Environ. Sci. Technol. 1996, 30, 2251-2258
Characterization of Two Major Toxaphene Components in Treated Lake Sediment G . A . S T E R N , * ,† M . D . L O E W E N , †,‡ B. M. MISKIMMIN,§ D. C. G. MUIR,† AND J. B. WESTMORE‡ Department of Fisheries and Oceans, Freshwater Institute, Winnipeg, Manitoba, Canada R3T 2N6, Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9, and Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
The structures of two environmentally significant toxaphene congeners, namely, a hexachlorobornane (HxSed) and a heptachlorobornane (Hp-Sed), from the sediment of two lakes located in Alberta, Canada, have been characterized by 1H NMR and mass spectrometries. Both lakes were treated in the early 1960s by the addition of toxaphene, as a pesticide, to the water column at low microgram per liter concentrations. Highest toxaphene concentrations were found in sediments of both lakes in slices dated to the early 1960s. In these slices, the chromatographic pattern resembled that of the toxaphene standard while, in more recent slices, the number of chlorinated bornane (CHB) peaks was greatly reduced, with the two most prominent peaks corresponding to 2-exo,3endo,6-exo,8c,9b(or 8b, 9c),10a-hexachlorobornane and 2-exo,3-endo,5-exo,6-endo,8c,9b(or 8b, 9c),10aheptachlorobornane. Hx-Sed has previously been identified as one of the major reductive dechlorination metabolites of toxicant B (2-exo,3-endo,6,6,8c,9b,10a-heptachlorobornane), one of the most toxic components of the technical mixture. Never before, however, has it been identified as a persistent contaminant in the environment.
Introduction Toxaphene is a complex mixture consisting of between 2 and 300 penta- to undecachlorobornanes (CHBs) and camphene/diene isomers (1, 2). Introduced in the United States in 1945 by Hercules Co. as a new insecticide to control a variety of insect pests, two-thirds of toxaphene production was used for insect control on cotton. It was also used on vegetables, small grains, and soy beans and for control of external insects on livestock (3, 4). In Canada and the United States, toxaphene was also used extensively * To whom correspondence should be addressed; telephone: 204984-6761; fax: 204-984-2403. † Freshwater Institute. ‡ University of Manitoba. § University of Alberta.
S0013-936X(95)00622-5 CCC: $12.00
1996 American Chemical Society
in fish eradication programs. This practice, however, was discontinued when it was found that toxaphene was extremely persistent and that lakes could not be successfully restocked for years after treatment (5-7). Prior to its ban in 1982 by the U.S. Environmental Protection Agency (8), toxaphene was the most extensively used pesticide in the United States and many other parts of the world. Global production has been estimated to be 1.33 Mt (9), with production in the United States (1946-1982) accounting for about 0.45 Mt (8, 9). Although no longer manufactured in the United States, toxaphene and similar products are still being used in Central and South America, Africa, Eastern Europe, the Indian subcontinent and in regions of the former USSR (8-10). In a recent paper by Miskimmin et. al. (11), the distribution of toxaphene in profundal sediment cores from two treated lakes located in Alberta, Canada, was examined, and the chromatographic patterns between recent and older sediments were compared. Peanut Lake (50°01′ N, 114°21′ W; mesotrophic) was treated with 7.5 µg L-1 toxaphene in September 1961, and Chatwin Lake (54°15′ N, 110°15′ W; eutrophic) was treated at 18.4 µg L-1 in October 1962. Gas chromatography, with electron capture detection (GCECD), showed that the maximum total toxaphene concentration in the sediment from both treated lakes occurred at depths representing the years of treatment (500 ng/g in Peanut Lake and 1602 ng/g in Chatwin Lake) and that their chromatographic profiles were very similar to that of technical toxaphene. This result indicated that the majority of the original toxaphene remaining at these depths (representing 1961/1962) was essentially unchanged after over three decades in anaerobic sediments. In more recent core slices, however, Chatwin Lake sediments were found to have a peak profile similar to those of Peanut Lake sediments of about the same age (∼1970) but were significantly different from the technical mixture. A shift toward earlier eluting, less chlorinated congeners was observed. Gas chromatography-electron capture negative ion mass spectral (GC-ECNIMS) selected ion chromatograms of toxaphene in the near surface sediment of Chatwin Lake revealed the presence of two major toxaphene congeners, a hexa- and a heptachlorobornane here referred to as Hx-Sed and Hp-Sed, respectively (Figure 1). A similar CHB pattern was observed in fish from the same lakes. We report here the isolation of Hx-Sed and Hp-Sed from 10 kg (wet wt) of near-surface sediment from Chatwin Lake and their structures as determined by 1H NMR and mass spectrometries.
Experimental Section Toxaphene Extraction and Isolation of Hx-Sed and HpSed. Approximately 10 kg (wet wt) of Chatwin Lake surface sediment (0-10 cm deep) was collected with a modified Ekman dredge and was stored in three 4-L brown glass bottles. This corresponded to 735 g (dry wt) of sediment after freeze drying. The freeze-dried sediment was refluxed, 100 g at a time, in 800 mL of dichloromethane (DCM) for 5 h. Extracts were treated with 1:1 fuming nitric/sulfuric acids to remove organic pigments and chlorinated aromatics (12), separated, and then treated with elemental copper to remove sulfur compounds. To isolate Hx-Sed and HpSed from other organochlorines, we used high-performance
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FIGURE 1. GC-ECNIMS selected ion chromatograms for hexa- and heptachlorobornanes in near surface sediment extracted from Chatwin Lake (Alberta, Canada) and in the toxaphene standard (11).
