Report
Congener-Specific Polychlorinated biphenyls (PCBs) consist of 209 distinct chlorinesubstituted biphenyl molecules (congeners), of which about 150 appear at significant levels in various commercial mixtures. PCBs were manufactured in the United States by Monsanto and marketed under the trade name Aroclor. Although U.S. production of PCBs ceased in 1977, their long-term stability, dispersion into the environment by prior uncontrolled releases lipophilicity (resulting in biomagnification up food chains) and suggestions of possible toxicity have caused concern dDout ineir impact on humans and the environment. L/ischarge of P L^Ds and sites contaminated by mem are thus ugntiy regulated; extensive research on their fate and transport, as well as on methods to destroy them, has been undertaken.
Comprehensive standard sets combined with improved capillary GC columnsanddetectors bring completecharacterizationof PCB mixtures within reach
Less comprehensive congener-specific In this Report, I do not focus on these PCB methods are designed to measure limited congener-specific PCB analyses or select lists of priority congeners. Euroaddress the details of sample preparation, pean regulationsfromthe Community cleanup, or proper instrument operation. Bureau of Reference (BCR) often mandate A comprehensive overview of PCB analyreporting levels of seven "indicator" consis is available in a recent book on the geners. The program for "Quality Assursubject by Erickson (3), and review artiance of Information for Marine Environcles (4-6) cover many aspects of congenermental Monitoring in Europe" recomspecific PCB analyses and contain extensive bibliographies. Instead, I will locus sn mends measurement of 31 congeners for which a certified standard solution is disthe requirements and methods for CQCS These activities require detailed analytitributed. The National Oceanic and AtmoPCB analysis. cal characterization of PCB mixtures. Bespheric Administration's National Status cause there are only ten possible levels of and Trends program for marine environchlorination, the mixtures contain many Nomenclature and Aroclor mental qualitv established a target list of isomers with the same number of chlorine distributions 20 congeners and the U S Environmental PCB nomenclature is summarized in Figatoms, and implementing a procedure to individually quantify most of the congeners Protection Agency (EPA) draft method ure 1. Each congener has a unique pattern present in a sample is challenging and labo- 1668 specifies measurement of 13 "coola- of chlorine substitution that can be desigcongeners (i) that have the potential nated by numbering each position around rious. This type of PCB analysis is sescribed as comprehensive, quantitative, and to bind to chlorinated dioxin receptors the rings. In 1980, Ballschmiter and Zell and from which proposed dioxin-like toxic (7) assigned each of these congeners a congener-specific (CQCS). "BZ number" between 1 and 209; Guitart The methods of choice for CQCS PCB equivalencies (2) may be estimated. The PCB congeners on these short lists tend et al. (8) later corrected these for the six analyses use high-resolution GC (HRGC) to represent those observed with the listed congeners. Alterations of PCB conon capillary columns with sensitive and greatest frequency in environmental sam- geners such as dechlorination or biodegselective detection by an electron capture ples or those of greatest significance forradation are often specific to particular detector (ECD), selected-ion-monitoring phenyl-ring, chlorine-substitution patMS (MS-SIM), or full-scan, ion trap MS estimating toxic equivalencies. Some earterns. These may be classified by refer(ITMS). Many, but certainly by no means her EPA regulatory methods (e.g., 8080 ence to the ortho- meta- or para- orientaall, of the congeners can be individually and 8081) required reporting PCBs as quantified. Aroclor mixture equivalents; however, the tions to the opposite phenyl ring of chlorine substituents on the ring in question draft revision of method 8082 also sugMost Aroclor PCB mixtures are numgests measurement of individual congey bered from 1221 to 1262 in which 1? indinero without specifying either . priority George Frame cates a 12-carbon biphenyl nuclpns and list or comprehensive analysis. General Electric 468 A
Analytical Chemistry News & Features, August 1, 1997
0003-2700/97/0369-468A/$14.00/0 © 1997 American Chemical Society
P C B Analysis Aroclor" congeners if these might be encountered. Alternatively, secondary standards of thoroughly characterized Aroclors or Aroclor mixtures, which have been historically easier to obtain than primary standards, can be used. For each peak in the chromatogram on the specific HRGC column (s) to be used, accurate assignments of all significant congeners and the weight percentages of PCB in the peak must be provided. The proportions of congeners of different chlorination levels (homologues) within each peak must be known if MS detectors are to quantify each of them in the same peak
