Environ. Sci. Technol, 1993, 27, 1811-1818
Determination of Chlorinated Furanones, Hydroxyfuranones, and Butenedioic Acids in Chlorine-Treated Water and in Pulp Bleaching Liquor Leif Kronberg' and Robert Franz6n
Department of Organic Chemistry, Abo Akademi University, Akademigatan 1, SF-20500, Turkulabo, Finland Compounds with structural similarities to the potent bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy2(5H)-furanone, MX, have been synthesized and quantitatively determined in extracts of chlorination-stage bleaching liquors (CBL) from pulp mills, chlorine-treated aqueous solution of fulvic acids, chlorinated natural humic water, and chlorine-disinfected drinking water. In addition, a preliminary screening of the mutagenic potency of the compounds was performed. The compounds were found in the extracts with the exception of a few compounds not detected in the drinking water extracts. The compounds induced mutagenicity in Ames tester strain TAlOO in tests performed without metabolic activation. All the compounds were found to be less potent mutagens than MX. The only compound which could be considered as a strong mutagen was 3-chloro-4-(chloromethyl)-5hydroxy-2(5H)-furanone (CMCF), although the revertants per nanomole of CMCF was only one-fifth of the revertants generated by MX. CMCF was found to account for at most 6% of the mutagenicity of the extracts. The contribution to the mutagenicity of the extracts of the other compounds was negligible.
Introduction Chlorinated organic compounds are formed during chlorine bleaching of pulp and during disinfection of drinking water by chlorine. Because of the possible environmental hazards possessed by many chlorinated compounds, much work has been devoted to the structural characterization of the compounds. The most abundant and frequently found chlorinated compounds are the trihalomethanes, haloacetonitriles, short-chain carboxylic acids, acetones, and phenolic compounds (1-5). Much attention has been paid to the presence of the trihalomethanes in drinking water and the possible healtheffects associated with the compounds (e.g., ref 6). In 1981,Holmbom et al. (7)reported on the identification of the chlorinated hydroxyfuranone 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone(MX) in bleached pulp mill effluents. MX was found to be one of the most potent direct-acting mutagens ever tested in Ames tester strain TA100. Later, Hemming et al. (8)detected MX in extracts of chlorinated drinking water. On the basis of an extended survey of MX in drinking waters in Finland, Kronberg and Vartiainen (9) estimated the compound to account for 15-57 % of the drinking water mutagenicity. In addition, MX has been detected in chlorinated drinking waters in the United States (IO), in the United Kingdom (111, in Canada (12), in Japan (13),and in the Peoples Republic of China (14). Recently, Kronberg et al. (15)reported the presence of 3-chloro-4-(dichloromethyl)-2(5H)-furanone(red-MX), (2)2-chloro-3-(dichloromethyl)-2-butenedioic acid (ox-MX) (Figure 11, and (E)-2-chloro-3-(dichloromethyl)-2-butenedioic acid (ox-EMX) in extracts of chlorinated waters. 0013-936X/93/0927-1811$04.00/0
@ 1993 American Chemical Society
MX exists in an open-chain form, as an oxobutenoic acid, at pH conditions above 5.3 (16,17).red-MX can be seen as an MX analogue where the aldehyde group of the open form of MX is replaced by an alcoholic group (reduced form of MX). In ox-MX, the aldehyde group has been replaced by a carboxylic group (oxidized NIX) and oxEMX represents the oxidized form of (E)-2-chloro-3(dichloromethyl)-4-oxobutenoic acid (EMX), the geometric isomer of MX. In previous studies, several chlorinated furanones and hydroxyfuranones have been synthesized in order to determine the structural features responsible for the mutagenicity of MX (18-20). Most of the compounds were found to be active mutagens, although less potent than MX. However, studies of the presence of these compounds in extracts of chlorinated water has not been undertaken. In case some of the compounds are present in chlorinated water in significantly higher concentrations than MX, they could account for considerable amounts of the total mutagenicity in the extracts. In this study, we have synthesized hydroxyfuranones with a dichloromethyl,monochloromethyl,or methyl group attached to the carbon at the 4-position in the furanone ring. A chlorine or hydrogen atom was attached to the carbon at the 3-position. Further, the corresponding furanones and butendioic acids were prepared (Figure 1). We carried out qualitative and quantitative determinations of these compounds in chlorination-stage bleaching liqtlours, in extracts of chlorinated fulvic acids, in extracts of chlorinated humic water, and in two extracts of chlorinedisinfected drinking water. In addition, we have performed a preliminary screening of the mutagenic potency of the compounds in Ames tester strain TA100.
