Environ. Sci. Technol. 1988, 22,1097-1103
(52) Ingold, K. U. Acc. Chem. Res. 1969, 2(1), 1. (53) Holm, T. R.; George, G. K.; Barcelona, M. J. Anal. Chem. 1987, 59(4),582. (54) Keene, J. P. Radiat. Res. 1964, 22, 14. (55) Balzani, V.; Carassiti, V. Photochemistry of Coordination Compounds; Academic: New York, 1970; p 145.
Bevington, J. C.; Toole, J. J . Polymer Sci. 1958, 28, 413. Ross, A. B.; Neta, P. Rate Constants for Reactions of Inorganic Radicals in Aqueous Solution; U.S. Department of Commerce: Washington, DC, 1979; NSRDS-NBS 65; pp 5-13.
Dorfman, L. M.; Taub, I. A,; Buhler, R. E. J. Chem. Phys. 1962, 36(11),3051.
Bhatia, K.; Schuler, R. H. J. Phys. Chem. 1974, 78, 2335. Richter, H. W.; Waddell, W. H. J. Am. Chem. SOC. 1982, 104(17),4630. Wallng, C.; El-Taliawi,G. M.; Johnson, R. A. J. Am. Chem. SOC.1974, 96(1),133.
Received for review March 25,1987. Revised manuscript received October 28,1987. Accepted March 28,1988. Mention of trade names in this paper is for descriptive purposes only and does not constitute an endorsement by the U.S. Geological Survey.
Identification and Quantification of the Ames Mutagenic Compound 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and of Its Geometric Isomer (€)-2-Chloro-3-(dichloromethyl)-4-oxobutenoic Acid in Chlorine-Treated Humic Water and Drinking Water Extractst Leif Kronberg* Department of Organic Chemistry, The University of Abo Akademi, SF-20500 Turku/Abo, Finland
Bjarne Holmbom and Markku Reunanen Laboratory of Forest Products Chemistry, The University of Abo Akademi, SF-20500 TurkuIAbo, Finland
Leena Tikkanen Food Research Laboratory, Technical Research Centre of Finland, SF-02 150 Espoo/Esbo, Finland
rn Mutagenic compounds in XAD extracts of chlorinated humic water were separated in two stages of fractionation by reversed-phase high-performance liquid chromatography. Analyses of mutagenic fractions by gas chromatographylmass spectrometry resulted in the identification of the strong Ames mutagen 3-chloro-4-(dichloromethyl)-5hydroxy-2(5H)-furanone (MX) and its geometric isomer (E)-2-chloro-3-(dichloromethyl)-4-oxobutenoicacid (EMX). Both compounds were also detected in extracts of chlorinated drinking waters. MX accounted for 50-100% of the activity of extracts of chlorinated humic water and for 20-50% of the activity of extracts of drinking water. E-MX exhibited at most one-tenth of the MX mutagenicity and accounted for 2% or less of the activity of the extracts of drinking water. However, because of the poor reproducibility of E-MX mutagenicity tests and the ability of the compound to isomerize to MX, there is uncertainty about the significance of E-MX in chlorinated waters. ~
~~~
Introduction The widespread occurrence of Ames mutagenic compounds in drinking water, disinfected with chlorine, is well documented (1,2). At present it is not known whether any human health risks are associated with the mutagens. The chemical identity of the principal mutagens needs to be known before their potential human health effects can be assessed (2). Knowledge of the chemical structure of the mutagens may lead to the elucidation of the mechanisms involved in their formation and allow the development of processes to minimize or prevent their production ( 3 ) . Moreover, it could contribute to our overall understanding This work was carried out at the Department of Organic Chemistry, The University of Abo Akademi. 0013-936X/88/0922-1097$01.50/0
of relationships between chemical structure and mutagenicity. It is generally assumed that the mutagenic compounds are products of the reaction of chlorine with humic substances (4-7). In a previous study we found similarities in the mutagenic activity of chlorine-treated natural humic water and drinking water, derived from surface water (8). The strong activity of extracts of acidified, chlorine-treated humic water makes these extracts suitable for the study of mutagens most likely to be present in drinking water. Although a number of mutagenic compounds have been identified in extracts of both drinking water and chlorinated humic acids, it is obvious that these compounds are responsible for only a minor proportion of the observed activity (7,9).However, in a recent study we reported the presence of the strong Ames mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone(MX) in extracts of chlorinated humic water and drinking water, and we found that MX accounted for significant proportions of the activity of some of the samples (IO). In this study we have investigated further the significance of MX in chlorinated water. At the same time, efforts were made to elucidate the structure of other mutagens formed during chlorination of humic substances. This was carried out by repeated fractionation of mutagenic extracts by reversed-phase high-performance liquid chromatography (RP-HPLC) followed by analysis by gas chromatography/mass spectrometry (GC/MS) of mutagenic fractions. The work resulted in the identification of (E)-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid (EMX), the geometric isomer of MX.
