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Analysis and characterization of polychlorinated hydroxybornanes as metabolites of toxaphene using polar bear model Lea Reger, Christoph Gallistl, Karl Skirnisson, and Walter Vetter Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02662 • Publication Date (Web): 07 Jul 2017 Downloaded from http://pubs.acs.org on July 7, 2017

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Environmental Science & Technology

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Analysis and characterization of polychlorinated hydroxybornanes as metabolites of

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toxaphene using polar bear model

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Lea Reger1, Christoph Gallistl1, Karl Skírnisson2, Walter Vetter1*

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1

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Germany

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2

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Iceland

Institute of Food Chemistry, University of Hohenheim, Garbenstraße 28, D-70599 Stuttgart,

University of Iceland, Keldur, Institute for Experimental Pathology, IS-112 Reykjavík,

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* Corresponding author

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Walter Vetter

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Phone: +49 711 459 24916

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Fax: + 49 711 459 24377

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Email: [email protected]

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ACS Paragon Plus Environment


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Abstract: Abiotic and biotic transformation of toxaphene (camphechlor) results in the

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selective enrichment of recalcitrant congeners while other, less persistent compounds of

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technical toxaphene (CTTs) are degraded. Up to now there is little knowledge on oxidation

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transformation of toxaphene. For instance, existence of hydroxylated CTTs (OH-CTTs) in

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authentic environmental and food samples was not proven yet. For this reason, we synthesized

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a mixture consisting of tetra- to heptachlorinated OH-CTTs and simplified it by

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countercurrent chromatography (CCC). Thereby, 227 OH-CTTs were detected in the CCC

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fractions (12 tetra-, 117 penta-, 81 hexa- and 17 heptachlorinated OH-CTTs), which was

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>50% more than detected before the fractionation. One CCC fraction consisting of only 18

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OH-CTTs was used to develop a sample clean-up method which aimed to remove CTTs,

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isobaric PCBs and sample matrix. The final clean-up procedure consisted of (i) gel

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permeation chromatography (GPC) and adsorption chromatography using (ii) deactivated and

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(iii) activated silica gel. Hence, up to 320 and 4,350 µg/kg lipid weight of octa- and

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nonachlorinated CTTs were detected in four liver samples and adipose tissue of polar bears,

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respectively. Furthermore, presence of one hexachlorinated OH-CTT isomer could be verified

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in the samples which was about 1% of the octachlorinated CTTs determined in the liver

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samples.

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INTRODUCTION

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Since its introduction in 1945, toxaphene was one of the most heavily used non-systemic

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organochlorine pesticides worldwide with a production rate of ~1.3 million tons from 1950-

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1993.1,2 The chloropesticide was produced by the chlorination of α-pinene/camphene, which

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results in a complex mixture of ~1,000 of more than 30,000 theoretically possible

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polychlorinated bornanes which are the predominant substance class in technical toxaphene

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(>90%). In addition, low contributions of polychlorinated bornenes, bornadienes, camphenes


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and dihydrocamphenes were detected in technical toxaphene.2-5 Toxaphene was found to be

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neurotoxic, as well as carcinogenic and mutagenic in mice and rats, and showed a high acute

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and chronic toxicity, especially on aquatic organisms.2,6-9 However, it is unknown whether

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compounds of technical toxaphene (CTTs) or their metabolites (or a mixture of both) was

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responsible for the observed effects. Production and use of toxaphene were stopped in several

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countries at different dates: in 1962 (Egypt), in 1975 (China), in 1986 (USA), in 1990 (former

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East Germany) and in 1992 (Brazil).1,7 Finally, since its classification as persistent organic

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pollutant (POP) by the Stockholm Convention in 2004, toxaphene is banned in most

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countries.10 However, toxaphene residues are still detected in food samples as well as in

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environmental samples all around the world.11-13 High toxaphene levels were determined in

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marine organisms, especially in fatty fish and marine mammals. E.g. Vetter et al. reported 5-

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1,460 µg/kg wet weight of CTTs in different seal species which was a higher level than

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hexachlorocyclohexane (4-660 µg/kg) and hexachlorobenzene (0.2-230 µg/kg) detected in the

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same samples.14-16 However, the toxaphene residue pattern in these higher organisms differed

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vastly from the complexity of the technical product.2,6 One octa- and one nonachlorobornane -

