Anal. Chem. 1980, 5 2 , 2257-2262 Knox, J. H.; Pryde, A. J. Chromafogr. 1975, 112, 171-188. Scott, R. P. W.; Kucera, P. J. Chromafogr. 1977, 142, 213-232. Scott, R. P. W.; Kucera, P. J . Chromatogr. 1979, 175, 51-63. Westerlund, D.; Theudorsen, A. J. Chromatogr. 1977, 144, 27-37. Tilly-Melin, A,; Askemark, Y.; Wahlund, K. G.; Schill, G. Anal. Chem. 1979, 5 1 , 976-983. McCorrnick, R. M.; Karger, B. L. J , Chromatogr., in press. Knox, J. H., poster session presented at The 3rd International Symposium on Column Liquid Chromatography, Salzburg. Austria, Sept 1977. Berendsen, G. E.; Schoenmakers, P. J.; deGalan, L.; Vigh, G.; VargaPuchony, Z.; InczBdy, J. J. Li9. Chromafogr., in press. HorvBth, C.; Lin, H. J . Chromatogr. 1976, 126, 401-420. Scott, R. P. W.; Kucera, P. J. Chromafogr. 1976, 125, 251-263. Hemetsberger, H.; Kellerman, M.; Ricken, H. Chromafographia 1977, IO, 726-730. Slaats, E. H.; Kraak, J. C.; Brugrnan, W. J. T.; Poppe, H. J. Chromatogr. 1978. 149, 255-270. van de Venne, J. L. M. Ph.D. Dissertatlon, Technische Hogeschool Eindhoven, Eindhoven, The Netherlands, 1979. Halasz, I.J. Chromatogr. 176, 122,3-16. Karch, K.; Sebestian, I.; DeVault, D. J . Am. Chem. SOC.1943, 65, 532-540. Peterson, D. L.; Helfferich, F. J . Phys. Chem. 1965, 69, 1283-1293. Helfferich, F. J. Chem. Hut. 1964, 41, 410-413. Helfferich, F.; Klein, H. "Multicomponent Chromatography"; Marcel Dekker: New York, 1970; Chapter 3. Zhukovitski, A. A. I n "Gas Chromatography 1964"; Goldup. A,, Ed.;
2257
Institute of Petroleum: London, 1965; pp 161-169. (26) Reilley, C. N.; Hiklebrand, G. P.; Ashley, Y. W. Anal. Chem. 1962, 34, 1198-1213. (27) Scott. R. P. W.; Scott, C. G.; Kucera, P. Anal. Chem. 1972, 44, 100-104. (28) hais, K.; Krejd, M. J. Chromatogr. 1974, 91, 161-166. (29) Engelhardt, H. "High Performance Liquid Chromatography"; SpringerVerlag: New York, 1979; p 127. (30) Helfferich, F.; Peterson, D. L. Science 1963, 142. 661-662. (31) Conder, J. R. in "Progress in Gas Chromatography"; Purnell, J. H., Ed.; Interscience: New York, 1968; p 209. (32) Tanaka. N.; Thornton. E. R. J . Am. Chem. Soc. 1976, 98, 1617-1619. (33) Tanaka, N.; Thornton, E. R. J. Am. Chem. Soc.1977, 99, 7300-7307. (34) Unger, K. K. "Porous Silica; Its Properties and Use as Support in Column Liquid Chromatography": Elsevier: Amsterdam, 1979; p 170. (35) Snyder, L. R. "Principles of Absorption Chromatography", Marcel Dekker: New York, 1968; Chapter 8. (36) Parris. N. A. J. Chromafogr. 1978, 149, 615-624.
RECEIVED for review May 12,1980. Accepted September 2, 1980. The authors wish to acknowledge the support of the National Science Foundation. Contribution number 72 from the Institute of Chemical Analysis.