Materials and Methods Water Samples. Drinking water, derived from surface water, was collected from the distribution system of three
0 1988 American Chemical Society
Environ. Sci. Technol., Vol. 22, No. 9, 1988 1097
I
CHLORIN41ED H W I C W T E R Water ac~difledto pH 2 and pessed over a column of XA3 518. Adsorbed miterial
Table I. Ion Peaks Used for SIM Quantification of Methylated MX and E-MX vs Methylated MBA compound
fragment ion
m/z
ration
M-OCH3
198.912 200.909 202.906 241.003 243.001 244.954 246.951 240.832
0.51
RFb
el-ted with ethyl ace:ate
MX X X EXTRACT (Hi")"
I
XU extract re-extracted with e t h e r
E-MX
M-OCH3
DIETHYL ETHER ERR4CT (H+'/E)*
MBA rclumr HPLC frectionat.an
I
M-CI
M-OCHB
1.00
0.55 0.54 0.35 1.00 0.95
0.57
1.60 (1.00)
Relative ion peak area ratios. ResDonse factor.
HPLC FRACTIOK C18A (mV/F/Cl8A)*
I
CHCli ,COOH
;c =c, o=c C'
Cg c c l m n
CI
CHCI, X
HPLC fracticnatian
I
Ho,Q*o
Figure 1. Procedure for purification and fractionation of mutagenic compounds present in chlorine-treated humic water. ( * ) = abbreviations used in Table 11.
towns in Finland. The waterworks used conventional treatment methods: alum coagulation followed by sand filtration and chlorine disinfection. The total chlorine residual in the samples was less than 0.1 mg/L. Immediately after sample collection, 4 M HC1 was added until pH 2.0 was reached. Natural freshwater, with a high content of humic substances (TOC = 25 mg/L), was collected from a lake situated in a marsh region in southwest Finland. The lake did not receive any industrial or municipal effluents. The water was chlorinated at a 1:l chlorine: TOC weight ratio under buffered conditions (potassium phosphate buffer) at pH 7.0 f 0.2. Chlorination was performed at ambient temperature in the dark with a freshly prepared aqueous solution of chlorine. After a reaction time of 60 h, the total chlorine residual was less than 0.1 mg/L and the water was acidified to pH 2.0 by addition of 4 M HC1. XAD Concentration Procedure. Ten liters of each water was passed through a column (2 X 20 cm) of XAD-4 and XAD-8 (1:l mixture) a t a flow rate of one bed volume/min (60 mL/min). Residual water in the column was removed with a gentle stream of nitrogen. The adsorbed organics were eluted in reverse direction with three bed volumes (180 mL) of ethyl acetate. The extract was concentrated by rotary evaporation, and finally its volume was adjusted to 0.5 mL ethyl acetate per liter of original water. Extraction and Fractionation of Mutagenic Compounds. The mutagenic material in the extract of chlorine-treated humic water was fractionated by the procedure depicted in Figure 1. The XAD extract was evaporated to dryness, dissolved in 20 mL of 0.02 M aqueous HC1, and reextracted with three 10-mL portions of diethyl ether. The combined ether extract was fractionated by RPHPLC. Fractionation on a CI8 semipreparative column (Macherey-Nagel, Nucleosil 7 Cl8, 10 X 250 mm) was followed by further fractionation on a C6 analytical column (Phase Sep, Spherisorb 5 C6, 4.5 X 250 mm, packed in our 1098
Environ. Sci. Technol., Vol. 22, No. 9, 1988
