Envlron. Sei. Technol. 1903, 17, 324-329
Middleton, P.; Kiang, C. S.; Mohnen, V. A. Atmos. Environ. 1980,14, 463-473.
Meyers, R. E.; Ziegler, E. N. Environ. Sci. Technol. 1978, 12, 302-309.
Winchester,J. W.; Leslie, A. C. D. Geophysical Monograph 26; American Geophysical Union: Washington, D.C., 1982; pp 250-256.
Received for review April 12,1982. Revised manuscript received December 20,1982. Accepted January 25,1983. This work was supported in part by the U.S. Environmental Protection Agency
and by the National Institute of Environmental Health Sciences by a predoctoral traineeship for R.J.F. as a portion of the Acid Precipitation Experiment (APEX)carried out at the National Center for Atmospheric Research (sponsored by the National Science Foundation). Although the research described in this report has been funded in part by the U.S. Environmental Protection Agency through interagency agreement EPA AD49-F-0-028-0 to the National Science Foundation, it has not been subjected to the Agency's required peer and policy review and therefore does not necessarily reflect the views of the Agency, nor does mention of any trade names or commercial products constitute endorsement or recommendation for use.
Structure Elucidation of 3-(2-Chloroethoxy)-l,2-dichloropropene, a New Promutagen from an Old Drinking Water Residue M. Wilson labor
Department of Environmental Health, Ketterlng Institute, University of Cincinnati Medical Center, Cincinnati, Ohio 45267
w A Salmonella TAlOO microsomal-dependent mutagen previously isolated from residue organics prepared from drinking water has oow been tentatively identified as a new chloroallyl ether. On the basis of Fourier transform infrared and nuclear magnetic resonance spectroscopies in conjunction with high-resolution mass spectroscopy, a confident structural assignment for this promutagen was elucidated to be 3-(2-chloroethoxy)-l,2-dichloropropene. The relationship of this compound to mutagens in other drinking water samples is discussed. Introduction A great variety of natural and synthetic chemicals find their way into the nation's drinking water supplies. Many of these organic chemicals may have no detrimental effects at low concentration (in the parts per billion range), but some compounds may have serious and substantial health effects, particularly in the long-term exposures possible from drinking water (1). Operationally, this broad spectrum of compounds is divided into two categories: (a) compounds of low solubility and with a volatility sufficient for rapid separation and identification; (b) relatively soluble compounds with low volatility that are not readily separated but are collected by extraction or concentration from water as complex mixtures of residue organics. A US. National Academy of Science-National Research Council Report (2) noted that all but about 10% of the volatile constituents of drinking water have been identified and can be quantitated. The NAS-NRC report (2) also stated that the nonvolatile organic fraction in drinking water comprises 90-95 % of the total organic material by weight, but isolation, identification, and risk assessment of the nonvolatile or residue organics are far more difficult. The strategies and methods employed in sampling for the nonvolatile organics in water were recently reviewed by Kopfler (3). For the toxicological assessment of such mixtures, initial characterization of the isolated residue mixtures has relied upon short-term bioassays of concentrated residues ( 4 ) . We have developed a general preparative procedure for the isolation of bioactive components from the residue organics (5). As currently applied (6, 7) this procedure features the Salmonella/microsome mutagenicity assay coupled with one or more separation runs on reverse-phase high-performance liquid chromatography (HPLC). Once mutagens have been suitably enriched in 324
Environ. Sci. Technol., Vol. 17, No. 6, 1983
subfractions, further separations are effected according to their particular chemical properties, using either further HPLC separations in normal or reverse-phase modes or preparative gas chromatography. On isolation of mutagenic components, structural elucidation can be accomplished by the application of chemical and physical techniques. For the development of this general procedure we utilized a sample of drinking water residue organics that had been prepared in the early 1960s by the carbonchloroform megasampler procedure of Middleton et al. (8). This paper reports the tentative identification of a Salmonella TAlOO microsomal-dependent mutagen that we isolated from that sample. The promutagen has a mutagenic specific activity of 4 X 105TAlOO revertants/mg assayed in the presence of Aroclor 1254 induced rat liver microsomes (5). Preliminary mass spectrometry results indicated the compound to be a polychlorinated aliphatic ether whose fragmentation pattern did not match any entries in the NIH-EPA library (5). Due to limitations in the quantity of promutagen isolated, identification