Chloramines V: Products and Implications of the Chlorination of Lysine

Environ. Sci. Technol. 1997, 31, 1680-1685

Chloramines V: Products and Implications of the Chlorination of Lysine in Municipal Wastewaters BARBARA CONYERS AND FRANK E. SCULLY, JR.* Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529-0126

The chlorination reactions of lysine (Lys) in water and wastewater were studied. 5-Chloraminopentanal (VIII) and 5-dichloraminopentanal (IX) are the main products at Cl2/ Lys molar ratios of 1 and 2 after 30-min contact time. Formation of N,N′-dichlorolysine (V) was suggested but not confirmed. 5-Dichloramino-N-chloropentanimine (XII) and 5-dichloraminopentanenitrile (XV) are the main producs at Cl2/Lys molar ratios of 4 and 5. At a Cl2/Lys molar ratio of 3, a compound believed to be 5-chloramino-N-chloropentanimine (XI) was observed, but its yield was small. Halflives of lysine chlorination products in model solutions were found to be as follows: 0.8 (V), 1.5 (XI), 23 (XII), >45 (VIII and IX), and 210 h (XV). The concentrations of lysine and its chlorination products after 30-min contact time were determined in a primary municipal wastewater. All have the potential to interfere with the proper measurement of disinfection levels in chlorinated wastewaters.

Introduction The bactericidal potential of a wastewater disinfected with aqueous chlorine is routinely determined by measuring the “combined residual chlorine” concentration. Because this parameter is usually a measure of the concentration of monochloramine, many treatment plant operators have come to rely on this measurement to produce a desired level of disinfection. Recently, we have reported disinfection interference in a wastewater in the apparent presence of sufficient combined residual chlorine (1). This interference was believed to be associated with the formation of poorly bactericidal organic N-chloramines (2-6). The problem is traced to the use of iodimetric methods (5-9) as a surrogate for disinfection effectiveness, because both organic chloramines and the far more effective bactericide monochloramine respond in the same manner to conventional methods for the measurement of disinfectant concentration. Because the formation and fate of specific organic N-chloramines has been poorly understood, we have been studying the reactions of organic amino acids with aqueous chlorine. The primary reactions are understood to be HOCl

HOCl

H2N-CHR-COO- 98 ClNH-CHR-COO- 98 [Cl2N-CHR-COO-] (1) We have demonstrated that both in model solutions and in wastewater the N,N-dichlorinated derivatives of the R-amino acids isoleucine, valine, phenylalanine, and methionine are unstable and rapidly decompose with loss of CO2 and chlorine * Corresponding author e-mail address: [email protected]. odu.edu.

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to form N-chloroaldimines (10-13). To understand the effect of reactive side chains on the chlorination reactions of amino acids, we have studied the reactions of lysine. Based on previous work, lysine might be expected to form the following types of chlorination products: an N-chloramino acid, an aldehyde, an N-chloroaldimine, and a nitrile (10-13). However, because lysine (I) contains an additional amino moiety that can become chlorinated and dichlorinated, the list of possible oxidation products is far more complex than those of the simple amino acids. More importantly, its aminopropyl side chain can react with aqueous chlorine to form chloramino derivatives that are likely to be long-lived in aqueous solution. In addition to R-N-chloro- (II) and R,R-N,N-dichlorinated lysine (III), there are 12 other possible products that might be expected to form (Table 1).

Experimental Methods Reagents. All chemicals were reagent grade or better. L-Lysine monohydrochloride (98%) and all other amino acids were obtained from Sigma Chemical Co. Hexamethylenetetramine (99+%) was obtained from Aldrich Chemical Co. Lancaster Synthesis Inc. was the source of 5-bromovaleronitrile. L-[4,5-3H]Lysine monohydrochloride in an aqueous solution (93 Ci/mmol, TRK.520, Batch 129) was acquired from Amersham Corporation. Preparation of all buffers, standard solutions, and chlorine demand free (CDF) water (14), the preparation and standardization of hypochlorite solutions, and the DPD/FAS analysis of residual chlorine concentration (15) were carried out as previously described. Instrumentation. GC/MS analyses were carried out on a Hewlett Packard 5880A GC interfaced with a Hewlett Packard 5970 Series quadrapole mass selective detector operated in the EI mode. Chloroform extracts were analyzed by GC (10:1 split injection) with an injector temperature of 200 °C and a column temperature program of 50 °C for 1 min followed by an increase of 10 °C/min to 250 °C. A DB-5 fused silica capillary column (25 mm i.d. × 30 m) with a 0.25 µm film thickness (J&W Scientific) was used. The scan acquisition rate was 1.71 scans/s from m/z 50 to m/z 300 with an electron multiplier voltage of 2000. High-resolution mass spectroscopic analysis of 5-aminopentanenitrile was completed by the Purdue University Mass Spectroscopy Center on a Kratos MS50 double-focusing mass spectrometer in the CI mode using isobutane with an ionizing energy of 70 eV and a source temperature of 250 °C. All other high-resolution GC/MS was carried out at the University of North Carolina at Chapel Hill Mass Spectrometry Center using a DB-1 column (30 m) and instrumentation described previously as GC/MS 2 (16). A Varian 400 Unity Plus NMR spectrometer was used to acquire structural information on chlorination products. The infrared spectrophotometer and the Waters HPLC system and solvents used in the separation of the chlorination products have been described elsewhere (10). Amino acid derivatives were detected using a Waters Associates Model 474 scanning fluorescence detector. Separations were carried out on a Whatman Partisil 5 ODS-3 analytical column with a dual-solvent system described previously (10) [A ) 90% water (containing 1% acetic acid)/ 10% acetonitrile, B ) 90% acetonitrile/10% water (containing 1% acetic acid)]. The solvent program (1 mL/min) consisted of a 5-min isocratic elution with 100% A followed by linear gradients, first to 60% A/40% B over 20 min, second to 50/50 A/B in the next 10 min, and lastly to 10% A/90% B over 5 min. Analysis of Chlorinated Model Lysine Solutions. A 1.43 mM solution of lysine was prepared in 25 mM NaH2PO4. The pH was adjusted to 7.0. Throughout the model studies, 15mL aliquots were chlorinated while stirring to chlorine-to-

