Environ. Sci. Technol. 1993, 27, 1211-1220
Enantioselective Determination of Chlordane Components, Metabolites, and Photoconversion Products in Environmental Samples Using Chiral High-Resolution Gas Chromatography and Mass Spectrometry Hans-Rudolf Buser' and Markus D. Muller
Swiss Federal Research Station, CH-8820 Wadenswil, Switzerland Enantiomer separation of various chlordane compounds, including heptachlor, the oxygenated metabolites heptachlor epoxide (HEP) and oxychlordane (OXY), and photoconversion products was investigated using chiral high-resolution gas chromatography and detection by electron ionization (EI) and electron-capture, negative ionization (ECNI) mass spectrometry (MS). In technical chlordane and in ambient air from Scandinavia, heptachlor and the chiral octa- and nonachlordanes were present in enantiomeric ratios of 1:l. The results indicate input of these compounds via aerial transport into the northern environment as racemic mixtures. Laboratory experiments using solid-phase photolysis by natural sunlight yielded caged and half-caged products from heptachlor and cis-chlordane, respectively. HEP was identified as a photooxidation product of heptachlor and formed in an enantiomeric ratio of 1:l. In contrast, incubation of heptachlor with rat liver homogenate (S9fraction) yielded predominantly one enantiomer of HEP, likely due to the stereoselectivity of the mixed-function oxidase system. Biota from the Baltic (fish, seal), Arctic (seal), and Antarctic (penguin) showed changed isomeric and enantiomeric compositions of the octa- and nonachlordanes as compared to technical chlordane, and the metabolites HEP and OXY present in enantiomeric ratios differing from 1:l. The finding of photoheptachlor and photo-cischlordanes in these species document that photoreactions of chlordane compounds play a role in the transformation of these compounds in the environment.
enantiomeric forms which differ in their biological properties. The two enantiomers of heptachlor (structures, see Chart I) and those of cis- and trans-chlordane were individually synthesized, and those of heptachlor showed different insecticidal activity (I5 , 16). The major metabolites HEP and OXY are also chiral, and the two enantiomers of HEP were individually synthesized (16). In previous studies, we reported on the enantiomer separation of several chiral chlordane components as well as on the results of enantioselective determinations in aquatic biota and human adipose tissue (17,181.However, in those studies several chiral components including heptachlor were not yet enantiomerically resolved. In this study, we report on the enantiomer resolution of additional chlordane compounds and on their presence in ambient air from Scandinavia. We also report on the enantiomeric composition of chiral products formed in model reactions by enzymatic (biotic) and photochemical (abiotic) processes. Furthermore, we document the presence of photoconversion products of some chlordane compounds in aquatic species from the Baltic, Arctic, and Antar ctic. Experimental Section
Chlordane, an important chlorinated pesticide consists of a complex mixture of various chemically similar components, predominantly hexa- to decachlorinated congeners (1-3). Among the major constituents are cisand trans-chlordane, trans-nonachlor, and heptachlor. Heptachlor is also a pesticide of its own with a similar-use pattern. The two pesticides were extensively used in some countries (US.) but reportedly not in others, e.g., not in Scandinavia ( 4 ) . Both pesticides are toxic and now considered possible human carcinogens (1). They have some potential for bioaccumulation. The fact that chlordane is detected in biota from remote areas such as the Arctic and Antarctic (5-7) points to global distribution mechanisms such as via long-range aerial transport (812). However, actual data on routes of entry of chlordane in such environments are scarce. Heptachlor and some chlordane components are metabolized in almost all organisms into two persistent epoxides, heptachlor epoxide (HEP) and oxychlordane (OXY) (13). HEP is also reported as an environmental metabolite of heptachlor (14). Several chlordane components including heptachlor are chiral and exist in two
Materials and Reference Compounds. cis-Chlordane, trans-chlordane, cis- and trans-nonachlor, heptachlor, HEP, heptachlor endo-epoxide (endo-HEP), and OXY were from Ehrenstorfer GmbH, Augsburg, FRG; a technical chlordane obtained in the 1950s from Maag, Dielsdorf, Switzerland, was also used for comparative analyses. Solutions of the reference compounds and the technical chlordane mixture in toluene (1-10ng/pL) were prepared and used for analysis. a-Cyclodextrin (a-CD) and 0-cyclodextrin (0-CD) were from Fluka (Buchs, Switzerland), and permethylated 0-CD (PMCD) was from Sigma (Buchs, Switzerland), PerethPECDI and ylated a-CD [hexakis(2,3,6-tri-O-ethyl)-a-CD, tert-butyldimethylsilyl-0-CD (BSCD) were prepared according to refs 19 and 20, respectively. The silicon compounds used for the preparation of the capillary columns were as previously described (I8). Ambient Air Samples. Ambient air samples of about 500 m3 (collection time approximately 24 h, sampling flow 20 m3/h) were collected in August/September 1991 by the Norwegian Institute for Air Research, NILU, Lillestram, Norway (courtesy M. Oehme), on the southern Norwegian coast using large-volume air samplers with polyurethane foam (22,221. The samples were extracted and cleaned up a t NILU. After initial analysis at NILU, the sample extracts were used in the present study. Sunlight Photolysis of Chlordane Compounds. Heptachlor, cis- and trans-chlordane, cis- and transnonachlor, and the technical chlordane were exposed to natural sunlight in the presence of air. Aliquots of 10-20 pg of an individual compound (800 pg in the case of
0 1993 American Chemical Society
Envlron. Sci. Technoi., Vol. 27, No. 6, 1993 1211
Introduction
0013-936X/93/0927-1211$04.00/0
Chart I. Structures of the Two Enantiomers of Heptachlor, Arbitrarily Assigned as Heptachlor-I and Heptachlor-28 CI
CI'
CI
heptachlor-1
CI'
heptachlor-2
Not necessarily correlatingwith elution on chiralHRGC columna. Mirror plane perpendicular through Cp and CS.
