Environ. Sci. Technol. 1992,26, 1998-2004
Ramana, A. M.S. Thesis, A new class of sorbents for selective removals of arsenic(V) and selenium(IV) oxy-anions. Civil Engineering Dept., Lehigh University, Bethlehem, PA
18015,1990. Sengupta, A. K.;Lim, L. AZChE J . 1988,34,2019-2029. Price list K,Table 7,Bio-Rad Laboratories, Richmond, CA,
1985. Grinstead, R. R.Hydrometallurgy 1984,12,387-400. Helfferich, F. G. Ion Exchange; Xerox University Microfilms: Ann Arbor, MI, 1961;Chapter 6. Fritz, W.Ph.D. Dissertation, University of Karlsruhe, Germany, 1978.
(10) Hoell, W. React. Polym. 1984,2,103. (11) Helfferich, F.J . Phys. Chem. 1965,69,1178. (12) Najm, I.; et al. J.-Am. Water Works Assoc. 1990,82,65. (13) Hand, D.; Crittenden, J. C.; Thacker, W. E. J . Environ. Eng. Diu., Am. Chem. SOC.Civ. Eng. 1983,109,82-101. (14) Loureiro, J. M.; Costa, C. A.; Rodrigues, A. E. Chem. Eng. Sci. 1988,43,1115-1124.
Received for review February 18, 1992. Revised manuscript received June 9, 1992. Accepted June 24, 1992. This study received partial financial support from the Environmental Protection Agency through Grant R-817438-01-0.
Identification of Compounds in Mutagenic Extracts of Aqueous Monochloraminated Fulvic Acid Rohinl Kanniganti, J. Donald Johnson, Loulse M. Ball, and M. Judith Charles"
Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7400
rn Although monochloramine has been considered as an alternative disinfectant to chlorine, little is known about the identity of the byproducts from its reaction with natural organics. In this study, byproducts in mutagenic extracts of monochloraminated aqueous fulvic acid were identified by high-resolution gas chromatography/mass spectrometry (HRGC/MS). These analyses resulted in the identification and quantification of the potent bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone (MX), and the related compounds, (E)-2chloro-3-(dichloromethyl)-4-oxobutenoicacid (EMX), (E)-2-chloro-3-(dichloromethyl)butenedioicacid (oxE M ) , and 2,3-dichIoro-4-oxobutenoic acid (mucochloric acid). The compounds MX, EMX, and ox-EMX accounted for 9%, 26%, and 2%, respectively, of the mutagenic activity of the monochloramination extracts. Several short-chain (C2-C9)aliphatic chlorinated organic acids, alcohols, and aldehydes were also identified. Of these, the alkenoic acids may be of toxicological significance because of their structural similarity to the open oxobutenoic form of MX. Introduction The search for a suitable alternative disinfectant to chlorine has been active ever since Rook in 1974 (1) demonstrated that chlorination of water that contains humic substances leads to the formation of trihalomethanes such as chloroform. Initial concern about chlorination was heightened by the discovery that large quantities of nonvolatile chlorinated compounds are also formed (2),some of which are mutagenic in the Ames assay. Activity in the Ames assay is cause for concern because the assay has been shown to be a predictor of carcinogenicity for many classes of compounds ( 3 , 4 ) . The mutagenic nonvolatile byproducts of drinking water chlorination that have been identified thus far are chlorinated acetones, propenals, acids, and hydroxyfuranones. The most potent of these is 3-chloro-4-(dichloromethyl)-5-hydroxy-2 (5H)-furanone (MX), which has been shown to contribute 20-50% to the mutagenic activity of chlorinated drinking waters (5-8). Other mutagenic byproducts identified are structural analogs of MX, (E)-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid (EMX), 3-chloro-4-(dichloromethyl)-2(5H)-furanone (red-MX), 2-chloro-3-(dichloromethyl)butenoic acid (ox-MX), and (E)-2-chloro-3-(dichloromethy1)butenedioic acid (ox-EMX). They are weaker 1988 Environ. Scl. Technol., Vol. 26, No. 10, 1992