required the utilization of trace analytical high-resolution techniques. Experimental Section Promutagen Isolation. High-performqnce liquid chromatography (HPLC) subfractions containing microsomal-dependent TAlOO mutagenesis were prepared and bioassayed as previously described (5). These fractions were found to contain two major components by gas chromatography (GC). This separation was accomplished on a Perkin-Elmer Model 900 flame ionization unit fitted with a glass 6 f t by 2 mm i.d. column containing 5% OV17 on 100/120 mesh Gas Chrom Q. The nitrogen carrier gas was flowing at 18 mL/min, and the temperatures of the injector, oven, and detector were at 280,160, and 350 "C, respectively. Data were continuously collected and analyzed by using a Spectra Physics Autolab I computing integrator, and chromatograms were displayed on a 10-mV recorder. To isolate the promutagen from these HPLC subfractions, preparative GC was carried out under the same conditions, except the instrument was fitted with a glass 6 ft by 4 mm i.d. column containing the same packing and a 15 to 1 stream-splitter at the column effluent. Typically, 10-20 injections, 10 p L each, of the bioactive HPLC subfractions were made, and GC fractions were collected by time. Under these conditions the promutagen
0013-936X/83/0917-0324$01.50/0
@ 1983 American Chemical Society
eluted between 6.5 and 8.3 min following injection. The split GC effluent containing the promutagen was collected in a manner similar to that described by Costanzo (9)using a 9-in. Pasteur pipet snugly inserted into the streamsplitter collection port. Other GC fractions were collected similarly at 2.0-5.0,5.&6.5,8.3-9.0, and 9.0-12.0 min. The pipets were eluted with 100-pL volumes of methylene chloride, and the presence of the promutagen was verified by bioassay of each eluate as previously described (5). Recovery of the promutagen in this preparative GC step was 65-75%, Quantitation was accomplished via external standardization using the GC column and conditions as first described. The external standard, methyl laurate of 99.98% purity from Emory Industries of Cincinnati, OH, was chosen because it had approximately the same retention time, 7.2 vs. 7.4 min, and a similar molecular weight, 214 vs. 188. A linear standard curve of peak area integrations vs. weight of methyl laurate in nanograms was used in quantitation. Infrared and Nuclear Magnetic Resonance Spectroscopies. Gas chromatography/Fourier transform infrared (GC/FT IR) analyses were carried out on a Hewlett-Packard Model 5710A FID-GC fitted with a Digilab FTS-15B spectrophotometer and Digilab GC/FT IR interface. Chromatography was accomplished on a 6 f t by 0.25 in. i.d. stainless steel column packed with 10% SE30 on 80/100 mesh Supelcoport. The helium carrier gas was flowing at 30 mL/min, and system components were at the following temperatures: isothermal column, 180 "C; injection port, 250 "C; FID detector, 300 "C; spectrophotometer light pipe, 200 "C; transfer line, 190 "C. GC data were continuously displayed via the 10-mV recorder, and IR data were continuously processed with a Data General 2/10 minicomputer of 32K core memory with a moving head disk for temporary data storage and digital magnetic tape capabilities for permanent data storage. Spectral searches were conducted via the Digilab FT IR instrument software against the EPA-GIFTS library of 2300 spectra (IO) and via computer time sharing against the IRGO commercial reference data base (Chemir Laboratories) of published IR spectra collections, which contain more than 150000 IR spectra (11). GC/FT IR injections consisted of solutions containing 200-400 ng of promutagen in 10 pL of methylene chloride. Comparison spectra were obtained by injection of 1.0 pL of neat bis(2-chloroethyl) ether (Matheson, Coleman and Bell) as a control. Verification of IR assignments was made from absorption frequencies observed in IR scans of 1,2,3-trichloropropene, 1,3-dichloropropene, and 2-chloroethyl vinyl ether, all obtained from Aldrich, and in an IR scan of (S)-2,3-dichloroallyl diisopropylthiocarbamate, the herbicide diallate, obtained as a reference standard from US.EPA. The IR spectra of these latter four compounds were obtained on neat samples between sodium chloride plates by using a Perkin-Elmer 599 infrared spectrometer. The samples were scanned from 3600 to 600 cm-l at 250 cm-l/min. Fourier transform 13C nuclear magnetic resonance spectra were obtained on a Varian Model CFT-20 NMR spectrophotometer system using a deuterium oxide lock signal. A 300-ng deuterochloroform solution of the purified promutagen was scanned at an acquisition time of 0.511 s for 997952 transients at a pulse width of 8 X 10* s. High-resolution lH NMR spectra were obtained on a JEOL Model JNM FX270 Fourier transform NMR system using water as a lock signal. A 2-pg hexadeuterodimethyl sulfoxide solution of the purified promutagen was scanned at an acquisition time of 3.033 s for 249 transients at pulse widths of 8 X lo* and 40 X lo4 s.