High-Resolution Gas Chromatography of the 22 Tetrachlorodibenzo-p-dioxin Isomers Hans Rudolf Buser * Swiss Federal Research Station, CH-8820 Wadenswil, Switzerland
Christoffer Rappe Department of Organic Chemistry, University of Umea, S-901 8 7 Umea, Sweden
The 22 tetrachlorodibenzo-p-dioxins (TCDDs) were synthesized in microgram quantities by a simple pyrolysis procedure from different potassium chlorophenates. The separation of these TCDD isomers was studied on high-resolution glass capillary columns with different stationary phases (Sllar IOc, OV-17, OV-101) and by use of mass spectrometric detection. Conditions were found that allowed the unambiguous asslgnment of many of these Isomers, including the very toxic 2378-TCDD. The determination of the various TCDD Isomers is illustrated in the analysis of samples from known contaminated areas in Seveso, Italy, and in eastern Mlssourl, and the method is also applied to the analysis of fish from the Tittabawassee River In Michigan and fly ash samples from municipal incinerators in Switzerland.
Polychlorinated dibenzo-p-dioxins (PCDDs) are a group of compounds that have been the subject of much concern in recent years. Some of these compounds have extraordinary toxic properties and are teratogenic, mutagenic, and potentially carcinogenic ( 1 ) . They are known as highly stable contaminants present in a variety of synthetic chemicals like the chlorinated phenols, phenoxy acids, and chlorobenzenes. PCDDs can be formed in substantial quantities in pyrolytic reactions from such common chemicals as the chlorinated phenols and chlorobenzenes (2, 3 ) . Recently, PCDDs have been identified in fly ash and emissions from municipal incinerators and industrial power facilities (4-8). 0003-2700/80/0352-2257$01 .OO/O
In all there are 75 positional PCDD isomers ranging from the mono- up to the octachloro compounds; there are 22 tetrachlorodibenzo-p-dioxins(TCDDs) alone. Positional isomers of the various PCDDs appear to vary greatly in acute toxicity and biological activity ( 9 , I O ) . The most toxic isomer seems to be 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378-TCDD) (11). This compound was involved in several industrial accidents and has caused severe environmental contamination, most recently a t Seveso, Italy (12). Highly sensitive analytical methodology is required to monitor levels of these compounds in environmental samples and to assess possible risks associated with the presence of these compounds. Detection limits in environmental and biological samples must be in the parts-per-trillion ( 10-l2) range (13). Furthermore, the analytical procedures should allow a differentiation between the various PCDD isomers. Many analytical techniques have been described for the analysis of PCDDs, the most specific methods making use of mass spectrometry (MS). In a series of papers we have reported on the application of high-resolution gas chromatography (HRGC) in combination with MS for the analysis of PCDDs and related compounds in various environmental, biological, and industrial samples (14-16). In this paper now, we report on the application of HRGC for the separation and identification of the various TCDD isomers. All 22 TCDD isomers were synthetically prepared from different chlorophenates using a simple pyrolysis procedure. These isomers were also synthesized by Nestrick et al. (17) using a more elaborate ex0 1980 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
Table I. HRGC Glass Capillary Columns Used in the Present Study column I I1 I11
stationary phase Silar 1Oc OV-17 ov-101
column efficiencyP tY Pe highly polar semipolar nonpolar
l,a m
55
50 50
mm 0.25 0.37 0.40
a 1 = length. i.d. = internal diameter of capillary column. T F = final column temperature. programmed conditions). temperature ("C) for naphthalene.
perimental setup. They used reversed- and normal-phase high-performance liquid chromatography (HPLC) for the separation and isolation of the TCDDs followed by packed column gas chromatography and mass spectrometry. T h e requirement of analyzing several fractions of a sample, in case knowledge on all PCDDs is desired, impairs the application of their method. In our approach, the sample purification was designed to yield a fraction containing the PCDDs as a group and final separation of the PCDD isomers was then achieved on highresolution glass capillary columns coupled to a mass spectrometer. None of the columns used gave complete separation of all the 22 TCDD isomers; however, conditions were found t h a t allowed the unambiguous assignment of many of these isomers, including the extremely toxic 2378-TCDD. T h e application of the method is illustrated in the analysis of samples from known contaminated areas in Seveso, Italy, and in eastern Missouri, and the method is also applied t o t h e analysis of fish from the Tittabawassee River in Michigan and to fly ash samples from municipal incinerators in Switzerland. This paper focuses on qualitative TCDD isomer identification, the quantification of these isomers was not yet thoroughly investigated.
