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Jan 23, 2003 - The method was used to determine the PCBs and pesticides in eggs of the black-crowned night heron (Nycticorax nycticorax), a bird which...
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Anal. Chem. 2003, 75, 1058-1066

Methodological Refinements in the Determination of 146 Polychlorinated Biphenyls, Including Non-Ortho- and Mono-Ortho-Substituted PCBs, and 26 Organochlorine Pesticides as Demonstrated in Heron Eggs Shaogang Chu,† Chia-Swee Hong,*,† Barnett A. Rattner,‡ and Peter C. McGowan§

Wadsworth Center, New York State Department of Health and School of Public Health, State University of New York at Albany, Albany, New York 12201-0509, U.S. Geological Survey, Patuxent Wildlife Research Center, 12011 Beech Forest Road, Laurel, Maryland 20708-4041, and U.S. Fish and Wildlife Service, Chesapeake Bay Field Office, 177 Admiral Cochrane Drive, Annapolis, Maryland 21401

A method for the determination of 146 polychlorinated biphenyls (PCBs), including four non-ortho- and eight mono-ortho-substituted congeners, and 26 chlorinated pesticides is described. The method consists of ultrasonic extraction, Florisil cleanup, HPLC fractionation over porous graphitic carbon (PGC), and final determination with GC/ECD, GC/MS, or both. Two PCB congeners (PCB 30 and PCB 161) and two polybromobiphenyls (2,4′,5tribromobiphenyl and 3,3′,4,4′-tetrabromobiphenyl) were used as surrogate standards to evaluate the analytical efficiency. Four PCB congeners, PCB 14 and PCB 159 for the first fraction, PCB 61 for the second fraction, and PCB 204 for the third fraction, were used as internal standards to monitor the GC performance. The retention behavior of PCBs and pesticides on the PGC column are discussed. The method was found to be reproducible, effective, and reliable under the operational conditions proposed and was applied successfully to the analysis of individual PCBs and chlorinated pesticides in black-crowned night heron (Nycticorax nycticorax) egg samples. Polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides are the most widespread and persistent environmental pollutants. Their massive and indiscriminate use, along with their high chemical stability, has led to the accumulation of residues of these compounds in a wide range of organisms, including plankton, fish, birds, marine and land mammals, and humans.1,2 Although they have been banned in industrialized countries for years and in some instances for decades, PCBs and OC pesticides * To whom correspondence should be addressed. Phone: 518-473-7299. Fax: 518-473-7689. E-mail: [email protected]. † Wadsworth Center. ‡ U.S. Geological Survey. § U.S. Fish and Wildlife Service. (1) Waid, J. S., Ed. PCBs and the Environments; CRC Press: Boca Raton, FL, 1986; Vol. 3. (2) Brouwer, A.; Ahlborg, U. G.; Rolaf, V. L. F. X.; Feeley, M. M. Chemosphere 1998, 37, 1627-1643.

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are still routinely found throughout the world and continue to cause many ecotoxicological problems. For years, eggs have been used for monitoring the environmental contaminants in breeding habitats of birds.3 Migratory birds can also be used as bioindicators if they arrive in an area well in advance of their breeding season. Agricultural, industrial, and point and nonpoint source pollution have been found to be the critical factors causing the deterioration of habitats that support birds.4 The harmful effects of PCBs and OC pesticides have resulted in mortality and reproductive problems in birds. In fact, the level of OC pesticides and PCBs in eggs may be closely correlated with effects on eggshell thinning and reproductive success and, thus, on the population dynamics of bird species.5-7 PCBs are accumulated via the food chain, and biotransformation of the original technical PCB mixtures has resulted in major changes of the congener composition. Because each of the PCB congeners displays a unique combination of physical, chemical, and biological properties, the measurement of individual PCB congeners is important for evaluating the toxic potential of the chemicals. PCB congeners vary in their biodegradation and bioaccumulation patterns and are of varying toxicities to birds. Because birds inhabit diverse ecosystems and feed on a variety of prey, their exposure may vary widely, both in degree and in type of PCBs. An understanding of the differences and similarities in congener patterns among related species is important in determining differences in exposure and metabolism.8,9 The importance of congener-specific analysis in environmental samples is becoming more evident as the toxicities of individual congeners are defined. For example, the total dioxin-toxic(3) Hebert, C. E.; Norstom, R. J.; Weseloh, D. V. C. Environ. Rev. 1999, 7, 147-166. (4) Rattner, B. A.; McGowan, P. C.; Hatfield, J. S.; Hong, C. S.; Chu, S. G. Arch. Environ. Contam. Toxicol. 2001, 41, 73-82. (5) Ohlendorf, H. M.; Fleming, W. J. Mar. Pollut. Bull. 1988, 19, 487-495. (6) Boumphrey, R. S.; Harad, S. J.; Jones, K. C. Arch. Envrion. Contam. Toxicol. 1993, 25, 346-352. (7) Rattner, B. A.; Melancon, M. J.; Rice, C. P.; Riley, W., Jr.; Eisemann, J.; Hines, P. K. Environ. Toxicol. Chem. 1997, 16, 2315-2322. (8) Focardi, S.; Leonzio, C.; Fossi, C. Environ. Pollut. 1988, 52, 243-255. (9) Mora, M. A. Environ. Toxicol. Chem. 1996, 15, 1003-1010. 10.1021/ac0205560 CCC: $25.00