Mass Spectrometry. Low-resolution electron impact (EI) spectra were routinely obtained on a Finnigan Model 4021 quadrupole mass spectrometer (MS) fitted with a Model 2000 INCOS data system as previously described (5). Prior to one group of low-resolution MS experiments, the promutagen was subjected to silylation or methylation in reactions conducted according to the instructions from the suppliers. Reagents used for these derivatization reactions were Sylon BTZ (Supelco, Bellefonte, PA) and METH-PREP 11 (Applied Science, State College, PA), respectively. Other MS analyses were obtained on a Kratos MS80 high-performance spectrometer. These data were continuously collected during MS runs and processed on a Data General NOVA/4C DS-55 data system. Computer interaction, data display, and output were via Hewlett-Packard systems. High-resolution MS analyses were performed in the presence of an internal mass standard, perfluorokerosene (PFK). The conditions of E1 spectra were as follows: ionizing current 1 X A; ionizing energy , 4 0 eV; accelerating voltage, 4 kV; scan range, m/z 20-600; scan speed, 5.5 s for 3000 resolution and 13 s for 7500 resolution; scan interval, 1s. Samples were introduced via either direct insertion probe or capillary GC. Two-hundred to four-hundred nanograms of promutagen in 5 pL of methylene chloride were evaporated into a capillary sample holder, which was placed in the shaft tip of the direct insertion probe. The probe was air cooled to 20 "C, the air shut off, and then the temperature of the probe increased at 10 "C/min while the total ion current was continually monitored for the appearance of m/z 189. When this ion was observed at approximately 120 "C, the temperature was held constant, and spectra were obtained. Capillary GC introduction of samples into the MS was via a Carlo Erba Series 4160 GC fitted with a SE54 30 m X 0.321 mm fused silica WCOT column, film thickness of 0.25 pm, obtained from J. & W. Scientific, Inc. GC conditions were as follows: injection temperature, 50 "C;oven temperature a t 50 "C for 75 s after injection and then programmed to 200 "C at 10 OC/min; separator temperature, 250 "C; helium carrier gas, 20 cm/s with a 20 mL/min flow makeup to the separator. Twenty-five to fifty nanograms of promutagen in 0.5 pL of methylene chloride was introduced by the cold on-column splitless injection technique of Grob and Neukom (12). After a 70-s solvent divert, the valve to the MS was opened. Isobutane chemical ionization (CI) spectra at loo0 resolution were obtained on samples introduced via capillary GC at an ionization A. Other GC/MS conditions were current of 1.5 X as described above. The Kovats retention index (13) of the promutagen was determined on the capillary SE54 column with MS detection using, as the standard, a mixture containing C,-C18 hydrocarbons (Supelco) at a concentration of 100 pg/mL each in methylene chloride. Five-hundred nanoliters of this standard solution was injected into the GC as described above. E1 spectra were acquired over a scan range of m/z 50-300 at a resolution of 1000, scan speed of 2 s, and scan interval of 0.75 s. Other GC/MS conditions were as described above. Twenty nanograms of promutagen in 0.5 pL of methylene chloride were injected, and E1 spectra were acquired under GC/MS conditions identical with these used for the standard hydrocarbon mixture. This sequence of GC/MS runs for these two samples was repeated twice under the same running conditions. The adjusted retention time for each compound was calculated from the spectral scan of the peak maximum for that component, and the promutagen was found to elute beEnviron. Sci. Technol., Vol. 17, No. 6, 1983
