Environ. Sci. Technol. 1996, 30, 1032-1037
Determination of Non- and Mono-ortho-Polychlorinated Biphenyls in Background Ambient Air A. LOPEZ GARCIA,† A. C. DEN BOER,‡ AND A . P . J . M . D E J O N G * ,‡ Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/ Julia´n Claverı´a No. 8, 33006 Oviedo, Spain, and Laboratory of Organic-Analytical Chemistry, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The Nederlands
An analytical procedure has been developed for the isomer-specific determination of polychlorinated biphenyls in background ambient air. The methodology uses Carbosphere activated carbon and isotope dilution HRGC/MS for the isolation and analysis of the toxic fraction of PCBs in the presence of all other, less or nontoxic congeners. This toxic fraction includes the non-ortho- and mono-ortho-PCBs, which recently were assigned by a WHO/IPCS expert group on toxicity as being the most toxic congeners. The method provides quantification limits ranging from 0.01 to 0.08 pg/m3. Mean atmospheric levels for the sum of these congeners varied between 3.0 and 5.0 pg/ m3 with some events with higher concentrations. PCB 126 typically present at 0.03 pg/m3 was found to contribute 77% of the total toxic equivalent values in air samples.
Introduction Polychlorinated biphenyls (PCBs) constitute a complex group of 209 congeners ranging from monochloro- to decachlorobiphenyl. The unique physical and chemical properties of PCB mixtures have led to an extensive use of these compounds in many industrial applications (1). Unfortunately, these properties coupled with the widespread use and improper disposal management have resulted in the contamination by PCBs of almost any compartment of the global ecosystem (2-6). This prevalent contamination by PCBs is due in part to their stable chemical nature and the fact that they can survive longrange transport through the atmosphere being an important pathway for the global distribution of these contaminants (7-11). Therefore, data on the occurrence of PCBs in the atmospheric environment are essential for the assessment * To whom correspondence should be addressed; fax: +31-30274-4424. † University of Oviedo. ‡ National Institute of Public Health and Environmental Protection.
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of regional air quality as well as for the implementation of clean air legislation and risk management programs. It has long been recognized that the selective congener analytical approach provides the most meaningful information regarding exposure assessment (12). Recent studies of structure-activity relationships in the toxicities of PCB congeners have revealed the profound effect of the chlorinated substitution patterns on the toxic response and on the induction of drug metabolizing enzymes in mammalian and avian species (13, 14). PCBs having none or one chlorine in the 2,2′ or 6,6′ (ortho) position of the phenyl ring and one or more m- and p-chlorines on each ring can assume a planar configuration (15), which makes them approximate stereoisomers of 2,3,7,8-tetrachlorodibenzop-dioxin (2,3,7,8-TCDD), the most toxic synthetic compound known (16). They are capable of producing the same type of toxic responses, such as dermal lesions, body weight loss, thymic atrophy, hepatic damage, immunotoxicity, teratogeniticy, reproductive deficits and carcinogenicity, and tumor promotion (17, 18). Collectively, polychlorinated dibenzo-p-dioxins and dibenzofurans and these dioxinelike PCBs elicit their toxic effects through a common mode of action, including the cytochrome P-450 1A1 and P-450 1A2 hemoproteins and their associated microsomal monooxygenases, which include aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities (19). It is well known that dioxin-like compounds appear as complex mixtures of congeners both in environmental and biological materials. In order to simplify risk assessment and regulatory control of toxic PCB congeners in those samples, the concept of toxic equivalents (TEQ) has been introduced. Each of the non-ortho- and mono-ortho-PCBs has been assigned a toxicity equivalent factor (TEF) based on its toxicity relative to 2,3,7,8-TCDD, which is assigned a TEF value of 1.0. The TEQ value of individual congeners in samples is obtained from the concentration found in the sample multiplied by its assigned TEF value. The total toxicity value follows from their sum or TEQ ) ∑ci TEFi, where ci is the concentration of individual PCB congeners and TEFi is the corresponding toxic equivalent factor. A TEF model for PCBs has recently been proposed by a toxicological expert group (20) in the framework of the International Programme on Chemical Safety of the World Health Organization (WHO/IPCS). Congeners included in this scheme together with their TEF values are shown in Table 1. This model is commonly used to determine the relative contribution of PCBs, PCDDs, and PCDFs to the overall ‘dioxin-like’ activity of halogenated aromatic contaminants in environmental samples (21) in vegetable oil (22) and in air samples (23-25). Two factors complicate the analysis of these toxic compounds in the environmental and biological matrices: (i) poorly resolved clusters of congeners on most commercial GC columns and (ii) concentrations ranging over several orders of magnitude lower than the most abundant congeners. In order to solve these problems, several analytical methods for isolation and determination of the non- and mono-ortho-PCB congeners have been described in the literature (26-29), with off- or on-line carbon chromatography being most frequently used.
