Environ. Sci. Techno/. 1995, 29, 2603-2609
Screening Assay for Dioxin=like Compounds Based on Competitive Binding to the Murine Hepatic Ah Receptor. 2. Application to Environmental Samples KEKE HU AND NIGEL J. BUNCE* Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N l G 2W1 BROCK G. CHITTIM, C O L L E E N H . M. T A S H I R O , B R I A N R . Y E O , B O N N I E J . SHARRATT, FIONA J , CAMPBELL, AND DAVID W. POTTER Wellington Laboratories, 398 Laird Road, Guelph, Ontario. Canada N1 G 3x7
A series of environmental samples, comprising fly ash, caustic wash water from regeneration of a petrochemical reforming catalyst, fish homogenates, soil, and pulp mill sludge, were analyzed for dioxinlike compounds by an assay involving competitive binding of a reference radioligand and an extract from the sample to mouse hepatic Ah receptor; the results were compared with GC/MS. The bioassay generally gave higher TEQ values than GUMS. Further analysis by GC/MS in most cases identified much of the additional Ah receptor-active material as coplanar PCBs and PAHs.
Introduction The presence in the environment of dioxin-likecompounds (I),which include polychlorinated congeners of the dibenzop-dioxin, dibenzofuran, biphenyl, naphthalene, azobenzene, and other families, continues to be of concern. The determination of the TCDD equivalent concentration (TEQ) of these contaminants in an environmental matrix is time consuming and labor intensive, and hence costly because it involves many steps. The preparation of the sample for analysis typically involves spiking the sample with an isotopicallylabeled dioxin surrogate such as [13C121-TCDD, followed by extraction of the PCDDlPCDF fraction into an organic solvent and multiple chromatographic procedures to isolate the PCDD/PCDF fraction. Separation and quantitation of the individual PCDD and PCDF congeners is then carried out using capillary GC/high-resolution MS, with the overall recovery determined by reference to the * Corresponding author; telephone: (519) 824-4120, ext. 3962; Fax: (519) 766-1499; e-mail address:
[email protected].
0013-936X/95/0929-2603$09.00/0
D 1995 American Chemical Society
isotopically labeled surrogate. Finally, the TEQ of the sample is obtained by the application of toxic equivalency factors (TEFsl (2) for the 17 PCDD and PCDF congeners that are chlorinated in the 2,3,7,and 8 positions and hence considered to be the most toxic. The complexity and cost of congener-specific analysis make it impractical to mount extensive screening or monitoring programs for dioxin-like substances in environmental matrices. In addition, the omission of other substances beside the 17 named PCDDs and PCDFs from the I-TEF scheme could cause the toxicity of an environmental sample to be underestimated. For example, the coplanar PCB congeners 77,126, and 169 have provisional TEFs for dioxin-like activity0.01,0.1, and 0.05, respectively (3).Because their concentrations in animal tissues may be orders of magnitude higher than those of PCDD/PCDFs (41,their toxic potential might be equally or more important than that of the 17 named PCDD and PCDF congeners. There is thus a need for an assay that would screen environmental samples for PCDDs, PCDFs, and related compounds to yield a TEQ value inclusive of the contributions of all dioxin-like contaminants. In the accompanying paper (3, we have proposed an assay based upon the competitive binding of the analyte and a reference radioligand to the intracellular Ah (aryl hydrocarbon) receptor protein. This method is attractive because the toxicity of dioxin-likecompounds is mediated through the Ah receptor (6, 7), and so the extent of binding to the receptor might serve as a surrogate measure of toxicity. Correlations between the strength ofbinding to the Ah receptor in vitro, enzyme induction in vitro, and toxic potency in vivo have been established for numerous classes of halogenated aromatic compounds (3). The assay is based on a previouslyreported competition method developed by Bradfield and Poland (8) for halowe dibenzo-p-dioxins. In the accompanying paper (3, extended the concept of Bradfield and Poland to polychlorinated congeners of several dioxin-like families and used the commercially available [3Hl-TCDDas the radioligand. Most importantly, we demonstrated additive behavior for a wide variety of mixtures of dioxin-like compounds, because a receptor binding assay would be invalid if one dioxin-like compound were to cancel out the binding effect of another. For synthetic samples, the assay affords the total TEQ of the sample, comprising all substances which interact with the Ah receptor (and which hence are potentiallytoxic),whether or not they have I-TEFs assigned and without preseparation of the mixture. However, the assay does not identify the specific dioxin-like compounds in the mixture. In this work, we apply the Ah receptor assay to actual environmental samples and compare the TEQ values with those obtained by GUMS. We also investigate the extent of chromatographic cleanup needed for successful application of the A h receptor assay to these samples.