EXPERIMENTAL SECTION Synthesis of TCDDs. The various TCDDs were prepared by microscale pyrolyses of different potassium chlorophenates under conditions we previously used for the preparation of a series of PCDDs (16, 18). TCDDs with a 2:2 chlorine substitution pattern ( 2 2 type isomers) were prepared by pyrolysis of individual trichlorophenates (tri-CPs) and various tri-CP combinations; 3:l type TCDDs were prepared by pyrolysis of mixtures of di- and tetrachlorophenates (di- and tetra-CPs). The chlorophenols used in the present study were of analytical quality from Fluka (Buchs, Switzerland); they were 2,3-, 2,4-, 2,5- and 2,6-dichlorophenol (di-CPs), 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, and 2,4,6-trichlorophenol (tetra-CPs). (tri-CPs), and 2,3,4,5- and 2,3,5,6-tetrachlorophenol The potassium salts of these tri-CPs and mixtures of di- and tetra-CPs were prepared with potassium hydroxide in methanol as previously described (18). Pyrolyses of these chlorophenates were carried out with quantities of about 1 mg placed in the tip of a glass re--tion tube (150 x 5 mm i.d.). The reaction tubes were plugged ith glass wool and a 5-cm layer of alumina. They were heated for 30-60 min to 300 "C. The exact conditions and the clean-up procedure for the TCDDs were as described previously (18). With yields in the microgram (pg) range the TCDDs prepared by this route were characterized by HRGC and MS without actual isolation. Additional Reference Samples. 1234-TCDD was the gift of K. Andersson, Research Institute of the Swedish National Defense, UmeB, Sweden; 2378-TCDD was obtained from Stickstoffwerke, Linz, Austria. In addition, samples of UV-photolyzed 123678- and 123789-hexa-CDD (19)and octa-CDD (16,20) containing tetra- and penta-CDDs formed by reductive dechlorination were used for reference purposes to obtain assignments of some of the TCDD isomers. Preparation and Purification of Environmental Samples. Seueso Soil. A soil sample of zone A, Seveso, Italy (courtesy of Givaudan Ltd., Dubendorf, Switzerland),taken after the accident in 1976 was extracted with n-hexane-acetone (1:l)and purified by partition and alumina chromatography as previously described
e
TE,="C 234.6 216.0 221.4
T F , d"C
N
240 250 250
192 000 152 000 138 000
K, TT (4.7, 100) (5.8, 80) (4.3, 90)
T E = elution temperature of 2378-TCDD (temperature N = theoretical plate number, K = capacity ratio, TT = test
L 2 2 t y w TCDDs
3 1
type TCDDs
Figure 1. Synthesis of tetrachlorodibenzo-pd~xins(TCDDs): 2:2 type TCDDs from the pyrolysis of trichlorophenates (tri-CPs) and 3: 1 type TCDDs from the pyrolysis of di- and tetrachlorophenates (di- and tetra-CPs). (15). A 1-mg soil aliquot (2 pL) in benzene was used for analysis. Missouri Horse Arena Soil. A 2-g soil sample from the Shenandoah Horse Arena, Missouri (courtesy of R. Kimbrough, Department of Health, Education and Welfare, Center for Disease Control, Atlanta, GA), was extracted with methylene chloride, the extract was filtered through alumina, taken up into n-hexane, and chromatographed on silica and alumina as described for fly ash samples (5,6). A 1-mg soil aliquot in benzene was used for analysis. Tittabawassee River Carp. A purified extract of a Tittabawassee river carp (courtesy of D. Stalling, Columbia National Fisheries Research Laboratory, Columbia, MO) was obtained. This extract had been purified by gel permeation and cesium silicate and carbon foam chromatography resulting in improved enrichment of planar aromatic contaminants (21). An aliquot in benzene corresponding to approximately 1 g of fish was used for analysis. Fly Ash from Municipal Incinerator. A 10-g sample of ash retained in the electrofilter of a municipal incinerator in Switzerland was extracted with methylene chloride and the extract purified by chromatography on silica and alumina as previously described (56).An aliquot corresponding to 0.4 g of fly ash was injected for GC-MS analysis. GC-MS Analysis. HRGC glass capillary columns (column characteristics given in Table I) were coupled via a platinum capillary interface leading into the ion source of a Finnigan 4000 quadrupole MS operated in the electron impact (EI) mode (70 eV, 250 OC). The samples (1-2 pL) were splitlessly injected (splitting valve and septum flush 45 