0013-936X/96/0930-1032$12.00/0
1996 American Chemical Society
TABLE 1
Assigned Toxic Equivalent Factors for Polychlorinated Biphenyls (20) IUPAC No.
structure
TEF
77 126 169
Non-ortho 3,4,3′,4′-TCB 3,4,5,3′,4′-PeCB 3,4,5,3′,4′,5′-HxCB
0.0005 0.1 0.01
105 114 118 123 156 157 167 189
Mono-ortho 2,3,4,3′,4′-PeCB 2,3,4,5,4′-PeCB 2,4,5,3′,4′-PeCB 3,4,5,2′,4′-PeCB 2,3,4,5,3′,4′-HxCB 2,3,4,3′,4′,5′-HxCB 2,4,5,3′,4′,5′-HxCB 2,3,4,5,3′,4′,5′-HpCB
0.0001 0.0005 0.0001 0.0001 0.0005 0.0005 0.00001 0.0001
170 180
Di-ortho 2,3,4,5,2′,3′,4′-HpCB 2,3,4,5,2′,4′,5′-HpCB
0.0001 0.00001
In this paper, we present an isotope dilution, highresolution gas chromatography/medium-resolution mass spectrometric method based on the method for the analysis of toxic coplanar PCBs, PCDDs, and PCDFs in milk described by van der Velde et al. (30). The modified method was applied to the environmental monitoring of non- and mono-ortho toxic PCBs in ambient air. Individual PCB congener concentrations obtained from the present study were used to determine their relative contribution to the total TEQ value. To our knowledge, these data represent the first time that TEQ quantities for airborne PCBs are reported.
Experimental Section Sample Collection. Air samples were taken in Bilthoven, a suburban residential area about 5 km east of the city of Utrecht, The Netherlands. The location was selected to establish common atmospheric concentrations for the toxic non- and mono-ortho-PCB congeners in residential not extra ordinary industrialized areas in the country. Air volumes of about 1000 m3 (60-70 h sampling time) were collected using a high-volume sampler LIB/P type III (Stranovsky GmbH, Essen, Germany) designed for simultaneous particulate and vapor collection. The sampler head was equipped with a Gelman A/E glass fiber filter (GFF) (Gelman Sciences, Inc., Ann Arbor, MI), where particles larger than 1 µm in diameter were retained, followed by two 11 cm diameter × 5 cm thick and 25 kg/m3 density polyurethane foam (PUF) plugs (Sunda & Co., Soesilfabrikk for Skumplastartikler, Gan, Norway) used to adsorb vapor or aerosol phase compounds. The second PUF plug was employed to check for possible breakthrough. Foam plugs were cleaned before use by Soxhlet extraction with dichloromethane (48 h) and petroleum ether (8 h), whereas glass fiber filters were used without any prior treatment. Prior to sampling, five 13C-labeled standards (Cambridge Isotope Laboratories, Woburn, MA) in amounts of 0.25 ng for [13C12]PCB 126 and 169 and 1.0 ng for [13C12]PCB 77, 118, and 105 in 100 µL of toluene/tetradecane (1:1, v:v) were applied to the GFF filter to control losses during the sampling and analytical procedure and to determine the overall recovery. Sample Extraction and Cleanup. Sample Extraction. After sample collection, the front PUF plug and the GFF
filter were carefully removed from the sampling head, combined, and extracted with dichloromethane (300 mL) for 16 h using a Soxhlet reflux apparatus. The second PUF plug and a laboratory blank, consisting of the clean and unused filter and plug, were extracted and analyzed parallel to each individual sample. Extracts obtained were reduced in volume to approximately 5 mL using a Kuderna-Danish apparatus and were subsequently extracted on a carbon column to isolate the toxic non- and mono-ortho-PCBs from non-planar PCBs. Sample Cleanup. The non (planar) and mono-orthoPCBs were separated from all other PCBs on a column packed with 1.2 g of Carbosphere (activated carbon, 80100 mesh, Chrompack, Middelburg, NL). Prior to use, columns filled with Carbosphere were cleaned by refluxing for 16 h with 25 mL of toluene and subsequently dried with a stream of nitrogen. Next, samples (5-mL extracts in dichloromethane) were quantitatively transferred to the columns, using a glass container attached to the top of the columns, followed by two 5-mL volumes of rinsing solvent of both sample tubes and containers, and they subsequently were washed with 130 mL of dichloromethane. In this step, the di- to tetra-ortho-PCB congeners are