325
I I
Table I. FT IR Spectral Summary (ClCH,CH,),-0, promutagen, cmcm3017 2979 2967 2948 2928 2883 2882 2843 2920 1740 1516 1460 1443 1444 1439 1367 1343 1305 1316 1259 1262 1205 1215 1128 1123 1050 1046 965 953 849 765 760 680 675
-
type of vibration C-H stretch (vinyl) C-H stretch (methylene, asym)
functional group assgmt = CH CH,
C-H stretch (methine, asym) C-H stretch (methylene, sym)
= CH =CH,
C-H stretch (vinyl) C-CI overtone C-H scissor (asym) C-H scissor (asym due to ether) C-H scissor (sym) C-H scissor (sym due t o ether) C-Cl bend C-H wag (methylene) C-0-C stretch C-C stretch C-H rock (methylene) =CH out-of-plane bend (trisubstituted olefinic) C-C1 stretch (asym) C-Cl stretch (syn)
c=c cc1
tween C14 and C16 on this SE54 capillary column. The Kovats retention index was determined to be 1462.
Results Infrared Analysis. Functional group information was derived by Fourier transform infrared (FT IR). Promutagen was examined in the gas phase following separation via GC; companion spectra of bis(2-chloroethyl) ether (BCEE) were obtained under identical conditions. Although vibrational and functional group assignments typically are interpreted from published IR spectroscopic data obtained on substances in the liquid phase, Sheppard and Simpson (14) have reported only very small if any frequency shifts occur on passage from gas to liquid phase. A summary of the data as to absorption frequencies, functional group assignments, and types of vibration is presented in Table I. Several similarities between the promutagen and BCEE were observed. Both compounds are chloro ethers containing methylene groups. It is noted that the methylene hydrogen stretch vibrations in the promutagen appear to be split, giving multiple peaks. This has been reported to be characteristic of methylene groups attached to a vinylic carbon (15). The asymmetric and symmetric methylene hydrogen deformations are shifted approximately 10-20 cm-l to lower frequencies for BCEE and approximately 20-30 cm-l for the promutagen. This shift has been observed in compounds having strongly electronegative atoms, e.g., C1 or 0, attached to the methylene group (16). For both compounds, asymmetrical and symmetrical deformations typical of methylene hydrogens on a carbon bonded to an oxygen were observed (17). Both compounds exhibited C-0-C stretch vibrations typical for ethers and the usual C-C1 stretching vibrations were observed in the 833-667-cm-’ region for both compounds. No specific correlation can be made between band position and location of chlorine atom(s) on’this basis (18). Additionally, a C-C1 overtone was observed for the promutagen at 1560 Cm-1. In contrast, several bands observed in the proniutagen were not present for BCEE. One of these, a medium-intensity band observed at 3017 cm-l, is in the range typical of the C-H stretch of a vinylic hydrogen (13). This absorbance frequency (3015-3020 cm-l) has been ascribed to vinylic hydrogen of the RR’C=CHR’’ type ( 1 7 ) . To verify this assignment, IR scans of similarly substituted 326 Environ. Sci. Technol., Vol. 17, No. 6,1983