Methodology Chemicals. Glycerol,sodium ethylenediamine tetraacetate (EDTA), Celite 545, and dithioerythritol (DTE) were purchased from Fisher Scientific Company (Fair Lawn, NJ). Dimethyl sulfoxide (DMSO) was purchased from the same
VOL. 29, NO. 10,1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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company in Nepean, ON. N-2-HydroxyethylpiperazineN-2-ethanesulfonic acid (HEPES)and bovine serum albumin (BSA) were purchased from Sigma Chemical Company (St. Louis, MO), hydroxylapatite and protein assay dye were from Bio-Rad Laboratories (Richmond,CA), Triton X-100 was from Terochem Laboratories Ltd. (Edmonton, AL), and activated carbon was from Anderson Development Company (Adrian, MI). Cytoscint (Liquiscint), commercially prepared scintillation fluids, were purchased from ICN Biomedicals Inc. (Irvine, CAI. [3Hl-2,3,7,8-TCDD, specific activity of 35 Cilmmol, was purchased from Chemsyn Company (Lenexa, KS). Organic solvents, e.g., toluene, dichloromethane (DCM),hexane, and cyclohexane, used for sample extraction and cleanup were purchased from Caledon Laboratories Ltd. (Georgetown, ON). Sample Selection and Preparation. Lake trout homogenates (Lake Ontario) were supplied by D. M. Whittle and D. B. Sergent, Fisheries and Oceans Canada; carp homogenates (Lake Huron, reference material) was supplied by M. Siu and S. Berman, National Research Council of Canada, Ottawa; fly ash 1 (Commissioners Street, Toronto, MSW incinerator) and fly ash 2 (Solid Waste Reduction Unit (SWARU) Hamilton, ON) were supplied by R. E. Clement, Ontario Ministryof Environmentand Energy (Toronto,ON); caustic wash water came from a petroleum refinery catalyst regeneration unit (Sarnia,ON);soil (electronicsalvageyard, Halifax Co., Nova Scotia) was supplied by R. Berrigan, Environment Canada; pulp mill sludge originated at an unnamed Canadian chlorine bleaching mill. Extraction Procedures. Samplesfor GClMSwere spiked with 13C12 surrogates before beginning extraction. Fish Samples. About 10 g of frozen homogenate was thawed at room temperature, mixed with anhydrous Na2SO4 (-100 g) in a glass mortar, and placed in an empty chromatographic column (4 cm i.d. x 25 cm long). Another layer (4 cm) of Na~S04was added, the mortar and pestle were rinsed three times with DCM, and the rinsings were transferred to the column, which was eluted with 300 mL of DCM. The eluate was evaporated to dryness and weighed to determine the “lipid content” of the samples. After dissolution in hexane (10 mL), 2.5 mL of the solution was removed for Ah receptor assay and labeled “sample after extraction”. The remaining solution was then transferred quantitatively to a GPC column (BioBeadsS-X3,3.5cm i.d. x 30 cm), which was eluted with 1:l DCMlcyclohexane. The first fraction (80 mL) contained the majority of the lipid and was discarded. The second fraction (150 mL) contained the analytes and was evaporated to dryness. A third eluate (100mL) was used to clean the column for the next set of extracts to be processed. The residue from the second fraction was dissolved in hexane (7.5 mL), and 2.5 mL of the sample was removed for Ah receptor assay and labeled “sample after GPC”. The remaining solution was chromatographed on 7.5 g of activated alumina (Bio-Rad AG 10) and covered with about 2 cm of anhydrous Na2S04 to absorb water, eluting with hexane (25 mL) and then with 60% DCMlhexane (30 mL). The second eluate was evaporated to dryness, hexane (5 mL) was added to the residue, and 2.5 mL of the solution was removed for Ah receptor assay and labeled “sample after alumina”. The remaining solution was chromatographed over a 8.5% (wl w) PX21 activated carbon on Celite, which had been precleaned using Soxhlet extraction with toluene (48 h) and then dried in a convection oven prior to use. The column (0.25 g) was prerinsed with 10-12 mL of toluene, 2604