liquid chromatography (HPLC) on a Nova-Pak HR C18 preparative column (Waters Scientific), employing an isocratic solvent system consisting of an acetonitrile/water (65:35, v/v) mixture (4.0 mL min-1). GC-ECNIMS (electron capture negative ion mass spectrometry) was used to analyze the various HPLC fractions for Hx- and Hp-Sed. Approximately 20-µg amounts of Hx- and Hp-Sed were isolated in this manner; these were more than sufficient for mass spectrometric studies. 1H NMR studies, however, required milligram amounts, and we therefore had to resort to isolating Hx- and Hp-Sed from the technical standard (Chem Service Ltd.). This was carried out using the same HPLC conditions as noted above. However, because of the complexity of the mixture, the HPLC fractions containing Hx- and Hp-Sed had to be further fractionated on Florisil [45 g; 1.2% (v/w) water deactivated]. Both congeners were eluted with hexane/DCM (85:15) in 1-mL aliquots. All solvents used were purchased from Caledon Laboratories Ltd. GC solvents were distilled in glass, while those used for HPLC were of HPLC grade. Reagent-grade sulfuric and fuming nitric acids were purchased from Fisher Scientific. 1H
NMR Spectrometry. 1H NMR spectra of the compounds, dissolved in perdeuteriobenzene (99.5 atom %, Aldrich Inc.), were recorded with a Bruker AMX500 spectrometer, operating at a frequency of 500 MHz. Difference decoupling and NOE experiments were performed with a digital resolution of 0.18 Hz. Mass Spectrometry. GC-EIMS (electron ionization mass spectrometry), GC-ECNIMS, and linked field scanning were performed on a Kratos Concept high-resolution mass spectrometer (EBE geometry) controlled by a Mach 3X data system. EI positive ion mass spectra were scanned from 35 to 450 Da at a scan rate of 1 s/decade. The ion source was maintained at a temperature of 170 °C, the trap current was 500 µA, the ion accelerating voltage was 8 kV, and the
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electron energy was adjusted for maximum sensitivity (∼50 eV) . Decompositions of selected ions in the first field-free region were identified by a series of linked-field scans, namely, B/E (product), B2/E (precursor), and CNL [constant neutral loss (daughter and parent)]. Ion decompositions were enhanced by collisional activation by introducing argon into the collision cell at a pressure to give approximately 50% attenuation of the m/z 231 ion of perfluorokerosene (PFK). Selected ion ECNIMS was performed at a spectrometer resolution of M/∆M ∼ 14 000. Methane was used as the moderating gas, and PFK was used as the mass calibrant. Optimum sensitivity was obtained at a gas pressure of ∼2 × 10-4 Torr as measured by the source ion gauge. The electron energy was adjusted for maximum sensitivity (∼180 eV), the ion accelerating voltage was 5.3 kV, and the ion source temperature was 120 °C. The following characteristic ions were monitored from the [M - Cl]- isotopic cluster of the hexa- to nonachlorobornane homolog groups: Cl6 308.9352, 310.9323; Cl7 342.8962, 344.8933; Cl8 376.8573, 378.8543; Cl9 410.8183, 412.8154. GC separations were performed with a Hewlett Packard Model 5890 Series II gas chromatograph on a 60 m x 0.25 mm i.d. DB-5ms (film thickness 0.25 µm) fused silica column (Chromatographic Specialities), which was connected directly to the ion source of the mass spectrometer. Helium was used as the carrier gas. Samples, made up in 2,2,4trimethylpentane (TMP), were run using splitless injection (2 min) with the injection port at 260 °C. The initial column temperature was 80 °C; at 2 min the oven was ramped at 20 °C min-1 to 200 °C, then at 2 °C min-1 to 230 °C then at 10 °C min-1 to a final temperature of 300 °C and held for 8 min. Electronic pressure programming was used to increase the pressure during the injection cycle and then to maintain a constant flow of 1 mL min-1 during the remainder of the run. All injections were made by a CTC A200SE autosampler under data system control. GC-ECD. Capillary gas chromatography with (63Ni) electron capture detection was carried out using an automated Varian 3400 gas chromatograph (Varian Instruments, Georgetown, ON). Samples were injected (splitless mode) on a 60 m × 0.25 mm i.d. DB-5 column (film thickness 0.25 µm) with an initial temperature of 100 °C then programmed at 15 °C/min to 150 °C and 3 °C/min to 265 °C. The carrier gas was H2 (about 1 mL/min) and makeup gas was N2 (40 mL/min). Structural Calculations. Relative stabilities of proposed structures and conformations were estimated with the Molecular Modeling Pro program (WindowChem Software Inc., Fairfield, CA).
Results and Discussion 1H NMR Spectrometry. The HRGC-ECD chromatograms of the fractions containing Hx- and Hp-Sed (Figure 2) reveal the presence of the isolates as the major peaks in their respective chromatograms plus some smaller peaks attributed to the presence of impurities (CHBs). Assuming similar response factors, Hx- and Hp-Sed are present at concentrations approximately 12 and 8 times greater, respectively, than that of the most abundant impurity in their respective fractions. The 500-MHz 1H NMR spectrum of Hp-Sed was essentially first order, while that of Hx-Sed exhibited second-order behavior for some protons. The presence of 12 individual proton signals in Hx-Sed and of 11 in Hp-Sed spectra (supported by integration) confirmed incorporation of six and seven chlorines, respectively. The
very small (