laboratory). The C18 column was eluted with a linear gradient from 30% methanol in water, acidified to pH 3.0 with orthophosphoric acid, to 100% methanol over a period of 30 min at a flow rate of 4 mL/min. The C6column was isocratically eluted with 0.05 M phosphate buffer at pH 4.0 at a flow rate of 1.0 mL/min. Collected fractions were acidified to pH 2.0 and repeatedly extracted with diethyl ether. The combined ether extracts were evaporated to dryness and the residues dissolved in ethyl acetate. Extract Derivatization and GC/MS Analyses. Prior to GC/MS analysis, XAD extracts and mutagenic fractions were evaporated to dryness and the residues methylated by 300 pL of 2% (v/v) H2S04in methanol for 1 h at 70 "C. The reaction mixture was neutralized by addition of 600 pL of 2% aqueous NaHC03 and extracted twice with 600 pL of n-hexane. The combined hexane extract was concentrated under nitrogen and injected into the GC. Quantitative determinations of MX and E-MX were carried out by reference to the internal standard mucobromic acid (MBA), added to the extracts and fractions at a concentration corresponding to 0.46 pg/L water equiv. The GC/MS analyses were performed on a Dani 3800 capillary gas chromatograph interfaced to a VG 7070E mass spectrometer. The GC/MS system was equipped with a fused silica capillary column (25 m, 0.22 mm i.d.) coated with SE-30 stationary phase (film thickness 0.25 pm). Helium (approximately 0.8 mL/min) was used as carrier gas, and the column oven temperature was programmed from 80 to 200 "C at 6 "C/min. The GC injector and the ion source temperatures were maintained at 250 and 200 "C, respectively. The ionization mode was electron impact (70 eV), and the resolving power was 1000. Lowresolution accurate mass determination was based on reference ions 126.9045 (I), 140.9201 (CHJ), 253.8089 (Iz), and 267.8246 (CH,I,) from diiodomethane. For quantitative purposes the mass spectrometer was operated in selected ion monitoring (SIM) mode (Table I). The standard SIM routine of the VGll-250 data system was used to record and compute the SIM data. Response factors for MX and E-MX ions vs MBA were calculated from the analysis of a standard mixture of MX, E-MX, and MBA in ethyl acetate at concentrations corresponding to 0.21,0.27, and 0.69 pg/L water equiv, respectively. The identification of MX and E-MX in actual samples was based on positive matching of retention times and relative ion peak area ratios. Procedure for Isolation of E-MX. MX was synthesized by the method developed by Padmapriya et al. (11). E-MX was formed as a byproduct in the synthesis. In order to isolate MX and E-MX separately, the pH of the reaction mixture was adjusted to pH 5.0 and MX was extracted by diethyl ether. (Further purification of MX was carried out by flash chromatography, see ref 11). The aqueous phase was acidified to pH 2.0, and E-MX was extracted by diethyl ether. The extract was evaporated
Table 11. Mutagenic Activity of Extracts of Drinking Water and of Extracts and Fractions of Chlorine-Treated Humic Water; MX and E-MX Concentrations and Theoretical Contribution to Observed Mutagenicity
sample" drinking water A
B C humic water A
B H W A, fractions HW/E HW/E/Cl8 total eluate C18A HW/E/C18A/C6 total eluate
mutagenicity net rev/Lb % recovered 3440 1190 3750
concn, ng/L MX E-MX
mutagenicity contributionC MX E-MX net rev/L % activity net rev/L % activity
26 15 67
37 8 41
670 390 1740
19 33 46
50 10 60
2
1
1
8720 10370
(100)
380 190
700 390
9850 4950
113 48
1040 580
12 6
8770
101
350
650
9070
103
960
11
7470 6670
86 76
3970 1500 5130
46 17 59
520
35
350
136 7000 270 CBB "Abbreviations for fractions of humic water (HW A) as in Figure 1. bCalculated by least-squares regression analysis of the linear portions of the dose response curves. Determined by multiplying the molar concentration with the specific TAlOO mutagenicity of the respective compound: MX, 5600 net revertants/nmol; E-MX, 320 net revertants/nmol.