s closed) and the column temperature was programmed as follows: 100 "C, 3 min isothermal, 20 OC/min to 180 "C, 2 OC/min to 240 or 250 "C (see Table I). Single- or multiple-ion detection ( m / e 320, 322, and 324) was used. Complete mass spectra (EI) for the samples were recorded by using a Finnigan 6115 data system (repetitively scanning m / e 35-500,1.5 s/scan). Isomer identification was based on coinjectionswith a selected number or all of the TCDD isomers and using single ion detection. Absolute retention times were used as guidance only. From the higher chlorinated PCDDs, only penta-CDDs may elute on the Silar 1Oc column in the TCDD elution range. Penta-CDDs have a low-intensity ion (M+ - 35 + 1)at the m / e values used for TCDDs; however, they are easily distinguished from TCDDs by the presence of the much more intense molecular ions (M+) a t m / e 354 etc.
RESULTS AND DISCUSSION PCDDs are formed in a twestep condensation process from ortho-halogen-substituted phenoxy anions. T h e reaction is best carried out by heating alkali metal salts of chlorophenols t o about 300 "C. We used this reaction in several previous investigations for the synthesis of a whole series of PCDDs ranging from tetra- u p to the octachloro compounds (18,22).
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
Table 11. 2:2 Type TCDDs Expected and Actually Observeda from the Pyrolysis of Individual Tri-CPs ret time
(mini \ ,
and elution temp isomer
("C) obsd on Silm 1 0 ~
1368137923781267128912691469-
27.85, 223.7 29.15, 226.3 33.25, 234.6 37.10, 240 39.60, 240 36.40, 240 35.40, 238.8
tri-CP pyrolyzed 235- 246- 245- 234-
236379
a
X
X
X
X
X X
x
X
X X
Major isomers italicized.
Pyrolysis of small amounts of polychlorophenates thus was found to be a simple and safe way to synthesize microgram amounts of PCDDs from readily available starting materials. TCDDs can be formed from the pyrolysis of tri-CPs or from combined di- and tetra-CPs (see Figure 1). In the first case, TCDDs with two chlorine substituents in each of the two carbon rings of the dioxin molecule are formed (2:2 type TCDDs, total of 13 isomers); in the latter case, TCDDs with three and one chlorine substituent in each ring are obtained (3:l type TCDDs, total of eight isomers) in addition to di- and hexa-CDDs (dimerization products of di- and tetra-CP, respectively). If required, 1234-TCDD can be prepared by pyrolysis of a mixture of 2-chloro- and pentachlorophenate; this isomer is the only 4:O type TCDD. Different isomers are formed, depending on the actual chlorophenates taken. Due to the Smiles rearrangement (16,23,24), some chlorophenates yield more PCDD isomers than expected from a simple condensation scheme. In this way, e.g., 246-tri-CP forms 1368and 1379-TCDD, the normal and the Smiles rearranged condensation products. In Table I1 we list the TCDDs expected from the pyrolysis of individual tri-CPs. There are seven 2:2 type TCDDs to be expected, and analysis of the pyrolyzed samples did in fact reveal this number of isomers. All these isomers were easily separated on the Silar 1Oc HRGC column. Assignment of 1368-TCDD as the first eluting and 1379TCDD as the later eluting isomer from the pyrolysis of 235(or 246-)tri-CP was based on photolysis experiments with two isomeric hexa-CDDs (19);from the same experiments, the assignments for 1267- and 1289-TCDD were made as the first and later eluting isomers from 234-tri-CP. Assignment of 1269- and 1469-TCDD from the pyrolysis of 236-tri-CP was based on photolysis experiments with octa-CDD in which the main reaction pathway was found to be via loss of chlorines from the lateral (2-, 3-, 7-, and 8-) positions leading to 1469-TCDD as the main TCDD isomer (16, 20, 22). T h e remaining 2:2 type TCDDs can be expected from the pyrolysis of various tri-CP combinations. As shown in Table I11 there are six additional isomers to be expected and this number of new isomers was in fact observed from these pyrolyses. Several of these isomers were studied in a previous
1279
1269
x a
i I
1
240
230
240
I
22o0c
23OoC
Flgure 2. Mass fragmentograms ( m l e 320; 55-m Silar 1Oc HRGC column) showing elution of (a) all 2:2 type TCDDs in a combined pyrolyzate sample of t r i p s and (b) all 3: 1 type TCDDs and 1234-TCDD
in a combined pyrolyzate sample.