Experimental Section Water Samples and Chlorination Procedure. The collection, chlorination, and extraction procedure of the fulvic acids (FA) and the natural humic water (HW) samples have been described previously (25). The drinking water samples (DW1 and DW2) were collected from the distribution system of two cities in Finland. The drinking water was derived from surface water with a TOC content of approximately 15 mg/L. Following alum flocculation and sand filtration, the TOC content of the water was approximately 6 mg/L. Prior to distribution, the water was disinfected by chlorine, 3-5 mg/L. The XAD extraction procedure of the drinking water samples was the same as described previously (15). The sample of chlorination-stage bleaching liquors (CBL) was derived from softwood (pine) kraft pulp which had been prebleached with oxygen. The subsequent chlorine bleaching was performed with 29 kg of Clz and 4.6 kg of ClOdt of pulp. The K number of the unbleached pulp was 18. The TOC content of the sample liquor was 370 mg/L. One liter of the acidic liquor (pH 1) was extracted with three portions of freshly distilled diethyl ether (3 X 200 mL). The combined extract was evaporated Environ. Scl. Technol., Vol. 27, No. 9, 1993
le11
FURANONE
HYDROXYFURANONE
-I__
B U T N E D D C ACID
Table I. IH Chemical Shifts in ppma Obtained for Selected Chlorinated 2(5I€)-Furanones and Butenedioic Acidsb 6H-i
compound R=CHCI,;
X=CI
red-MX
MX
OX-MX
R = CH,CI;
X = CI
redGMCF
CMCF
oxGMCF
R=CH,;
X=CI
red-MCF
MCF
ox-MCF
R = CHCI,;
X=H
reddCMF
dCMF
R = CHzCI; X r H
red-mCMF
mCMF
ox-mCMF
R=CI;
red-MCA
MCA
ox-MCA
X=CI
Flgure 1. Compound structures and abbreviations used.
6H.3
red-MCFc red-MCAc red-MBAC OX-CMCF~ OX-MCF~ ox-mCMFd 7.57 (s,lH)
others
6H-6
6H-6
4.80 (s,2H) 4.90 (8, 2H) 4.89 (9, 2H)
2.05 (8, 3H) 4.51 (s, 2H) f 2.15 (s, 3H) 1: 3.56 (s, 2H) 9.3 br.s, COOH 8.3 br.s,
OX-MCA~
COOH OX-MBA~
7.9 br.s,
COOH
to dryness, and the residue was redissolved in an exact volume of ethyl acetate. Compound Synthesis. 3-Chloro-4-(dichloromethyl)5-hydroxy-2 (5H)-furanone (MX) was synthesized and purified as described by Padmapriya et al. (21). The compounds 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)furanone (CMCF),3-chloro-4-(chloromethyl)-2 (5H)-furanone (red-CMCF) 4- (chloromethyl)-5-hydroxy-2(5H)furanone (mCMF),4-(chloromethyl)-2(5H)-furanone (redmCMF), 4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (dCMF), and 4-(dichloromethyl)-2(5H)-furanone(reddCMF) (Figure 1)were synthesized according to methods described by LaLonde et al. (22). 3-Chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) was prepared in a manner analogous to the preparation of MX, except that 1,l-dichloropropanone was used in the Wittig condensation instead of l,l,3,3-tetrachloropropanone. Purification on a silica gel column with ethyl acetate-hexane-acetic acid (50:50:0.1) as eluent gave the pure compound as a yellow oil in 45% total yield. This compound has also been prepared by Ishiguro et al. (23) and Streicher (20). The furanones, 3,4-dichloro-2(5H)-furanone(red-MCA), 3,4-dibromo-2(5H)-furanone(red-MBA), and 3-chloro-4methyl-2(5H)-furanone (red-MCF), were obtained by reduction of the corresponding hydroxyfuranones with aluminium isopropoxide in 2-propanol at 70 "C (15).The final purification of the compounds was carried out on a silica gel column with dichloromethane-hexane (3:l).The yields were 24% of red-MCA, 35% red-MBA, and 5% red-MCF. The compound red-MX had been prepared previously (15). The butenedioic acids, 2-chloro-3-(chloromethyl)butenedioic acid (ox-CMCF),2-chloro-3-methylbutenedioic acid (ox-MCF), 3-(dichloromethyl)butenedioic acid (ox-dCMF), 3-(chloromethyl)butenedioic acid (ox-mCMF), 2,3dichlorobutenedioic acid (ox-MCA), and 2,3-dibromobutenedioic acid (ox-MBA), were prepared by oxidation of the corresponding hydroxyfuranones with fuming nitric acid (15). Recrystallization of the crude products from CH2C12 gave pure ox-CMCF, ox-MCF, ox-MCA, and oxMBA in yields of 28,32, 50, and 72 %, respectively. The butenedioic acid ox-dCMF was formed in trace amounts only, and the pure compound could not be isolated from the reaction mixture. The compound ox-mCMF was formed as a byproduct during the preparation of dCMF and could be separated from dCMF by crystallization from CH2C12. NMR data of the compounds not reported in the literature previously are presented in Tables I and 11,and the mass spectra of the furanones and the methyl 1812 Environ. Sci. Technol., Vol. 27, No. 9,1993
*
Relative to TMS at 6 = 0.00 ppm. For the numbering of the protons see Figure 1. Dissolved in CDC13. Dissolved in CDC13 + acetone-&. e Dissolved in CDC13 + DMSO-& f Resonance signal of the carboxylic proton could not be observed due to the presence of water in the sample.