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2-exo,3-endo,5-exo,6-endo,8,8,10,10-octachlorobornane (B8-141317) and 2-exo,3-endo,5-

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exo,6-endo,8,8,9,10,10-nonachlorobornane (B9-167917) - were found to be particularly

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recalcitrant in marine mammals.18,19 Transformation of the many hundred less persistent

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CTTs may occur abiotically by UV light.20,21 Comparatively little is currently known about

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the

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dechlorination,18,26 hydroxylation via the phase-I-reaction of the detoxification metabolism

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represents one plausible degradation pathway, which has already been shown for similar

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xenobiotics.3 Chandurkar and Matsumura (1979) verified the formation of five hydroxylated

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metabolites by enzymatic transformation of toxaphene in rat livers under laboratory

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conditions.22,23 Furthermore, in-vitro experiments with toxaphene-spiked microsomes of

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marine mammals, e.g. sperm whales (Physeter macrocephalus) and harbor seals (Phoca

mechanisms

of

biotic

transformation

of

toxaphene.22-25


Next

to

reductive


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vitulina) resulted in toxaphene transformation along with the tentative detection of one mono-

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and one trihydroxylated metabolite formed after incubation with NADPH.24 Further studies

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on hepatic microsomes of a harbor seal (Phoca vitulina) and a grey seal (Halichoerus grypus)

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reported enzymatic metabolism of CTT congeners into their hydroxylated derivatives.25

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However, hydroxylated CTTs (OH-CTTs) have not been detected so far in authentic

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environmental samples.27 This may be due to the lack of suitable analytical standards because

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synthesis of OH-CTTs results in a complex mixture of hundreds of compounds which has to

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be simplified by means of a time-consuming process.27 Furthermore, OH-CTTs can be easily

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misinterpreted as polychlorinated biphenyls (PCBs) in gas chromatography with mass

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spectrometry (GC/MS) chromatograms due to their isobaric character (same nominal mass,

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same retention time).27 Due to their highly developed metabolism system which is known to

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generate hydroxylated PCBs (OH-PCBs) as well as hydroxylated polybrominated biphenyls

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(OH-PBBs) and polybrominated diphenyl ethers (OH-PBDEs), polar bears (Ursus maritimus)

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were selected for analytical purposes within the presented study.28-31

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The aim of this study was to develop a method for the verification of the occurrence of

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OH-CTTs in environmental samples. For this purpose, an OH-CTT mixture consisting of

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>200 compounds was synthesized according to Kapp and Vetter,27 followed by fractionation

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with countercurrent chromatography (CCC). CCC is an all liquid based (semi-) preparative

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instrumental chromatography method typically used for the isolation of natural products32,33

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which was previously also successfully used in the field of polyhalogenated compounds.34,35

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The resulting, less complex OH-CTT mixture was used to develop a sample cleanup method

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which provided a full separation of matrix compounds as well as isobaric PCBs which show

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the same molecular ion and hence would interfere the analysis by gas chromatography with

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electron-capture negative ion mass spectrometry (GC/ECNI-MS). This method was finally

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applied to analyze adipose tissue and livers of polar bears.


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MATERIALS AND METHODS

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Chemicals and standards. For synthesis of the OH-CTT-mix, sulfuryl chloride (≥97.0%,

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Sigma Aldrich, Steinheim, Germany) and L-bornyl acetate (Merck, Darmstadt, Germany)

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were used. Furthermore, n-hexane (HPLC grade, ≥95.0% for CCC experiments; residue

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analysis grade, ≥99.0% for all other experiments), cyclohexane (≥99.5%) and ethyl acetate

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(≥99.5%, distilled prior to use) were obtained from Th. Geyer (Renningen, Germany). A 1:1

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(v/v) mixture of cyclohexane and ethyl acetate was further purified by azeotropic distillation

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(C/E mixture, 46:54, w/w). Pyridine (for HPLC, ≥99.9%, distilled prior to use), 2,2,3-

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trimethylpentane (iso-octane, for pesticide residue analysis), toluene (for pesticide residue

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analysis) and cholesterol (>99.0%) were obtained from Sigma Aldrich (Steinheim/Seelze,

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Germany). Silica gel 60 (for column chromatography, also from Sigma Aldrich) was activated