to dryness and subjected to flash chromatography on a silica column (Kieselgel60, 40-60 pm particle size) eluted was chloroform-methanol (8:2). Fractions containing E-MX were collected, combined, and concentrated. HPLC separation on the semipreparative column was used for the final purification. The column was eluted with 20% acetonitrile in water adjusted to pH 2.8 with orthophosphoric acid, flow rate 3.0mL/min. The E-MX fraction was collected, acidified to pH 2.0,and extracted by diethyl ether. The solvent was evaporated to give 7.4 mg of E-MX [resulting from the hydrolysis of 2.25 g of 2,4,4-trichloro-3-(dichloromethyl)crotonicacid]. The compound was considered pure as no impurities were detected in the 'H nuclear magnetic resonance (lH NMR) spectrum, by MS analysis by a temperature-programmed direct inlet probe, or by analyses on C18and c6 analytical HPLC columns. The IH NMR spectrum was recorded with a Jeol GX 400 Fourier transform NMR spectrometer on a CDCl:, solution of approximately 700 pg of E-MX. Assay for Mutagenic Activity. Mutagenicity assays were performed according to the Ames test method (22). Salmonella typhimurium tester strain TAlOO without metabolic activation (S9mix) had previously been found to be the most sensitive assay and was therefore used throughout this work (8). Extracts and fractions of chlorinated humic water were tested in ethyl acetate, while the solvent of the extracts of drinking water was changed to dimethyl sulfoxide (DMSO) immediately before testing. Three or four dose levels were used, with two plates per dose, and each sample was tested at least twice. The standard deviation of the mean revertant number was usually within &lo%, and the number of spontaneous revertants was 90-120. The number of revertants induced by the positive control, sodium azide, was 500-600 for 1 pg and 1400-1600 for 5 pg,
Results The XAD extracts of drinking water samples and of chlorine-treated humic water exhibited mutagenic activity in strain TAlOO (Table 11). Reextraction of the extract of chlorine-treated humic water, using diethyl ether, did not cause any loss in mutagenicity. Fractionation by RP-HPLC using the C18 column resulted in 76% of the original activity eluting in fraction Cl8A (Figure 2A and
H20 PH 3 70%
MeOH 100%
Fraction Ret.time
1 b
10
20
30
39 min
E
F:
zB
E
Fraction
Ret. time 0
-
min
Figure 2. (A) C,, column HPLC separation of reextracted chlorinated humic water (HW/E) and collected mutagenic fraction (C,,A). (B) C, column HPLC separation of fraction CI8A. Fraction C,A and CB, were mutagenic. Table 11). Following HPLC fractionation using the (26 column two distinct peaks of mutagenicity were evident; the peak at shorter retention time (fraction c6A)accounted for 17% and the peak of less polar organics (fraction C6B) accounted for 59% of the original activity of the XAD extract (Figure 2B and Table 11). Other chromatographic fractions were nonactive at the dose levels assayed. HPLC analysis of pure MX, using the c18 and c6 columns, showed that MX eluted in fractions and C6B, respectively. Fraction C6B, collected from subsequent samples, was analyzed for MX by GC/MS. The mass chromatogram of the most abundant MX fragment ion at m/z 147 (formed by loss of CHClz from the molecular ion) was generated (Figure 3). The mass spectrum obtained at the retention time of the MX standard (scan 493) was Environ. Sci. Technol., Vol. 22, No. 9, 1988 1099
Ncmlnal m:2
UIfferenre between Measured and C a i c u lared m i 2 L'aluei, mu
Exact m/z
276
275 9 7 2 1
245 247 249
244.9119
2.3
216 9110 246.9160
-2.6
S f r u r t u r e af Mdii FreQenf
Formation
c8H1104c13
M
C&OjCl)
U-C'I30
i
1.4
493
100
c,nci2 ,coocn, c=c, n ccnO
i
0
200
600
400
Scan
110
').