Table 11. '3C Chemical Shifts in ppma for Selected Chlorinated 2(5H)-Furanones and Butenedioic Acidsb 6C.i
compound red-MCFC red-MCAC red-MBAC
6 ~ . ~
168.6 165.6 166.7
6c.3
6c.4
6c.a
6c.s
119.8 121.2 114.7
156.1 148.9 143.6
72.1 70.9 74.2
12.6
6C-2,S
b3,4
compound OX-CMCF' ox-MCFf ox-mCMFe OX-MCA~ OX-MBA~
6C-6
162.gd 163.4d 167.4" 173.4d 167.4" 172.gd 162.1 1.62.9
130.8d 135.6d 127.3d 135.gd 130.4" 136.7d 130.6 125.3
39.0 20.7 34.0
Relative to TMS a t 6 = 0.00 ppm. For the numbering of the carbons see Figure 1. Dissolved in CDCl3. The signals were not unambiguously assigned. e Dissolved in CDCl3 t acetone-&. f Dissolved in CDCL + DMSO-&. (I
derivatives of the hydroxyfuranones and the butenedioic acids are shown in Figure 2. The compound 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid, MCA) and 3,4-dibromo-5-hydroxy2(5H)-furanone (mucobromic acid, MBA) were obtained from commercial sources (Aldrich-Chemie, Steinheim, Germany). Full-scan mass spectra of the compounds were recorded using a Hewlett-Packard 5971A mass selective detector (MSD) coupled to a Hewlett-Packard 5890 gas chromatograph. The GC was equipped with a HP-1 fused silica capillary column (25 m X 0.20 mm i.d., film thickness 0.33 pm). The NMR spectra were recorded with a Jeol GX 400 Fourier transform NMR spectrometer. Derivatization and GC/MS Procedures. The procedures for methylation of the extracts for GC/MS determination of hydroxyfuranones and of butenedioic acids have been described previously (15). The determinations of the furanones were carried out on underivatized extracts. The selected-ion monitoring (SIM) mode GC/MS analyses were carried out on a Dani 3800 capillary gas chromatograph interfaced to a VG 7070E mass spectrometer. The ionization mode was electron impact, and the resolving power of the MS was 1000. The GUMS system
red-CMCF
red-mCMF
reddCMF
I,,
1 _ ~ ,132 ,,,_
CMCF
dCMF
mCMF
red-MCF
i, , [;, MCF
r
OX-CMCF
ox-dCMF
'T'
red-MCA
MCA
I
"j ox-mCMF
OX-MCF
OX-MCA
,
I
mh
m/z
d Z
dz
Figure 2. Partial mass spectra of furanones, methylated hydroxyfuranones, and methylated butenedioic acids.
was equipped with a HP-1(25 m X 0.20 mm i.d., film thickness 0.33 pm) fused silica capillary column. A DB-5 (60 m X 0.25 mm id., film thickness 0.25 pm) capillary column was used for the determination of ox-MX, oxEMX, ox-CMCF, and ox-dCMF. The introduction of the samples to the GC was carried out in split mode with a split ratio of 1:lO. The column oven temperature was programmed from 100 to 180 "C at 6 "C/min. Analyses of the butenedioic acids ox-CMF, ox-MCF, and ox-MCA were carried out using the Hewlett-Packard GC/MSD system equipped with the HP-1 fused silica column. The samples were injected splitless with the split valve closed for 1min. The column oven temperature was programmed from 40 "C for 2 min to 100 "C at 20 "C/min and then at 6 "C/min to 170 "C. The ion peaks monitored for SIM qualitative and quantitative determination of compounds are listed in Table 111. The response factor for the most abundant ion of each analyte vs the ion of the internal standard MBA, red-MBA, or ox-MBA was calculated from the analyses of standard mixtures (single-point calibration). The identification of the analytes in the extracts was based on positive matching of retention times and relative ratios of ion peak areas.
