Occurrence of brominated flame retardants, polycyclic musks, and chlorinated naphthalenes in seal blubber from Antarctica: Comparison to organochlorines Alessandra Schiavone a, * , Kurunthachalam Kannan b,c , Yuichi Horii b,c , Silvano Focardi a , Simonetta Corsolini a a Department of Environmental Science ‘‘G. Sarfatti”, University of Siena, via P.A. Mattioli, 4, I-53100 Siena, Italy b Wadsworth Center, New York State Department of Health, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, USA c Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, USA In the last few years there has been increasing interest in nam- ing additional toxic substances to the list of ‘‘dirty dozen” persis- tent organic pollutants (POPs) identified by the United Nations Environment Programme (UNEP). Other compounds that have the characteristics of POPs have been identified. These include bro- minated flame retardants such as polybrominated diphenyl ethers (PBDEs), perfluorooctanesulfonate (PFOS) and polychlorinated naphthalenes (PCNs). Although these ‘new’ compounds have been in use for several decades, they are referred to as emerging contaminants. Polychlorinated naphthalenes (PCNs) have physico-chemical properties largely similar to those of PCBs and have been used in similar industrial applications as dielectric fluids, engine oil addi- tives, cable insulators, lubricants, and heat exchange fluids (Falan- dysz, 1998). PCNs are produced as complex technical mixtures (such as Halowaxes and Nibren waxes) and have also been found in PCB formulations (Yamashita et al., 2000) and combustion-re- lated emissions such as those of waste incinerators (Falandysz, 1998). A number of PCN congeners and Halowax formulations have been shown to induce drug-metabolizing enzymes (Blankenship et al., 2000; Villeneuve et al., 2000). PCNs have been detected in air, water, sediments, soils and biota (Corsolini et al., 2002; Helm et al., 2008; Ishaq et al., 2003; Kannan et al., 2002). Very little is known regarding the presence of PCNs in Antarctica. Another class of contaminants of interest is polycyclic musks (Kannan et al., 2005; Reiner et al., 2007). Synthetic musks are man- ufactured and used in a wide variety of personal care products, such as perfumes, skin cream, deodorants, soaps and detergents. Among the various classes of synthetic fragrances, polycyclic musks such as Galaxolide (HHCB) and Tonalide (AHTN), have been shown to elicit antiestrogenic effects in various bioassays (Schreurs et al., 2004). Environmental exposure and potential toxic effects of polycyclic musks are a cause for concern. Synthetic fragrances, including the polycyclic musks, have been found in air, sediment, crustaceans, mussels, and fishes from freshwater and marine envi- ronments (Peck and Hornbuckle, 2004; Fromme et al., 2001; Nak- ata et al., 2007). Many of these novel chemicals can be found in remote regions of the world, including the polar regions, due to their propensity to get disbursed by atmospheric or hydrospheric transport. Marine mammals accumulate elevated levels of bioaccumulating contam- inants, and therefore are valuable in biomonitoring studies. In this study, blubber of seals from Antarctica was analyzed to determine concentrations of PCBs, OC pesticides, PBDEs, PCNs and polycyclic musks. Current concentrations of emerging pollutants in seal blub- ber from Antarctica are reported for the first time. Four blubber of Antarctic fur seal (Arctocephalus gazella) pups were collected between January and February 2004, at Livingston Island, South Shetland, Antarctic Peninsula, (62°39 0 S, 60°30 0 W) (Fig. 1). Samples of Antarctic fur seal pups were taken from the car- casses found dead during a season (2004/2005) of particularly high seal neonatal mortality. Sampling location, liver weight, gender, body weight and body length of seals were recorded. All tissue samples were kept in polyethylene bags and immediately frozen at 60 °C until analysis. Age of the subjects, on average, was 1 month for seal pups (range: stillborn-2 months) (Table 1). Lipid content of the blubber samples ranged from 2% to 67% (mean: 49%). OC pesticides, PCBs, PBDEs, PCNs and polycyclic musks were analyzed following the methods described elsewhere (Kannan et al., 2000, 2005; Loganathan et al., 1995; Johnson-Restrepo et al., 2005; Reiner and Kannan, 2006), with some modifications. Approximately 4 g of the tissue samples were homogenized with anhydrous sodium sulphate and extracted in a Soxhlet apparatus for 16 h using dichloromethane and hexane (3:1; 400 mL). Before Soxhlet extraction, samples were spiked with deuterated 7-acet- yl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (d 3 - AHTN) (Dr. Ehrenstorfer GmbH; Augsburg, Germany). The extract was rotary-evaporated at 40 °C to 5 mL, and an aliquot (0.5 mL) was used for the determination of fat content by gravimetry. The remaining extract was spiked with 13 C-labeled PCB congeners 28, 52, 101, 153, 138, 180, 209 and 13 C-labeled PBDE congeners 28, 47, 100, 99, 154, 153, 183 as internal standards. Lipids were re- moved from the samples by gel permeation chromatography (GPC) using a Bio-beads S-X3 (Bio-Rad Laboratories, Hercules, CA, USA) packed glass column (380 mm  22 mm i.d.). The extract was passed through a silica gel packed cartridge (100–200 mesh; Aldrich, Milwaukee, WI, USA) for cleanup. The solvent was concen- trated to 1 mL and 100 ll was reserved for musk compounds anal- ysis. The remain 900 ll were then purified by passage through a series of layers of silica gel (Davisil, 100–200 mesh, Aldrich, Mil- waukee, WI). Samples purified were rotary-evaporated to 1 mL for PCBs, PBDEs and OCs analysis. The remaining samples were passed through a glass column (10 mm i.d.) packed with 1 g of sil- ica gel impregnated activated carbon (Wako Pure Chemical Indus- tries, Osaka, Japan) for the separation of PCBs from PCNs. A procedural blank was included every ten samples and when needed, results were blank corrected. PCBs, PBDEs and musk compounds were identified and quanti- fied using a gas chromatograph interfaced with a mass spectrome- ter (GC–MSD, Agilent Technologies 6890 GC and 5973 Series MSD). The GC column was a Restek (Bellefonte, PA, USA) Rxi-5 ms fused silica capillary column (30 m  0.25 mm i.d.). Concentrations of HHCB, AHTN, and HHCB–lactone were determined using an exter- nal calibration curve. PCB and PBDE congeners were monitored at the two most intense ions of the molecular ion cluster. An equiva- lent mixture of Kanechlor (KC 300, 400, 500, and 600) with known PCB composition was used in the identification of PCB congeners. Quantification of PCB congeners was based on external calibration standards containing known concentrations of di- through nona- CB congeners. Concentrations of individually resolved peaks of PCB isomers were summed to obtain total PCB concentrations. Total PCBs represent the sum of all di- through nona-chlorobiphenyl congeners. Nine major PBDE congeners 28, 47, 66, 85, 99, 100, 153, 154 and 138 were quantified in this study. Total PBDE concentrations represent * Corresponding author. Tel.: +39 577 232 882; fax: +39 577 232 2806. E-mail address: schiavone4@unisi.it (A. Schiavone). Baseline / Marine Pollution Bulletin 58 (2009) 1406–1419 1415