Flgure 3. Mass chromatogram at rn / z 147 and mass spectrum recorded at scan 493.
Table 111. E-MX Isomerization in Acetonitrile Solutions Exposed to Sunlight
time, h 0 5 10 20
E-MX solution concn, ng/mLn MX E-MX total 0 260 470 590
a Determined
720 430 260 90
720 690 730 680
i l I
I
I
fraction CGA concn, ng/La MX E-MX total 0
370
370
340
20
360
by GC analyses of methylated aliquots.
Lm
1200
18ce 80."
identical with the mass spectrum of the MX standard (Figure 3). The presence of MX in fraction C6Bwas thus confirmed. The mass spectrum recorded at scan 925 in the total ion current chromatogram of fraction C6A was identical with the mass spectrum of one of the byproducts formed during MX synthesis (Figure 4). Direct inlet MS analyses of the isolated, underivatized byproduct resulted in the tentative identification of the geometric isomer of MX, (E)-2chloro-3-(dichloromethyl)-4-oxobutenoicacid (E-MX) (Figure 5). A suggested scheme of the main fragment pathways of the compound is inserted in Figure 5. In the 'HNMR spectrum of E-MX two resonance signals of equal intensity were observed (and not present in a blank sample passed through the same purification procedure). The signals at 6 5.59 and 6 9.99 were assigned to the protons in the CHClz and CHO groups, respectively. High-resolution studies showed the signals to be doublets with a coupling constant J = 1.0 Hz due to long-range coupling between the protons. The resonance signal from the carboxyl proton was masked by water. Support for the structural interpretation was obtained by observing the sunlight-induced isomerization of the isolated byproduct and of the compound in fraction C6Ato MX (Table 111). The mean mutagenic activity calculated for five tests of MX in ethyl acetate was 5600 f 500 net revertants/ nmol. On the basis of the MX activity and MX quantifications, it was possible to explain all of the observed activity in fraction C6B and in the original XAD extract 1100
Environ. Sci. Technol., Vol. 22, No. 9, 1988
Figure 4. Total ion current chromatogram of fraction CBA, mass spectrum recorded at scan 925, and proposed structure of main fragment ions.
of chlorinated humic water by the presence of MX (Table 11). In the other chlorinated humic water sample, MX accounted for 50% and in the drinking water samples for 20-50% of the total activity. The E-MX concentration in fraction C6Awas 350 ng/L, and the mutagenic response of the fraction was 1500 net revertants/L water equiv (Table 11). When E-MX was chromatographed with the c6 column and the E-MX peak collected and tested for mutagenicity, the E-MX response was 900 net revertants/nmol. This response was high enough to explain all the activity in fraction C6A. However, when mutagenicity tests were carried out on pure E-MX, dissolved in ethyl acetate, the test results showed poor reproducibility. The mean E-MX activity and the standard deviation of the mean, calculated for seven assays, was 320 f 250 net revertants/nmol. On the basis of this number, E-MX accounted for 35% of the activity of fraction C6A and for 1 2 % and 6%, respectively, of the activity in the extracts of humic water A and B (Table 11). In the extracts of drinking water, the concentration of E-MX was similar to the corresponding MX concentration and E-MX accounted for 2 % or less of the observed mutagenicity (Table 11). When the mutagenic activity of MX, dissolved and stored in DMSO at room temperature, was monitored over 32 days, a time-dependent decrease in MX mutagenicity
Table IV. Stability Experiments with DMSO Solutions of MX, HW/E Extract, and HW/E Extract Spiked with MX net revertants" as a function of time, days sample
0
2
4
8
16
32
mean f SD
% activity covered by MX
MX HW/Eb HW/E + MXc
5700 8220 18300
2600 8100 16190
1400 12270 19390
1200 12790 18890
800 15550 19200
700 13890 21090
11800& 304 18840 f 160
77 91
+
"MX, net revertants/nmol; HW/E and HW/E MX, net revertants/L. Calculated by least-squares regression analysis of the linear portion of dose response curves. *MX concentration = 350 ng/L. CHW/Espiked with 315 ng/L MX.