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by heating for 24 h at 130 °C. Acetonitrile (ACN, gradient grade for HPLC, ≥99.9%) and

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methanol (gradient grade for HPLC, ≥99.0%) were both purchased from VWR chemicals

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(Darmstadt, Germany) whereas n-butanol (≥99.0%) and sodium sulfate (water-free, p.a.,

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≥99.0%) were both from Carl Roth (Karlsruhe, Germany). A mixture of N,O-

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bis(trimethylsilyl)-trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) (99:1

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(v/v)) was obtained from Supelco (Bellefonte, PA, USA). Demineralized water was produced

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in-house by means of an ELGA PURELAB Classic Ultrapure water system (Celle, Germany).

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A mix of the polychlorinated biphenyl (PCB) congeners PCB 28, 52, 99, 101, 114, 118, 123,

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138, 141, 144, 149, 153, 170, 180, 187, 194, 196 and 201 (Dr. Ehrenstorfer, Augsburg,

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Germany) was prepared in iso-octane (PCB-mix, Table S1, Supporting Information).

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Individual CTT standards (B8-1413, B7-515, B9-1679, B9-1025) were from Dr. Ehrenstorfer

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(Augsburg, Germany). The internal standards perdeuterated α-hexachlorocyclohexane (α-

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PDHCH, >98%) and 6’-MeO-BDE 66 (BCIS, >99%) were synthesized in-house.36,37



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Synthesis of an OH-CTT-mix. An OH-CTT-mix was synthesized by photochlorination

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of L-bornyl acetate according to Kapp and Vetter (Figure S1, Supporting Information).27 L-

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bornyl acetate (2.0 g) was diluted in 30 mL sulfuryl chloride and irradiated with a water-

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cooled TQ150 medium pressure mercury vapor UV lamp (150 W, Heraeus/Peschl Noblelight,

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Hanau/Germany). An aliquot of 25 mL was taken after 24 h irradiation. Further clean-up

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steps, including cleavage of the acetate, were performed according to Kapp and Vetter27 and

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resulted in 1.6 g OH-CTT synthesis feedstock.

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Countercurrent chromatography (CCC). CCC fractionation was performed with an

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AECS Quikprep MK8 instrument (AECS, London, United Kingdom) using the setup of

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Hammann et al. (Figure S1, Supporting Information)40 Separations were performed with

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bobbin 1 (total column volume 238 mL). The solvent system (n-hexane, ethyl acetate, water,

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methanol, 40:10:40:10, v/v/v/v)41 was used in tail-to-head mode. The mobile phase was

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transported with a flow rate of 4 mL/min. Stationary phase retention (Sf) was 214 mL. An

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aliquot of the synthesis mixture (790 mg), dissolved in the solvent system, was injected into

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the CCC system. After 169.1 min, 70 consecutive fractions of 7.2 mL were collected.

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Fractions were evaporated to dryness and re-dissolved in 1 mL methanol.

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Gas chromatography coupled with electron-capture negative ion mass spectrometry

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(GC/ECNI-MS). The Agilent 7890A/5975C GC/MS system (Waldbronn, Germany) was

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equipped with an HP5-MS UI column (30 m x 0.25 mm internal diameter, 0.25 µm film

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thickness, J&W Scientific, Folsom, CA, USA). Sample solutions (1 µL) were injected via a

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7693A autosampler (Agilent) into a Gerstel CIS-4 PTV injector (Mülheim, Germany)

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operated in pulsed splitless mode (25 psi/min, 1 min). The injector temperature was ramped

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from 80 °C (0.01 min) at 500 °C/min to 300 °C (2 min), then cooled with 10 °C/min down to

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260 °C and held until the end of the run. GC oven program started for 1 min at 50 °C, then,

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the temperature was raised with 10 °C/min to 300 °C (4 min). The transfer line, quadrupole


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and ion source temperatures were set at 300 °C, 150 °C and 150 °C, respectively. Helium

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(purity 99.9990%, Westfalen, Münster, Germany) was used as carrier gas with a constant flow

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rate of 1.2 mL/min. Methane (purity 99.9995%, Air Liquide, Bopfingen, Germany) was used

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as reagent gas (2 mL/min) for measurements in GC/ECNI-MS full scan (m/z 50-550) and

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selected ion monitoring mode (SIM) mode (Tables S3, S4, Supporting Information). The

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internal standard (ISTD) α-PDHCH (10.7 ng/µL in 10 µL iso-octane) was added to each CCC

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fraction before measurements in full scan mode. Peak heights were normalized to α-PDHCH.