technical chlordane) were dissolved in ethanol (50 pL) and placed in small (volume = 300 pL) quartz vials (23). The solvent was evaporated by vacuum with gentle heating and axial rotation by hand. This resulted in deposition of materials as a thin film on the walls of the quartz vials; some droplet formation was still visible. The vials were exposed to sunlight on July 30-August 3, 1992, at Wadenswil for total exposure periods up to 24 h. Unexposed vials were kept in the dark as controls. Residues in the control and in the exposed vials were then dissolved in 100 pL of toluene, and 1-pL aliquots were analyzed. In the case of technical chlordane, 1 pL of a further (1:lOO) dilution was analyzed. Biological Samples Analyzed. Herring oil from Baltic herring (Clupea harengus, Gulf of Bothnia) and tissues of female salmon (Salmo salar, Ume river at Stornorrfors, Sweden; muscle tissue), Baltic grey seal (Halichoerus grypus, Swedish southeastern coast; composite of liver tissue), and juvenile Adelaide penguin (Pygoscelis adelis, Ross Island, Antarctica) were analyzed. These samples as well as human adipose tissue of a male American were the same as in previous studies (2 7,181;they were extracted and cleaned up at the Institute of Environmental Chemistry, University of Umeb, Sweden, according to published methods (24). Additionally, tissue from a harp seal (Pagophilus groenlandicus) collected from Greenland was analyzed. This particular sample was extracted and cleaned up at NILU. Aliquots of 2 pL corresponding to 200-400 mg of tissue of aquatic samples and to 20-40 mg of human adipose tissue were used for analysis. Enzymatic Conversion of Heptachlor, Octachlordanes, and Nonachlordanes. These reactions were carried out similarly as described in ref 25. Liver of untreated Sprague-Dawley female rats was disintegrated in 3 vol of cold 0.1 M phosphate buffer (pH 7.4) with a Potter homogenizer. The homogenate was centrifuged for 10min at 9OOOg. Portions of 500 p L of the supernatant were mixed with 4.5 mL of 0.1 M phosphate buffer (pH 7.4) containing NADP, glucose 6-phosphate, and glucose6-phosphate dehydrogenase at concentrations of 0.5 mM, 4 mM, and 1unit/mL, respectively. Approximately 80 pg of a chlordane compound dissolved in 40-80 pL of ethanol was added, and the mixture was incubated at 37 "C with occasional shaking. Aliquots of 1 mL were removed immediately after mixing and after time intervals of up to 2 h. These aliquots were immediately extracted with small portions of n-hexane (total 3 mL); the extracts were passed through small columns of silica (0.5 g of silica gel 60, Merck, Darmstadt, FRG, in 5-mm i.d. Pasteur pipette), eluted with 5 mL of 30% dichloromethaneln-hexane, and then carefully concentrated. Aliquots of 1-2 pL of 100ML were used for analysis. HRGC-MS Analysis. A VG Tribrid double-focusing magnetic sector hybrid mass spectrometer (VG Analytical, Manchester, England) was used for analyte detection and 1212 Envlron. Scl. Technol., Vol. 27, No. 6, 1993
-
Table I. Ions Monitored* ( m / z Values) in SIM Experiments for Chlordane Components, Metabolites, and Photoproducts E1 ECNI
Heptachlor, Photoheptachlor (C10H5C17) 334.852 336.849 269.813 297.868 299.865
E1 ECNI
Octachlordanes (CioHsCls) 370.829 372.826 374.823 405.798 407.795 409.792
E1 ECNI
Photo-cis-chlordanes (CloH&ls) 370.829 372.826 374.823 297.868 299.865
E1 ECNI
Nonachlordanes (CloH&19) 404.790 406.787 439.759 441.756 443.753
E1 ECNI
384.808 419.777
E1 ECNI
HEP, endo-HEP (CloH&170) 350.847 352.844 354.841 385.816 387.813 389.810
a
271.810
OXY (CioH4ClsO) 386.805 421.774 421.774 423.771
Up to 12 ions were monitored simultaneously (0.5 s/scan).
identification. The ion source was operated in either the electron ionization (EI, 70 eV, 180 "C)or in the electroncapture, negative ionization (ECNI, 50 eV, 140 "C) mode. A modified chemical ionization source was used in these ECNI experiments. This source allowed the use of neat argon as a buffer gas (0.5-1 X mbar, as measured by the ion gauge). Full-scan mass spectra (mlz 50-500, 1.16 s/scan, resolution MIAM = 500) were recorded for analyte identification in all samples. Analyses were then repeated with selected ion monitoring (SIM) for increased sensitivity and optimal enantiomer/isomer separation using up to 12 ions simultaneously (0.50 s/scan; see Table I). A lockmass of 451.974 (ECNI) or 413.978 (EI) from perfluorotributylamine was used in the SIM experiments. Concentrations in the biological samples were estimated from SIM chromatograms in comparison to those of known quantities of the reference compounds. All samples were comparatively analyzed using an achiral and three chiral HRGC column systems (see Table 11). The chiral columns were made with the chiral selectors (PMCD, BSCD, or PECD) dissolved in the polysiloxane stationary phases (PS086 or OV1701). A commercial, achiral25-m SE54 fused silica column was also used. The actual column dimensions and operating conditions are listed in Table 11. All samples (2 pL in toluene) were oncolumn injected at 100 "C. Data acquisition and retention time measurements were started at 140 "C (PS086/BSCD, OV1701/PECD, SE54) or 120 OC (PSO86/PMCD). Enantiomers were denoted with annex-1 and annex-2 for earlier and later eluting enantiomers, respectively. Enantiomeric ratios (ER) were defined as ER = p d p z , whereby PI = peak area of enantiomer-1, and p2 = peak area of enantiomer-2. Average and range from two replicate measurements are reported. Compound identification was done initially on the achiral SE54 HRGC column using the reference compounds available and using published retention data (retention indexes), in particular the data reported by Dearth and Hites for the minor chlordane components (3). MC compounds refer to the work by Miyazaki et al.