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equivalents (TEQs)10 for PCBs in eggs, which are mainly due to high concentrations of congener 126, may reflect the trophodynamics of toxic PCBs in fish-eating birds and may explain the low hatching success and embryo deformities in their populations.4,7,11 Low rates of metabolic clearance of congener 126 in gulls may explain the change in the relative concentrations of nonortho PCBs in gull eggs.12 The high concentration of congener 126 in fish-eating birds means that the potential for toxic effects in these species is disproportionate to the total PCB concentration.13-16 Although levels of PCBs have been determined in bird tissues or eggs for almost three decades, congener-specific analysis of PCBs is still challenging. The numbers of PCB congeners and pesticides found in the environment are difficult to separate and analyze; however, ecotoxicologists need accurate data of individual PCBs and pesticides. To solve this problem, less comprehensive congener-specific PCB methods were suggested to measure the selected lists of priority congeners. European regulations from the Community Bureau of Reference (CBR)17 identified seven “indicator” congeners that could be used in environmental monitoring programs as a result of their high concentrations in technical mixtures and their recalcitrance. The program for “Quality Assurance of Information for Marine Environmental Monitoring in Europe”18 recommended measurement of 31 congeners. The National Oceanic and Atmospheric Administration’s National Status and Trends program19 for marine environmental quality established a target list of 20 congeners. In recent years, increasing attention has been centered on the aryl hydrocarbon hydroxylase (AHH)-inducing potency of those PCBs that have two para chlorines and at least two meta chlorines and that show the same type of toxicity as 2,3,7,8-TCDD. Considerable toxicity is attributed to the few non-ortho-substituted and some of the monoortho-substituted PCBs, which are termed “coplanar and semicoplanar” PCBs. Because of the ultratrace levels at which coplanar and some semicoplanar PCBs exist, they are subject to measurement bias because of interference, including that from other PCBs or pesticides that may exist at much higher concentrations. Changes in congener pattern may reveal information about the relationship between PCBs’ structures and their environmental fate. Congener-specific responses have been demonstrated for microsomal enzyme induction, estrogenic or anti-estrogenic effects, thyroid hormone alterations, brain dopamine depletion, environmental occurrence, and other effects.20 Therefore, a (10) Van den Berg, M.; Birnbaum, L.; Bosveld, A. T. C.; Brunstro¨m, B.; Cook, P.; et al. Environ. Health Perspect. 1998, 106, 775-792. (11) Rattner, B. A.; Hoffman, D. J.; Melancon, M. J.; Olsen, G. H.; Schmidt, S. R.; Parsons, K. C. Arch. Environ. Contam. Toxicol. 2000, 39, 38-45. (12) Pastor, D.; Ruiz, X.; Barcelo´, D.; Albaige´s, J. Chemosphere 1995, 31, 33973411. (13) Kubiak, T. J.; Harris, H. J.; Smith, L. M.; Schwartz, T. R.; Stalling, D. L.; Trick, J. A.; Sileo, L.; Docherty, D. E.; Erdman, T. C. Arch. Environ. Contam. Toxicol. 1989, 18, 706-727. (14) Ankley, G. T.; Nieemi, G. J.; Lodge, K. B.; Harris, H. J.; Beaver, D. L.; Tillitt, D. E.; Schwartz, T. R.; Giesy, J. P.; Jones, P. D.; Hagley, C. Arch. Environ. Contam. Toxicol. 1993, 24, 332-344. (15) Yamashita, N.; Tanable, S.; Ludwig, J. P.; Kurita, H.; Ludwig, M. E.; Tatsukawa, R. Environ. Pollut. 1993, 79, 163-173. (16) Metcalfe, T. L.; Metcalfe, C. D. Sci. Total Environ. 1997, 201, 245-272. (17) Wells, D. E.; Echarri, I. Int. J. Environ. Anal. Chem. 1992, 47, 75-97. (18) Wells, D. E. Mar. Pollut. Bull. 1994, 29, 143-145. (19) http://ccmaserver.nos.noaa.gov/NSandT/New_NSandT.html. (20) Gerstenberger, S. L.; Gallina, K.; Dellinger, J. A. Environ. Toxicol. Chem. 1997, 16, 2222-2228.

complete analysis of PCBs, including coplanar and noncoplanar congeners, and pesticides in a complex matrix is required. For many congeners, an unequivocal separation from other compounds has been achieved, and it has been possible to validate the method with appropriate reference materials. However, the determination of some congeners in environmental samples at ultratrace levels (microgram per kilogram or lower) still remains a problem. In most cases, the amounts of coplanar PCBs are often quite close to the limit of detection, and recovery measurement is one of the most difficult and ill-defined aspects of trace organic analysis. Although most of the congeners in Aroclor mixtures can be analyzed by multidimensional gas chromatography (MDGC) using a heart-cut technique, it is not appropriate to use this technique to separate PCBs that are present at concentrations that differ by 3 orders of magnitude or more, or that occur in environmental matrixes containing many compounds other than PCBs. A method for complete analysis of PCBs, including coplanar and noncoplanar congeners, and OC pesticides in eggs is described. The method was used to determine the PCBs and pesticides in eggs of the black-crowned night heron (Nycticorax nycticorax), a bird which has been used as a biomonitor of potentially contaminated wetlands and estuaries.4 EXPERIMENTAL SECTION Chemicals. All individual PCB congeners (the IUPAC No. nomenclature is used in this study),21 PCB calibration mixtures, 3,3′,4,4′-tetrabromobiphenyl, 2,4′,5-tribromobiphenyl, and pesticides were purchased from AccuStandard Inc. (New Haven, CT). The nine PCB calibration mixtures provide all 209 congeners with 10 µg/mL of each congener in isooctane. All solvents used were nanograde from Burdick & Jackson (Muskegon, MI). Florisil (Silica Company, Berkeley Springs, WV), 60/100 mesh, was activated at 130 °C for 8 h in oven and then deactivated with 2% water. Standards. A PCB calibration standard containing the mix of five calibration mixtures (AccuStandard PCB congener mix nos. 1-5, which contain 39, 36, 27, 22, and 20 PCB congeners, respectively, occur in Aroclors, excluding PCBs 126 and 169) with 20 ng/mL each of 144 PCB congeners in isooctane was used for conventional PCB analysis. A separate calibration standard including 10 ng/mL each of four coplanar (PCBs 77, 81, 126, and 169) and eight semicoplanar PCBs (PCBs 123, 118, 114, 105, 167, 156, 157, and 189) in isooctane was used for coplanar and semicoplanar PCB analysis. The surrogate solution contains 20 ng/mL each of PCBs 30, 161, and 2,4′,5-tribromobiphenyl and 1 ng/mL of 3,3′,4,4′tetrabromobiphenyl in isooctane. The internal standard solution contains PCBs 14 and 159, with 500 ng/mL of each in isooctane for fraction 1; 500 ng/mL of congener 61 in isooctane for fraction 2; and 100 ng/mL of congener 204 in toluene for fraction 3. The surrogate and internal standards that were chosen were those that would not likely occur in the respective fractions. Safety. The dioxin-like PCBs should be treated as a potential health hazard. Exposure to these compounds should be reduced to the lowest possible level. These compounds were handled using essentially the same techniques employed in handling PCDD/Fs recommended by EPA Method 1613.22 Sampling. Black-crowned night heron (N. nycticorax) eggs were collected from a contaminated site in Baltimore Harbor, MD, (21) Cochran, J. W.; Frame, G. M. J. Chromatogr., A 1999, 843, 323-368.