investigation (22); some were also observed from the photolysis of the two hexa-CDDs (19). 1278-TCDD and 1469-TCDD had identical retention times on the Silar 1Oc HRGC column; however, these isomers were easily separated on OV-17 or OV-101 (see later). Assignment of 1268-TCDD as the first eluting, and 1279-TCDD as the later eluting isomer from the pyrolysis of the 246- and 234-tri-CP combination again was based on the photolysis experiments
Table 111. Additional 2 : 2 Type TCDDs from the Pyrolyses of Various Tri-CP Combinationsn ret time (min) and tri-CP combinations ( 1 : l ) pyrolyzed elution temp ("C) isomer obsd on Silar 1Oc 245- + 234- 245- + 235- 245- + 236- 246- + 234- 235- + 236127 813781478126812791369a
35.40, 238.8 30.65, 229.4 33.00, 234.1 32.30, 232.8 34.00, 236.1 31.45, 231.0
X
X X X
Isomers observed in addition to the TCDDs from the individual tri-CPs as listed in Table 11.
X X
X X
X
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
Table IV. 3:l Type TCDDs from the Pyrolysis of Di- and Tetra-CP Combinations retention time (min) di- and tetra-CP (1:1)combinations pyrolyzed and elution temp ("C) isomer obsd on Silar 1Oc 23- + 2345- 25- + 2345- 24- + 2356- 26- + 235612361239123711238-
34.05, 35.85, 33.55, 33.75. 31.60;
236.1 239.7 235.1 235.5 231.6 31.60, 231.6 33.60, 235.2 33.65, 235.3
124711248124611249-
X X X X X X
X X 2371
I
1488
1378
b
a
I
I
240
230
Figure 3. Mass fragmentograms ( m / e320) of and ( c ) 50-m OV-101 HRGC columns.
,
2;ooc
220
210°C
I
I
230
2m
2:ooc
a composite sample shc)wing elution of all 22 TCDD isomers on (a) 55-m Silar lOc, (b) 50-m OV-17,
of the two hexa-CDDs mentioned above (19) (1367- and 1389-TCDD in ref 19 are identical with 1268- and 1279-TCDD of this study, respectively). In Figure 2a we show a chromatogram of a composite sample of these pyrolyzates containing all 13 2 2 type TCDD isomers analyzed on a 55-m Silar 1Oc HRGC column. All isomers gave the characteristic mass spectral pattern (22) in the lower mass range differing from the pattern of the 3:l and 4:O type TCDDs. TCDDs with 3:l type substitution are obtained from the pyrolysis of mixtures of di- and tetra-CPs (see Figure 1). In Table IV we list all the 3:l type TCDDs expected from combinations of various di- and tetra-CPs. Again, analysis of the pyrolyzed samples revealed the presence of the expected number of isomers. Each of these pyrolysis experiments gave a pair of TCDD isomers. The isomers from 23-di- and 2345-tetra-CP were most easily separated, the other pair of isomers were more difficult to separate. Assignment of 1236and 1239-TCDD (from 23-di- and 2345-tetra-CP) as the first and later eluting isomer, respectively, again was based on the photolysis study of the two hexa-CDDs: 1236-TCDD was exclusively formed from 123678-hexa-CDD,1239-TCDD from 123789-hexa-CDD(19). 1237-TCDD and 1238-TCDD (from 25-di- and 2345-tetra-CP) were separated on Silar 1Oc but not on OV-17 or OV-101. However, full assignment of the first and later eluting isomer was not yet possible; both isomers can be expected from the photolysis of each of the hexa-CDDs mentioned above. 1246-TCDD and 1249-TCDD (from 26-diand 2356-tetra-CP) were only partially separated on Silar lOc, and 1247- and 1248-TCDD (from 24-di- and 2356-tetra-CP) were found to be the most difficult pair of isomers to separate. In the latter case, only the Silar 1Oc column under very slow programming conditions (0.5 OC/min) showed the presence of two isomers (shoulder and increased peak width) in the pyrolyzate. Full assignment of these peaks was again not yet possible. In Figure 2b we show a chromatogram of a composite pyrolyzate sample containing all the 3:l type isomers in addition