Table 11. HRGC Columns Used column, chirality
polysiloxane, stationary phase
chiral selector, (contenta)
column material, dimensions
column tempb (rate)
1, chiral 2, chiral 3, chiral
PS086 PS086 OV1701 SE54
PMCD (10%) BSCD (25%) PECD (30%) none
glass, 20-m, 0.30-mm i.d. glass, 16-m, 0.30-mm i.d. fused silica, 12-m, 0.25-mm i.d. fused silica, 25-m, 0.32-mm i.d.
100-120-250 "C (2 "Cimin) 100-140-250 "C (3 "Cimin) 100-140-250 "C (3 "Cimin) 100-140-250 "C (3 OC/min)
4, achiral
Injection, intermediate, and final hold temperatures; program rate between 0 Concentration relative to polysiloxane stationary phase. intermediate and final hold temperature listed; program rate between injection and intermediate temperature, 20 "Cimin.
(26). These identifications were supported from full-scan E1 and ECNI mass spectra.
Results and Discussion Enantiomer Resolution Using Chiral HRGC and Detection by Mass Spectrometry. Several major chlordane components (heptachlor, cis- and trans-chlordane) and the metabolites HEP and OXY are asymmetric and, thus, exist in two enantiomeric forms. trans-Nonachlor and cis-nonachlor, additional constituents of technical chlordane, are achiral. Previously, the enantiomer resolution of several chiral compounds was achieved by chiral HRGC with 6-CD (torus with seven linked D-glucopyranose units) derivatives (PMCD, BSCD) as chiral selectors (I7, 18). Enantiomer resolution was different for each of the compounds with some more easily resolved than others. Some chiral compounds such as heptachlor still remained unresolved. Apparently, the stereochemistry of an analyte and of the chiral selector influences guest-host interaction and thus separation. Presently, no means are known to predict enantiomer resolution from structural considerations. An additional chiral HRGC column using an a-CD (torus with six linked D-glucopyranose units) derivative as a chiral selector (PECD) showed enantiomer resolution of heptachlor. All columns showed excellent chromatographic performance (high effective plate numbers, high inertness) and could be used in a broad temperature range (70-250 "C) for a wide variety of environmental pollutants. For instance, all polychlorinated biphenyls (PCBs, monoto decachlorinated congeners) were eluted under temperature-programmed conditions. Each chiral HRGC column had its capabilities for some enantiomer separations, but none resolved all enantiomers desired (see Table 111for enantiomer resolution of some analytes). For instance, heptachlor is only resolved on OVl'IOlIPECD, but this column neither resolved OXY nor component MC5. The latter compounds, however, were resolved on PS086/BSCD (18). Some components, e.g., cis-chlordane, are resolved on all columns to some degree but may coelute with enantiomers of other components. A careful selection of the column for a particular separation thus is necessary, and all three columns were finally required for an enantioselective determination of all compounds studied. In particular, PSO86/PMCD was used for the enantioselective determination of octachlordanes, PS086/BSCD for HEP and OXY, and OV1701/ PECD for heptachlor and others. Attempts to prepare chiral columns by mixing different chiral selectors were unsuccessful. For instance, a column (PS086) containing both PECD and BSCD did not separate the enantiomers of heptachlor and endo-HEP, although these compounds were enantiomerically resolved on columns with PECD or BSCD alone. Although the chromatographic performance of such columns was still excellent, chiral recognition apparently is not simply additive.
Table 111. Enantiomer Resolution (R Valuesa) of Selected Chiral Chlordane Compounds on Different Chiral HRGC Columns chiral HRGC columnb compd (chiral) heptachlor trans-chlordane cis-chlordane component MC5 component MC7 component U82 component MC6 HEP endo-HEP OXY photo-heptachlor photo-cischlordanes
PS086/PMCD PS086/BSCD OV1701IPECD 0.0 1.0 0.8 1.3 2.4 0.0 0.0 0.0 0.0 0.0 0.2 0.0
1.2 0.0 0.9 0.0 0.0 0.0
0.0 2.1 1.0 4.8 ndc 0.5 1.2 1.2 1.2 1.2 3.6 0.0
0.0
1.2 4.3 0.0 0.7 0.0
a R values defined as R = (tl - tz)/(wl+ WZ),whereby t l and tz are the retention times of enantiomer-1 and -2, respectively,and w1 and w2 are the peak widths at half-height; R = 0.0 indicates unresolved, R = 1 indicates approximately 95% resolved, and R > 1 indicates fully resolved enantiomers. Column dimensions and operating conditions, see Table I and text. nd = not determined. ~
~~
Because individual enantiomers were not available, the elution order of enantiomers and their absolute configurations are presently not known. Furthermore, the elution orders of enantiomers cannot be assumed to be identical on any chiral column (27). However, for cischlordane and HEP, and some biological samples enriched with one or the other enantiomer, we could establish that the elution orders were the same on OV1701/PECD and PS086/BSCD. Both ECNI and E1 MS were used for detection of chlordane components. Typically, Me- (ECNI) and (M C1)+ions (EI),and sometimes additional (e.g., retro-DielsAlder, RDA) fragment ions, were monitored. Several compounds, e.g., photoheptachlor and the photo-cischlordanes (see later), yield intense (M - C1)+ fragment ions in EI, but no M'- ions under our ECNI conditions. These photocompounds, however, can be monitored in ECNI using the (M - 2HC1)'- and (M - 3HC1)'- fragment ions (CIoH3C15,m/z 298), respectively, although these ions are less characteristic and are also formed from other chlorinated hydrocarbons. Most of the chlordane compounds investigated showed extensive fragmentation under E1 as well as under ECNI conditions. The extent of these fragmentations may differ significantly between isomers. The relative response among isomers and the isomer profiles observed for a particular sample may thus differ significantly depending whether E1 or ECNI was used. However,the mass spectra of enantiomers of chiral compounds are identical (E1mass spectra of the two enantiomers of heptachlor are shown in Figure la,b). Therefore, the relative response of Environ. Sci. Technol., Vol. 27,
No. 6, 1993
1213
10
1
100
100
'g a
roo,
c
LOO
272
I
BO
b
tC8
70
60
60
50
50
Ifcc*
40
40 30 20 10 0
/
331
237
M274 k
3bO
340
460
Table IV. Composition of Technical Chlordane Used
congener
heptachlor trans-chlordane cis-chlordane component MC5 component MC6 trans-nonachlor cis-nonachlor ratio trans-/cis-chlordane
C17 Cls Cls Cls c19 c19
wt %
c19
of total mixturen 10.0 f 0.8 14.5 f 0.3 13.0 f 0.3 3.6 f 0.1 0.5 f 0.1 6.0 f 0.4 1.6 f 0.3 1.12
a Data relative to reference compounds available; average response factors used for components MC5 (trans- and cis-chlordane) and MC6 (trans- and cis-nonachlor);average and range from two replicate determinations listed.