the last two digits indicate the weight percentage of chlorine. Data from a recent exhaustive CQCS study (9) of a wide range of Aroclors make it possible to color code the congeners in the matrix of Figure 1 to the maximum weight percentages observed in any Aroclor over this chlorination range. Comparison of the relative abundances with respect to the single-ring chlorine-substitution patterns along the sides of the matrix reveals that some patterns are much less likely to appear than others. The 52 congeners in purple cells almost all contain either 3 5- or 2 4 6substituted rings or are located near the lower right corner of the matrix where differences between the number of chlorines on each ring increase Substitution
of chlorines meta- to phenyl or other chlorines is unfavored; and 3,5- or 2,4,6- -rngs would require multiple instances of that process. These and other systematics of substitution pattern abundances in Aroclors are consistent with the theory of electrophilic aromatic chlorinations (9), and the relative absence of the congeners with these chlorine substitution patterns from commercial mixtures leads us to characterize them as "non-Aroclor" PCBs. Requirements for analyses
Three considerations are paramount for efficient development and validation of a CQCS PCB analysis. First, appropriate standards are necessary. Ideally, these consist of complete sets of primary congener standards, including "non-
Second, GC detectors possessing high selectivity and sensitivity for the PCBs must be used. This requirement stems from the complex biological or environmental matrices from which the congeners are often extracted at very low concentrations, sometimes less than ppb levels. Thus the more universal and less sensitive flame-ionization detector is used much less often than ECDs, which have exceptional sensitivity to multiply chlorinated compounds. The MS-SIM or ITMS detectors have sensitivities somewhat lower than an ECD but they have greater selectivity for PCBs and sometimes can distinguish and individually measure homologues that may coelute on a particular HRGC column
Finally, and most importantly, capillary HRGC columns capable of separating a substantial proportion of the congeners are necessary. Ideally one would like to choose from a wide variety of stationary phases capable of withstanding programming to temperatures high enough to readily elute all congeners and for which retention and resolution information is available for all PCB congeners.
Analytical Chemistry News & Features, August 1, 1997 4 6 9 A
Report ECD response factors for congeners not measurable in the Aroclor standards. Other authors have published more or less comprehensive congener assignments to peaks eluting on other HRGC columns, each of which might possess a special capability for measuring particular lists of congeners. In 1994 one of die two U.S.-licensed synthesizers of PCB congeners (AccuStandard) offered all 209 as individual standards (14). A set of diese was purchased, and a worldwide consortium of 12 laboratories was organized to obtain complete retention information on 27 HRGC systems using 20 stationary phases (15). The congener distributions in six Aroclors were calculated by combining data from 18 of these systems (16); a follow-up study using several systems determined to be optimal for CQCS PCB analysis completely characterized 17 lots of Aroclors encompassing eight chlorination levels (9) Figure 1 . Matrix of ring-chlorine substitutions and relative PCB congener abundances in Aroclors.
Milestones
In 1984, Mullin and colleagues published the retention times on a 50-m SE-54 capillary and the ECD relative response factors for all 209 PCB congeners, many of which had been synthesized and characterized for this purpose (10). The following year he measured the weight percentages of all congeners in a 20:10:7:6 blend of Aroclors 1221:1016:1254:1262 by analysis on two HRGC columns and assigned them to the peaks eluting on 5% phenyl 95% methylsilicone stationary phase capillaries This work established a secondary Aroclorbased standard for calibrating CQCS PCB analyses After convening a workshop to train analysts in using the standard he renpatpH the prnrpw for a 2"v18:18 blend of Aroclors 1232:1248:1262 mass 1
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standard to workers developing CQCS PCB methods and has updated the calibration for new lots of the Aroclors, but the details i
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have never been formally published (11)In the late 1980s, Wagner and Carnai
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han characterized eight individual Aroclors from 1221 through 1262 and made 470 A
congener assignments for an HRGC column with a 100% methyl silicone phase (DB-1), forming the basis for quantitative calibration in the GE "118-peak" CQCS PCB analysis used by many researchers characterizing microbial alterations of congener distributions. Their tables were restricted to components above ~ 0.5 weight percentage and were only formally published in 1996 (12). Figure 2 shows the Green Bay Standard mixture of Aroclors analyzed on a DB-1 (ECD) system with the numbers of the peaks in the 118peak system displayed below the baseline and each significant PCB congener's BZ numbers assigned above the peak in which it elutes. In 1989 Schulz and co-workers (13) used heart-cutting 2D GC to isolate all components of eight commercial mixtures (four Aroclors and four Clophens, European analogues of Aroclor products) and produced tables of weight percentage distributions for congeners present at > 0.05%. Many workers developing CQCS PCB analyses have used these tables to characterize secondary Aroclor standards, employing 5% phenyl, 95% methyl silicone phases and the tables in Reference 10 to assign congeners to peaks and to estimate