mutagens than MX, and together they contribute less than 10% to the mutagenicity of chlorinated drinking and humic waters (6-11). Monochloramine has gained importance as a possible alternative or secondary disinfectant in drinking water treatment in view of the human health hazards posed by chlorination byproducts. Although it has poorer bacteriocidal and virucidal capabilities (12),it has the advantage of producing much lower quantities (less than 3%) of trihalomethanes than chlorine (13). In addition, monochloramine produces only 9-49% as much total organic halide (TOX) as chlorine, and the TOX that is formed is more hydrophilic and larger in molecular size (14).Information regarding the potential human health hazards associated with the use of monochloramine is very limited. Although several researchers have identified mutagenicity in monochloraminated humic waters as well as drinking waters (5, 15-17), few have been able to identify byproducts leading to this mutagenicity. The only byproducts identified prior to this work were MX and EMX (5). A need thus exists to further investigate the effect of monochloramine on the formation of potentially harmful byproducts, especially that of MX and its analogs. The objective of this research was to identify the byproducts in mutagenic extracts of aqueous monochloraminated fulvic acid. Fulvic acid was chosen as a model precursor because it accounts for approximately 45% of the organic carbon in natural water. In addition, the products of aqueous chlorination of fulvic acid and chlorination byproducts found in drinking water have been found to be similar (18). Experimental Methods Monochloramination of Fulvic Acid. Monochloramine solutions were prepared by the method of Johnson and Overby (19). Ammonia and chlorine were allowed to react in 3:l molar ratio. Equal volumes of desired concentrations of NaOCl and NH&l in 0.05 M phosphate buffer, pH 8.0, were mixed with stirring over an ice bath. Chlorine and monochloramine concentrations were determined by the DPD ferrous titrimetric method (20). The fulvic acid used in this study had previously been isolated from Lake Drummond, VA, waters by the method of Thurman and Malcolm (21). It had a low ash content and a carbon content of 48.8% (22). Monochloramine (300 mg/L as Clz)and fulvic acid (254 mg/L as C) were allowed to react at a molar C1 to C ratio
0013-936X/92/0926-1998$03.00/0
0 1992 American Chemical Society
of 1:5 at room temperature, with stirring. The reaction mixture was maintained at pH 8.0 over the reaction period of 96 h to ensure that monochloramine was the only chloramine species in solution. The monochloramine residual a t the end of 96 h was 31 mg/L as Clz. No quenching agents were used prior to extraction or concentration due to the uncertainty of the effects of quenching agents on the extracted compounds. Fulvic acid (254 mg/L as C) was chlorinated with NaOCl (300 mg/L as Clz) at pH 7.0 for 48 h at a molar C1 to C ratio of 1:5. The chlorine residual at the end of 40 h was 40 mg/L as Clz. Again, no quenching agents were used. Extraction was by liquid/liquid extraction using diethyl ether. Products in the resulting extracts were then quantified so that a comparison of the amounts of specific byproducts of monochloramination and chlorination could be obtained at comparable doses (C1 to C molar ratio). Extraction of