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and from a relatively uncontaminated reference site in southern Chesapeake Bay (Holland Island, MD) in 1998. The details of the study sites and sample collection were described in a previous paper.4 Egg samples were evaluated and then frozen in chemically clean jars (ICHEM Research, New Castle, DE) at -10 °C before chemical analysis. Extraction and Cleanup. The egg samples were homogenized with a tissuemizer (Kika Works, Inc., Wilmington, NC) for ∼5 min. A subsample (2-5 g) was weighed into a 100-mL centrifuge bottle together with 1 mL of the surrogate standard. A 3-mL portion of methanol was added, and the mixture was shaken well and equilibrated for 20 min. Anhydrous sodium sulfate (20 g) was added to the sample, which was then extracted with 30 mL of hexane by sonication (Sonicator W-385, Heat Systems, Inc., Farmingdale, NY) for 5 min and then centrifuged at 2500 rpm for 10 min. The aqueous residue was extracted twice more with 30 mL of hexane. The lipid content of each egg sample was determined by accurately weighing the residue of hexane extract following solvent evaporation. The remaining extract was concentrated to ∼2 mL in a Kuderna-Danish (K-D) evaporator on a steam bath. Then the extract was quantitatively transferred to a 1-cm-diameter glass column containing 10 g of 2% deactivated Florisil topped with 2 g of anhydrous sodium sulfate. The column was eluted with 10% dichloromethane in hexane, and the first 90 mL of the eluate was collected. The 90-mL fraction was concentrated and solvent-exchanged to isooctane to ∼2 mL with the K-D apparatus, and it was further concentrated under a gentle stream of nitrogen to ∼100 µL. Fractionation by HPLC. A porous graphitic carbon (PGC) HPLC column (100 × 4.7 mm, 7-µm particle size, Hypercarb, Keystone Scientific, Inc., Bellefonte, PA) was used for the separation of PCBs according to the number of chlorines in the ortho positions. The PGC column was fitted with a 7040 Rheodyne switching valve to enable back-flushing of the column. The column was eluted with hexane at 1 mL/min for 6 min, collecting the first fraction that contained 2-4 ortho-substituted PCBs, most pesticides, and surrogate PCBs 30 and 161. Then, the column was eluted with 50% dichloromethane in hexane for 16 min, collecting the second fraction that contained mono-ortho-substituted PCBs, some other pesticides, and surrogate 2,4′,5-tribromobiphenyl. After the second fraction was collected, the column was back-flushed with toluene at 1 mL/min for 20 min, collecting the third fraction that contained non-ortho-substituted PCBs, hexachlorobenzene (HCB), and surrogate 3,3′,4,4′-tetrabromobiphenyl. The first and second fractions were volume-reduced and solvent-exchanged to isooctane to the appropriate volume (1 mL in most cases), and the third fraction was concentrated to 2∼4 mL by the rotary evaporator and further concentrated to ∼100 µL by a gentle nitrogen stream. The volume was accurately measured by weighing the solution. Instrumental Analysis for PCBs. The appropriate internal standard was added into the three fractions. All analyses were performed on an HP 6890 gas chromatograph (GC) equipped with a split/splitless injector system and a 63Ni electron capture detector (22) Environmental Protection Agency. Method 1613. Tetra- through OctaChlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS. EPA 821B94005a; U.S. Environmental Protection Agency, Office of Water Engineering and Analysis Division: Washington DC, , October 1994.

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(ECD), fitted with a 60-m × 0.25-mm i.d., 0.25-µm film thickness of DB-XLB fused-silica column (J & W Scientific Inc., Folsom, CA). Helium was used as carrier gas. The injector and detector temperatures were 280 °C and 350 °C, respectively. The column temperature was maintained at 50 °C for 2 min, then programmed to 150 °C at 15 °C/min, then increased to 285 °C at 1.2 °C/min and held for 10 min. The determination of coplanar PCBs was confirmed on an HP 5972 mass selective detector (MSD). ECD-derived concentrations of ortho-substituted PCBs that coeluted with pesticides were corrected by subtracting the concentrations of coeluting pesticides from the GC/MSD analysis. Analysis of Chlorinated Pesticides. Pesticides were coextracted with PCBs in the extraction of egg samples and were fractionated into three fractions on a PGC column. Most of the 26 pesticides were eluted in the first fraction, but β-BHC, δ-BHC, endosulfan II, endrin aldehyde, and endosulfan sulfate were eluted predominantly in the second fraction, and HCB was eluted only in the third fraction. The analysis of pesticides in the first fraction was carried out with an HP GC 6890 equipped with HP 5972 MSD and a DB-XLB column (60-m × 0.25-mm i.d., 0.25-µm thickness). The GC parameters were the same as those for analysis of PCBs, except that the injection volume was 2 µL instead of 1 µL, and the GC/MSD interface temperature was 280 °C. Selected ion monitoring (SIM) was used for quantification. The criteria for identification of a pesticide were retention time and the ratio of two selected ions. The pesticides in the second and third fractions were quantified by GC/ECD without interference from PCB congeners. Total concentration of each pesticide was the sum of its concentrations in both the first and second fractions, except that the HCB concentration was only from the third fraction analysis. All PCB and pesticide concentrations were calculated using the internal standard method and were corrected by surrogate recoveries. Quality Control and Quality Assurance. Precision and accuracy of the method were evaluated by replicate analyses of four spiked chicken eggs. One replicate sample per 10 samples was analyzed. Acceptable precision between duplicate sample analyses for PCBs and pesticides is 30%, expressed as relative percent difference (RPD). Surrogate standards and laboratorycontrolled spike were used to monitor analytical recoveries (accuracy). Recoveries of each PCB, pesticide, and surrogate standard must be 50% < R < 120%. The method detection limits (MDLs) for selected PCBs and pesticides were determined using the selected standard mix at a low concentration (5 ng/g for 2-4 ortho-substituted PCBs and pesticides; 0.5 ng/g for coplanar and semicoplanar PCBs) in a procedural spike. A mean value and standard deviation for each congener in the mix were established across seven spikes. The MDLs were defined as 3 times the standard deviation. A hexane blank and calibration standard were run once per six samples. The calibration standard that was included at the beginning of the run set was also used to evaluate peak resolution and column performance. Spiked samples consisted of matrix blanks spiked with representative levels of selected PCBs and pesticides and were carried through analytical procedures similar to those for actual samples. The method blank consisted of a hexane blank carried through the entire analytical procedures