Mutagenicity Tests. The preliminary screening of the mutagenic potency of the synthesized compounds was performed by use of the Salmonella typhimurium tester strain TA 100without metabolic activation (24). Just prior to the assay, the compounds were dissolved in dimethyl sulfoxide. Five dose levels were used with two plates per dose. The mutagenicity of the compounds, expressed as revertant numbers per nanomole, was calculated by leastsquares regression analysis of the linear part of the doseresponse curve. The number of revertants induced by the positive control, sodium azide, was 500-600 for 1 pg and 1400-1600 for 5 pg, and the number of spontaneous revertants was 90-120.
Results S t r u c t u r a l Assignments of Synthesized Compounds. The spectral characteristics of the compounds synthesized according to the methods described by LaLonde et al. (22)and Ishiguro et al. (23)were in all essential features identical with the previously published data. In the lH NMRspectra of the furanones, the resonance signals of H-5 protons appeared at 4.80-4.90 ppm (Table I). The protons in the methyl group of red-MCF gave a signal at 2.05 ppm. In the 13CNMR spectra of the furanones, the Envlron. Sci. Technol., Vol. 27, No. 9, 1993
1813
Table 111. Ion Peaks Used for Compound Qualitative and Quantitative Determinations compound furanone red-MX red-CMCF
ret timea (min)
fragment ion
13.80
M-C1
13.42
M - CHO M+
red-dCMF
13.07
M - CHO
red-mCMF
11.77
M+ M - CHO
red-MCF
10.56
M+ M+
red-MCA
9.39
M+
red-MBA hydroxyfuranone MX
M-Br
13.00
M - OCH3
15.44
C
CMCF
M - OCH3
13.52
C
M-CO dCMF
13.18
M - OCH3
mCMF
12.03
M - CHzCl M - OCH3
MCF
11.02
M - OCH3 M-CO
10.41
M - OCH3
MBA butenedioic acid OX-MX
14.07
M - OCH3
17.00
M - CH30H
OX-EMX
17.00
M - C1
OX-CMCF
14.96
M - OCH3 M - CH30H
MCA
M - OCH3 ox-dCMF
14.97
M - CH30H M - OCH3
ox-mCMFMsD
12.13
OX-MCFMS~
11.66
M - COzCH3 M-C1 M - OCH3 M - CH30H M - OCH3
ox-MCAMSD OX-MBA
12.47 16.15
M - OCH3 M+ M79~8l-OC&
relative peak area ratiosb FA HW DW1
m/z
std
164.95 166.95 136.96 165.96 167.96 136.96 138.95 165.96 103.00 104.99 132.00 132.00 134.00 151.94 153.94 160.92
1.00 0.62 1.00 0.23 0.14 1.00 0.53 0.22 1.00 0.35 0.02 1.00 0.34 1.00 0.65
1.00 0.58 1.00 0.33 0.17 1.00 0.36 0.19 nd nd nd nd nd 1.00 0.62
1.00 0.62 1.00 0.45bg 0.23 1.00 0.26 0.13 nd nd nd nd nd 1.00 0.61
1.00 0.62 1.00 0.27 0.15 1.00 0.47 0.17 nd nd nd nd nd 1.00 0.67
1.00 0.33 na na na na na na nd nd nd nd nd nd nd
1.00 0.59 1.00 0.21 0.13 1.00 0.55 bg nd nd nd nd nd 1.00 0.80bg
198.91 200.91 202.91 164.95 166.95 167.98 169.97 164.95 166.95 168.95 113.02 130.99 132.99 130.99 134.02 136.01 150.94 152.93 240.83
0.59 1.00 0.58 0.76 0.91 1.00 0.62 1.00 0.75 0.20 1.00 0.81 0.41 0.61 1.00 0.31 1.00 1.08
0.35 1.00 0.65 0.68 0.66 1.00 0.95 1.00 0.77 bg nd nd nd 0.91bg 1.00 0.35 1.00 0.92
0.48 1.00 0.75 0.80 0.82 1.00 0.70 1.00 0.72 0.16 nd nd nd 0.68 1.00 0.32 1.00 1.10
0.54 1.00 0.66 1.03bg 1.31bg 1.00 0.55 1.00 0.66 0.44bg nd nd nd 0.52 1.00 0.28 1.00 1.06
0.50 1.00 0.60 1.44bg 1.78bg 1.00 0.66 nd nd nd na na na 0.38 1.00 0.20 1.00 0.95
0.57 1.00 0.59 0.77 0.83 1.00 0.35 nd nd nd na na na nd nd nd 1.00 1.12
227.92 229.91 224.97 226.97 228.92 193.96 195.95 194.96 196.96 193.95 195.95 194.95 196.95 133.00 157.00 161.00 160.00 162.00 161.00 163.00 180.95 182.95 211.95 270.85
1.02 1.00 1.00 0.66 0.50 0.92 0.73 1.00 0.62 1.oo 0.68 0.91 0.52 0.24 1.00 0.19 0.46 0.18 1.00 0.25 1.00 0.60 0.14
0.98 1.00 1.00 0.69 0.53 0.90 0.70 1.00 0.62 1.00 0.82 0.77 0.56 0.29 1.00 0.22 0.45 0.16 1.00 0.24 1.00 0.63 0.18
1.15 1.00 1.00 0.66 0.50 0.86 0.66 1.00 0.63 1.00 0.71 0.64 0.55 0.38 1.00 0.32 0.46 0.17 1.00 0.24 1.00 0.64 0.18
1.10 1.00 1.00 0.67 0.50 0.91 0.76 1.00 0.64 1.00 0.98 bg 0.66 0.29 1.00 0.27 0.48 0.22 1.00 0.29 1.00 0.74 0.23
0.88 1.00 1.00 0.76 0.56 bg bg bg bg 1.00 0.57 0.82 0.57 bg 1.00 0.45 0.55 0.18 1.00 0.34 1.00 0.73 0.27
0.95 1.00 1.00 0.65 0.52 0.78 0.67 1.00 0.82 1.00 0.56 1.11 0.59 bg 1.oo 0.35 0.40 0.23 1.00 0.33
CBL
DW2
1.00
0.53 0.15