eluted and discarded. After being dried with a stream of nitrogen, the column was placed in a soxhlet reflux unit and back-flush extracted with 25 mL of toluene for 4 h to recover the nonand mono-ortho-PCB fraction. This fraction was concentrated down to 1-2 mL by a nitrogen stream in isomantle at 110 °C and then carefully evaporated to dryness under a gentle stream of nitrogen at room temperature. Next, the residue was reconstituted in 5 mL of hexane and further purified on an alumina column as described by van der Velde et al. (30). The final extract was evaporated to dryness and redissolved in toluene (100 µL) containing 10 ng/mL of a syringe standard ([13C12]PCB 80). An aliquot of 1 µL was normally used for GC/MS analysis. Gas Chromatography/Mass Spectrometry Parameters. GC/MS analyses of non- and mono-ortho toxic PCB congeners were carried out on a VG70SQ mass spectrometer (VG Analytical, Manchester, U.K.) connected to a Hewlett Packard HP5890 gas chromatograph (Hewlett Packard, Palo Alto, CA). Instrumental control, data acquisition, and data processing were done by a VG 11/250 data system (VG, Manchester, U.K.). Gas chromatographic separation of samples was carried out on a 50-m fused silica capillary column of 0.2 mm i.d., coated with a 0.11-µm film of 5% diphenyl polydimethylsiloxane (HP Ultra-2, Hewlett Packard). Helium was used as carrier gas at a linear velocity of 30 cm/s. An aliquot of 1 µL of the sample was injected using an all-glass falling needle injector (solid injector, Fa. Koppens, Best, The Netherlands). The injection port temperature was 275 °C. The GC/MS interface temperature was set to 290 °C. The GC was temperature programmed from 70 to 200 °C at a ramp of 25 °C/min, then from 200 to 250 °C at 3 °C/min, hold for 5 min, and finally from 250 to 290 °C at 5 °C/min and maintained isothermal for 3 min. The mass spectrometer was operated in electron impact ionization mode with an ionization energy of 31 eV and a source temperature of 275 °C. The resolution (10% valley) was 3000 at m/z 381.98 of perfluorokerosene (PFK). Quantification of Non- and Mono-ortho Toxic PCBs. The analytes were detected by recording the two most abundant ions of the molecular chlorine isotope cluster of PCBs, their 13C -labeled analogues and the syringe standard [13C ]12 12 PCB 80 (Table 2). The total number of ions to be analyzed
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TABLE 2
TABLE 3
Ions and m/z Values Used for Analysis of Native and Deuterated PAHs with GC/MS Selected Ion Monitoring
Average Recovery for Some Di-ortho-, Mono-ortho-, and Non-ortho-PCB Congeners from the Carbosphere Column
compound
ion
m/z
PCB 105, 114, 118, 123, and 126
M M+2 M+2 M+4 M+2
289.9224 291.9195 325.8805 327.8775 359.8415
M+4 M+2 M+4 M M+2 M+2 M+4 M+2 M+4
361.8386 393.8025 395.7996 301.9626 303.9597 337.9207 339.9177 371.8817 373.8788
PCB 77
PCB 156, 157, 167, and 169 PCB 189 [13C12]PCB 77, 80 [13C12]PCB 105, 118, and 126 [13C12]PCB 169
isotope ratio 0.78 1.55 1.24 1.04 0.78 1.55 1.24
was divided over three groups of six ions (excluding one ion from PFK in each group serving as a lock or reference mass) each. Quantification was carried out by comparison of the intensity ratio of native PCBs to the corresponding 13C -labeled standards as obtained in samples and in a 12 standard mixture; respectively PCBs 77, 126, 169, 118, and 105 were quantified relative to their stable isotope analogue. PCBs 123 and 114 were quantified relative to [13C12]PCB 118; PCB 167 was quantified relative to [13C12]PCB 126; and PCBs 156, 157, and 189 were quantified relative to [13C12]PCB 169. Prerequisites for positive identification of congeners of interest were as follows: (a) The retention time of the analyte should match ((1 s) the retention time of its stable isotope labeled analogue or the corresponding homologue. (b) The isotope ratio should be within (10% of theoretical value of the chlorine isotope distribution pattern. (c) The response for both the analyte and the labeled analogues should have a signal/noise ratio of 3:1 or greater.