CH2 CH,O CH, CH,O CH,C1 CH, COC CHt RR C=CHR“ cc1
cc1
olefins were made. 1,2,3-Trichloropropene and (S)-2,3dichloroallyl diisopropylthiocarbamateshowed absorption frequencies at 3015 and 3020 cm-‘, respectively, for the vinylic hydrogen stretch. The intensities of these bands were of intensities comparable to that observed for the promutagen. In contrast to these data, IR scans of a disubstituted olefin, 1,3-dichloropropene, and a monosubstituted olefin, 2-chloroethylvinyl ether, showed absorption frequencies in the 3050-3110-~m-~ range, which is typical for the vinylic hydrogen stretches of these substitution types (17). A weak carbon-carbon double bond stretch was observed for the promutagen at 1740 cm-l, which is higher frequency than usual for a vinylic bond. Also, a weak vinylic carbon-carbon stretch was observed for 1,2,3-trichloropropene in the same region at 1730 cm-l. For the thiocarbamate herbicide, a strong carbonyl stretching frequency was observed at 1665 cm-l, but as a shoulder to this peak a weak absorbance was observed at 1740 cm-l. This possibly could be the vinylic carbon-carbon stretch. 1,3-Dichloropropeneand 2-chloroethyl vinyl ether showed their vinylic carbon-carbon stretch absorbance as medium-intensity bands at 1640 and 1622 cm-l, respectively. Therefore the trisubstituted olefins, promutagen included, appear to have the carbon-carbon stretch absorbance frequencies at values higher than those for disubstituted and monosubstituted olefins. However, vinylic halogen substituents are known to shift this stretch to higher frequencies (14,17, 18),and the presence of these substituents on the vinylic carbons also contribute to this effect (17). The decrease in intensity for this absorption band observed for the promutagen, for trichloropropene, and for diallate is also typical of trisubstituted vinylic groups (14, 17). The frequency assignment for the promutagen vinylic bond was confirmed by the presence of a medium-intensity band at 849 cm-’, which is in the 790-850-cm-l range typical of the C-H deformation of a trisubstituted olefin (17,19). Both the trichloropropene and diallate had this typcial medium-intensity band at 835 and 825 cm-l, respectively. Several general features are absent from the IR spectrum of the promutagen. No bands were observed to indicate the presence of a methyl group, particularly not the distinctive C-H deformation at 1380-1375 cm-l (14,17). NO 1248-cm-’ band was observed for an allylic methylene halogen (18) of the type C=C-CHC1. This band was
Scheme I
76
HRMSA=O.rlmmu
40
111
1
M/E
t
-HCL
+C ~ H ~ C L M/E
50
'5
100
I
153 1 II I
125
150
1, 175
200
I 225
mle Figure 1. Mass spectrum of the promutagen. The HRMS refers to the difference between the measured exact mass and that calculated from the molecular formula.
Table 11. High-Resolution Mass Spectrometry Data m / z exact mlz exact massa emp form mass'" emp form 92.9952 C,H,CIO 188.9739 C,H,CI,O 78.9976 C,H,ClO 152.9875 C,H,C1,0 75.0028 C,H,Cl 140.9937 C,H,Cl,O 48.9886 CH,C1 C,H,Cl, 110.9897 a Referenced to perfluorokerosenes.