enantiomers and, thus, enantiomeric ratios are identical and, thus, independent of detection modes. Enantiomer Composition of Technical Chlordane and Synthetic Reference Compounds. Detailed analyses of some octa- and nonachlordanes in a technical chlordane and their enantiomeric composition were reported previously (17, 18). In Table IV we list the concentrations of the major components of this technical chlordane as determined from E1 SIM data in comparison to the reference compounds available. In Figure 2, E1 SIM chromatograms (mlz 272 and 373) show the elution of heptachlor and some octachlordanes in this sample using the chiral OV1701/PECD column. The chromatogram reveals the presence of both enantiomers of heptachlor as a racemic mixture (ER = 0.97 f 0.01). This column also separated cis-chlordane enantiomerically and confirmed its presence as a racemic mixture (ER = 0.99 f 0.021, but it did not resolve the enantiomers of trans-chlordane and component MC5. Previous data, however, showed that these components and other chiral components were present as racemic mixtures (ER values ranging from 0.95 f 0.05 to 1.01 f0.01) (17). A sample of heptachlor (purity >99%) was also found to be racemic (ER = 0.98 f 0.011, as were all the other chiral reference compounds available (see Experimental Section). The technical chlordane contained neither the epoxides (HEP, endo-HEP, and OXY) nor the photoproducts 1214
Envlron. Scl. Technol., Vol. 27, No. 6, 1993
I
Hh
Figure 1. E1 mass spectra of the two enantiomers of heptachlor (M'+ = mlz370, CI,) separated on the chiral OV1701lPECD HRGC column: (a) heptachlor-1 (earlier elutlng), (b) heptachlor-2 (later eluting enantiomer). Absolute configurations not known. Note the presence of RDA fragment Ions C5Clg*+(mlz 270) and C5H5CI'+(mlz 100).
component
MC5
Figure 2. E1 SIM chromatograms showing elution of (a) heptachlor (mlz 272) and (b) octachlordanes (mlz 373) In the technical chlordane mixture, using the chiral OV1701lPECD HRGC column. Note the resolution of heptachlor (ER = 0.97) and cis-chlordane (ER = 0.99) into enantiomers: trans-chlordane and component MC5 not enantiomerically resolved on this column. Abbreviations: cC8 and tC8 for cis- and trans-chlordane, hepta for heptachlor: for others, see text.
(photoheptachlor and photo-cis-chlordanes, see later) in detectable amounts (detection limits, 0.05 % of total mixture). The ratio trans-/cis-chlordane was 1.12 and, thus, close to the ratios reported for other technical materials (1,281. The absolute concentrations of these two constituents in our formulation (see Table 11),however, are somewhat lower than in others (28). Enzymatic Epoxidation of ChlordaneComponents. It is well-established that the cytochrome P-450-linked mixed function oxidase (MFO) system of most species is capable of epoxidizing unsaturated and aromatic compounds (29). Such metabolic reactions play a significant role in the formation of compounds with mutagenic activity from a whole series of xenobiotics. Using the enzyme system from the rat liver, we investigated the transformation of heptachlor and other chlordane components into epoxides. As biotic transformations, these processes likely are enantioselective. The MFO-catalyzed epoxidation of racemic heptachlor yielded HEP in yields up to 10% (2-h reaction time). Detailed analysis of the reaction mixtures using the chiral OV1701/PECD column revealed the formation of both enantiomers but with a significant excess of HEP-1. E1 SIM chromatograms (mlz 272 and 353; see Figure 3) show that predominantly heptachlor-1 was converted into HEP1. Assuming no further products being formed, the enantiomeric ratio of HEP (ER = 3.94) should be expected to be reciprocal to that of the remaining heptachlor (ER = 0.353; reciprocal value, 2.84). As indicated, this was roughly, though not exactly, observed. Surprisingly, similar ER ratios of heptachlor and HEP were found immediately after mixing (reaction time 1-2 min) and later in the experiment (up to 2 h), although overall conversion into HEP increased during these periods. A possible explanation of this finding may be that the reaction is inhomogenous (incompletely dissolved compounds, adsorption processes). Nevertheless, the data clearly confirmed the enantioselective nature of this enzymatic reaction. No endo-HEP was observed in these experi-
bepta-Z
3070
a
HEP-1
0 , 8:'OO
,
,
,
,
.
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,
.
,
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,
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.