Byproducts. The entire sample was acidified to pH 2.0 with HC1 and immediately extracted with diethyl ether. Mutagenic compounds were isolated only at this low pH (23) as demonstrated by activity in the Ames assay (24). Prior to extraction, the reaction mixture (2-L volume) was divided into 250-mL aliquots to allow analyses of the sample for bacterial mutagenicity, total organic halide (TOX), and total organic carbon (TOC) (in duplicate) prior to mass spectrometric analysis. The results of the analyses were used to estimate the amount of chlorinated mutagenic product in the sample. Each of the aliquots was extracted successively using liquid/liquid extraction, with 40,20, and 20 mL of diethyl ether. The extracts were stored in ether at -20 "C. Internal Standardization and Derivatization. The choice of derivatizing agent depended on its ability to methylate MX and its analogs. Diazomethane does not methylate MX, while sulfuric acid in methanol does not methylate the diacidic analogs of MX (25). Boron trifluoride in methanol was found to successfully derivatize MX as well as its oxobutenoic and diacidic analogs. Diethyl ether extracts were spiked with [13C6]benZOiC acid (50 pg/pL) as an internal standard prior to methylation. They were then taken to dryness under a stream of N2gas and redissolved in 250 pL of methanol containing 14% (v/v) boron trifluoride. The mixture was allowed to react for 12 h in a 70 "C mineral oil bath, then neutralized with 3 mL of 2% (v/v) NaHC03, extracted twice with 250 pL of hexane, and concentrated to 100 pL under a stream of N2 gas. Pure standards of selected analytes were derivatized similarly. High-Resolution Gas Chromatography/Mass Spectrometry. High-resolution gas chromatography/ mass spectrometry (HRGC/MS) analyses were performed on a Hewlett-Packard 5890 gas chromatograph interfaced to a VG70-250SEQ mass spectrometer (resolving power 1000 or 10000; 10% valley definition). All experiments were conducted with a DB-5, 30-m-length, 0.25-mm-i.d., 0.25-pm film fused-silica capillary column. Full-scan analyses were conducted by using a GC temperature program of 50 "C for 1min followed by a temperature rise of 2.5 "C/min to 150 "C and 5 "C/min to 300 "C. A temperature program of 50 "C for 1 min and a rise of 10 "C/min to 300 "C was used in analyses conducted by selected ion monitoring (SIM). Low-Resolution Mass Spectrometry. Mass spectra were obtained under electron ionization (EI) conditions at an electron energy of 70 eV, 200-PA trap current, 250 "C source temperature, and resolving power of 1000, scanning the magnet from 500 to 50 amu at 1 s/decade.
Table I. Ions Monitored by High-Resolution Mass Spectrometry
compound
ion type
exact mass
methyl deriv of MX M - OCH3 198.9121 methyl deriv of EMX methyl deriv of OX-EMX methyl deriv of mucochloric acid
200.9094 M - OCH3 244.9539 246.9510 224.9721 M - C1 226.9691 M - C1 146.9848 148.9818
re1 theor retentn isotope time, min ratio 1.67
100:98
1.93
10067
1.80
100:67
1.15
100:33
Selected-Ion-MonitoringMass Spectrometry. Selected-ion-monitoring analyses were performed under instrument conditions similar to those for full-scan analyses except that a resolving power of 10000 was used. The internal standard was [13C6]benzoicacid (50 pg/pL) and the recovery standard for quantification of MX was anthracene-dlo (25 pg/pL). The compound [13C6]benzoicacid was chosen as an internal standard because, to our knowledge, it is the only 13Cisotopically labeled aromatic compound with an acidic group (COOH) on the molecule that is commercially available. The ions