similar to those for samples. The method blank and matrix spike were run once per 10 samples. Validation of a new method should include an intercomparison of extraction efficiencies and cleanup steps and an intercalibration of the whole procedure. As an alternative to the intercomparison exercises, it would be possible to use a suitable reference material, but currently, only nonplanar PCBs are certified in environmental matrixes. RESULTS AND DISCUSSION Extraction. Accurate trace organic analysis begins with quantitative extraction of the analytes from the sample. This method involves precipitation of protein in the egg samples with methanol23 and subsequent ultrasonic extraction with hexane. During method development, the efficiency of the extraction procedure was evaluated by the analysis of a spiked egg sample. The extraction efficiency of the procedure was assessed relative to a well-established Soxhlet extraction method, which has been successfully applied to the determination of PCDD/Fs, PCBs, and OC pesticides in bird eggs.12,24 Our results indicated that similar concentrations were obtained using the two extraction methods. However, of the two extraction procedures, the sonication procedure was the more economical and less time-intensive. Cleanup. The cleanup step is an important procedure, especially in biological samples that contain large quantities of lipids. The most effective cleanup method for removing lipids involves the use of concentrated sulfuric acid,8 which can remove more than 90% of lipids in samples by a relatively simple technique. Combined with other cleanup steps or even used as the only cleanup step for some environmental samples, it is widely applied in the analysis of PCBs. However, it should be applied cautiously, because it completely or partially destroys many labile compounds. Investigation of potential losses of PCB and pesticides during sulfuric acid cleanup have demonstrated little or no loss of the PCBs but unsatisfactory losses for some OC pesticides. Bernal et al.25 evaluated 84 pesticides and, as expected, found recoveries ranging from quantitative for some pesticides, such as DDT, DDE and HCH, to not detected for some compounds, such as endrin and dieldrin. A large gel permeation chromatography (GPC) column packed with biobeads SX-2 or SX-3 can remove up to 500 mg of lipid,26 but elution will require up to 300 mL of solvent, which may increase the background residue and the MDL. In the method described here, the cleanup of lipid is performed with column chromatography of Florisil alone. The results show that lipids are removed efficiently if the sample loading is no more than the ratio of 1:2 of sample weight (wet weight of egg) vs Florisil. All of the PCB congeners and 26 chlorinated pesticides are completely eluted with 90 mL of 10% dichloromethane in hexane, and the eluate is suitable for the following HPLC fractionation. (23) Association of Official Analytical Chemists (AOAC). Organochlorine and Organophosphorus Pesticides, General Multiresidue Methods, Method 29.001. In Official Methods of Analysis of the Association of Official Analytical Chemists, 14th ed.; Horwitz, W., Ed.; Association of Official Analytical Chemists: Washington, DC, 1984; pp 533-540. (24) Scharenberg, W.; Ebeling, E. Chemosphere 1998, 36, 263-270. (25) Bernal, J. L.; del Nozal, M. J.; Jime´nez, J. J. J. Chromatogr. 1992, 607, 303309. (26) Erickson, M. D. Analytical Chemistry of PCBs, 2nd ed.; CRC Lewis Publishers: Boca Raton, FL, 1997; pp 215-221.

Surrogate and Internal Standards. Recovery measurements constitute one of the more difficult and ill-defined aspects of trace organic analysis. For determination of the efficiency of extraction, it is necessary that the analyte be bound to the matrix in a manner similar to how it exists in the environment. The natural binding would occur via feeding. Although addition of a known amount of the analyte to the matrix prior to extraction followed by a subsequent analysis does not precisely measure the extraction efficiency of naturally bound analytes, this type of spiked sample analysis will determine the accuracy and precision of the subsequent analytical steps. Isotope dilution mass spectrometry (IDMS) is a more elegant method to overcome the problem of sample recovery.16,26-29 In this method, a 13C-labeled isotope of the analyte is added to the sample at the beginning of the analysis, and the ratio of labeled and unlabeled compounds is determined by MS. This technique eliminates the need for recovery measurements and automatically accounts for any loss in the determination. However, the cost and availability of labeled compounds and the need to use MS limit the use of this method. An alternate method is to use a PCB congener that is not present in the real sample as the surrogate. Wells et al.17 used PCB 209 as the surrogate, and 2,4-dichlorobenzyl hexyl ether and 2,4-dichlorobenzyl dodecahexyl ether as internal standards in their PCB analysis. Congener 209 is often found in environmental samples, especially in biota because of the high bioconcentration factor for highly chlorinated PCBs, but it is not usually found in industrial products or sediments. In our investigation, congener 209 was found in almost all egg samples, thus making it unsuitable as a surrogate for the determination of PCBs in bird eggs. Burgess et al.30 used congener 198 (2,2′,3,3′,4,5,5′,6-octachlorobiphenyl) as a surrogate to determine the concentration of PCBs in three isolated phases: colloidal, dissolved, and particulate. Unfortunately, this congener is usually found in technical mixtures and is consequently found in environmental samples. Bo¨hm et al.31 used PCB 169 as a surrogate to determine coplanar PCBs in food. It is true that this congener, exhibiting the maximum chlorine substitution possible for a coplanar conformation, has only been detected so far in Aroclor 1260 at 0.05% level, 32 and in most environmental samples, the concentration is very low. However, considering that this congener is the main coplanar PCB of concern in ecotoxicological investigations, it is not really suitable as a surrogate for the determination of coplanar congeners. Although many PCB congeners do not exist in environmental samples, only a few are suitable for use as surrogates. For the congener to be suitable as a surrogate, it should be resolvable from other PCB congeners in GC analysis. The fact that surrogates are added to the sample prior to extraction and are subsequently separated into three fractions makes the choice even more complicated. Gerstenbeger et al.20 used congeners 11, 14, 65, and (27) Patterson, D. G., Jr.; Lapeza, C. R.; Baarnhart, E. R.; Groce, D. F.; Burse, V. W. Chemosphere 1989, 19, 127-134. (28) Kuehl, D. W.; Butterworth, B. C.; Libal, J.; Marquis, P. Chemosphere 1991, 22, 849-858. (29) Loos, R.; Vollmuth, S.; Niessner, R. Fresenius’ J. Anal. Chem. 1997, 357, 1081-1087. (30) Burgess, R. M.; McKinney, R. A.; Brown, W. A. Environ. Sci. Technol. 1996, 30, 2556-2566. (31) Bo ¨hm, V.; Schulte, E.; Thier, H. P. Z. Lebensm. Unters. Forsch. 1991, 192, 548-550. (32) Schwartz, T. R.; Tillitt, D. E.; Feltz, K. P.; Peterman, P. H. Chemosphere 1993, 26, 1443-1460.