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Flgure 3. E1 SIM chromatograms showlng enantioselectlve formatlon of HEP from racemic heptachlor by the rat MFO enzyme system (30mln sample analyzed), using the chiral OV1701IPECD HRQC column. (a) Chromatogram (mlz 272) showing depletion of heptachlor-1 (ER = 0.35); (b) chromatogram (mlr 353) showing predominant formatlon of HEP-1 (ER = 3.94); note the absence of endo-HEP. Abbreviations: see Figure 2 and text.
menta. Apparently the reaction is highly stereoselective leading exclusively to the exo(cis)-epoxide. The results prove that both enantiomers of HEP are biologicaltransformation products of heptachlor, and they clearly show that the rat liver MFO system is enantio- and stereoselective. Interestingly, HEP-1, the major product formed, was also the major enantiomer present in human adipose tissue, whereas in all the aquatic species investigated (fish, seal, penguin) HEP-2 was more abundant (18). The data provide evidence for preferential biotransformation routes among different species. Under our experimental conditions, none of the octaand nonachlordanes showed any formation of epoxides (HEP, endo-HEP, and OXY). However, hydroxylated derivatives were observed but not further investigated. Metabolic conversion of octa- and nonachlordanes into epoxides by the rat MFO appears to be much slower than that of heptachlor under these conditions. Photoconversion Products of Chlordane Compounds. The photochemical transformation of chlordane compounds has been reported in detail (30-35). These abiotic processes are expected to be nonenantioselective. Solid-filmphotolysis yielded late-eluting photoconversion products from heptachlor and cis-chlordane, which were identified by MS and retention data (31, 32) as photoheptachlor and photo-cis-chlordanes with caged and halfcaged structures, respectively (see Charts I1and 111).Both photoproducts are chiral. Analogous products were not observed from trans-chlordane or cis- and trans-nonachlor. Whereas it is known that the trans-series of compounds yield much smaller amounts of such photoproducts (33), the reason for their absence from cis-nonachlor in our experiments remains unknown. It should be pointed out that photodegradationof chlordane componentsmay also, via other pathways (e.g., reductive dechlorination),proceed to products not detected in our study. Heptachlor, when exposed to sunlight for 4 h, yielded photoheptachlor (-2%) and smaller amounts of HEP (-0.2%). In Figure 4a,b E1 SIM chromatograms (m/z
Flgure 4. E1 SIM chromatograms showing nonenantloselectlve formatlon of photoproductsfrom racemic heptachlor by natural sunlight (4-h exposed sample analyzed), uslng the chlral OVlPOllPECD HRQC column. (a) Chromatogram (mlr 268) showing elution of heptachlor and photoheptachlor, ER = 1-00 and 038, respectlvely; (b) chromatogram (mlr 353) showlng formatlon of HEP, ER = 0.99. Note the formatlon of trace amounts of embHEP. Abbrevlatlons: photo for photoheptachlor; for others, see Flgure 2 and text.
Chart 11. Formation of Photoheptachlor (Caged) from Heptachlor&
* CI
F'
CI
^.
sunlight
CI
*
c1-
Y
heptachlor photoheptachlor (chlral) (caged,chiral) a Intramolecular (2:2) cycloaddition reaction; cage formation through C2-C6and c3-c6 bond formations. CI
Chart 111. Formation of Two Isomeric Photo-cis-chlordanes (Half-Caged) from cis-Chlordanea
ciCI
CI@
cl@l CI
4
(2,5)-photo-cis-chlordane
'
CI
I/" CI CI
cis-chlordane
(chirai)
(half-caged,chiral)
sunlight
clj&z+ CI (1,5)-photo-cis-chlordane
(half-caged.chiral) Half-cage formation (among other possibilities) through C2-C5 and C & ,bond formations, respectively.
268 and 353)using the chiral OV1701/PECDcolumn show the separation of heptachlor, photoheptachlor, and HEP into their enantiomers. Although enantiomer resolution of photoheptachloron this column is incomplete, a racemic mixture is indicated. This was confirmed by reanalysis on PS086/BSCD,which fully separated these enantiomers. Envlron. Scl. Technol., Vol. 27, No. 6. 1993
1216
Figure 5. E1 and ECNI mass spectra of photoheptachlor and photo-c/s-chlordane, using the chlral PS086lBSCD HRGC column. (a, c) E1 and ECNI mass spectra of photoheptachlor-1 (M"', M'- = mlz 370), respectively, (b, d) E1 and ECNI mass spectra of first-eluting photo-c/s-chlordane isomer (M", Ma- = mlz 406), respectlvely. Note presence of intense (M CI)+ fragment ions (mlr 335 and 371, respectlvely) In EI, and (M - 2HCI)'- and (M - 3HCI)- fragment ions (mlz 298) in ECNI, respectively. Note also the presence of RDA fragment Ions at mlz 270 (C5Cls)'+ and mlz 100 (CsH5CI)'+ in EI.
-
The results prove that both photoproducts are formed in enantiomeric ratios of 1:1, consistent with a nonenantioselective transformation of racemic heptachlor. The chromatogram in Figure 4b also revealed the presence of small amounts of endo-HEP (=O.Ol%), present as a racemate. The photochemical oxidation of heptachlor apparently leads to the exo(cis)-epoxide with very little of the endo(trans)-isomer formed. The two enantiomers of photoheptachlor gave identical E1 and identical ECNI mass spectra. In Figure 5a,c we show E1 and ECNI mass spectra of one of these enantiomers (photoheptachlor-1); neither of the spectra shows the presence of molecular ions. The most intense ions in E1 and ECNI were (M - C1)+ (mle 335, C16) and (M 2HC1)'- (m/z 298, C15), respectively. As observed with heptachlor, the E1 mass spectrum (Figure 5a) shows the presence of RDA fragment ions (mlz 100and 270),although at lower intensity. In case of photoheptachlor, these RDA fragmentations require prior breakage of the cage-forming bonds. Octa- and nonachlordanes, when exposed to sunlight for up to 24 h total exposure, yielded no detectable quantities of HEP, endo-HEP, and OXY (detection limits 0.05%). Apparently photoconversion of the octa- and nonachlordanes to these epoxides is negligible under our experimental conditions. cis-Chlordane when exposed to sunlight for 24 h yielded twolate eluting photoproducts in an approximate 2:l ratio (PSOSG/BSCD,see Figure 7a, later). These two compounds gave practically identical E1 and identical ECNI mass spectra. Reanalysis on the achiral SE54 column also revealed two partially resolved peaks, indicating that the two compounds are isomeric rather than enantiomeric. According to the reaction mechanism proposed (32-341, several isomeric photoproducts are possible which are formed via CZ-CS,Cz-Ce, c1-c5, and C& bridging, all with intramolecular hydrogen transfer. All these structures are asymmetric, and the compounds are thus chiral. Previous work (33,34)suggested that the major products are formed via CZ-Cs and ( 2 1 4 5 bridging (see Chart 111). However, presently we are unable to assign the exact 1216