a Column, HP-1,25 m X 0.20 mm i.d., film thickness 0.33 pm; carrier gas, He a t 0.40 mL/min; oven temperature program, 40 "C for 2 min, then at 20 "C/min to 100 "C, and finally at 6 "C/min to 170 "C; splitless injection, split valve closed for 1min; retention time of air peak, 2.00 min. b bg = interference from background; nd = not detected; na = not analyzed; MSD = analyzed by the mass selective detector. The peak area is affected by the presence of a fragment ion formed by cleavage of M - (H + CO).
signals a t 165.6-168.6 ppm were assigned to the carbonyl resonances (Table 11). The resonance signals at 114.7121.2 and 143.6-156.1 ppm were assigned to the olefinic carbons C-3 and C-4, respectively (22). The signals at 70.6-74.2 ppm were considered to be due to the C-5 1814
Envlron. Sci. Technol., Vol. 27, No. 9, 1993
resonance, and the methyl carbon in red-MCF appeared at 12.6 ppm. The protons in the methyl and chloromethyl group of ox-MCF, ox-mCMF, and ox-CMCF gave resonance a t 2.15, 3.56, and 4.51 ppm, respectively (Table I). The smaller
shift of the CH2C1 protons in ox-mCMF compared to oxCMCF reflects the electron-withdrawing effect of the chlorine at C-3. The olefinic proton in ox-mCMF appeared at 7.57 ppm. The proton in the carboxyl group of OXCMF, ox-MCA, and ox-MBA gave signals at 9.3,8.3, and 7.9 ppm, respectively. Because of trace amounts of water in the NMR samples of ox-CMCF and ox-MCF, the definitive resonance fields of the carboxylic protons could not be determined. In the l3C NMR spectra of the butenedioic acids, the signals at 162.1-173.2 ppm were assigned to the carboxylic carbons while the signals at 123.3-136.7 ppm were assigned to the olefinic carbons (Table 11). In the mass spectra of the furanones, the molecular ion was clearly observable (Figure 2). The most abundant fragment ion appeared 29 mass units lower than M+ and is consistent with cleavage of CHO. The mass spectra of the methylated hydroxyfuranones showed a fragment ion due to loss of H' from the molecular ion. These compounds formed a strong mass peak 31 mass units lower than the molecular ion. The mass peak was attributed to cleavage of OCH3 from M+. In addition, CMCF and MCF formed abundant ions with masses 29 and 28 units lower than M+. These fragments were consistent with the loss of CO from [M - 11*+ and M+ ions, respectively. The mass spectra of the methyl derivatives of the butenedioic acids showed an ion peak corresponding to the molecular ion, with the exception of the mass spectrum of ox-mCMF where M+ could not be observed. Cleavage of OCH3 produced an abundant fragment ion from these compounds. The compounds ox-CMCF, ox-dCMF, and ox-MCF produced an additional ion by cleavage of HOCH3 from M+. Quantitative Analyses. The ions used for SIM mode GC/MS analyses were selected from the mass spectra of the furanones, methylated hydroxyfuranones, and methylated butenedioic acids (Figure 2). The hydroxyfuranones MX, CMCF, MCF, and MCA were detected in all extracts with the exception of DW2 where MCF could not be observed (Table I11 and Figure 3). In addition, the compound dCMF could be detected in the extracts of CBL, FA, and HW, while the compound mCMF could not be observed in any extract. The dominating hydroxyfuranone in CBL was MCA which was found at a concentration of 67 pg/L. In this sample, MX and CMCF were observed in concentrations of about 10 pg/L, and MCF was found in concentrations of about 1 pg/L. Also in the FA extract, MCA was found to be the major hydroxyfuranone (2.5 pg/L) followed by MX (1.3 pg/L), CMCF (0.6 pg/L), and MCF (0.2 pg/L). In the HW and the DW1 extracts, the hydroxyfuranones were found in a rather narrow concentration range of 0.1-0.35 pg/L and of 20-60 ng/L, respectively. In the DW2 extract, MX was the major hydroxyfuranone (50 ng/L), while CMCF and MCA were found in 5 times lower concentrations. The furanones red-MX, red-CMCF, red-dCMF, and redMCA were detected in all extracts with the exception of DW1 where red-MCA could not be observed and analysis of red-dCMF was not performed. No signs were found for the presence of red-MCF and red-mCMF in any extract. The furanones were observed in a rather narrow concentration range in the CBL, FA, and HW extracts. In CBL, the compounds were detected in amounts of a few micrograms per liter, in FA the compounds were found in approximately 10 times lower amounts, and in HW the concentrations were lowered by an other factor of 10. In