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GC/ECNI-MS analysis of individual CCC fractions was based on the two most abundant

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isotope peaks of the molecular ion ([M]-) of tri- to hepta-chlorinated OH-CTTs (Table S5,

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Supporting Information). Due to possible overlapping with [M-Cl]- or [M-HCl]- fragment ions

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of higher chlorinated isomers, it was necessary to investigate the relative isotope peak ratio of

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the two highest [M]- isotope peaks and to compare this to the theoretical isotope pattern from

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tri- to heptachlor-isomers. Deviation up to 5% was accepted.

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Gas chromatography coupled with electron ionization mass spectrometry (GC/EI-

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MS). The Agilent 6890/5973 GC/MS system (Waldbronn, Germany) was equipped with a

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guard column (2 m x 0.53 mm internal diameter, deactivated with 1,3-diphenyl-1,1,3,3-

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tetramethyldisilazane, BGB Analytics, Boeckten, Switzerland) connected to a Rtx-1 column

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(15 m x 0.25 mm internal diameter, 0.1 µm film thickness, Restek, Bellefonte, PA, USA).

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Injection of sample solutions (1 µL) was by means of a 7693 autosampler system (Agilent)

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into a cool-on-column injector. The GC oven was initially set for 1 min to 60 °C, then the

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temperature was raised at 10 °C/min to 250 °C (5 min), then at 5 °C/min to 300 °C (0 min)

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and finally at 30 °C/min to 350 °C (10 min). The transfer line, quadrupole and ion source

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temperatures were set at 350 °C, 150 °C and 230 °C, respectively. The injector port

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temperature was set to track the oven temperature. GC/EI-MS full scan spectra were recorded

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from m/z 50-800 after solvent delay of 7 min.



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Samples and sample extracts. Fish oil capsules “Omega-3 1000” (tetesept Pharma,

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Frankfurt am Main, Germany) were used as fish oil simulation matrix for development of the

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clean-up procedure. For this purpose, 500 mg fish oil was spiked with 150 µL PCB-mix only,

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150 µL OH-CTT-mix (CCC fraction #27, 65.9 min) only, or with both mixtures (Table S2,

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Supporting Information). Liver samples and adipose tissue were available from four polar

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bears (Ursus maritimus) which swam malnourished from East-Greenland to Iceland in 2008,

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2010, and 2011.38 Lipid extraction has been described in detail in a previous study.38 Briefly,

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~15-20 g of polar bear livers samples and ~2.3 g of polar bear adipose tissue were lyophilized,

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followed by open-vessel microwave-assisted extraction (FOV-MAE).38,39 Liver fat extracts

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were stored in dark at -20 °C diluted in C/E-mixture (46:54, w/w) until use.

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Gel permeation chromatography (GPC). Spiked fish oil, polar bear liver extracts as

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well as sample blanks (Table S2, Supporting Information) were dissolved in 5 mL C/E-

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mixture (46:54, w/w) and chromatographed by means of an AccuPrep MPS system (ANTEC,

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Sindelsdorf, Germany) which featured a 41 cm length, 1.5 cm i.d. column filled with Bio-

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Beads SX-3. Polyhalogenated compounds were eluted with C/E-mixture (flow rate 5

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mL/min)42 into GPC fraction 2 (20 - 40 min) which was evaporated nearly to dryness. Then,

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the solvent was changed to iso-octane and made up to 1 mL with iso-octane.

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Adsorption chromatography on deactivated silica gel (column A). The whole GPC

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extract (see previous section) was further purified on a 1 cm internal diameter glass column

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filled with 1 g silica gel deactivated with 1.5wt% water which was covered with a small layer

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of anhydrous sodium sulfate (pre-dried for 24 h at 550 °C) using the protocol of Specht and

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Tillkes for pesticides of different polarity (Figure S2, Supporting Information).43

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Fractionation of samples started with 8 mL n-hexane (A1), followed by 8 mL n-

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hexane/toluene (65:35, v/v, A2), 16 mL toluene (A3), 8 mL toluene/acetone (99:1, v/v, A4*),

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8 mL toluene/acetone (95:5, v/v, A4) and 8 mL toluene/acetone (80:20, v/v, A5) (Figure S2,


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Supporting Information). Each fraction was concentrated to 1 mL. Aliquots (20 µL) of all

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fractions were silylated with BSTFA/TMCS for 30 min at 60 °C in a pre-heated sand bath.40

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Silylated sample solutions were re-dissolved in methanol and measured by GC/EI-MS.