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166 as surrogates to determine PCBs in fish, with the rationale that these congeners were not commonly found in the environment. However, these congeners are not suitable for surrogates in our method, which requires a congener group fractionation. Di-ortho-substituted congeners, PCBs 30 (2,4,6-trichlorobiphenyl) and 161 (2,3,3′,4,5,6-hexachlorobiphenyl), not found in egg samples, identified as pure peaks in GC, and separated into the first fraction by HPLC, were used as surrogates for the first fraction in our method. For the second fraction, 2,4′,5-tribromobiphenyl, having one bromine at the ortho position, and having an elution behavior similar to that of mono-ortho-substituted PCBs on the PGC column, was chosen as the surrogate. This compound can be separated into the second fraction as a pure GC peak, although the response factor is not as high as that of trichlorobiphenyl. On the basis of the same consideration, 3,3′,4,4′-tetrabromobiphenyl was chosen as the surrogate for the third fraction. Because the GC internal standard is added just before the GC analysis, and because PCB congeners will have already been separated into different fractions, leading to fewer congeners in each fraction, the choice of an internal standard is much simpler. In our method, congeners 14 and 159, congener 61, and congener 204 were used as the internal standards for the first, second, and third fractions, respectively. PCBs 14, 159, and 61 were not found in the egg samples and can be determined as pure peaks by GC. Congener 204 in the sample had been separated into the first fraction, and there was no interference with the added PCB 204 in the third fraction. HPLC Fractionation. The conventional cleanup method for PCBs and OC pesticides results in two fractions after elution over silica columns, a PCB and one pesticide fraction.33 This separation is normally adequate when the predominant PCBs are to be determined. However, with the exception of PCBs 118, 105, and to a minor extent, 156, all coplanar and semicoplanar PCBs are present at substantially lower concentrations than are the remaining PCB congeners. Because the range of PCB concentrations is normally too large for all congeners to be measured directly (without additional dilution or concentration) and because some of the key PCBs cannot be resolved on a single GC column, it was therefore necessary to separate the non- and mono-ortho PCBs into different groups. In some papers, congeners 77, 126, and 169 in environmental samples are reported to occur in concentrations as high as those of other common congeners. These unusually high concentrations of coplanar PCBs reported in environmental samples are doubtful and may be due to the unsuccessful separation from coeluted PCBs (with possible falsepositive signals), resulting in the overestimated toxicity. For example, PCBs 110 and 81 can be separated on a DB-XLB column at the same concentration, 20 ng/mL (the resolution is about 1.0), but in environmental samples, the concentration of congener 110 is more than 100 times higher than the concentration of congener 81, and thus, it is impossible to quantitatively analyze congener 81 at the tail of the huge peak of congener 110. MDGC is a very useful technique to separate coeluted congeners with comparable concentrations. However, it is not appropriate to use this technique to separate PCBs that have concentrations that differ by a factor of more than 600.34 (33) Erickson, M. D. Analytical Chemistry of PCBs, 2nd ed.; CRC/Lewis Publishers: Boca Raton, FL, 1997; pp 191-197.

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A few preseparation methods for determination of non-orthosubstituted PCBs have been proposed. Low-pressure liquid-solid chromatographic techniques with various sorbents are generally used, but not all of them are suitable for coplanar PCB analysis because of the problems of reproducibility and standardization. Although Florisil has been used to separate coplanar PCBs from the dominant ortho PCBs, and although the recoveries of the coplanar PCBs 77, 126, and 169 are very high, two problems are encountered. The first is the coelution of PCB 81 with DDE, and the second is that the separation does not provide a complete resolution of coplanar PCBs.12 For the analysis of non- and monoortho PCBs, the advantage of using activated charcoal is that it had a high affinity for organic compounds, even at the ultratrace level, and it is inexpensive, readily available, and easy to use. However, the recovery of coplanar PCBs from active carbon at the trace level in environmental samples is not always fully quantitative.35 Furthermore, activated charcoals are not able to completely separate the non-ortho PCBs from some orthosubstituted PCBs.36 In particular, the coelution of congener 110 with PCB 77 and of congeners 129 and 178 with 126 are sufficient to prevent the quantification of PCBs 77 and 126. Thus, data for congeners 77 and 126 obtained by these methods are likely to be overestimated unless MDGC or other techniques are used. High-performance liquid chromatography (HPLC) is a useful technique for the preseparation. It is often used for the prefractionation of PCDDs and PCDFs, for which PGC columns and 2-(1pyrenyl)ethyldimethylsilylated silica (PYE) columns are often used. Wells et al.17 used the PYE HPLC column eluting with a single eluant to determine non-ortho-, mono-ortho-, and higherortho-substituted PCBs in marine mammals. They found that with this technique, the elution order of the PCBs is not dependent upon the degree of ortho chlorine substitution alone, because a number of the more highly chlorinated PCBs coeluted in the second and third fractions. These results are similar to those in the earlier work of Haglund et al.,37 who found that retention tends to increase with the degree of chlorination and to decrease with increasing number of ortho chlorines. An off-line HPLC (PYE)HRGC method is described by Ramos et al.38 for the unambiguous determination of 41 PCB congeners, including coplanar congeners and chiral congeners. Another disadvantage of the PYE HPLC column is that it cannot tolerate lipid in the samples.39,40 If a lipidcontaining sample is injected into the PYE column, the retention behavior of PCBs/pesticides will change, leading to a shift in retention time. This makes the congener fractionation process nonreproducible. Because PGC exhibits the inherent pH stability and the chemical homogeneity that a bonded-silica phase cannot, it is most applicable to the separation of positional and (34) Hess, P.; de Boer, J.; Cofino, W. P.; Leonaards, P. E. G.; Wells, D. E. J. Chromatogr., A 1995, 703, 417-465. (35) Jansson, B.; Andersson, R.; Asplund, L.; Litzen, K.; Nylund, K.; Sellstrom, U.; Uvemo, U. B.; Wahlberg, C.; Wideqvist, U. ; Olsson, T. O. M. Environ. Toxicol. Chem. 1993, 12, 1163-1174. (36) Kannan, N.; Petrick, G.; Schulz, D.; Duinker, J.; Boon, J.; Van Arnhem, E.; Jansen, S. Chemosphere 1991, 23, 1005-1076. (37) Haglund, P.; Asplund, L.; Jarnberg, U.; Jansson, B. J. Chromatogr. 1990, 507, 389-398. (38) Ramos, L.; Hernnandez, L. M.; Gonzalez, M. J. Anal. Chem. 1999, 71, 7077. (39) Tuinstra, L. G. M. Th.; van Rhijn, J. A.; Roos, A. H.; Traag, W. A.; van Mazijk, R. J.; Kolkman, P. J. W. J. High Resolut. Chromatogr. 1990, 13, 797-802. (40) de Boer, J.; Stronck, C. J. N.; van der Valk, F.; Wester, P. G.; Daudt, M. J. M. Chemosphere 1992, 25, 1277-1283.