RH+*+ M
-+
+
+ MH+ R- + MH+ R
These ionization mechanisms have been reported for a number of other aliphatic ethers (20). Additional compound classes also have given an apparent molecule ion at M + 1 (20). The m / z 189 ion was observed in E1 spectra from a variety of both low- and high-resolution analyses, although the ratio of intensities for m/z 189 to 141 or 111 varied between instruments. Additional support for this interpretation was obtained upon examination of the promutagen under CI conditions using isobutane as the ionizing gas: the m/z 189 ion was present, no additonal ions of higher mass appeared, but some changes in lower mass fragments were observed. On the basis of the assignments of the other fragments as shown in Figure 1and Table 11, plus the interpretation of an M 1ionization mechanism for the origin of m/z 189, a partial structure proposed for the promutagen is
+
observed for both 1,2,3-trichloropropene and 1,3-dichloropropene as an absorbance of medium intensity. No absorbance frequencies characteristic of nitrogen (N-C, N-H) were observed for the promutagen. Mass Spectrometry. For further elucidation of the structural relationship of these functional groups, the promutagen was examined by MS. In numerous experiments using different instruments, the compound was introduced via packed or capillary column GC or by direct probe. Spectra were obtained in the E1 or CI modes and under conditions of low or high resolution. Pretreatment by methylation or silylation reagents did not change the observed E1 spectra of the compound. Spectra representative of low-resolution runs on a Finnigan 4020 have been published for the promutagen (5). A typical Kratos MS80 high-resolution spectrum is shown in Figure 1. Measured exact masses and their corresponding emperical formulas are given in Table I1 for key fragments in the spectra. The highest m/z fragment observed occurred at 189 with an attendant isotopic cluster typical of three chlorines. Since the presence of nitrogen could not be inferred, the possibility was considered that this odd mlz arose from a higher molecule weight species. The most likely possibilities, M-C1 or M-CH2C1, would show m/z values of 224 or 238 + 203, respectively. A computer search of the total ion chromatogram was conducted for these ions (203,224, and 238) vs. the appearance of mlz 111, but they were not detected. Therefore, the mlz ion at 189 is probably an M + 1 ion, resulting from the following ionization processes: RH+ + M
75
(1) (2)
The C2HC12portion of the molecule could be one of two olefinic groups, either C12C=CH or ClHCl=CCl. Some of the observed fragments, Table 11, could arise as follows. The m/z 153 ion would be from the loss of HC1 from the m/z 189 parent ion. Likewise, the loss of CHCl from the parent ion would give m/z 141. A possible origin of the m / z 75 base peak, Figure 1and Table 11, could be via m/z 111 as shown in Scheme I. Nuclear Magnetic Resonance. On the basis of the MS and FT IR data, the nature of the bonding in this olefinic fragment cannot be determined. To resolve this question, the compound was examined by Fourier transform lH and 13CNMR spectroscopy. A preliminary proton spectrum was obtained on a Me2SO-d, solution containing approximately 2 pg of the compound. It was evident that this sample contained methylene chloride, arising from the isolation procedure, and some water, contained in the Me2S0. These contaminants masked many features of the spectrum, but two important peaks were observed with intensity ratios of 2 to 1: triplet at 6 6.32 (J = 5 Hz) and a singlet at 6 7.72. The triplet was coupled to two protons masked by the interfering peaks. This triplet was decoupled by irradiation in the vicinity of the broad methylene chloride peak centered at 6 4.49. No definite assignment could be made for this peak, but it is probably one of the two methylene groups on the ethyl side of the promutagen ether. The other main peak, observed for the promutagen at 6 7.72, is in the region of an olefinic hydrogen (21). The appearance of this proton as a singlet peak supports the structure ClHC=CCl rather than C12C=CH for the propylene side of the ether. No definitive stereochemical Environ. Sci. Technol., Vol. 17, No. 6, 1983
327
Table 111. 13C Fourier Transform Nuclear Magnetic Resonance chem shift, 6 obsd 150.0 145.6 79.6 79.0 45.6
calcd 149.3 143.3 79.6 78.6 48.6
assgmt =CCl ClHC= CH,O( propenyl) OCH,(ethyl) CH,Cl