73 loo]
I
mo
Figure 5. Mass spectrum and suggested main fragmentation pathways of E-MX. (*) = fragmentation observed by constant parent ion ( B I E ) and/or constant daughter ion ( B 2 / E )scans.
was noted (Table IV). A similar experiment conducted with DMSO solutions of the HW/E extract and of the HW/E extract spiked with MX showed the activity in both solutions to be stable throughout the experiment. Neither was any loss of activity observed in parallel experiments where ethyl acetate was used as solvent.
Discussion Numerous studies on mutagenic activity in drinking waters have been carried out on extracts obtained at neutral or ambient pH (I,2, 13,14). However, we have previously found that the TAlOO activity of acid extracts of chlorine-treated humic water and of some drinking water exceeded the activity of neutral extracts by a factor of 10 ( 4 1 5 ) . Therefore, our work on structural elucidation of major mutagens has focused on mutagens with acid properties. Other research groups have also observed strong activities in extracts of acidified water (16-19), and recently Ringhand et al. (20) demonstrated that sample acidification does not produce mutagenic artifacts. Water extracts are too complex to carry out successful qualitative analyses of mutagens, and some form of separation/fractionation is required. In studies where mutagens in neutral drinking water extracts have been fractionated, the original activity has been distributed among several subfractions, and further fractionation has suffered from difficulties in detecting the diluted activity (21-23). However, Meier et al. (24) found that when the muta-
genicity of the extract of strong acids, obtained from an aqueous solution of chlorinated humic acids was fractionated by C18column HPLC, the activity was concentrated in two distinct subfractions: the fraction collected in the middle of the chromatographic run being twice as active as the fraction which eluted later. Maruoka (25) applied thin-layer chromatography (TLC) for the fractionation of mutagens produced by chlorination of humic substances. Most of the activity was located in the fraction containing the strongly retained polar constituents, while minor activity was observed in the fraction containing less polar constituents. In the present work the initial separation of mutagens in the acid XAD extract of chlorinated humic water was carried out by liquid-liquid extraction by diethyl ether at pH 2. The mutagenic activity was quantitatively transferred to the ether phase, while much of the yellow high molecular weight material was nonextractable. At the same time a decrease in the Ames test toxicity was achieved. When the ether extract was fractionated on the CIScolumn, most of the injected activity was recovered in one single fraction, Cl& Previously, we carried out fhther isolation of the mutagens in fraction C18A by high-performance size exclusion chromatographic (HPSEC) and TLC methods (26). However, the TLC method suffered from poor recovery of mutagenicity and low reproducibility, while the HPSEC method was limited by low sample capacity. Therefore these methods have been replaced. Molndr et al. (27) developed a technique called hydrophobic chromatography for RP-HPLC analyses of low molecular weight carboxylic acids. The retention,of the acids is enhanced by the use of aqueous eluents at pH values low enough to suppress the degree of ionization of the acid solute. The technique, when applied to fraction C18Awith 0.05 M phosphate buffer at pH 4.0 as eluent, was very useful. Although most of the injected material did not elute from the column under these conditions, approximately 60% of the injected activity eluted within 25 min. Furthermore, this activity was collected in two distinct fractions, C6A and C6B,of such purity that GC/MS was successfully applied for subsequent analyses. During GC/MS analyses