Environ. Sci. Technol., Vol. 27, No. 6, 1993
structures due to the lack of reference materials. The two isomers are well-separated on all three chiral HRGC columns whereby the elution orders were reversed on PS086/PMCD and OV1701IPECD. None of the chiral columns separated the isomers enantiomerically. E1 and ECNI mass spectra of the first eluting photocis-chlordane (PSO86/BSCD) are shown in Figure 5b,d. As with photoheptachlor, no molecular ions were observed for both of the photo-cis-chlordanes. In EI, the major fragment ion is (M - C1)+; RDA fragment ions (m/z 100 and 270) are present. In ECNI, the major fragment ion is (M - 3HC1)'- at m/z 298 (C10H&lb*-). There are thus similarities between the mass spectra of photoheptachlor and those of the photo-cis-chlordanes. Photoheptachlor and the photo-cis-chlordanes are isomeric to the parent compounds. Monitoring these photocompounds in E1 using the (M - C1)+ ions is more selective than when using ECNI, because the most abundant fragment ions in ECNI (C10H&lb*-, m/z 298) are relatively noncharacteristic (see above). Due to the absence of Me- ions in ECNI, these compounds were not detected in our earlier studies on octa- and nonachlordanes (17, 18). Technical chlordane when exposed to sunlight for 16 h yielded photoheptachlor (=0.5%) and HEP (-0.5%) in enantiomeric ratios of 1:land the two photo-cis-chlordanes (-1%)in a 2:l ratio. About 70% of the heptachlor was decomposed. Additionally, a very small amount of OXY (=0.05%) was formed as a racemate whereby its source remained unknown. Additional products were detected but not yet investigated due to the complexity of the chromatograms obtained. Occurrence of Chlordane Components in Ambient Air. Four ambient air samples from Norway were analyzed enantioselectively. Previous analyses of the same samples revealed the presence of cis- and trans-chlordane and other chlorinated hydrocarbons (a- and 7-HCH, HCB) in a typical summer pattern (35). The samples were judged to be representative for the sampling time and sampling location.
Table V. Amountsa (pg) of Chlordane Components Detected In Ambient Air (Norway, Aug/Sept 1991) and Enantiomeric Ratios of Chiral Components Detected
compoundb
no. 1,8111-12191
heptachlorc$ trans-chlordanecsd cis-chlordanec,e trans-nonachlor cis-nonachlor ratio trans-/cis-chlordane
1600 (0.97) 900 (0.97) 2300 (0.97,0.99)
sample (date) no. 2,8/15-16/91 no. 3,8/22-23/91 1000 (1.02)
700 (0.99,1.02) 1200 (1.02, 1.04)
1600
300 0.37
1000
400 0.63
3700 (0.92, 1.03) 3100 (0.99,0.94) 4700 (0.99) 2800 400 0.66
no. 4,9115-16/91 500 (0.98) 200 (0.98,0.98) 400 (1.06) 300
300 ~0.5
Amounts in pg of the total sample (about 500 m3) as received; small but unknown aliquot removed for prior analyses; detection limits, -100 pg in total samples, 20.2 pgim3. * Additionally, components MC5, MC6, and U82 detected, but not quantified. Enantiomeric ratios determined on OVli'Ol/PECD; trans-chlordane on PS086IPMCD. d ECNI SIM data. e E1 SIM data.
Flgure 6. E1 SIM chromatograms showing elution of (a) heptachlor (mlz 272) and (b) octachlordanes (mlz 373) in ambient air, Norway, using the chiral OV 1701/PECD HRGC column. Note the presence of racemic heptachlor, ER = 1.03, and racemic cis-chlordane, ER = 0.99;transchlordane, not enantiomerically resolved on this column. Abbreviations: see Figure 2 and text.
The samples were reanalyzed using all three chiral HRGC columns and using E1 and ECNI. The presence of cis- and trans-chlordane was confirmed, and additional components of technical chlordane were detected by one or the other technique. In particular, heptachlor, cis- and trans-chlordane, and trans-nonachlor were detected by EI, and components MC5, MC6, and U82 and cisnonachlor were detected additionally by ECNI. These findings leave little doubt that all compounds originate from technical chlordane. In Table V we list the approximate amounts of the major chlordane components detected in the four samples. The ratio trans-/cis-chlordane in these samples ranged from 0.37 to 0.66 and was thus in the range listed for summer ambient air (22). These ratios are lower than the ratio in the technical material (ratio 1.12, see above) and indicate depletion of trans-chlordane during the summer period, presumably by photolysis (22). In Figure 6, E1 SIM chromatograms (OV1701/PECD; m/z 272, 373) show the presence of heptachlor and octachlordanes in one of the samples. Both, heptachlor and cis-chlordane are enantiomerically resolved, and enantiomeric ratios of 1:lare indicated. trans-Chlordane is not enantiomerically resolved on this column, but reanalysis on PS086/PMCD confirmed its presence as a racemate (see Table IV for enantiomeric ratios in each sample). The chromatograms suggested components MC5 and MC6 present as racemates, but levels were too low for an accurate determination of enantiomeric ratios. Signals from additional components are apparent in the chromatograms. These data indicate that all chiral chlordane components are present in Scandinavian ambient air in enantiomeric ratios of 1:l. This finding suggests that mainly abiotic processes such as photolytic or chemical
degradation are responsible for the changed isomeric composition of these chlordane components, particularly for the decreased levels of trans-chlordane during the summer periods. Occurrence of ChlordaneCompoundsin Biological Environmental Samples. The presence of octa- and nonachlordanes, HEP and OXY, and the enantiomeric composition of some chiral compounds in aquatic species from the Baltic (fish, grey seal) and Antarctica (penguin) and in human adipose tissue were previously reported (17, 18). Changed isomeric profiles with some minor components of the technical mixture showing much higher abundance in the biological samples were observed. Changed enantiomeric ratios of some of the components were ascribed to enantioselective biological processes taking place prior to or after uptake by the species (17). None of the species showed the presence of endo-HEP (18).
