm i.d., 0.1 µm film thickness, Varian Inc.) for T4-H6CDDs/DFs. Individual congeners were monitored by a selective ion monitoring mode where the two most intensive ions of the molecular ion cluster were scanned, except for P5CDDs at ions of [M]+ and [M + 2]+. All the congeners were quantified using the isotope dilution method to the corresponding 13C12-congeners, if the isotope ratio was within 15% of the theoretical ratio, and the peak area was more than 5 times the noise or procedure blank level. The detection limit for individual congeners was 0.05-1.0 pg/g for the lipid weight basis for PCDDs/DFs and non-ortho coplanar PCBs and 10 pg/g for mono-ortho coplanar PCBs (Table 1). Quality Control. For every five samples, a laboratory blank was incorporated in the analytical procedure. Recoveries for the 13C12-labeled PCDDs/DFs and coplanar PCBs, which were added prior to GPC column cleanup, were within 40-120%. The concentrations were corrected based upon the recoveries of 13C12-labeled compounds. Toxic Equivalents. The 2,3,7,8-TCDD toxic equivalents (TEQs) were calculated from mammalian toxic equivalency factors (TEFs) and concentrations of individual PHAH congeners (17). Statistical Analysis. Statistical analyses were performed using StatView (Ver. 4.5, Abacus Concepts, Inc.). Regression analysis was carried out to examine the relationships between age and PHAH concentrations and the relationships of PHAH burdens between blubber and liver. Correlations between hepatic total TEQs and liver/blubber distribution ratios of PHAH congeners were examined by Spearman’s rank correlation test. P values below 0.05 were regarded as significant. Concentrations below the detection limit were treated as half of the detection limit reported for respective congeners.

Results and Discussion Concentrations, TEQs, and Their Toxicological Significance. Total concentrations of PCDDs/DFs and coplanar PCBs showed a range of 440-1400 ng/g fat wt (370-1200 ng/g wet wt) and 500-5500 ng/g fat wt (16-400 ng/g wet wt) in blubber and liver, respectively (Table 1). In both tissues, concentrations of total mono-ortho coplanar PCBs were the most dominant followed by total non-ortho PCBs. Concentrations of total PCDFs and PCDDs were much lower than total coplanar PCB concentrations. Congener-specific analysis of PCDDs and PCDFs showed that 1,2,3,7,8-P5CDD and 1,2,3,7,8P5CDF were higher than the highly chlorinated congeners including H7CDD, O8CDD, H6CDF, H7CDF, and O8CDF. Among mono-ortho coplanar PCB congeners, the residue level of PCB118 was the highest, and both PCB123 and PCB189 were less accumulated congeners. As for non-ortho coplanar congeners, PCB126 was the most abundant, whereas PCB169 was the least. Comparison of concentrations in the livers of male and female seals clearly revealed that males accumulated more of most congeners than females. The sex-difference in PHAH levels was more pronounced in adult animals than in the young, suggesting maternal transfer to pups. TEQ levels in the blubber and liver were in the range of 210-920 pgTEQ/g fat wt (180-800 pgTEQ/g wet wt) and

TABLE 1. Concentrations (pg/g fat wt) of PCDDs/DFs and Coplanar PCBs in the Blubber and Liver of Baikal Seals blubber male (n ) 7) congener fat (%)

mean 89

SD 3.9

min 84

liver male (n ) 13) max 95

mean

SD

5.3

liver female (n ) 23)

min

max

1.6

3.3

96 970 170 400 110 6.5 5.9 1700

15 36 5.9 9.4 2.9 3.6 4.0 77

68 490 330 12 6.2 1.9 1.4 0.91 0.80 0.77 890 2600

53 110 68 0.9 1.7