monitored for each of the analytes, MX, EMX, ox-EMX, and 2,3-dichloro-4-hydroxy-2(5H)-furanone (mucochloricacid) are shown in Table I. Ions monitored for the internal and recovery standards were the M+ ion (m/z 142.076) of methyl [13C6]benzoateand the M+ ion (m/z 188.1410) of anthracene-dlo, respectively. A dwell time of 25 ms and a delay time of 10 ms were used in all analyses. Quantification of MX. The rationale for the method used to quantify MX has been discussed elsewhere (26). Briefly, a calibration curve was constructed from the analysis of standard solutions that contained 50,100,250, or 500 pg/pL MX and the internal and recovery standards. A response factor of the methyl derivative of MX to isotopically labeled benzoic acid was obtained from the calibration data so that the concentration of MX in the sample extracts could be determined. The response of isotopically labeled methyl benzoate (50 pg/pL) to the recovery standard anthracene-dlo (25 pg/pL) was also obtained to calculate the recovery of methyl benzoate. Anthracene& was added to the derivatized sample just prior to analysis. In the recovery calculation we make the assumption, based on previous data (26), that the derivatization yield of methyl benzoate is the same as the methyl derivative of MX. The measured concentration of MX was corrected for recovery (77 f 7%) as well as the purity of the standard material, which was determined to be 84% in our previous investigation (26). Large variations in response factors were observed when the MX standards and the internal standard were derivatized together. For this reason, standard solutions of MX were derivatized individually and then mixed with previously methylated internal standard. However, extracts of the monochloraminated and chlorinated fulvic acid were derivatized together with the internal standards. The difference in response, if any, between samples and standards has not been accounted for in the calculations. Semiquantification of the Byproducts. The internal standard used in the semiquantitative analyses performed in the full-scan mode was decafluorobiphenyl. This compound was added (10 ng/pL) to the derivatized sample just prior to analysis. Semiquantitative analyses were performed by comparing the response of the m f z 83 ion of Environ. Sci. Technol., Voi. 26, No. 10, 1992
1999
Table 11. Byproducts Identified in the Mutagenic Extract of Monochloraminated Fulvic Acid GC peak no. 2 5
8 18 20 23 26 27 29 31 32 42
compound structure
base peak, m/z
A. Methyl Derivatives of Saturated Acids CHClzCOOCH3b+ 59 CH3CC12COOCH3b 97 CC13COOCH3bsd 59 (COOCH3)CHClCCl(OCH3)2b 109 (COOCH~)CHC~CH~CHZ(OCH~) 59 (COOCH3)CClzCOOCH~ 59 CCl3CH(OCH3)CH2COOCHSb 163 CHClz(CH2)4COOCH3 167 (COOCH3)CHC1CHC1COOCH3b*e 59 CHC12(CH2)2CH(CH3)COOCH3b 115 CH2Cl(CH2)2CCl(CH3)COOCH3b 77 (COOCBH~)CH(CH~Cl)CH~CCl(OCH~)~ 123
three most abundant peaks m / z (% re1 intens) 83 (74), 85 (58), 63 (16) 59 (84), 99 (66), 61 (36) 117 (20), 119 (19), 82 (12) 59 (94), 111 (57), 133 (39) 77 (51), 79 (34), 69 (26) 141 (40), 113 (31), 143 (26) 161 (99), 125 (98), 103 (92) 169 (61), 139 (47), 59 (35) 61 (29), 135 (20), 74 (18) 55 (431, 59 (37), 167 (15) 59 (951,149 (82), 122 (80) 125 (34), 195 (29), 135 (29)
identifn statusn
cs cs
TP T T T T T
cs
T T T
B. Methyl Derivatives of Unsaturated Acids 11 21 24 28 30 33 35 38 39 40 41 43 44 45
123 147 59 147 151 181 59 59 79 79 59 59 75 74