Harp seal from Greenland revealed now a very similar though not identical isomer composition as previously observed for grey seal from the Baltic (17,18). A very similar enantiomeric composition of the major chiral components (component U82, HEP, OXY) was indicated, despite the fact that the two species originate from largely different geographical areas. In Figure 7b-d, E1 SIM chromatograms (PS086/BSCD; m/z 373) of aquatic species (salmon, harp seal, penguin) show the elution of octachlordanes and other components (see below). The isomer profiles of salmon and penguin shown here differ from those shown earlier (18),because earlier ECNI was used for detection. Heptachlor was not detected in any of these samples. This is not unexpected since heptachlor is rapidly converted into HEP and other metabolites by most organisms. It is generally not detected in aquatic species (36). Photoheptachlor, however, was detected by E1 SIM (m/z 335,337) using PS086/PMCD in all aquatic species with smaller amounts in the fish (herring, salmon) and increased amounts in the warm-blooded species (harp and grey seal, penguin); trace amounts were also detected in human adipose tissue. Full-scan E1 mass spectra confirmed its presence in all samples. As an example, the E1 mass spectrum of photoheptachlor thus identified in penguin is shown in Figure 8a. The total ion chromatogram indicated its presence in this particular sample at a concentration level of about 20 nglg. PS086/PMCD did not separate the two enantiomers of photoheptachlor but was still used because less interference from other, coeluting components was experienced (component US2 still partially coeluting). On PS086/ BSCD, good enantiomer resolution was observed, but Environ. Sci. Technol., Vol. 27, No. 6, 1993 1217
photo-cis
n
0:
,
,
, , , ,
20100
, , ,
22100
,
24100
100
~~
x
Lb,+
26100 -x4.00
28:OO
I
cC8-2
7
1
photo-cis
:: U82 20 10 0
20:oo
A
, , , ,
22100
, ,
24100
,
,
,
,
,
26100 -x2.00
,
, ,
, , , , ,
28i00
,
30:OO
,
, , ,
TIME
Figure 8. E1 mass spectra of (a) photoheptachlor (M*+ = m/z 370; not enantiomerically resolved), identified in Antarctic penguin, and (b) photo-cis-chlordane (M" = m/r 406; first-elutlng isomer) identified In harp seal, Greenland, using the chirai PSO86/BSCD HRGC column. Note the presence of signals from a coelutlng pentachlorobiphenyl (Ma+ = m/z 324) in spectrum b.
Figure 7. E1 SIM chromatograms (m/z 373) showing elution of octachlordanes and photo-cis-chlordanes, using the chiral PS086/ BSCD HRGC column, in (a) referencematerial (24-h sunlight photolyzed cls-chlordane), (b) Baltlc salmon, (c) Arctic harp seal, Greenland, and (d) Antarctic penguin. Note the presence of the two photo-cischlordanes in all aquatic species. Signals for nonachlors present in chromatograms b-d. Abbreviations: photo-cis for photo-cis-chlordanes; for others, see Figure 2 and text. Peak marked by X comprized of trans-chlordanel, cls-chlordane-1, and MC5-2.
photoheptachlor-1 was interfered by trans-nonachlor, and photoheptachlor-2 by component MC6 (see E1 SIM chromatograms mlz 337 in Figure 9a-c). Enantiomeric ratios of photoheptachlor could thus not be determined with precision. However, preliminary data indicate that photoheptachlor-2 is more abundant in penguin (see Figure 9c). An enantiomeric ratio different from 1:lin this sample and the fact that racemic photoheptachlor is formed by photolysis indicate that this compound still underwent some biological degradation. The two isomeric photo-cis-chlordanes were detected in all aquatic species (see E1 SIM chromatograms mlz 373 using PS086/BSCD in Figure 7) in a ratio very similar to that observed from the direct sunlight photolysis of cischlordane (see Figure 7a). Full-scan E1 mass spectra confirmed the presence of both compounds, as illustrated in Figure 8b for the major isomer in Arctic seal. Photocis-chlordanes, however, were not detected in human adipose tissue. The concentration of photo-cis-chlordanes in the aquatic species was estimated at about one-tenth of the total concentrations of octachlordanes (5-80 nglg, see ref 17). 1218 Envlron. Sci. Technol., Vol. 27, No. 6, I993
Flgure 9. E1 SIM chromatograms (mlz 337) showing the presence of photoheptachlorin(a) Baltic herrlng,(b)Baltlcsalmon, and (c)Antarctic penguin, usingthe PSO86/BSCD HRGC column. Notethat enantiomer-1 and -2 are resolvedbut interfered from trans-nonachlor and component MC6, respectively. Abbreviations: see Figures 2 and 4, and text.
Conclusions Application of chiral HRGC toward the enantioselective detection of chiral chlordane compounds in environmental samples is again documented. The chiral columns used showed excellent gas chromatographic behavior and allowed the analysis of a broad range of environmental contaminants. Each column had its merits, but none resolved the enantiomers of all the chiral chlordane compounds studied.