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Adsorption chromatography on activated silica gel (column B). A 500 µL aliquot of

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fraction A3 (see previous step) of all samples was fractionated on activated silica gel

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according to Kapp and Vetter27 (Figure S2, Supporting Information). Elution was achieved

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with 48 mL n-hexane (B1), followed by 50 mL n-hexane/ethyl acetate (90:10, v/v, B2) and

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50 mL ethyl acetate (B3) (Figure S2, Supporting Information). Each fraction was evaporated

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and made up to 500 µL with iso-octane. After addition of 10 µL of the ISTD (200 ng BCIS),

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sample solutions were analyzed by GC/ECNI-MS.

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RESULTS AND DISCUSSION

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Synthesis, purification and analysis of OH-CTTs as comparative standards.

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Synthesis of OH-CTTs. Elemental analysis (details are presented in the Supporting

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Information) of the unfractionated OH-CTT synthesis mixture attested an average carbon

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content of 22.5% (average chlorine content of 75.0%) and a molecular formula of

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C10H6.8Cl11.2O. Upscaling by a factor of five compared to the synthesis procotol of Kapp and

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Vetter27 provided 1.6 g OH-CTTs, i.e. suitable amounts for semi-preparative CCC separation.

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According to GC/ECNI-MS, the unfractionated synthesis mixture consisted of at least 148

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tetra- to heptachlorinated OH-CTTs (retention time covering 17.3 to 23.0 min on a 30 m HP5-

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MS UI column).

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CCC procedure. The complex OH-CTT synthesis mixture was considered unsuited for a

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detailed composition analysis and for its application in method development. Therefore, CCC

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was used for initial fractionation of the OH-CTT synthesis mixture. Partitioning coefficients ACS Paragon Plus Environment


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(KU/L values) were determined for ten tri- to heptachlorinated OH-CTTs, which covered the

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whole elution range in the GC/ECNI-MS chromatogram (tR = 13.5-22 min) (Figure 1). KU/L

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values of these ten OH-CTTs were determined in 25 different solvent systems (Table S6,

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Supporting Information), and the most promising results were obtained with the solvent

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system n-hexane/ethyl acetate/methanol/water (40:10:40:10, v/v/v/v) of Friesen and Pauli41

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which resulted in KU/L values between 0.45 and 1.96 of the ten OH-CTTs. Using this solvent

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system, the complexity of individual fractions was drastically reduced by CCC. In several

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occasions, isomers with the same GC retention time were detected in different CCC fractions,

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which indicated a higher complexity of the synthesis product than was anticipated from the

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inspection of the GC/ECNI-MS chromatogram. In detail, 227 different OH-CTTs were

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detected in the CCC fractions (12 tetra-, 117 penta-, 81 hexa- and 17 heptachlorinated OH-

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CTTs), which was >50% more than detected before the fractionation. Finally, CCC fraction

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#27, consisting of only 18 OH-CTTs, was selected for method development.

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GC/ECNI mass spectra from PCBs and OH-CTTs. The unchlorinated backbones of

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OH-CTTs (borneol, C10H18O) and PCBs (biphenyl, C12H10) have the same nominal mass of

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154 u. Likewise, molecular ions (M-) of PCBs and OH-CTTs of the same chlorination degree

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are isobaric and cannot be distinguished by low resolution MS.27 In addition, GC retention

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time ranges are also virtually the same (e.g. heptachlorinated OH-CTTs: 21.1-23.0 min,

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heptachlorobiphenyls: 21.1.-22.5 min), both substance classes may be mixed with each other

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in environmental samples. However, distinct differences exist in the GC/ECNI-MS

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fragmentation patterns of PCBs and OH-CTTs (Figure 2). In general, OH-CTTs fragmented

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more extensively than PCBs. For instance, GC/ECNI-MS mass spectra of penta- to

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heptachlorinated OH-CTTs generally featured at least one fragment ion which was more