Table 1. PCB Congeners and Pesticides Analyzed in This Study and Their Fractionation by PGC peak no.a

compdb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84

1 4 + 10 9 R-BHC 19 14d 30c 18 δ-BHC 17 27 24 16 32 54 53 heptachlor 51 45 46 73 69 52 48 49 aldrin 104 + 47 75 44 59 42 71 41 64 103 + 40 100 oxychlordane heptachlor epoxide 93 95 91 o,p′-DDE 92 84 90 + 101 γ-chlordane R-chlordane 99 endosulfan I trans-nonachlor 119 + 83 97 87 p,p′-DDE 136 + 117 115 dieldrin 154 + 85 110 o,p′-DDD 151 82 135 144 endrin 147 149 109 134 o,p′-DDT 131 165 146 161c p,p′-DDD 153 + 132 179 141 176 137 130 164 p,p′-DDT 138

structure

fraction

peak no.a

compdb

1 1 1,2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1,2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1,2 1 1

85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 1 2 3 4 5 6 7 8 9 10

163 129 + 178 158 175 187 183 159d 128 + 185 174 202 177 201 + 171 173 methoxychlor 197 172 180 193 200 191 170 199 mirex 190 196 203 208 207 + 195 194 205 206 209 2 3 7 6 5 8 12 34 29 β-BHC 26 25 31 28 δ-BHC 33 + 20 22 35 37 67 61d 63 74 70 66 56 60 tribromobiphenylc 124 123 118 122 114 endosulfan II 105 endrin aldehyde endosulfan sulfate 167 156 157 189 HCB 14 13 15 81 77 126 204d 169 tetrabromobiphenylc

2 22′ + 26 25 22′6 35 246 22′5 22′4 23′6 236 22′3 24′6 22′66′ 22′56′ 22′46′ 22′36 22′36′ 23′5′6 23′46 22′55′ 22′45 22′45′ 22′466′ + 22′44′ 244′6 22′35′ 233′6 22′34′ 23′4′6 22′34 234′6 22′45′6 + 22′33′ 22′44′6 22′356 22′35′6 22′34′6 22′355′ 22′33′6 22′34′5 + 22′455′ 22′44′5 23′44′6 + 22′33′5 22′3′45 22′345′ 22′33′66′ + 234′56 2344′6 22′44′56′ + 22′344′ 233′4′6 22′355′6 22′33′4 22′33′56′ 22′345′6 22′34′56 22′34′5′6 233′4′5 22′33′56 22′33′46 233′55′6 22′34′55′ 233′45′6 22′44′55′ + 22′33′46′ 22′33′566′ 22′3455′ 22′33′466′ 22′344′5 22′33′45′ 233′4′5′6 22′344′5′

a According to GC retention time order in each fraction. the predominant fraction.

b

IUPAC no.21

c

Surrogate standards.

structure

d

fraction

233′4′56 22′33′45 + 22′33′55′6 233′44′6 22′33′45′6 22′34′55′6 22′344′5′6 233′455′ 22′33′44′ + 22′3455′6 22′33′456′ 22′33′55′66′ 22′33′4′56 22′33′45′66′ + 22′33′44′6 22′33′456 22′33′44′66′ 22′33′455′ 22′344′55′ 233′4′55′6 22′33′4566′ 233′44′5′6 22′33′44′5 22′33′455′6′ 233′44′56 22′33′44′5′6 22′344′55′6 22′33′455′66′ 22′33′44′566′ + 22′33′44′56 22′33′44′55′ 233′44′55′6 22′33′44′55′6 22′33′44′55′66′ 3 4 24 23′ 23 24′ 34 2′35 245 23′5 23′4 24′5 244′ 2′34 + 233′ 234′ 33′4 344′ 23′45 2345 234′5 244′5 23′4′5 23′44′ 233′4′ 2344′ 24′5 2′3455′ 2′344′5 23′44′5 2′33′45 2344′5 233′44′ 23′44′55′ 233′44′5 233′44′5′ 233′44′55′ 35 34′ 44′ 344′5 33′44′ 33′44′5 22′344′566′ 33′44′55′ 33′44′

1 1 1,2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1,2 1 1,2 1 1 1 1,2 1 1 1 1 1 1 1 1 2,3 2,3 2 2 2 2 2,3 2 2 2 2 2 2 2 2 2 2 2,3 2,3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3