assignment is made for the chlorine positions around the olefin bond. However, the lack of multiplicity of this singlet, as would be expected for long-range coupling to the methylene, suggests the chlorines are in a cis configuration. A 13C FT NMR spectrum of a chloroform-d solution containing approximately 300 ng of promutagen showed five peaks. These data are summarized in Table 111. Peaks for two carbons were observed in the olefinic range at 6 150.0 and 145.6, two in the aliphatic carbon-oxygen range at 6 79.6 and 79.0, with the other at 6 45.6 in the aliphatic carbon-halogen range (22). The structural assignments were confirmed by theoretical chemical shifts calculated according to the principles of Dorman et al. (23) for alkenes utilizing the method outlined by Cooper (21). The presence of a gem-dichlorovinyl group in this compound was ruled out by the 13Cchemical shift data, neither of the predicted peaks, 6 152.3 for a C12C= carbon nor 6 140.3 for a =CH carbon, were observed. Discussion and Conclusions On the basis of the data presented, a confident structural assignment for the promutagen is 3-(2-~hloroethoxy)-l,2dichloropropene. Studies of the synthesis of the promutagen have been initiated in our laboratory to confirm this structural assignment. This compound is unique in that no citation appears in Chemical Abstracts or Beilstein. Three structurally related ethers of the empirical formula C6H7C130were referenced in Chemical Abstracts: 3-ethoxy-1,1,3-trichloropropene, 3-ethoxy-1,1,2-trichloropropene, and 3- (2,2,2-trichloroeth0xy)propene (24-26). None of these compounds has the 1,2-dichloropropenylstructural moiety, and their bioeffects in mutagen assays have not been reported. However, this moiety is present in the widely used herbicide diallate ((S)-2,3-dichloroallyldiisopropylthiocarbamate). Diallate and two other (S)-chloroallylthiocarbamates, triallate and sulfallate, have been characterized as promutagens in Salmonella assays (27-30). In studies to be reported elsewhere, we have shown that in terms of specificity for the Salmonella tester strains and of specificity for microsomal activation, these three herbicides are similar to 3-(2-chloroethoxy)-l,2-dichloropropene.Diallate is most similar in its mutagenic potency; triallate and sulfallate, which are not 1,2-chloropropenyl compounds, are less active.
We have a sample of residue organics from raw water that was collected by Middleton et al. (8) as a companion to the finished water residue organics from which the promutagen was isolated. That raw water residue contains mutagenic activities that, in terms of tester strains and microsomal activation, are similar to the finished water residue sample. Fractionation studies are being conducted according to our procedures (5) to determine if identical or chemically related mutagens are involved. The environmental source of the isolated promutagen will also be investigated. The compound has not been noted in MS studies of current drinking water residue 328 Environ. Sci. Technol., Vol. 17, No. 6 , 1983
organics (31),and the mutagenicity of recent drinking water samples typically does not require such microsomal activation (4, 6 , 7 ) . Acknowledgments
I am grateful to S. Baxter, J. Brooks, E. Fauod, K. Jayasimhulu, J. MacGee, and J. Pustinger for assistance in obtaining data and to W. E. Coleman for professional suggestions. J. C. Loper and R. A. Day are gratefully acknowledged for intellectual input throughout the course of the work. Prereview of this paper by F. Kaplan and W. H. Glaze is appreciated. This publication is dedicated to the late Aaron A. Rosen. Registry No. 3-(2-Chloroethoxy)-l,2-dichloropropene, 84987-77-9; water, 7732-18-5.
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(24) Nesmeyanov, A. N.; Freidlina, R. K. H. Zakharkin, L. I. Dokl. Akad. Nauk. SSSR 1954,97,91-94; Chem. Abstr. 1955,49,8793. (25) V'yunov, K. A; Zhukova, T. I.; Sochilin, E. G.; Smorygo, N. A. Zh. Org. Khim. 1976,11, 2331-2335. (26) V'yunov, K. A.; Garabadzhiu, A. W.; Zhukova, T. I.; Zhi. linskaya, T. D.; Sochilin, E, G. Zh.Org. Khim. 1978, 14, 1187-1193. (27) Delorenzo, F.; Staiao, N.; Silengo, L.; Cortese, R. Cancer Res. 1978, 38, 13-15. (28) Rosen, J. D.; Segall, Y.; Casida, J. E. Mutat. Res. 1980, 78, 113-119.
(29) Douglas, G. R.; Nestman, E. R.; Grant, C. E.; Bell, R. D. L.; Wytsma, J. M.; Kowbel, D. J. Mutat. Res. 1981, 85, 45-56. (30) Sikka, H.C.; Florczky, P. J . Agric. Food Chem. 1978,26, 146-148. (31) Coleman, W. E. US.Environmental Protection Agency Health Effects Research Laboratory, personal communication, 1981. Received for review May 3, 1982. Accepted January 24, 1983. This work was supported by U.S.Environmental Protection Agency Grants CR806872 and CR808603.