125 (64), 60 (191, 95 (12) 149 (35), 59 (30), 53 (11) 143 (59), 145 (57), 73 (31) 59 (42), 149 (31), 53 (13) 160 (51), 59 (48), 163 (32) 183 (69), 59 (601, 87 (33) 129 (40), 141 (36), 169 (32) 167 (87), 191 (86), 169 (53) 191 (85), 59 (73), 167 (53) 167 (43), 159 (421, 191 (41) 167 (39), 191 (86), 169 (53) 79 (67), 201 (51), 203 (48) 59 (45), 187 (31), 215 (30) 101 (58), 59 (51), 147 (37)
22
C. Ethers and Acetals 87 93 93 107 123 CH3CH2CCl(OCH3)CH2CH(CH3)2 107 CH2(OCH3)CH20CH2CHCl-CCl=CH2 89
107 (68), 55 (47), 57 (39) 107 (42), 57 (34), 95 (32) 107 (64), 57 (40), 95 (38) 109 (31), 127 (21), 56 (14) 141 (74), 143 (47), 125 (33) 121 (53), 97 (34), 109 (27) 59 (84), 112 (57), 131 (57)
1 3 4 6 9 10 12 15 25 34 36 37
D. Methyl Derivatives of Alcohols and Aldehydes CH2C1CH(OCH3)2b 75 93 (67), 95 (27) CH2ClCH2CH(OCH&2b 75 107 (28), 57 (21), 109 (9) CHZClC(OCH3)2CH3 107 57 (82), 89 (81), 109 (44) CH3CHClCH&H(OCH3)2 75 121 (E),85 (E),114 (9) CHC12CH(OCH3)2 75 127 (23), 77 (18), 129 (14) CH,ClCH(CH3)CH(OCH3)2 89 121 (78), 123 (25), 53 (11) CH,CHClCH2CH(OCH3)2 75 59 (52), 55 (43), 73 (22) CHC12CH&H(OCH3)2 89 141 (sa), 143 (61), 63 (59) CHClzC(CH3)2CHClCH(OCH3)2 123 89 (73), 175 (49), 177 (46) CH2(OCH3)CH&Cl=CClCH(OCH3)2 75 169 (72), 171 (49), 123 (22) C(CHg)pClC(OCH3)2CH3 89 179 (27), 119 (26), 101 (18) CH2ClCH2CH(CH3)CH(OCH3)CH(OCH,), 161 119 (go), 101 (87), 123 (86)
7 13 14 16 17 19
CH2ClOC(CH3)zCH2(OCH3) CH2ClOCH2CHClCH2(0CH3) CH2C10CH(CH3)CHzOCHzCl CH&lCCl(OCHS)CH2CH3 CH2CICCI(OCH3)CH2(0CH3)
T T T T
T T T T T T T
cs
T T T
T T T T T T T T T T T T
T T T T T T
T, tentative identification based solely on interpretation of E1 spectrum; TP, tentative identification supported by comparison with published spectrum (29);CS, identification confirmed by comparison with authentic standard. *Compound or ita isomer has been previously observed as a byproduct of drinking or humic water chlorination (2, 9, 31-34, 36). CMethylderivative of dichloroacetic acid. dMethyl derivative of trichloroacetic acid. e Methyl derivative of dichlorosuccinic acid. 'Methyl derivative of ox-EMX. #Compounds structurally similar to the oxobutenoic form of MX.
dichloroacetic acid, the m/z 117 ion of trichloroacetic acid, the m/z 183 ion of dichlorosuccinic acid, and the m/z 225 ion to the response of the m/z 334 ion of decafluorobiphenyl. Semiquantification of EMX, ox-EMX, and mucochloric acid was performed by using selected-ion-monitoringdata and was based on the relative response factor obtained from the analysis of a single methylated standard of each analyte (250, 50, and 250 pg/pL, respectively) that contained 50 pg/pL [13C6]benzoicacid.
Results and Discussion Structural Assignments of the Byproducts. The total ion chromatogram resulting from the analysis of the methylated, pH 2.0, ether-extractable monochloramination 2000
Envlron. Sci. Technol., Vol. 26, No. 10, 1992
extract is shown in Figure la. Forty-five compounds were identified by interpreting the electron-ionization mass spectra (Table 11). Structural assignment relied on recognition of spectral patterns of methylated compounds. Alcohols, acids, and aldehydes are derivatized to esters, ethers, and acetals. ROH ROCH, (27)
-
R(OCH3)2 (27) RCOOH RCOOCH, (27) R(CO0H)Z R(COOCHJ2 (27) RCOH RCH(OCH3)z (27,28) Identification of the compound is generally based on the +
+
-
-+
a
2
I I4
'f
'? I
44
IS b
A Retention Time Figure 1. Total ion chromatograms of the methylated extracts of (a) monochloramlnated fulvic acid and (b) corresponding blank containing fulvic acid. Numbered GC peaks refer to the byproducts in Table 11. IS,internal standard. The chromatograms are normalized to the largest peaks. Thus, the internal standard peak in (a) is not visible, although present.