to 1234-TCDD (the only 4:O type isomer) from a reference sample. In this case, the Silar 1Oc HRGC column did not separate all isomers; however, these isomers are separated from 2378-TCDD. All the 3:l type isomers and also 1234-TCDD showed the typical mass spectral patterns in the lower mass range that allowed a differentiation of these isomers from the 2:2 type isomers mentioned previously (22). Figure 3 shows chromatograms of a composite pyrolyzate sample containing all 22 TCDD isomers analyzed on the Silar lOc, OV-17, and OV-101 HRGC columns. Whereas 2378TCDD can be uniquely assigned by using the Silar 1Oc column (see Figure 3a), this isomer is coeluting with 1279-TCDD and is only partially separated from 1469-TCDD on OV-17 (see Figure 3b). On OV-l01,2378-TCDD is coeluting with 1237and 1238-TCDD and only partially separated from 1239TCDD (see Figure 312). It should be pointed out that for the separation of 2378-TCDD from all the other TCDD isomers, a rather long (55 m) and narrow (0.25 mm id.) Silar 1Oc HRGC column was used; shorter and wider columns of this type did not fully separate 2378-TCDD from 1234-TCDD (14) and 1237-, 1238-, 1246-, and 1249-TCDD. In Table V we list all 22 TCDD isomers and we indicate which of these can be uniquely assigned on each HRGC column used in the present study. As indicated in this table and as also seen in Figure 3a, Silar 1Oc allows the highest number of isomers to be unambiguously assigned including the extremely toxic and therefore mmt important 2378TCDD. Some additional isomers can be assigned on the other columns, e.g., 1278,1279-, and 1239-TCDD and 1236-TCDD on OV-101 and OV-17, respectively, but for several isomers an unambiguous assignment on either of these HRGC columns is not possible. However, some of these isomers may be determined together with their Smiles-related counterparts (e.g., 1247and 1248-TCDD)or they may be determined by a differential quantification such as for 1469-TCDD (on Silar lOc, 1469TCDD plus 1278-TCDD; on OV-101, 1278-TCDD alone). Applications. In the following, we illustrate the application of HRGC in combination with MS for the identification of
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
2261
Table V. TCDD Isomer Assignments Obtainable on Different HRGC Columns isomers ( X ) that can be uniquely assigned on Silar 1oc
TCDD isomer
12671268126912781279128913681369-
(see
Figure 3c) X
X X
1378
X X X X X
1378-
137914691478237 8123612371238123912461247124812491234-
ov-101
!=e substitution Figure OV-17 (see type 3a) Figure 3b) 2: 2 X X
X X X X X
X X X X X
X
I
I
240
230
m/e 320 1
220°C
Flgure 5. Mass fragmentogram ( m / e320) of contaminated soil from the Shenandoah Horse Arena in eastern Missouri showing the elution of TCDDs from a 55-m Silar 1Oc HRGC column.
X
X X
3: 1
X X
X
4:O
240
2378-TCDD
xY 8 1378
J i J
230
220%
Figure 6. Mass fragmentogram ( m / e 320) of a purified extract of a river carp (Tittabawassee River, Michigan) showing the elution of 2 3 7 8 T C W and other chlorinated pollutants from a 5 5 m Sihr 1Oc HRGC column.
A
I
I
240
230
m/e 320
/
I
220%
Flgure 4. Mass fragmentogram ( m / e 320) of a Seveso soil extract showing the elution of TCDDs from a 55-m Silar 1Oc HRGC glass capillary column.