Air transport is considered to be one of the principal routes of entry of anthropogenic compounds into remote areas such as the Arctic and Antarctic (cold-trapping in polar regions) (IO). The finding of chlordane components in ambient air from nordic locations provides an atmospheric link for the entry of these compounds into such an environment; it may also point to current uses of the pesticides. All chlordane components (heptachlor, octachlordanes) detected in ambient air were present as racemic mixtures, suggesting that only abiotic (chemical, photochemical) processes are involved in the decomposition of some of the components such as trans-chlordane during long-range transport in the atmosphere. However, we were unable to detect photoconversion products of chlordane components in these samples. Heptachlor may originate from two sources, technical chlordane and as a pesticide of its own. Both sources are racemic. As previously pointed out (51, relative contributions from the two sources are difficult to assess because of the similar-use pattern of the products. The finding of similar proportions of heptachlor relative to the octa- and nonachlordanes in ambient air as in technical chlordane may suggest technical chlordane as a primary source of heptachlor. Heptachlor was not detected in any of our biological samples, which is consistent with its rapid metabolization to HEP and other metabolites. Photoheptachlor and two isomeric photo-cis-chlordanes were detected in biota from the Baltic, the Arctic, and the Antarctic regions. Previously, photoheptachlor has been tentatively identified (ascomponent U1) in similar samples (5),but to the best of our knowledge photo-cis-chlordanes were not previously reported in aquatic samples. The finding of these photoproducts in biota from areas remote to most sources of these chemicals is interesting. Their presence clearly shows that photochemical reactions play a role in the transformation of chlordane components in the environment. The much lower levels of such photoproducts in human adipose tissue may point to a different route of exposure of this individual than of the aquatic species and to exposures more closely to a source of technical chlordane, possibly in chlordane-treated homes. Both the caged (photoheptachlor) and the half-caged products (photo-cis-chlordanes) appear to be much more accumulating in biota and more stable under environmental conditions than the parent compounds, heptachlor and cis-chlordane. Additionally, the photoproducts reportedly have higher acute toxicities to several vertebrate species than the parent compounds (37), which further increases their importance. The results show that HEP is an enzymatic as well as a photochemical product of heptachlor whereby predominantly one enantiomer (HEP-1)is formed by the biological pathway and racemic HEP by the photochemical pathway. Both pathways are highly stereoselective leading to the exo(cis)-isomer. It remains difficult to estimate the relative contributions of the biological and the photochemical conversion to the residues of HEP observed in the aquatic species. Significant contributions from both such processes were implicated in the environmental fate of other marine pollutants (hexachlorocyclohexane isomers) (38). Previously, HEP was largely attributed to in vivo epoxidation of heptachlor in animal tissues (14).This assumption is supported by the finding that HEP in all aquatic species and in the human tissue is present not as a racemic mixture but in enantiomeric ratios clearly
different from 1:l. OXY was observed in all aquatic species and in human adipose tissue. OXY is a major metabolite of octa- and nonachlordanes in many organisms and in cell cultures, and enantioselectivity of its formation was indicated through the metabolism of trans-chlordane in pigs (39). However, OXY was not observed as a metabolite of octaand nonachlordanes in our study using rat liver homogenate. We do not yet know which cytochrome P-450linked enzyme system is required for this metabolism and whether induction of these enzymeswill lead to measurable quantities of OXY. Additional investigations are required to obtain amore profound knowledge on such mechanisms. Acknowledgments We thank C. Rappe, Institute of Environmental Chemistry, University of Umeb, Umeb, Sweden, for the aquatic and the human adipose tissue samples. The receipt of the ambient air samples and the harp seal sample from M. Oehme, NILU, Lillestrerm, Norway, is gratefully acknowledged. We also thank H. Poiger, Institute of Toxicology, University of Zurich, Switzerland, for discussions and carrying out the described enzymatic reactions, and H. Seed, VG Analytical, Manchester, England, for construction of a modified ECNI source. Literature Cited WHOIIARC. Occupational Exposures in Insecticide A p plication, and some Pesticides; IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 53;World Health Organization, International Agency for Research on Cancer: Lyon, 1991;pp 115-177. Savage, E. P.Rev. Enuiron. Contam. Toxicol. 1989,110, 117. Dearth, M. A.; Hites, R. A. Enuiron. Sci. Technol. 1991,25, 245-254. Andersson, 0.; Linder, C.-E.; Olsson, M.; Reutergirdh, L.; Uvemo, U.-B.; Wideqvist, U. Arch. Enuiron. Contam. Toxicol. 1988,17,155-765. Norstrom, R. J.;Simon, M.; Muir, D. C. G.; Schweinsburg, R. E. Enuiron. Sci. Technol. 1988,22,1063-1071. Muir, D. C. G.; Norstrom, R. J.; Simon, M. Enuiron. Sci. Technol. 1988,22,1071-1079. Kawano, M.; Inoue, T.; Hidaka, H.; Tatsukawa, R. Chemosphere 1984,13,95-100. Gregor, D. J.; Gummer, W. D. Enuiron. Sci. Technol. 1989, 23,561-565. Bidleman, T. F.; Wideqvist, U.; Jansson, B.; Soderlund, R. Atmos. Enuiron. 1987,21,641-654. Bidleman, T.F.; Patton, G. W.; Walla, M. D.; Hargrave, B. T.; Vass, W. P.; Erickson, P.; Fowler, B.; Scott, V.; Gregor, D. J. Arctic 1989,42,307-313. Oehme, M.; Mano, S. Fresenius Z. Anal. Chem. 1984,319, 141-146. Atlas, E.; Giam, C. S. Science 1981,211,163-165. Nomeir, A. A,; Hajjar, N. P. Rev. Enuiron. Contam. Toricol. 1987,100,1-22. Lu, P.-Y.; Metcalf, R. L.; Hirwe, A. S.; Williams, J. W. J. Agric. Food Chem. 1975,23,961-973. Miyazaki, A.; Sakai, M.; Marumo, S. J. Agric. Food Chem. 1980,28,1310-1311. Miyazaki, A.; Hotta, T.; Marumo, S.; Sakai, M. J. Agric. Food Chem. 1978,26,915-971. Buser, H. R.; Miiller, M. D.; Rappe, C. Enuiron. Sci. Technol. 1992,26,1533-1540. Buser, H. R.; Muller, M. D. Anal. Chem. 1992,64,31683175. Keim, W.; Kohnes, A.; Meltzow, W. J. High Resolut. Chromatogr. 1991,14,507. Environ. Sci. Technol., Voi. 27, No. 8, 1993
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Received for review November 16, 1992. Revised manuscript received March 2, 1993. Accepted March 3, 1993.