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then inverted,and further rinsedwith, in the followingorder, benzenelethyl acetate (1:L 2 mL), DCMlcyclohexane (1:L 2 mL), and finally hexane (2 mL);the rinses were discarded. The hexane extract of the analytes was applied to the column, followed by a hexane (1mL) rinse of the container. The column was then eluted with DCMlcyclohexane (l:l, 2 mL) followed by bemenelethyl acetate (l:l,2mL); the eluate was discarded. After all these rinses, the column was again inverted and eluted with 20 mL of toluene, and the eluate was evaporated to dryness. Hexane (2.5 mL) was added, and the solution was labeled “sample after carbon”. Other Samples. Fly ash and soil samples were first dried at 40 “C in a convection oven, and a 10-20-g subsample was Soxhlet-extracted for 20 h with toluene. The extract was concentrated by solvent exchange into nonane and then subjected to the multilayer column chromatography procedure described below. The petrochemical catalyst wash water samples (1 L) were extracted with 3 x 70 mL of DCM using a separatory funnel. The combined DCM extracts were evaporated to dryness and then subjected to multilayer column chromatography as described below. The pulp mill sludge and two of the soil sampleswere treated by acid extraction prior to chromatography. The concentrated extract in nonane was diluted with hexane (10 mL) and transferred to a solvent-prerinsed, screw-capped culture tube with a Teflon-lined cap and then extracted with concentrated sulfuric acid (3 mL). The hexane layer was separated from the acid layer by centrifugation and then chromatographed. Multilayer column chromatography was used for all the samples listed above. Into a clean, dry glass chromatographic column (2 cm i.d. x 25 cm long) plugged with glass wool were introduced the following sorbents (frombottom to top): silica (1 g), NaOH/silica (2 g), silica (1 g), H2S04/ silica (2 g), and silica and anhydrous sodium sulfate (2 g). The column was prerinsed with hexane (30mL),the extract was quantitatively transferred to the top of the column using hexane rinses (2 x 5 mL), and the column was eluted with 120 mL of hexane. The eluate was evaporated to dryness and taken up in 7.5mL of hexane. A 2.5-mLsample was removed and labeled “sample after multilayer”. The remaining solution was chromatographed over alumina and carbon as described for the fish tissue. HRGC/HRMS Analysis. GClMS analysis was based on U.S. EPAMethod 1613. The eluate from the carbon column was concentrated by solvent exchange in nonane, and internal standards were added prior to injection. The HRGClHRMS system was a Hewlett Packard 5890 Series I1 capillary GC (60 m DB-5 column), coupled to a VG 70SE magnetic sector MS, with a DEC 3100 Model 38 workstation using the OPUS 2020 operating system. C57BL16N Mouse Hepatic Cytosol. The C57BLl6N mouse cytosol was prepared from the liver of immature male mice (average weight 20 g) by the method previously described (3 and was stored in small aliquots at -70 “C. The buffer, HEGD, contained 1 mM HEPES, 1 mM EDTA, 10%(vlv)glycerol,and 1mM DTE at pH 7.60. For maximum stabilizationof the hepatic cytosolicTCDD-specificbinding components, DTE was added the day of use. Protein concentration was determined by the method of Bradford (9) using Bio-Rad protein assay dye reagent and BSA as a standard. Bioassay Protocol. The hexane extract (2.5 mL) was transferred quantitatively to a vial and evaporatedto dryness