of fraction C6B, the mass spectrum of MX was collected at the retention time of MX standard. This finding provided final verification of the presence of MX in the sample of chlorinated humic water. In the chromatographic systems applied by Meier et al. (24) and Maruoka (25) for the fractionation of mutagenicity in extracts of chlorinated humic substances, MX retention might, according to our interpretation, overlap with the retention time observed for the major mutagenic fraction in their work. This is supported by the observation made by Meier et al. that their fraction contained compounds with mass fragmentation patterns consistent with hydroxyfuranone structures. One of the mass spectra of fraction C6Awas interpreted as the methyl derivative of the geometric isomer of MX. In the isomer, E-MX, the aldehyde group is situated trans Environ. Sci. Technol., Vol. 22, No. 9, 1988
1101
aq KHCO,
CHCIz \ /
c=c
CI /
A
\COOH
-
/COOH
‘~‘‘2
Isomerisation
c=c o=c/
‘CI
H ‘
o=c\H
E-MX
Ring closure
MX
Flgure 6. Formation of MX and E-MX from 2,4,4-trichloro-3-(dichloromethy1)crotonic acid and E-MX isomerization.
to the carboxyl group, and therefore, ring closure (e.g., formation of the hydroxyfuranone structure) is hindered. E-MX exists as a highly water-soluble carboxylic acid and is poorly retained on the c6 column. Theoretically, E-MX should be formed as a byproduct in the synthesis of MX. In the last step of the synthesis, the dichloromethyl group cis to the carboxyl group in 2,4,4-trichloro-3-(dichloromethy1)crotonic acid is transformed into an aldehyde group, and after ring closure, MX is obtained (Figure 6). Transformation of the trans dichloromethyl group should also be possible, resulting in E-MX. In fact GC/MS analysis of the methylated crude reaction mixture showed the presence of a compound identical with the compound in fraction C6A. The result of direct inlet MS analysis of the isolated byproduct strongly supports a structure representing the M X isomer, (E)-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid. Additional support for this structure was provided from lH NMR analyses and from the observation of sunlight-induced isomerization to MX. Later we observed that when E-MX was stored in water at pH 2.0, a t 40 “C, the compound quantitatively isomerized to MX within 12 days (28). Nestmann et al. (29, 30) reported solvent-dependent variations in the mutagenic activity of trichloroacetic acid and some chloropropanones. They also demonstrated that DMSO was, in some cases, an inappropriate solvent because of chemical interactions with solutes (e.g., trichloroacetic acid and hexachloroacetone). Horth (31) carried out an experiment where MX was stored in DMSO for several days, and variations in the mutagenicity were recorded. The experiment showed a clear time-dependent decrease in the mutagenicity of MX. We carried out a similar experiment in our laboratory and obtained results which confirmed those of Horth. On the other hand, the activity of the H W / E extract and the HW/E extract spiked with MX was stable in DMSO throughout the stability experiment, although MX was present in concentrations high enough to account for most of the activity of the extracts. In the DMSO solution of pure MX chemical interactions take place between the solvent and MX, but in the complex extracts MX seems to be in some 1102
Environ. Sci. Technol., Vol. 22, No. 9, 1988