Combined Bioassay-Chemical Fractionation Scheme for the Determination and Ranking of Toxic Chemicals in Sediments Martln R. Samoiloff,*t James Bell,$ Detlef A. Birkholz,$G. R. Barry Webster,§ Evelyn G. Arnott," Rock Pulak," and Alicia MadridL Department of Zoology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2, Environmental Protection Service, Western and Northern Region, Northern Forest Research Centre, Edmonton, Alberta, Canada, Pesticide Research Laboratory, Department of Soil Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2, and Manitoba Research Council, Industrial Technology Centre, Winnipeg, Manltoba, Canada R2J 3T4
A protocol for the chemical fractionation of sediments and biological testing of these fractions has been developed. Fractions obtained were directly tested for toxicity by using both the Salmonella typhimurium test and the Panagrellus rediviuus test. When applied to sediments from Tobin Lake, Saskatchewan, this method demonstrated that the major toxic constituents of the sediment were neutral compounds that eluate from Florisil columns by 1:l hexane-dichloromethane. This most toxic fraction contained none of the priority toxic chemicals. These testa demonstrate agreement between the two biological assay systems.
Introduction The conventional approach to environmental toxicology involves parallel but distinct "arms length" activities of chemists on the one hand and biologists and toxicologists on the other hand. The chemist establishes what chemicals are present in a particular contaminated environment, while the biologist or toxicologist establishes the toxic effects of individual chemical species. Much of environmental toxicology involves the determination of the presence and levels of previously established toxic chemical species, usually detected one chemical or chemical family at a time. However, real aquatic ecosystems are seldom contaminated with a single chemical issuing from a single source. Rather, there is an extensive multiplicity of compounds and sources contaminating most aquatic systems. Any system in close proximity to human activity will contain literally thousands of contaminating chemicals and their byproducts. The major problem in such systems is to determine which specific components pose the greatest long- and short-term risks to biological systems, including man. The question of what chemicals are present is secondary; the primary objective is to establish which compounds in a particular system pose the greatest risk. 'Department of Zoology, University of Manitoba. Northern Forest Research Centre. Department of Soil Science, University of Manitoba. Industrial Technology Centre.
*
Toward this objective, we are developing methods involving chemical fractionation of environmental samples, coupled with biological testing of the fractions, as a means of establishing which components of the contaminated system produce the greatest toxic effects. Biological assays are utilized as an analytical method of establishing the risk potential of each fraction. The initial methods involve (1)the extraction of contaminants from environmental samples, (2) the fractionation of the extracts on the basis of differential solubility, (3) the bioassay of each fraction to ascertain the relative toxicity of each, and (4) the identification of the components of the major toxic fraction. Ultimately, a series of subfractionations of those fractions determined by this method to be the most toxic would be utilized to pinpoint the exact chemicals producing the greatest risk. We are using a protocol in which biological assays of broad classes of chemical fractions from sediments function as analytical means to localize and rank potential risk. Two bioassay methods are utilized in this study. The Salmonella typhimurium test (1,2)was used as a standard indicator of mutagenesis. A developmental assay (3) using the nematode Panagrellus rediuivus was used to detect lethal, semilethal, developmental, and mutagenic effects. These bioassays, as adjuncts to chemical fractionation, have permitted us to establish the characteristics of the major contaminants in sediments from two sites on Tobin Lake, Saskatchewan. We have selected as sources of contaminants sediment samples from Tobin Lake, on the Saskatchewan River. The Saskatchewan River, upstream of Tobin Lake, is exposed to contaminants from a broad spectrum of human activities: agriculture, mining, petrochemical industries, pulp and paper mills, and municipalities. Tobin Lake represents a major sink for these contaminants. From this extensive range of sources of contamination, we are attempting to focus on those compounds that represent the greatest potential risk. In this report, we present the methodologies and initial toxicity determinations. The work reported here represents only a single component of the Tobin Lake project. The other two components of the study involve the bio-
0013-936X/83/0917-0329$0 1.50/0 0 1983 American Chemical Society
Environ. Sci. Technol., Vol. 17, No. 6, 1983 329