TABLE 1
Summary of Bioassay and 6C/MS Analysis of Environmental Samples after Last Stage of Chromatographic Cleanup TEQ (named PCDDP) GCMS
sample
bioassay
Fly ashes 1 and 2 Fly ashes 3 and 4 petrochemical wash lake trout A lake trout B carp I and II carp I II soil I soil II soil 111 pulp mill sludge
240 ppb 3 PPb 640 ppt 720 ppt 760 ppt 1100 ppt < 380 ppt 800 ppb 1100 ppb 22 PPb 500 ppb
110 ppb 0.4 ppb 250 ppt 72 PPt 76 P P ~ 19 PPt 19 PPt 2 PPb 1 PPb 1 PPb 7.5 ppb
TEQ (coplanar PCB) GCMS
total PCDDlF GCNS
ND ND ND 720 ppt 150 ppt 40 PPt 40 PPt 14 PPb 2 PPb 18 PPb
53 PPb 11 ppb 13 PPb
PAH < 1 ppb PAH, 5 ppb PAH, 2 ppt
54 ppb 37 PPb 20 PPb
organochlorines, > 250 ppb organochlorines, =- 250 ppb PAH, 320 ppb PAH, 1200 ppb PAH, 170 ppb
under Nz, and the residue taken up in lOOpL of DMSO. An approximate EC5o for each sample was obtained by using a broad range of concentrations prepared by serial 10-fold dilution; a more precise EC50 was then secured from a second series of solutions prepared by serial 3-fold dilution 1 order of magnitude on each side of the approximate EC50. Ten microliters of the DMSO solution was used to prepare each “dilution series”, and the dilution factor (DF)was the ratio “concentration of original extract in DMSO/concentration of solution in DMSO”. The incubation protocol was described in detail previously (5). The incubation mixtures were prepared by adding 1.0 mL of cytosol to a mixture of 10 pL of 100 nM radioligand and 10 pL of the extract or diluted extract. Nonspecific binding was estimated by running control incubations containing 10pL of 100 nM radioligand and 10 pL of 20 pM TCDF. Percent specific binding (%SB) was obtained as (100 x SB for a given concentration of competitor + SB for incubations containing no competitor). %SB was plotted against loglo(DF) in order to determine the value DF50 at which %SB = 50%. The dioxin equivalent concentration (DEQ) (in nmol L-l) was the product (1.2 nh4 x DF50 x 1001, where 1.2 nM is the experimental EC50 of unlabeled TCDD (5), and the factor 100 refers to the fact that 1OpL of the original extract was diluted to 1.0 mL in preparing the incubation mixture. This DEQ applies to the extract that was assayed, not to the original soil, fish, etc. The TEQ (in ng) of the original sample was calculated using eq 1, in which the factor 1.0 x L reflects the fact that 1OOpLof DMSO extract were prepared, and 322 is the molar mass of TCDD in g mol-’. Equation 2 relates the TEQ of the DMSO extract (corresponding to a dose of TCDD) to the TEQ of the original sample (corresponding to a concentration of TCDD). TEQ = DEQ (nM)x (1.0 x
L) x (322 g mol-’) (1)
TEQ, sample, ppb = TEQ, extract, ng (2) mass of sample used to prepare extract, g In the data in this paper, each original sample provided four DMSO extracts, and therefore the mass of sample in eq 2 corresponds to one-quarter of the mass actually taken.
Results and Discussion All experimental results are summarized in Table 1,which gives the bioassay after the last stage of chromatography, along with the TEQ obtained by GClMS for comparison
: 5
IO
-oioo
o.doo
.