assumption that the methoxy moiety (or moieties) arises from methylation of hydroxy, carboxylic, and aldehydic groups on the molecule (Table 11). If however, the structure of the proposed compound (prior to derivatization) does not appear to be stable in natural waters, then we assume that the methylated analog is the byproduct. Thus, the ethers and acetals presented in Table IIC are very likely to exist as the methylated forms in the underivatized extract. This is because the cy-chloro ethers and chloro acetals shown are more stable and thus are more likely to exist in nature than their corresponding alcohols and acid chlorides. It is not possible to differentiate between a derivatized geminal diol and an aldehyde, as both compounds would possess the group CH(0CH3)2after methylation. For this reason, methyl deriva-
tives of alcohols and aldehydes are grouped together in Table IID. Mass spectral interpretation was based on the observation of characteristic losses and fragment ions (methylated portions in italics above) in the mass spectra of the methylated compounds. Typically, losses of OCH3 from the molecular ion were observed in the vast majority of the mass spectra. Losses of 59 amu (COOCHJ from the molecular ion or high-intensity fragment ions at m / z 59 were indicative of acidic compounds. Similarly, a base peak or otherwise high intensity fragment at m/z 75 [CH(OCHJ,] is indicative of an aldehyde or a geminal diol. The assignments of four byproducts identified in the mutagenic extracts of monochloraminated fulvic acid (Figures 2-4) are discussed in detail below. One of these Envlron. Sci. Technoi., Voi. 26, No. 10, 1992 2001
50
75
100
125
150
175
200
225
250
0 50
75
100
125
da
150
175
200
---* 250
225
mil
Flgure 2. E1 mass spectrum of the methyl ester of 2,3,4-trichlorobutenoic acid (GC peak 24).
Flgure 4. E1 mass spectrum of methylated 2,3dichloro-3-butenyl 2-hydroxyethyl ether (GC peak 22).
CH(OCH& fragment. Such a fragment has previously been observed in the spectrum of methylated EMX and resulted from the addition of two OCH, groups on the aldehyde group during methylation. In this case however, the fragment may have also originated from the methylation of a geminal diol. Thus, the underivatized compound can be either 2,3-dichloro-2-pentene-l,1,5-triol or 2,3-dichloro-5-hydroxy-2-pentenal. We present the trans configuration of the molecule. It is impossible however, based on the data, to differentiate between the cis and trans configurations of this compound. In Figure 4, a weak molecular ion with a two chlorine isotopic cluster can be observed at m / z 198. The ion fragment at m/z 167 also contains two chlorine atoms and 59:' d S
Flgure 3. E1 mass spectrum of methylated 2,3dichloro-2-pentene1,1,5-triol or 2,3dichloro-5-hydroxy-2-pentenal (GC peak 34).
compounds, 2,3,4-trichlorobutenoic acid, has previously been identified as a byprodud of humic water chlorination (8). The rest are novel to monochloramination. The mass spectrum presented in Figure 2 does not exhibit a molecular ion. A base peak at m/z 59 is present in the mass spectrum and indicates that the parent compounds are methyl esters of carboxylic acids. In Figure 2, the molecular mass of 202 amu can be deduced from the ion fragments at m/z 143 and 167 by proposing that they
$ a I a ;
CMzC+~Crn3 14+---!+59
arise from simple a cleavage and subsequent loss of COOCH, and the allylic Cl, respectively, from the molecular ion. These losses are justified by the three and two chlorine isotopic clusters at m/z 143 and 167, respectively. Thus the compound was tentatively identified as the methyl ester of 2,3,4-trichlorobutenoic acid. The spectrum presented in Figure 3 was interpreted on the basis of the ions at m/z 75 and 179 and the twochlorine isotope pattern at m / z 169. As previously explained, the base peak at m/z 75 is characteristic of the
c-! H3CO-CH2-CH2@W(@333)2 4 7 5 169 2002
Environ. Sci. Technol., Vol. 26, No. 10, 1992
'acl
HSCO~2-CH&O-CHZ~-&CH2 L+ I67