TCDD isomers. Major emphasis was kept on qualitative isomer identification; the quantification was not yet thoroughly investigated. The levels of TCDDs in the contaminated samples reported on here were estimated t o be in the low parts-per-billion (lo*) t o parts-per-million (lo*) range. However, we feel that the method should also be applicable to samples with TCDDs in the parts-per-trillion concentration range. PCDDs were observed in soil, plants, and animal tissue a t Seveso, Italy, after an accident in a chemical plant producing 245-tri-CP nearby (12). In Figure 4, we present a chromatogram of an extract of soil from zone A (high contamination zone) showing the elution of TCDDs from the 55-m Silar 1Oc HRGC column. The major isomer was found to coelute with 2378-TCDD, the minor with 1378-TCDD; no other TCDDs were found to be present.
Both isomers found had the expected mass spectra for 2:2 type TCDDs (22)and their retention times also matched those of 2378- and 1378-TCDD on the OV-17 and OV-101 HRGC columns. Previously we had reported the identification of these TCDDs based on retention data on OV-17 and OV-101 columns (15,221;however, a t that time we did not have all the TCDD isomers available. In 1971, some horse arenas in eastern Missouri were contaminated with TCDDs (25). This contamination resulted from the improper disposal of waste oils from a 245-tri-CP and hexachlorophene producer. The contamination in soil was reported as high as 30 ppm. I t resulted in a human poisoning and caused the death of horses and other animals. Horses continued to die, even after removal of most of the top soil. In Figure 5, we present a chromatogram of soil from the Shenandoah Horse Arena in Missouri analyzed on the 55-m Silar 1Oc HRGC column. The chromatogram reveals the presence of two TCDD isomers. The major isomer was identified as 2378-TCDD; the minor isomer was identified as 1378-TCDD. These identifications were confirmed by reanalysis on the OV-17 and OV-101 HRGC columns. Complete mass spectra again were in agreement with those of 2:2 type TCDDs. The soil extract also contained significant quantities of 124689-hexachloroxanthene(26). The findings support the accusation of contamination by residues from a hexachlorophene producer. In Figure 6 we show a chromatogram of a purified extract of a river carp from the Tittabawassee River in Michigan. This river flows adjacent t o a chemical plant producing various chlorinated products including 245-tri-CP. In a previous study a series of fBh samples from various rivers in the United States
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 14. DECEMBER.1980
estimated a t only 1% of the total TCDD amount.
ACKNOWLEDGMENT
1231 /1238
c
The authors are indebted t o D. Stalling, Columbia, MO, R. Kimbrough, Atlanta, GA, and to Givaudan Ltd, Dubendorf, Switzerland, for the various environmental samples and to K. Andersson, UmeB, Sweden, and Stickstoffwerke, Linz, Austria, for 1234- and 2378-TCDD, respectively.
76'1249 1247/1248
179
1368
LITERATURE CITED
/
( I ) IARC Internal Technical Report 78/001; International Agency for Re-
1378
1289
d I
I
240
230
m / e 320
u+L-tL I
220oc
Flgure 7. Mass fragmentogram ( m l e 320) of a fly ash sample from a municipal incinerator in Switzerland showing the elution of TCDDs from a 55-m Silar 1Oc HRGC column.