-
o.;oo
other components GCNS
I.dO0
I
: Fly O i h I a l l a sarhn:
A
boo
2.d00
2.;00
3.doo
3 . L
Logarithm of Dilution Factor
: Fly a i h Ill d l i r e e r h n ;
a
-
: a
Fb osh I1 o f t w ~ w b o n :
: fly o i h IV olter c0rMn
FIGURE 1. Graphical determination of TCDD TEQs of fly ash fractions after carbon column cleanup.
and other analytical data obtained by GUMS. Detailed results appear in later tables. Fly Ash. PCDDs and PCDFs form whenever chlorinecontaining materials are combusted, and they adsorb physically to the fly ash (10). Fly ashes I and I1 (duplicates) were taken from the CommissionersStreet municipal solid waste incinerator, Toronto, Ontario, now closed because of concerns about its high emission levels; Fly ashes I11 and IV (duplicates)were taken from the more modem Hamilton (Ontario) Solid Waste Reduction Unit (SWARU). Each sample yielded four extracts for analysis each derived from 2.5 g of the original fly ash. The TEQ values obtained by both bioassay and GUMS of fly ash from the Commissioners Street MSWI were almost 2 orders of magnitude greater than those from the SWARU: see Figure 1, which compares the curves of log DF vs %SB for fly ash samples after the last carbon column cleanup. This is consistent with the higher temperature of incineration achieved in the SWARU, because high combustion temperatures greatly disfavor PCDD and PCDF formation (11). The TEQ values of the Commissioners Street fly ash declined by less than a factor of 2 as the chromatographic cleanup progressed (Table 2). For this matrix, the Ah receptor-based bioassay could be carried out without extensive prior cleanup of the extracts. The TEQ of these samples determined by GUMS was about 110 ppb, about half the value obtained by the bioassay (240 ppb, average). Congener-specific analysis by GClMS (Table 3) revealed that the PCDDs and PCDFs were highly chlorinated, typical VOL. 29,
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TABLE 2
TABLE 4
TCDD TEQs of Fly Ash Samples (ppb) samples
detection TEQ PCDDs/ TEQ limit PCDW (bioassay) (bioassay) (GCIMS)
fly ash I after extraction after multilayer after alumina after carbon
394 301 283 224
fly ash I1 after extraction after multilayer after alumina after carbon
355 265 29 1 360
TGDD TEQs of Petrochemical Catalyst Wash Water Samples (ppt) PAHs (GCIMS)
0.3
samples
115
NDb < 1.09 ppb
0.3
lab blank (I and 11) after extraction 4.64 after multilayer < 0.3 after alumina 4.66 after carbon < 0.3 fly ash 111 after extraction 18.0 after multilayer 13.8 14.4 after alumina after carbon 3.40 fly ash IV after extraction 38.7 after multilayer 30.8 after alumina 27.4 after carbon 2.83 lab blank (111 and IV) after extraction < 0.3 < 0.3 after multilayer after alumina < 0.3 after carbon 0.8
109
ND < 0.65 ppb
1
788 762 407 407
182
3
PCODPCDF levels in Caustic Wash Water Samples
0.3
(PPt) Iwo8-15
W8-16
total TCDD total pentaCDD total hexaCDD total heptaCDD OCDD
NDs (0.2) 10.75b 1017 2772 274’
ND (0.2) ND (0.2) 1.5* 13.82 34.6’
total PCDDs total TCDF total pentaCDF total hexaCDF total heptaCDF OCDF
66315 48915 133616 454712 3548’
505 80.414 435’3 262112 47424 2415’
total PCDFs
16520@ 1718363
1029344 1034349
congeners 0.30
3.6
0.43
6.9
0.3
total PCDDs/PCDFs
Congener Concenttations in Commissioners Street Fly Ash Samples (ppb) I-TEF
fly Ash I
fly Ash II
1 0.5 0.1 0.1 0.1 0.01 0.001 0.1 0.5 0.05 0.1 0.1 0.1 0.1 0.01 0.01 0.001