the drinking water samples, the compounds were found in concentrations around 10 ng/L with the exception of red-dCMF, which was found at a concentration of 80 ng/L (DW2). In all extracts, the compound red-MCA was found in the lowest amounts of the furanones. The butenedioic acids studied were present in every extract. However,the ion peaks used for the determination of ox-CMCF were obscured by interfering peaks in the ion chromatograms of DW1, and the presence of the compound is questionable in this extract. In CBL and in FA, the compound ox-MCA was clearly the most abundant diacid followed by ox-EMX and ox-MCF. These two compounds were found in approximately equal amounts. The other butenedioic acids were found in CBL in a concentration range 2.5-23 pg/L and in FA in a quite narrow concentration range of 0.33-0.54 pg/L. In the HW sample, oxEMX was the major diacid followed by ox-MCF, while the concentration of the other acids ranged from 0.1 to 0.3 pg/L. In the drinking water samples, ox-MCF was the predominant diacid. In these extracts, the other butenedioic acids were present at concentrations of 10-50 ng/L. The concentrations of the hydroxyfuranone dCMF and the butenedioic acid ox-dCMF could not be accurately determined due to the lack of pure standards. Anyhow, the concentrations of these compounds in the extracts could not be significantly higher than the concentration of MX or ox-MX since the ion peak area of the major fragment ion was lower than the peak area of the MX or ox-MX major fragment ions. Mutagenic Potency of the Compounds. The studied compounds were found to generate mutagenicity in Ames tester strain TAlOO (Table IV). The hydroxyfuranones MX and CMCF were almost 100 times more potent mutagens than their furanone analogues, while a factor of activity difference of roughly 20 was observed between the other hydroxyfuranones and their furanone analogues. The butenedioic acids ox-CMCF, ox-MCF, and ox-MCA were 2-10 times less active than their furanone analogues. On the other hand, ox-mCMF generated more than 10 times higher activity than red-mCMF. The TAlOO mutagenicity recorded for CBL, FA, HW, DW1, and DW2 was 1000,50,20,3,and 3 revertants/mL, respectively. On the basis of MX concentration and MXspecific mutagenicity (5600 revertants/nmol), the compound was calculated to account for 20, 67, 44, 43, and 43% of the total activity in CBL, FA, HW, DW1, and DW2, respectively. A similar calculation of the activity contribution of CMCF showed the compound to be responsible for 5 % CBL, 6 % FA, and 3 9% HW, DW1, and DW2 extract mutagenicities. The activity contribution of the other compounds was negligible.
Discussion In the chlorinated hydroxyfuranone MX, a CHClz group and a C1 atom are bound to the furanone ring carbons at the 4-position and the 3-position, respectively. However, structurally related compounds with a CHgCl or a CH3 group instead of the CHC12 group and with or without the C1 atom at the 3-position could possibly also be formed in reactions of chlorine with lignin and humic material. If this is the case, one could also expect the formation of the corresponding furanones and dicarboxylic acids (by analogy to the formation of red-MX and ox-MX (15)). This work shows that many of these compounds are in fact formed in the chlorination processes. Environ. Sci. Technol., Vol. 27, No. 9, 1993
1815
FURANONES
HYDROXYFURANONES
I/
15 I
BUTENEDIOIC ACIDS
20
801
15 10
5
0
L'II 20
81
M 40
15
$
I 210
5 ;:
$10 10
10
0
Flguro 9. Concentrations of chlorinated furanones. hydroxyfuranones,a M butenediolc ackJs in pulp bleaching liquws (CBL). in chlorinated aqueous solutions 01 luivlc acids (FA). In chlorine-mated natural humic water (HW). and In two samples 01 drinking water (DWI and DW2): na = not analyzed; nd = not detected: bg = datermination of compwnd not possible due to Interference from background
Table 1V. TAlOO Mutagenicity of the Studied Compounds.