Results and Discussion Evaluation of PCB Fractionation Procedure. The binding affinity of chlorinated aromatic compounds to the activated surface of carbon is attributed to the planar structure of these compounds. It has been shown that planar compounds like polychlorinated dioxins and furans bind very strongly to the activated surface of carbon particles. Similar properties were found for PCBs possessing a quasi-planar configuration like the laterally (PCBs 77, 126, and 169) and the mono-ortho-substituted congeners. Coplanarity will decrease by substituents at the ortho positions (2,2′ or 6,6′) of the biphenyl ring system. Mono-ortho-substituted PCBs, however, demonstrated some affinity sufficient enough to separate them from congeners having two or more substituents at the ortho positions. In addition to coplanarity, the retention on activated carbon is governed by the number of electronegative substituents on the biphenyl skeleton (31). Thus, a mixture of standards containing non-, mono-, and di-ortho-PCBs, having four to seven chlorine substituents, was used to establish the elution pattern from the Carbosphere activated carbon column. The percent recoveries for the various PCBs in the dichloromethane fraction and in the toluene fraction are given in Table 3.
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recovery (%)
congener IUPAC No.
type
amount (ng)
fractiona
fractionb
PCB 52 PCB 74 PCB 60 PCB 101 PCB 77 PCB 118 PCB 114 PCB 153 PCB 105 PCB 138 PCB 126 PCB 167 PCB 156 PCB 157 PCB 180 PCB 169 PCB 189
di-ortho mono-ortho mono-ortho di-ortho non-ortho mono-ortho mono-ortho di-ortho mono-ortho di-ortho non-ortho mono-ortho mono-ortho mono-ortho di-ortho non-ortho mono-ortho
175 78 51 112 40 92 59 109 65 92 36 52 37 48 79 41 32
78.9 NDc ND 78.2 ND ND ND 73.3 ND 67.1 ND ND ND ND 63.9 ND ND
3.7 69.1 72.4 6.6 79.7 78.3 80.1 9.4 85.5 9.4 91.6 85.5 87.0 87.1 14.9 90.7 89.4
a Wash of column (130 mL of dichloromethane). extract in toluene. c ND, not determined.
b
Back-flush reflux
The data show clearly that the non- and mono-orthoPCBs are fully retained by the column after being washed with dichloromethane. However, some di-ortho-PCBs could not be completely removed by this treatment, particularly the higher substituted congeners. As the affinity to the carbon surface increases with increasing chlorine substituents, this will give rise to the increasing fraction of di-ortho-PCBs in the toluene eluate for higher substituted congeners. Recoveries in toluene ranged from 6% for the penta-chlorinated congeners to approximately 15% for hepta-chlorinated di-ortho congeners. The addition of a small percentage (1% v/v) of toluene to the rinsing solvent did not improve the fractionation process, but rather resulted in unwanted and poorly controllable losses for the lesser chlorinated mono-ortho congeners during this step. Gas Chromatography/Mass Spectrometry. Nonpolar polysiloxane capillary columns of the SE-54 type stationary phase have been most frequently used for the separation of complex PCB mixtures. Mullin et al. (32) reported the unique separation of 187 congeners with only 11 overlapping pairs (22 congeners) in the chromatogram. Pairs, within the toxic planar PCB group, that are difficult to resolve on various chromatographic stationary phases are described by Larsen et al. (33). Contrary to electron capture detection, mass selective detection enables the congener-specific detection of some of these unresolved congeners. This will be possible when the co-eluting compounds have a different molecular mass, e.g., for nonisomers. In this study, pretreated samples as described did not contain unresolvable congeners after MS detection. Electron impact (EI) spectra of PCBs comprise an abundant molecular ion cluster and a small number of fragment ion clusters. The major fragment corresponds to the loss of Cl2 ([M - 70]•+), and a less abundant fragment corresponds to the loss of a Cl radical ([M - 35]+). The first two ions appear at an even mass to charge ratio (m/z) in the spectrum, the latter at an odd m/z value. It is obvious that, for co-eluting congeners possessing a different number of chlorine, the lower substituted congener cannot produce interfering signals at
a
a
b b
c
c FIGURE 2. Separation of PCB 77 (a) and 110 (b) in an air sample and the absence of hexa-chlorinated PCBs in the GC elution window (c). TABLE 4
FIGURE 1. Partial GC/MS mass chromatograms from the analysis of an air sample. Shown are the detection and separation of PCB 123 and 118 (a). Pannel b shows the elution of an unidentified, probably hexa-PCB of which the [M - Cl] ion cluster caused a slight interference (
