Environ. Sci. Technol. 1992, 26, 1838- 1840
Meeussen, J. C. L.; Temminghoff, E. J. M.; Keizer, M. G.; Novozamsky, I. Analyst 1989, 114, 959-963. Keizer, M. G. E C O S A T , a computer program f o r the calculation of speciation in soil-water s y s t e m ; Department of Soil Science and Plant Nutrition, Wageningen Agricultural University: Wageningen, The Netherlands, 1991. Lindsay, W. L. Chemical equilibria in soils; Wiley-Interscience: New York, 1979.
(12) Beck, M. T. Pure A p p l . Chem. 1987,59, 1703-1720. (13) Dutch Ministry of Housing Physical Planning and Environment. Soil Clean Up (Interim) Act, 1983.
Received f o r review February 12, 1992. Revised manuscript received M a y 27,1992. Accepted M a y 29,1992. T h i s work was funded by the Netherlands Integrated Soil Research Programme ( N o . 8946).
Isolation and Identification of Two Major Recalcitrant Toxaphene Congeners in Aquatic Biota G. A. Stern,t D. C. G. Mulr,*st C. A. Ford,+ N. P. Grift,+E. Dewallly,$T.
F. Bidleman,§ and M. D. Wallas
Freshwater Institute, Department of Fisheries and Oceans, Winnipeg, Manitoba, R3T 2N6 Canada, Department de Sante Communautaire du Centre Hospitalier de I'Universit6 Laval, Ste.-Foy, Quebec, G I V 2K8 Canada, and Chemistry Department, University of South Carolina, Columbia, South Carolina 29208 Introduction Toxaphene (polychlorinatedcamphene; PCC) is a major organochlorine pesticide contaminant in fish and marine mammals in North American and European waters due to substantial use for insect control on cotton and other crops in the 1960s and 1970s (1-5). The profile of toxaphene peaks observed, in fish and mammal samples, by high-resolution capillary gas chromatography (HRGC) differs from the analytical standard presumably due to differential uptake and metabolic transformation of only selected congeners (6, 7). In marine mammals and fish livers up to 20 major PCC peaks can be distinguished but 2, which we have previously referred to as T2 (an octachlorocamphene) and T12 (a nonachlorocamphene), predominate (2-4). Although the structures of about 20 toxaphene components in the technical material have been elucidated (5), the identities of these two dominant PCC components in aquatic biota have not been reported previously. We report here the first isolation and identification of the structures of T2 and T12 by use of IH NMR spectroscopy and mass spectrometry. Experimental Section Isolation of T2 and T12. Beluga whale (Delphinapterus leucas) blubber from Hudson Bay animals was selected as a source of T2 and T12 because of its elevated PCC levels (4). PCCs and other organochlorines were separated from the lipid using polymeric film dialysis (8). A 100-g sample of beluga whale blubber was blended with 150 mL of hexane, the hexane was evaporated, and the lipid was added to a 55-cm length of "layflat" polethylene tubing (5.1 cm wide and 51 pm thick; Cope Plastics, Inc. Fargo ND) that had previously been rinsed twice with hexane. The tubing was placed in the dialysis chamber (27.4 cm X 7.7 cm X 24.0 cm, A1 foil covered to exclude light) with 3 L of hexane and was allowed to dialyze for 24 h at room temperature (ca. 24 "C). The hexane in the dialysis chamber was then syphoned off and its volume reduced to approximately 5 mL with a rotary evaporator; the hexane was saved for reuse. The dialysis extraction was repeated for another 24 h using the recycled hexane. Twenty extracts (total of 2 kg of blubber) were combined *To whom correspondence should be addressed Freshwater Institute, Dept. of Fisheries and Oceans, 501 University Cres., Winnipeg MB R3T 2N6 Canada. 'Freshwater Institute. 4 Universit6 Laval. University of South Carolina. 1838 Environ. Scl. Technol., Vol. 26, No. 9, 1992
and dialyzed, as above, on further time to remove as much residual lipid as possible. After rotary evaporation to a volume of 10 mL, the concentrated extract was fractionated by chromatographyon Florisil[45 g; 1.2% (v/w) water deactivated]. T2 was eluted with hexane (80%)along with PCBs, chlorobenzenes, 4,4'-DDE, and mirex, and T12 was eluted (90%) with hexane-DCM (85:15) with most other PCCs along with chlordane-related compounds and 4,4'DDT. T2 and T12 were isolated from other organochlorines by HPLC on a Nova-Pak HR CISprep column (Waters Scientific, Missisauga, ON, Canada) using an isocratic solvent system consisting of a methanol-water (9O:lO) mixture (4.0 mL/min). Analysis. A Varian 6000 capillary gas chromatograph, with electron-capture detection (GC-ECD) and a 60 m X 0.25 mm i.d. Rt,-5 column with Hz carrier gas, was employed to analyze the various HPLC fractions for T2 and T12. Operating conditions have been described previously ( 2 , 4 ) . Quantitation of T2 and T12 by GC-ECD was accomplished by f i s t determining the weight percent of each peak in the total ion chromatogram (electron impact GCmass spectrometry) of a toxaphene standard. The response factors were verified by comparison with purified octa- and nonachlorobornanes isolated from the technical material by HPLC (Stern and Muir, unpublished data). lH NMR spectra were recorded with a Bruker AMX500 spectrometer, operating at a frequency of 500 MHz. Difference decoupling and difference NOE experiments were performed with a digital resolution of 0.18 Hz. GC-electron capture negative ion mass spectrometry (ECNIMS) was performed on a VG-7070E-HF double-focusing mass spectrometer of EB geometry coupled to a HewlettPackard Model 5890 GC equipped with a 60 m X 0.25 mm i.d. DB-5 column (He carrier gas). Methane was used as the reagent gas, electron energy was typically 100 eV, and emission current and ion acceleration voltage were kept at 200 pA and 6 keV, respectively. The ion source temperature was 175 OC. Results and Discussion The 500-MHz IH NMR spectra of T2 and T12 were essentially first order with chemical shifts (Table I) and coupling constants (Table 11) characteristic of the norbornane ring system (9). Except for impurities introduced during isolation (resonances