furanone red-MX red-CMCF red-MCF red-dCMF red-mCMF red-MCA
mutagenicity data (revertantslnmol) this work literature
hydroxyfuranone
mutagenicity data (revertantalnmol) this work literature
8016
177'8
Mx
moo30
11 0.46
4.918
CMCF MCF dCMF mCMF MCA
1000 11 not tested0
0.5
0.09 0.26
notreported not reported 0.318 0.17l8
3.8 3.6
6,30019,13,000" 57918,lZWB 1.52, 10' not reported 3.9'8 3.3%. €0"
butenedioic acid ox-MX ox.CMCF ox-MCF ox-dCMF ox-mCMF ox-MCA
mutagenicity data (revertantslnmol) this work literature nonmutb 1.6
0.28 not testedc 1.6 0.12
not reported not reported not reported not reported not reDorted not reported
The number in superscript corresponds to the reference number. Nonmutwenie at doses UP to 6000 ng. Commund not Dure.
Most of these compounds have not previously been detected in bleaching liquors or in water treated with chlorine. In addition to MX, only the compounds oxMCA and MCA have been determined, and ox-MCF has been tentatively identified inCBL (25,26). Tbecompound 1816 Envirm. Sci. Techml., Voi. 27. No. 9. 1993
ox-MCA has previously been identifiedinchlorine-treated water containing humic substances, and Norwood et al. (27) and de Leer et al. (28)reported the compound to be a major fulvic acid chlorination product. Also,a hydroxyfuranone with a CHClz group a t C-5 (3,4-dichloro-5-
Scheme I
cf
‘Cl MX, open form
(dichloromethyl)-5-hydroxy-2-furanone) has been reposted to be present in abundant amounts in CBL and in extracts of chlorinated aquatic humic acids (29). In a previous study, Kronberg et al. (30)identified EMX, the geometric isomer of MX, in chlorinated water. In this work, we have not carried out analysis of the E-isomers of the hydroxyfuranones. It is likely that also the E-isomers of the studied hydroxyfuranones and butenedioic acids are present in the extracts. The naturally occurring lignin, humic acid, and fulvic acid macromolecules consist of numerous subunits, including various phenols. It is likely that the phenolic subunits are responsible for the production of the hydroxyfuranones, furanones, and butenedioic acids. Recently, Peters (31)found MX and red-MX to be formed from 3,5-dihydroxybenzaldehydeand 3,5-dihydroxybenzyl alcohol, respectively, and suggested a reaction mechanism for the formation of the furanones (SchemeI, adopted from ref 31). He further envisaged a reaction pathway for the formation of ox-EMX from 3,5-dihydroxybenzoic acid. Previously, 3,5-dihydroxybenzoic acid has been shown to be a major phenolic moiety of aquatic fulvic acids (32).A similar mechanism can be envisaged for the formation of the compounds CMCF, red-CMCF, and ox-CMCF from the same 3,5-dihydroxyphenols. In this case, the carbon at para-position to one of the hydroxyl groups is converted to a monochloromethyi group instead of a dichloromethyl group. By analogy, MCF could be obtained from 3,5dihydroxytoluene (orcinol),a compound known to produce chloroform upon chlorination (32). In this reaction, one of the carbonsin ortho-position to the methyl group should be transformed to an aldehyde group or a hydrate, possibly via a dichloromethyl group. This aldehyde group makes the ring formation possible and is in part represented in the furanone ring by the C-5 hydroxyl group. Boyce et al. (33)have reported ox-MCF to be formed upon chlorination of orcinol. This reaction requires that the aldehyde group is oxidized to a carboxylic group. Resorcinol would form MCA and ox-MCA in reactions by a mechanism analogous to the one proposed for MCF and ox-MCF formation from oricinol. de Leer et al. (28) found that ox-MCA is also formed upon chlorination of 3,5-dihydroxybenzoic acid. It has been shown that phenols without the metaarrangement of hydroxyl groups also form hydroxyfuranones upon chlorination (34, 35). Thus, additional amounts of the compounds may be produced by other mechanisms. A comparison of molar amounts of compounds formed per mole of organic carbon in the effluent and in the water solution of fulvic acids shows that lignin is less prone to produce the compounds in question than the fulvic acids are. Only the compounds MCA and ox-MCA were formed in higher amounts of lignin carbon than fulvic acid carbon. However, another explanation to this finding might be a degradation of the compounds due to the heavy chlorination during bleaching. Previous work in our laboratory