Analytical Chemistry News & Features, August 1, 1997
Illustrative applications Complete understanding of microbial PCB dechlorination processes often demands CQCS PCB analyses. The details of such processes have been reviewed by Bedard and Quensen (17). Figure 3a illustrates die power of the technique on samples of sediment from a site contaminated primarily by Aroclor 1260. Data from analyses similar to the one displayed in the chromatogram in Figure 2 were transformed in Figure 3a into mole percentages of PCB in each peak of die 118-peak system. (In the discussion that follows, die reader is cautioned not to lose track of the difference between the GC peak numbers and the BZ numbers of the congeners eluting within the peaks. Both are displayed on the chromatogram in Figure 2, and the congener structures are keyed by the BZ numbers in the matrix of Figure 1.) The black bars in Figure 3a represent the current distribution of congeners in the sediment, which is similar to that of Aroclor 1260 with admixture of a small portion of Aroclor 1254. Careful quantitative comparisons with pure Aroclor samples suggested the likelihood that some alteration of the originally deposited distribution might have taken place (18). The six small peaks identified with black dots are the key to confirming that some dechlorination had in fact occurred MS-SIM
was used to show that these peaks are primarily composed of "non-Aroclor" congeners 96,103,100,150,154, and 140 in GC peaks 41,43, 44, 55,62, and 70, respectively. In some cases the assigned "non-Aroclor" congener could potentially coelute with an Aroclor congener of a lower chlorination level. Such a coelution does not interfere with the MS-SIM measurement of the higher homologue, which
is monitored at its molecular ion cluster masses. Quantitation and congener assignments to these peaks are ensured by supplementing the Aroclor calibration mixture with the appropriate "nonAroclor" congeners. The next step in the research investigation of these samples displaying partial natural dechlorination was an in vitro experiment in which addition of BZ #70 (25-
34) stimulated rapid selective microbial para-dechlorination to BZ #26 (25-3)) which resulted in induction of selective para-dechlorination of several congeners present in the original distribution (19). This experiment resulted in the pattern of red bars in Figure 3a, which reveals decreases in peaks losing some more chlorinated congeners and corresponding increases in the peaks containing the ex-
Figure 2. GC-ECD chromatogram of 25:18:18 Aroclors 1232:1248:1262 on DB-1 column. GE 118-peak system numbers are below the peaks; PCB congener BZ numbers are above the peaks. Analytical Chemistry News & Features, August 1, 1997 471 A
Report pected products of a para-selective dechlorination. Specifically, the increases in peaks 31,32,37,51, and 65 correspond to products of dechlorination of major congeners comprising peaks (53 and 75), 54, 82, (77 and 102), and 93, respectively. Because the CQCS PCB analysis being used is quantitative, the individual parent/ child molar balances can be closed, thus elucidating and verifying the proposed selective para-dechlorination mechanism. A quite different kind of dechlorination selectivity prevails in the photolysis of PCBs (20), in which the primary route is loss of ortho chlorines. I have observed that BZ #74 (2454) in peak 46 is exceptionally prone to conversion to BZ #37 (344) in peak 38 when exposed to sunlight. This might be a sensitive indicator of photolytic alteration if a CQCS PCB analysis that could reliably detect and quantify these particular congeners was used. Thermal processes such as incineration of PCBs or organic material in the presence of chlorine may produce rearrangements of their chlorine substitu-
tion patterns or result in de novo synthesis of PCB congeners, yielding many "non-Aroclor" congeners and distributions radically different from those observed in Aroclor products. The evaluation of the extent to which such processes contribute to the environmental burden of PCBs requires studies using the best possible CQCS PCB analyses, calibrated with the full range of congeners. The difficulty of implementing such analyses may have resulted in overlooking small but significant contributions from such sources. Another category of microbial alteration of PCB congener distributions is aerobic microbial degradation Unlike dechlorination the process does not convert one congener into another but selectively performs oxidative phenyl ring
nated higher Aroclor. Figure 3b shows the results of an in vitro experiment (21) on an initial Aroclor 1242 distribution exposed to a particularly potent PCBdegrading microbial strain. The quantities of congeners in the peaks are expressed in absolute molar concentrations instead of mole percentages because CQCS PCB analysis does not measure the non-PCB degradation products and therefore cannot support closure of mass balance in this kind of process. Most of the PCBs in the earlier eluting less chlorinated peaks have been degraded whereas those in peaks bered above 45 are more resistant Peak 46
openinp which eliminates the PCB altncrpther Rpraii