Internal standards. Numbers in bold indicate

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Table 2. Selected Ions of Organochlorine Pesticides Used for Qualitative and Quantitative Analysis peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 1 2 3

compd R-BHC PCB14c PCB30b δ-BHC heptachlor aldrin oxychlordane heptachlor epoxide o,p′-DDE γ-chlordane R-chlordane endosulfan I trans-nonachlor p,p’-DDE dieldrin o,p′-DDD endrin o,p′-DDT PCB161b p,p’-DDD p,p’-DDT PCB159c methoxychlor mirex β-BHC δ-BHC PCB61c tribromobiphenylb endosulfan I endrin aldehyde endosulfan sulfate HCB PCB204c tetrabromobiphenylb

ions a

Table 3. Recoveries and Method Detection Limits for Selected PCBs and Pesticides

compd name

spiked level (ng/g wet wt)

PCB4 PCB18 PCB52 PCB44 PCB101 PCB87 PCB183 PCB201 PCB180 PCB207 PCB194 PCB209 R-BHC heptachlor aldrin oxychlordane o,p′-DDE R-chlordane endosulfan trans-nonachlor p,p′-DDE dieldrin o,p′-DDD endrin o,p′-DDT p,p′-DDD methoxychlor mirex PCB28 PCB123 PCB114 PCB167 PCB156 PCB157 PCB189 PCB81 PCB77 PCB126 PCB169 HCB

16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 1.67 1.67 1.67 1.67 1.67 1.67 1.67 1.67 1.67 1.67 16.7

fraction

181, 219 222, 224 256, 258 181, 219 100, 272 263, 265 115, 185 353, 355 246, 248 375, 373 375, 373 237, 277 409, 407 246, 318 263, 277 235, 237 263, 281 235, 237 360, 362 235, 237 235, 237 360, 362 227, 228 272, 274 181, 219 181, 219 292, 290 390, 392 237, 207 250, 345 272, 387 284, 286 464, 466 470, 472

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3

a Numbers in bold are ions used for quantitative analysis. b Surrogate standard. c Internal standard.

stereoisomers in samples of biota. After Creaser et al.41 first used PGC to successfully separate PCBs, PCDDs, and PCDFs, this column has been widely applied for analysis, including that of coplanar PCBs. Hong et al.42,43 developed a rapid separation process with a PGC column for the isolation of mono- and nonortho-substituted PCB congeners, with hexane as the eluting solvent. Bo¨hm et al.31 used the PGC column and separated the coplanar from other congeners with gradient elution using cyclohexane and toluene. Zebuhr et al.44 used an automated HPLC coupled column system consisting of an amino column and a PGC column for the analysis of PCDD/PCDFs, PCBs, and polycyclic aromatic compounds (PACs). The separation is based on the retention of planar or near-planar molecules by the graphitic surface of the adsorbent. Nonplanar molecules are either unretained or have a limited retention, whereas non-ortho-substituted PCBs can assume a coplanar configuration more readily than can the ortho-substituted isomers, thus allowing a stronger interaction with the planar PGC structure. Retention on the PGC stationary phase is also highly influenced by the polarity of the eluant. With (41) Creaser, C. S.; Al-Haddad, A. Anal. Chem. 1989, 61, 1300-1302. (42) Hong, C. S.; Bush, B.; Xiao, J. Chemosphere 1992, 24, 465-473. (43) Hong, C. S.; Calambokidis, J.; Bush, B.; Steiger, G. H.; Shaw, S. Environ. Sci. Technol. 1996, 30, 837-844. (44) Zebuhr, Y.; Naf, C.; Bandh, D.; Broman, D.; Ishaq, R.; Pettersen, H. Chemosphere 1993, 27, 1211-1219.

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a

fraction

% recoverya (n ) 4)

RSD (%)

MDL (ng/g wet wt)

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3 3 3

73 50 65 67 110 82 93 77 81 74 60 78 69 70 94 89 94 97 99 90 108 99 90 107 102 100 51 82 78 116 91 108 112 106 99 93 106 105 92 72

14.4 14.3 4.0 2.9 9.0 12.7 12.5 3.0 3.1 2.2 6.1 12.5 22.0 5.7 2.4 4.4 3.2 4.0 10.2 3.4 8.7 3.0 2.4 7.1 3.7 8.2 8.2 2.9 19.6 6.6 7.8 7.2 3.3 7.3 2.2 11.3 9.7 9.8 13.6 28.8

1.09 0.05 0.43 0.48 0.02 0.59 0.36 0.56 0.62 0.53 0.54 0.53 0.78 0.19 0.43 0.49 0.74 0.70 0.61 0.70 0.77 0.70 0.35 0.86 0.67 0.53 0.62 0.57 0.69 0.43 0.49 0.22 0.36 0.41 0.41 0.03 0.04 0.04 0.03 1.05

Recovery corrected to surrogate recovery.

use of a suitable eluant, the separation between the planar PCBs and other coeluted PCBs on the PGC column will be improved. PCBs and pesticides in the PGC fractionations, along with their corresponding surrogate and internal standards, are listed in Table 1. In fraction 1, which is eluted with hexane, the congeners with two or more chlorines substituted on the ortho positions and some of the mono-ortho-substituted PCBs with lower chlorine substitution are eluted in the first fraction. Some congeners, which have two ortho-substituted chlorines on one benzene ring, may not elute completely into this fraction, especially when the concentration is high, for example, congeners 110 (233′4′6), 164 (233′4′5′6), 158 (233′44′6), 193 (233′4′55′6), 191 (233′44′5′6), and 190 (233′44′56). In fraction 2, which is eluted with 50% dichloromethane in hexane, most of the mono-ortho PCBs and some minor di-ortho-substituted PCBs are eluted. Some non-ortho-substituted PCBs having no more than three chlorine substitutions (e.g., congeners 2 (3), 3 (4), 12 (34), 35 (33′4), and 37 (344′)) coeluted into this fraction. The third fraction contained non-ortho-substituted PCBs. Although it is not easy to explain the elution pattern observed with simple adsorption theory, it is clear that the retention of PCBs

Figure 1. GC/ECD chromatograms of fractions from PGC preseparation of a black-crowned night heron (N. nycticorax) egg sample. Refer to Table 1 for the peak label.