can thus be deduced to arise from the loss of OCH, from the molecular ion. The only plausible structure that explains this ion as well as the ions at m/z 59 and 89 is 2,3-dichloro-3-butenyl 2-hydroxyethyl ether. Identification of Monochloramination Byproducts by E1 Full-Scan Mass Spectrometry. The majority of the compounds listed in Table I1 may be present in a different isomeric form. The identification of a few compounds was confirmed by comparing the mass spectra and the relative retention times of the compound in the monochloramination extract and a pure standard or by comparison with published spectra (29). The majority of the products identified in this experiment are aliphatic ethers and esters derived from the methylation of chlorinated mono- and diacids, alcohols, and aldehydes. More than half of these compounds are acids that comprise approximately equal numbers of saturated and unsaturated species. The number of diacids (four)is low in comparison with those produced by chlorination. This observation, along with that of the paucity of aromatics and abundance of aliphatics in this study, can be explained by the poor oxidant ability of monochloramine in comparison with chlorine. Although the byproducts of chlorination are mostly aliphatic, presumably resulting from ring cleavage and oxidation of ring side chains, several substituted aromatic species have also been found. Monochloramine has previously been shown to be less reactive toward phenolic species and is thus not likely to cause either ring rupture or direct substitution. Instead, monochloramination probably results in primarily aromatic side-chain oxidation and substitution products (30).
Table 111. Concentration of MX and Structurally Related Compounds Produced by Chlorination and Monochloramination
compound
concn," nglpL monochlorchlorination amination*
MX
118 f 12
60 f 3
EMX
14000 f 1100
OX-EMX
110000 f 7800
mucochloric acid dichloroacetic acid trichloroacetic acid dichlorosuccinic acid
880 f 310
3100 f 1,800 (26%) 5000 f 1 (2%) 500 f 600 80000 11000 239000
(9%)
Values shown are the mean of two replicates f standard deviation. *Values in parentheses are the percent contribution to mutagenicity. The contribution to mutagenicity was calculated on the basis of 5600, 320, and 7.8 net revertants/nmol TA100-S9 mutagenicity for MX, EMX, and ox-EMX, respectively (25, 36). The TAlOO-SQmutagenicity of the extract of monochloraminated fulvic acid was 17476 revertanta/L (23).
The byproducts presented are all chlorine-substituted compounds. Nonchlorinated compounds were observed (for example, benzoic acid) but not listed because they were also found in the fulvic acid blank (Figure lb). No nitrogen-containing compounds were identified. This latter observation was surprising as we expected the production of nitriles and amides from the reaction of the amino functional group on monochloramine with components of fulvic acid. It may be that these compounds were present below the detection limit of full-scan EI, or that the low molecular weight nitriles rapidly oxidized and converted to the corresponding acid and nitrogen gas during reaction (31). Fifteen of the 45 chemicals listed in Table 11, mostly chlorinated acids, have previously been identified as byproducts of aquatic humic and fulvic acid chlorination (2, 9,25, 31-35). Among these were di- and trichloroacetic acids, dichlorosuccinic acid, and ox-EMX. The identification of these compounds was confirmed by comparing the mass spectra of these compounds in the sample extract with the mass spectra of a pure standard. Di- and trichloroacetic acids and dichlorosuccinic acid were present at concentrations of 80, 11, and 239 pg/L, respectively (Table 111). Novel compounds that are reported here are ethers, alcohols, and alkenoic acids. The latter may be the most interesting because of their structural similarity to the open form of MX. The structures corresponding to GC peaks 44 and 45 may have resulted from the methylation of oxo acids (indicated by g in Table IIB) and may thus exhibit mutagenic behavior similar to that of EMX. However, their prohibitive chain length diminishes the possibility of isomerization to a ring form. It is difficult to determine the relative health hazards of the chemicals identified as data in the toxicological literature on only the chlorination products have received attention. Recent work has shown that ox-EMX is weakly mutagenic (7.8 net revertants/nmol) in the Ames assay (36);di- and trichloroacetic acids, although not mutagenic (37),have been shown to induce hepatic tumors in B6C3F1 mice (38). Several alkenoic acids that are structurally similar to open and oxidized forms of MX may be of toxicological significance. Identification and Quantification of MX, EMX, ox-EMX, and Mucochloric Acid by E1 SIM Mass