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abundant than [M]- while [M]- was generally the base peak of PCBs. Abundant GC/ECNI-MS

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fragment ions of OH-CTTs corresponded with the loss of Cl/Cl2 and/or HCl/HCl2 27 (e.g. m/z

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357, m/z 321, and m/z 284 for heptachlorinated OH-CTTs, Figure 2a-c). In contrast, mass ACS Paragon Plus Environment

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spectra of PCBs featured predominantly [M+n*H-n*Cl]- fragment ions (e.g. m/z 290, m/z 324,

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and m/z 358 in the case of heptachlorobiphenyls, Figure 2d-f). A further diagnostic feature of

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OH-CTTs was the formation of [HCl2]-/[Cl2]- fragment ions which were not noticed in mass

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spectra of PCBs. In addition, OH-CTTs showed fragment ions at m/z 248 (Cl7-OH-CTTs),

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m/z 250 (Cl6-OH-CTTs), m/z 252 (Cl5-OH-CTTs) and m/z 254 (Cl4-OH-CTTs) caused by

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elimination of four, three, two and one HCl unit, respectively, which was not observed in the

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corresponding GC/ECNI-MS spectra of PCBs (Figure 2). Hence, the final GC/ECNI-MS-

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SIM method was based on two isotope peaks of [M]- of OH-CTTs along with specific ions

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(m/z 248/250 for Cl7-OH-CTTs, m/z 250/252 for Cl6-OH-CTTs and m/z 252/254 for Cl5-OH-

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CTTs) which enabled the unequivocal differentiation of OH-CTTs and PCBs (Table S3, S5,

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Supporting Information). Boon et al. tentatively identified one nonachlorinated OH-CTT

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metabolite after incubation of liver microsomes in an in-vitro experiment.24 Despite having

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different degrees of chlorination, the GC/ECNI-MS mass spectrum of this compound and the

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OH-CTTs in our synthesis product showed similar fragmentation. For instance, fragment ions

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of the series [M-Cl-n*HCl]- were observed in either case. Since the display of the literature

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spectrum only covered m/z 250-390,24 no statements can be made about the molecular ion and

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the presence of characteristic [HCl2]-/[Cl2]- fragment ions or fragment ions at m/z 248-254.

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Extraction, purification and analysis of polar bear tissues.

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Removal of lipid components. GPC removed ~99% of lipid matrix from the

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polychlorinated compounds (here: PCBs, CTTs and OH-CTTs). Yet, GPC fraction 2 still

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contained ~5 mg lipids, in particular cholesterol, which would lead to chromatographic

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interferences and matrix effects in GC/MS measurements. Fractionation of GPC fraction 2

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into six fractions by absorption chromatography on deactivated silica gel (column A) eluted

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~78% of OH-CTTs mainly into fraction A3 and the remaining share of the OH-CTTs in

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fraction A4-A5 together with cholesterol (Figure S3a, Supporting Information). Extension of



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the elution volume of fraction A3 (from 8 to 16 mL) and lowering the solvent strength of

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fraction A4 (by reducing the share of acetone, modified fraction A4*) (Figure S3b,

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Supporting Information) finally allowed to separate OH-CTTs from cholesterol (Figure S3b,

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Supporting Information), while ~10% OH-CTTs were lost (A4*-A5, Figure S3b, Supporting

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Information) which was deemed acceptable.

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Removal of PCBs. The bulk of PCBs (mainly eluting into fractions A1 and A2) and

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OH-CTTs (mainly in fraction A3) was also separated from each other by column A. However,

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fraction A3 still contained several PCBs (6 mass%). This share was removed on activated

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silica gel (column B, Figure S2, Supporting Information).27 With this procedure, PCBs were

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exclusively detected in fraction B1 (48 mL solvent system) while the OH-CTTs in CCC

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fraction #27 eluted into fraction B2 (17%) and fraction B3 (83%).

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CTTs in polar bear adipose tissue and liver. GC/ECNI-MS analysis of all four polar

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bears indicated the presence of octa- and nonachlorinated CTTs in adipose tissue (205-

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4,350 µg/kg lipid weight) but only octachlorinated CTTs in liver (65-320 µg/kg lipid weight)

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(Table 1). CTT levels in adipose tissue of the polar bears were about one order of magnitude

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higher than those reported in the literature (

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