4.6 37.7 47.0 55.3 119 279 395 24.6 16.2 34.8 102 46.1 63.4 3.0 2 48 15.5 55.6
4.1 38.6 43.8 50.2 105 258 350 28.5 14.5 31.4 104 41.4 57.5 2.4 237 13.9 50.8
ofcombustion sources (12). PAHs,whichmight be expected to form under conditions of incineration, were below the level of detection. The total TEQ of the SWARU fly ash samples ranged from 20 to 40 ppb in the initial extracts. It dropped to about 3 ppb after the carbon column cleanup, but remained approximately 1 order of magnitude greater than the value obtained by GCIMS. This indicated the presence of substances additional to the 17 named PCDDs and PCDFs, some of which could be removed during chromatography. 2606
324 4
~~~
TABLE 3
2,3,7,8-TCDD 1,2,3,7,8-pentaCDD 1,2,3,4,7,8-hexaCDD 1,2,3,7,8,9-hexaCDD 1,2,3,6,7,8-hexaCDD 1,2,3,4,6,7,8-heptaCDD OCDD 2,3,7,8-TCDF 2,3,4,7,8-pentaCDF 1,2,3,7,8-~entaCDF 1,2,3,4,7,8-hexaCDF 1,2,3,7,8,9-hexaCDF 1,2,3,6,7,8-hexaCDF 2,3,4,6,7,8-hexaCDF 1,2,3,4,6,7,8-heptaCDF 1,2,3,4,7,8,9-heptaCDF OCDF
4 5.45 x 103 1.84 x lo3 914 881
TABLE 5
a PCDDs/PCDFs are the 17 named PCDDs/PCDFs that have I-TEF values assigned. ND = not detected.
congeners
8908-15 after extraction after multilayer after alumina after carbon 8908-16 after extraction after multilayer after alumina after carbon
detection TEQ PCDDs/ TEQ limit PCDFsa PAHs (bioassay) (bioassay) (GCIMS) (GCNS)
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a ND, not detected. bThe superscript represents the number of congeners.
Congener-specificanalysis by GCIMS showed the presence of other tetra- to hepta-chlorinated PCDDs and PCDFs (totalling 9.9 and 13.1 ppb) and PAHs (3.6 and 6.9 ppb, respectively). The TEQs of these “other”compounds cannot be estimated because no TEF values have been established. PetrochemicalCatalystWashWater. PCDFs are formed during the reactivation of the catalysts used in catalytic reforming of petroleum (13) and pass out with the caustic wash water. The samples were taken in 1989from a catalyst regeneration system at a petrochemical refinery and weighed 890 and 966 g, respectively (about 1 L). One-fifth of each sample was prepared for GUMS analysis; the remainder provided four extracts after liquid-liquid extraction and chromatography. The TEQ values obtained by bioassay were 2-3 times greater than those found by GCI MS and dropped by less than an order of magnitude as cleanup progressed (Table4). In agreement with the results of Beard etal. (131,the major components in both caustic wash water samples were PCDFs rather than PCDDs. Congener-specific analysis by GCIMS revealed numerous other PCDDIPCDF congeners in absolute (not TEQ) amounts of 17 .ppm (47 congeners) and 10 ppm (37 congeners, Table 51, along with miscellaneous other substances such as hexachlorobenzene, anthraquinone, dichloroanthracene (or dichlorophenanthrene), hexachloronaphthalene, trichlorofluoranthene (or trichloropyrene), nona- and decachlorobiphenyls, and low levels of PAHs.
TABLE 6
TABLE 7
TCDD TEOs of Fish Tissue Samples (ppt)
Fish Tissue Samples TER (GC/MS)
TEO samples
trout A1 after extraction after GPC after alumina after carbon trout A2 after extraction after GPC after alumina after carbon trout
(bioassay) 104 103
89
658c
103 103 44
120
108
184
310 1.85 1.61 1.36 773
103 103 103
1.31 948 750 1.10
103 103
19
41
19
40
19
41
418 2.57 1.46 1.19 1.21
103 103 103 103 380
561 722