way protected against attack from DMSO. Since there was uncertainty about DMSO interactions with MX and other organics in mutagenic extracts, we preferred to store our extracts and to test them in the Ames assays, in ethyl acetate. However, the extracts of drinking water had to be tested in DMSO because of toxicity problems in ethyl acetate. In the work reported by Holmbom et al. (32) MX mutagenicity was determined in several contract laboratories. The reported activity in strain TAlOO ranged from 2800 to 10 000 net revertants/nmol. We previously reported that MX induced 2710 revertants/nmol (10). This number is probably too low as it was obtained from a solution in DMSO that was 2 days old. In ethyl acetate the mean MX activity calculated for five assays was 5600 net revertants/nmol. The number is in good agreement with the 6000 revertants/nmol, approximately, reported by Padmapriya et al. (11). In Table I1 we calculated the theoretical contribution of MX to the observed Ames mutagenicity of extracts and fractions. MX accounted for all of the activity in fraction C6B. Consequently, no other major mutagens eluted with the fraction. MX accounted for 50-100% of the observed activity in the extracts of chlorine-treated humic water and for 20-50% of the observed activity in the extracts of drinking water. Because of the poor reproducibility of the mutagenicity assays of E-MX, we were not able to estimate accurately the amount of activity represented by E-MX. A rough estimate made on the basis of the mean activity of 320 net revertants/nmol showed only a few percent of the mutagenicity in the extracts of drinking water to be due to E-MX. An evaluation of the significance of E-MX is further complicated by the ability of the compound to isomerize to MX, thereby causing a substantial increase in the mutagenicity. Isomerization could take place when E-MX is tested in the Ames assay, and thus the observed activity of the compound would be in fact due to small amounts of MX formed on the plates. At present it is not known whether conditions which favor E-MX isomerization are to be found in the drinking water distribution system or during drinking water consumption. In conclusion, more work on the chemical and mutagenic properties of E-MX has to be carried out before the significance of the compound can be estimated.
Acknowledgments We thank R. Sjoholm for the lH NMR analyses. Registry No. MX, 77439-76-0; E-MX, 115340-67-5.
Literature Cited (1) Loper, J. C. Mutat. Res. 1980, 76, 241-268. (2) Kool, H. J.; van Kreijl, C. F.; Zoeteman, B. C. CRC Crit. Rev. Environ. Control 1982, 12, 307-357. (3) Ridgway, J. W. Water Supply 1983,1, 511-518. (4) Bull, R. J.; Robinson, M.; Meier, J. R.; Strober, J. EHP, Environ. Health Perspect. 1982, 46, 215-227. (5) Meier, J. R.; Lingg, R. D.; Bull, R. J. Mutat. Res. 1983, 118, 25-41. (6) Coleman, W. E.; Munch, J. W.; Kaylor, W. H.; Streicher, R. P.; Ringhand, H. P.; Meier, J. R. Environ. Sci. Technol. 1984, 18, 674-681. (7) Fielding, M.; Horth, H. Water Supply 1986, 4, 103-126. (8) Kronberg, L; Holmbom, B.; Tikkanen, L. Vatten 1985,41, 106-109. (9) Meier, J. R.; Ringhand, H. P.; Coleman, W. E.; Munch, J. W.; Streicher, R. P.; Kaylor, W. H.; Schenck, K. M. Mutat. Res. 1985, 157, 111-122. (10) Hemming, J.; Holmbom, B.; Reunanen, M.; Kronberg, L. Chemosphere 1986,15, 549-556.
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Received for review August 11, 1987. Accepted April 18, 1988. This work was supported by The Academy of Finland.
NOTES Total Organic Carbon Concentrations in Acidic Lakes in Southern Norway? Arne Henriksen,” David F. Brakke, and Stephen A. Norton Norwegian Institute for Water Research, Postboks 33, Blindern, 03 13 Oslo, Norway
w Regional surveys in Norway demonstrate that most of the lakes have low concentrations of total organic carbon (TOC); 60% of the lakes had TOC < 2 mg/L and 90% had TOC < 6 mg/L. There was no apparent relationship between lake water pH and TOC. For the 1005 lakes sampled throughout Norway in 1986, organic anions represent 6 mg/L might be classed humic colored, although the characteristic yellow-brown color of humic lakes is niost noticeable at higher concentrations of TOC. The same report ( 4 ) used by Krug et al. (5) also contained measurements of TOC for some of the lakes. These
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