Spectrometry. Identification of MX, EMX, ox-EMX, and mucochloric acid was based on the comparison of relative retention times of the compounds in the standard solutions and the sample extract and comparison of the measured and theoretical ratio between ions in the chlorine isotope cluster (Table I). The concentrations of the compounds are shown in Table 111. Chlorination was observed to produce approximately twice as much MX (118 f 11 ng/L) as monochloramination (60f 3 ng/L). A previous study has shown that chlorination produces up to 4 times as much MX as chloramination and also reports higher MX levels for both oxidants than are reported here (5). In addition, MX was found to contribute 24% of the activity of the extract, nearly 3 times that (9%) reported in the present work. The remainder of the compounds quantified were detected in concentrations that were 8-63 times higher than the concentration of MX in monochloramination extracts and 10-653 times higher than MX in chlorination extracts. Monochloramination produced 3.1 f 1.8 pg/L EMX, a quantity that was a factor of 4 less than that produced by chlorination in this study. Backlund et al. (5) observed the same ratio of EMX production between chloramination and chlorination, but found lower concentrations of EMX than were found in this study. In addition, they found EMX to contribute only 2% to the total activity of the extract in contrast to the 26% found in this study. A pH-sensitive equilibrium has previously been shown to exist between MX and its open oxobutenoic form (8, 10). Thus, the use of a reaction pH that was 1unit higher than that used in the Backlund study (5) could have resulted in a shift in the equilibrium toward the production of the oxobutenoic acid tautomer, which may have subsequently caused an increase in the formation of EMX. The compounds ox-EMX and mucochloric acid were present at 5 pg/L and 500 f 600 ng/L, respectively, in the extracts of monochloraminated fulvic acid. Chlorination produced more than 20 times the amount of ox-EMX and about twice as much mucochloric acid as monochloramination. Mucochloric acid (39) and ox-EMX (35) have both been found to be weakly mutagenic (1.3 and 7.8 net revertants/nmol, respectively. Despite its occurrence in large concentration in the extracts, ox-EMX contributes only about 2% to the mutagenic activity of the monochloramination extract. The contribution of mucochloric acid is negligible. Conclusions Forty-six novel byproducts of monochloramination have been identified by using HRGC/MS analyses in the fullscan and SIM modes. Monochloramination produces smaller quantities of some known mutagens than does chlorination. These compounds, MX, EMX, and ox-EMX, together account for about 37% of the total mutagenic activity of the monochloramination extract. The novel alkenoic acids identified in the monochloramination extracts may be toxicologically significant owing to their structural similarity to the open forms of MX and may account for the mutagenicity not explained by MX and its structural analogs. Acknowledgments
We thank G. Dean Marbury, Gong Chen, and Richard Lelacheur for their help. Registry No. MX, 77439-76-0; EMX, 115340-67-5;ox-EMX, 126572-80-3; (Z)-HO~CC(Cl)=C(Cl)CHO,87-56-9; Cl&HC02CH3, 116-54-1; C13CC02CH3, 598-99-2; H3COCO(CHC1)&02CH,, Envlron. Sci. Technol., Vol. 26, No. 10, 1992 2003
62173-55-1; H~COCOC(CHC12)=C(Cl)CO&!H~, 142800-03-1; dichloroacetic acid, 79-43-6; trichloroacetic acid, 76-03-9; dichlorosuccinic acid, 42342-97-2; monochloroamine, 10599-90-3.
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Received for review January 6,1992. Revised manuscript received June 11, 1992. Accepted June 12, 1992. W e thank Dr. Paul Ringhand and the U.S. Environmental Protection Agency’s Health Effects Research Laboratory (CR 814900) for supporting this work.