has shown that MX is gradually lost when subjected to a water solution of chlorine (36). The humic material in natural humic water consists mainly of fulvic acids. Therefore, the higher concentration of MX, CMCF, and MCA in the FA extract than in the HW extract may be explained by the difference in the chlorination pH and the higher stability of the compounds at acid pH (37, 38). The lowest quantities of the compounds were observed in the samples of drinking water, and this is in accordance with the low amount of humic carbon subjected to chlorination in the drinking water treatment plants. When counted on the amount of carbon subjected to chlorination, it seems that the organic material which most readily forms these compounds is removed during alum flocculation in the water treatment plants. The amount of mutagenicity generated by the compounds is in accordance with previous reports (Table IV). The compound CMCF should be considered a strong mutagen although its activity is five times lower than the activity of MX. Since CMCF was found in concentrations lower than those of MX, the compound accounted for 6 5% or less of the observed activity in the extracts. This is comparable to the activity contribution of chlorinated acetones (4). The other compounds did not contribute to any significant extent to the mutagenicity of the extracts. The difference in mutagenicity observed for hydroxyfuranones and furanones stresses the importance of the presence of the C-5 hydroxyl group in the ring form or the aldehyde group in the open-chain form. It has been discussed whether it is the ring form or the open form of MX which is responsible for the mutagenicity of the compound. Lalonde et al. (39)compared the mutagenicity of MX, red-MX, and 2-(dichloromcthyl)-3,3-dichloropropenal (TCB) and came to the conclusion that the ring form should be the major mutagen. The higher activity of MX compared to red-MX was explained by the presence of the electronegative C-5 hydroxyl group. However, this hydroxyl group is responsible for the ring opening of MX and is transformed to an aldehyde group during ring opening. It is common knowledge that aldehyde groups are easily attacked by nucleophiles. In the nucleic acid bases, cytosine, adenine, and guanine, free amino groups are present and have been shown to react with aldehyde groups of mutagens such as 2-bromopropenal(2-bromoacrolein) and chloroacetaldehyde (40,41). If MX is attacked by amino groups of nucleic acid bases, the initial reaction product would be the same carbinolamine [-NH-C(OH)-I no matter whether the attack takes place at the aldehyde group of the open form or at the carbon at the 5-position in the ring form of MX. Thus, identical DNA adducts, which could possibly mediate the mutagenicity of MX, would be formed from the open and ring form of the compound. The answer to the question of the form of MX, which is the major mutagen, might be enlightened Environ. Sci. Technol., Vol. 27, No. 9, 1993
1817
when the products of MX reaction with nucleic acid bases are identified. The mutagenicity of the furanones and of the butenedioic acids is probably mediated by an interaction with nucleic acid bases different from that of the hydroxyfuranones. Conclusions
This study shows that in addition to MX several other structurally related hydroxyfuranones are produced upon chlorine bleaching of pulp and chlorine treatment of water containing humic substances. Although the compounds were found to generate mutagenicity in Ames tester strain TAlOO, their mutagenicity and their concentration in the extracts were too low for rendering the compounds significant mutagens. The mutagenicity contribution of the most active compound, CMCF, was at most 6 % ,which is approximately equal to the contribution of chlorinated acetones. The hydroxyfuranones were found to be 20100 times stronger mutagens than the corresponding furanones and butenedioic acids. This finding shows that the presence of the C-5 hydroxyl group is of critical importance for the mutagenicity of chlorinated hydroxyfuranones. Acknowledgments
This work was supported by a research grant from the Maj and Tor Nessling Foundation. We thank Dr. Lema Tikkanen at the Technical Research Center of Finland for conducting the Ames assays and Professors Osmo Hormi and Bjarne Holmbom for helpful discussions. We also would like to thank Mr. Markku Reunanen for performing the GC/MS analyses and Mrs. Paivi Pennanen for the NMR analyses. Literature Cited Stevens, A. A,; Moore, L. A.; Slocum, C. 9.; Smith, B. L.; Seeger, D. R.; Ireland, J. 6. In Water Chlorination: Chemistry, Environmental Impact and Health Effects; Jolley, R. L., Condie, L. W., Johnson, J. D., Katz, S., Minear, R. A., Mattice, J. S., Jacobs, V. A., Eds.; Lewis: Chelsea, MI, 1990; Vol. 6, p 579. Peters, R. J. B.; de Leer, E. W. B.; de Galan, L. Water Res. 1990, 24, 797.
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Received for review September 17, 1992. Revised manuscript received February 10, 1993. Accepted May 11, 1993.