were analyzed for the presence of PCDDs and other chlorinated pollutants by using negative chemical ionization mass spectrometry (NCIIMS) (27). Of all the fish samples analyzed only those from the Tittabawassee River revealed the presence of PCDDs (tetra- to octachloro compounds). Reanalysis of one of these positive fish samples now shows the presence of 2378-TCDD. The mass spectra for the major component shown was in agreement with that of a 2:2 type TCDD. Additional peaks a t m l e 320 present in the chromatogram were not due to TCDDs but to other chlorinated pollutants (M+ = 390, Cl,; M+ - C1, = 320). Reanalysis of this sample on OV-17 and OV-101 confirmed the finding of 2378-TCDD as the only TCDD isomer present. Finally in Figure 7 is a chromatogram of a fly ash sample from a municipal incinerator in Switzerland. This particular sample was shown to contain PCDDs and other chlorinated pollutants (5, 6 ) . The chromatogram reveals a complex isomeric mixture with at least 17 TCDDs present. The same isomeric pattern was observed in other fly ash samples. The major isomers identified are 1368-, 1379-, 1237-(or 1238-)TCDD making up more than half of the total TCDD amount present. Apparently, 2378TCDD is present at a very low level
search on Cancer: Lyon, France, 1978. (2) Rappe, C.; Marklund, S.; Buser, H. R.; Bosshardt, H.-P. Chemosphere 1978, 7,269-281. (3) Buser, H. R. Chemosphere 1979, 8 . 415-424. (4) Olie, K.; Vermeulen, P. L.; Hutzinger, 0. Chemosphere 1977, 6 , 455-459. (5) Buser, H. R.: Bosshardt, H.-P. Mttt. Geb. Lebensmtttelunters.tfyg. 1978, 69, 191-199; Chem. Abstr. 1979, 9 0 , 141762. (6) Buser, H. R.; Bosshardt, H.-P.; Rappe, C. Chemosphere 1978, 7, 165-172 .- - . . - . (7) Eiceman. G. A.; Clement, R. E.; Karasek, F. W. Anal. Chern. 1979, 51, 2343-2350. (8) The Chlorinated Dioxin Task Force, "Trace Chemistries of FlraA Source of and Routes for the Entry of Chlorinated Dloxlns Into the Envlronment"; Dow Chemical. Midland, MI. 1978. (9) Poland, A.; Glover, E.; Kende, A. S. J. Biol. Chem. 1976, 251, 4936-4946. (IO) Mc Connell, E. E.; Moore, J. A.; Haseman, J. K.; Harris, M. W. Toxicol. Appl. Pharmacol. 1978, 44, 335-356. (11) Moore. J. A. Ecol. Bull. 1978, No. 27. 134-144. (12j Homberger, E.; Reggiani, G.; Sambeth, J.; Wipf, H. K. Ann. Occup. wg. 1979, 22, 327-370. (13) Baughman. R.; Meselson, M. EHP Environ. Health Perspect. 1973, 5 . 27-35. (14) Buser, H. R. Anal. Chem. 1976, 4 8 , 1553-1557. (15) Buser, H. R. Anal. Chem. 1977, 49, 918-922. (16) Buser, H. R. Ph.D. Thesis, University of Umea, Urn&, Sweden, 1978. (17) Nestrick, T. J.; Lamparski, L. L.; Stehl, R. H. Anal. Chem. 1979, 51, 2273-2279. (18) Buser, H. R. J . Chromatogr. 1975, 174, 95-108. (19) Buser, H. R. Chemosphere 1979, 8 , 251-257. (20) Buser, H. R. J. Chromatogr. 1976, 129, 303-307. (21) . . Stallina. D. L.: Smith. L. M.: Petty, J. D. ASTM SDec. Tech. Pub/. 1979, STP 6-86, 302-323. (22) Buser, H. R.; Rappe, C. Chemosphere 1978, 7,199-21 1. (23) Gray, A. P.; Cepa, S. P.; Cantrgll, J. S. Tetrahedron Lett. 1975, 33, 2873-2876. _. . ~. (24) Kende, A. S.;Decamp, M. R. Tetrahedron Lett. 1975, 33, 2877-2880. (25) Carter, C. D.; Kimbrough. R. D.; Liddle, J. A,; Cline, R. E.; Zack, M. M.; Barthel, W. F.; Kcehler, R. E.; philips, P. E. Science 1975, 188, 738-740. (26) Gdthe, R.; Wachtmeister, C. A. Acta Chem. Scand. 1972, 2 6 , 2523. (27) Tondeur, Y.; Dougherty, R. C.; Rappe, C.; Buser, H. R. Proceedings of 27th Annual Conference, American Society for Mass Spectrometry, Seattle, WA, 1979. ~
RECEIVED for review April 15, 1980. Accepted September 8, 1980. Presented in part a t the workshop on Impact of Chlorinated Dioxins and Related Compounds on the Environment, Rome, Italy, Oct 22-24, 1980.