on PGC is dependent on the number of ortho chlorines more than those of the PYE stationary phase. On the PYE stationary phase, some di-ortho-substituted congeners, including congeners 194 (22′33′44′55′) and 170 (22′33′44′5), eluted after mono-orthosubstituted congeners.38 The elution pattern of pesticides on PGC was also evaluated. Most of the 26 organochlorine pesticides analyzed were eluted in the first fraction. However, endosulfan-II, endrin aldehyde, endosulfan sulfate, and predominant β-HCH and δ-HCH were eluted in the second fraction. HCB eluted exclusively in the third fraction as a result of its flat structure and the π electronic structure of the benzene ring portion of HCB molecule. GC and GC/MS Determination. It is not possible to separate all the 209 PCB congeners with a single capillary column. MDGC has been applied to supplement a confirmation when the complete GC separation is difficult and when MS has been of limited confirmatory use because of the similarity of the mass spectra of PCB isomers. The multidimensional technique has considerable potential, but it has not yet been fully refined to “heart-cut” the chromatographic peaks and to quantitate each peak with an

internal standard. The variability in retention time on the precolumn, poor accuracy of heart-cuts, long run times, difficulty of using the internal standard, and cost of MDGC have restricted this technique to only a few applications. At this time, most PCB analysis is performed with off-line rather than on-line preseparation. The HPLC fractionation can solve some of the coelution problems when the congeners coeluting by GC are separated into different fractions. Hess et al.34 have reviewed the methods for analysis of nonand mono-ortho PCBs. Cochran and Frame21 have reviewed the high-resolution GC of PCBs. From the compiled data (the retention and coelution of 209 PCB congeners on the various GC columns), many stationary phases have been compared for the separation of PCB congeners.45 In particular, the DB-XLB column shows a low number of coelutions (34 out of the 209 congeners), and in many cases, these coelutions can be resolved by MS.46 Because of the preseparation by HPLC PGC, some congeners have been resolved into different fractions, and thus, only 13 pairs (45) Frame, G. M. Fresenius’ J. Anal. Chem. 1997, 357, 701-713. (46) Frame, G. Anal. Chem. 1997, 69, 468A-475A.

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of the 146 congeners coeluted. In the first fraction, 12 pairs of congeners coeluted on the column. Seven of the 12 pairs can be quantitatively analyzed by GC/MSD,46 if it is necessary. In the second fraction, only one pair, congeners 33 and 20, coeluted on the column. There were no congeners in the third fraction coeluted on the column, which benefitted from the preseparation and high resolution of the DB-XLB capillary column. This is important, because TEQs in ecotoxicological investigations are primarily concerned with the coplanar PCB congeners10,47 that eluted in the third fraction. Coplanar PCBs 77, 81, 126, and 169 may contribute to most toxic effects associated with PCB contamination in birds because of its greater potency relative to other congeners.10 For the pesticide analysis, seven pesticides coelute with PCBs on the DB-XLB column, (e.g., endosulfan I with PCB 99, p,p′-DDE with PCB 136 and 117, dieldrin with PCB 154 and 85, endrin with PCB 147, o,p′-DDT with PCB 134, p,p′-DDT with PCB 138, and mirex with PCB 199). Although these pesticides coeluted with PCBs on the column, they can be analyzed by GC/MSD with SIM mode. Table 2 listed the selected ions for qualitative and quantitative analysis for pesticides. With the selected ion and retention time, the quantification of pesticides was performed without interference from the coeluting PCBs. The recoveries for the PCBs and pesticides in the three fractions were checked by spiking into chicken eggs with selected common PCBs and pesticides at a concentration of 16.7 ng/g, and coplanar and semicoplanar PCBs at 1.67 ng/g, as shown in Table 3. The mean recovery for the ortho PCBs and pesticides in the first fraction was 84%, with a range of 50-110%. In the second fraction, the mean recovery for the mono-ortho PCBs was 101%, with a range of 78-116%. In the third fraction, the recovery of coplanar PCBs was 99%, with a range of 92-106%, whereas for HCB, the recovery was 72%. The averaged relative standard deviation is 9.8% for 23 selected PCBs and 17 pesticides spiked. This method has been applied to black-crowned night-heron (N. nycticorax) egg samples collected from Baltimore Harbor and elsewhere in the Chesapeake Bay. The samples were treated

according to the extraction and cleanup procedures described above. GC chromatograms from the three fractions collected from HPLC (PGC) analysis of the sample extract are shown in Figure 1. Some data, including concentrations of coplanar, semi-coplanar, and total PCBs, and eight chlorinated pesticides, total TEQs from PCBs, and the relation between contaminant concentrations and reproductive success, were recently published.4 The detailed results, including concentrations of individual PCBs and pesticides, individual PCB TEQs, and the patterns of PCB accumulation in heron eggs, will be published in a separate paper.

(47) Metcalfe, C. D.; Haffiner, G. D. Environ. Rev. 1995, 3, 171-190.

AC0205560

1066

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CONCLUSIONS The described method is suitable for the analysis of non-, mono-, and di- to tetra-ortho PCBs and OC pesticides in eggs, because it shows good reproducibility and low detection limits. With slight modification in sample preparation, this method could be applied to nearly any environmental matrix (e.g., for sediment, to eliminate protein precipitation with methanol, and to incorporate removal of sulfur prior to Florisil cleanup). The HPLC fractionation using a PGC column offers an excellent and reproducible method for separation of non-ortho PCBs from the remaining PCBs, and it is not sensitive to lipids that are remaining in the sample extracts. All of the current list of 172 analytes (146 PCB congeners and 26 OC pesticides) could be detected and quantified with this method, which facilitates the possibility of later accurate analysis of these compounds, given that some (congeners that have the potential to bind to chlorinated dioxin receptors and from which proposed dioxin-like toxic equivalencies may be estimated) have been assigned toxic equivalent factors,10 but others have not. ACKNOWLEDGMENT The authors thank the Chesapeake Bay Environmental Effects Committee Toxic Research Program and the Virginia Sea Grant College Program of the National Oceanic and Atmospheric Administration for providing financial support. Received for review December 13, 2002.

September

9,

2002.

Accepted