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TABLE 1. Possible Products of the Chlorination of Lysine (I)

FIGURE 1. Total residual chlorine concentration and percent yields of products and unreacted lysine remaining 30 min after lysine model solutions (1.43 × 10-3 M in 0.025 M phosphate buffer, pH 7) were inoculated with [3H]lysine and chlorinated to increasing Cl2/Lys molar ratios. Polar products include all compounds that elute in the void volume with lysine. lysine molar ratios (Cl2/Lys) of 1, 2, 3, 4, and 5. The chlorinated solutions were incubated in the dark at ambient temperature for 30 min prior to analysis by HPLC. As the peaks eluted from the detector, the eluent was collected and analyzed for the presence of active chlorine by the DPD/FAS method as described elsewhere (11). The quantitation and distribution of the chlorination products were determined by mixing a 15-mL aliquot of the model solution with a solution of tritiated lysine in the same manner as described previously for a solution of tritiated valine (11). Each aliquot was chlorinated to one of the five Cl2/Lys molar ratios described above. Each was incubated for 30 min in the dark at room temperature and fractionated by HPLC; the fractions were assayed by liquid scintillation counting in a manner similar to what has been described previously (10). Radiochromatograms were generated as previously reported (10, 11) with an average recovery from the column of 87 ( 7% of the radioactivity applied. This was a measure of the mass balance of the reaction. GC/MS Analysis of Chlorinated Model Solutions. Lysine model solutions were chlorinated to Cl2/Lys molar ratios of 2, 3, and 4 before they were analyzed by HPLC. The solutions were extracted with 1 mL of CHCl3 and dried over anhydrous Na2SO4. A 2-µL injection of each extract was analyzed. The extract was then shaken with 1 mL of CDF water, and the aqueous layer was re-analyzed by HPLC. Synthesis of 5-Aminopentanenitrile (XIII). 5-Aminopentanenitrile was synthesized in 26% overall yield from 5-bromovaleronitrile by a method described elsewhere (1719). High-resolution GC/MS showed a single peak: m/z 99.0919 (99.0922 calculated). The IR (thin film): 3400 and 3325 (m, N-H), 2250 (s, CtN stretching), 1600 (m, N-H) cm-1; 1H NMR (CDCl ) (ppm) 2.62 (t, CH , J ) 6.8 Hz), 2.26 (t, CH , 3 2 2 J ) 7.2 Hz), 1.61 (m, CH2), 1.47 (m, CH2), 1.01 (s, NH2); 13CNMR (CDCl3) (ppm): 119.6 (CtN), 41.1 (CH2), 32.4 (CH2), 22.7 (CH2), and 16.9 (CH2). Synthesis of N,N-Dichloraminopentanenitrile (XV). Solutions of 5-aminopentanenitrile in 0.25 M NaH2PO4 (pH 7.0) were chlorinated with 2 equiv of aqueous chlorine and incubated in the dark for 30 min before they were extracted and analyzed by GC/MS and NMR. Identification of 5-Dichloramino-N-chloropentanimine (XII) and 5-Dichloraminopentanenitrile (XV) in Chlorinated Model Solutions. A 100-mL aliquot of the model solution was chlorinated with 4 equiv. After incubating in the dark for 30 min, the sample was analyzed by HPLC and extracted

with approximately 1.5 mL of CDCl3. After drying the extract over Na2SO4, 1H and 13C spectra were recorded. Both the proton and carbon NMR spectra revealed a mixture of compounds XII and XV. Characteristics of Wastewater. The primary wastewater used in this study was collected December 12, 1994, from a secondary treatment plant described as plant 2 in an earlier work (20). Handling and storage of the wastewater has also been described (20). The Hampton Roads Sanitation District Commission Laboratory determined the total Kjeldahl nitrogen (TKN) and ammonia concentrations by the automated phenate method (15). Amino Acid Analysis. Reagents and solvents for the reverse-phase HPLC analysis of amino acids in the wastewater using precolumn derivatization with o-phthalaldehyde are described elsewhere (21-23). The elution program was similar to that previously described (21-23) but was optimized to isolate lysine. Standard curves were prepared for lysine and other amino acids typically found in wastewater. Working standard concentrations ranged from 1.43 × 10-7 to 2.86 × 10-6 M. The correlation coefficient for each standard curve was greater than or equal to 0.991. Chlorination of Wastewater. The 30-min breakpoint curve was determined according to procedures outlined in earlier work (10-12). The quantitation of the lysine chlorination products was determined by inoculating 15.0-mL aliquots of the wastewater with a stock solution (0.7 µCi/mL) of tritiated lysine in the manner described previously for analysis of isoleucine chlorination products (10). Solutions were chlorinated, fractionated, and assayed as described previously (10).

Results Model Solutions. (A) Breakpoint Curve. Model solutions of lysine (1.43 mM) were chlorinated to various Cl2/Lys mole ratios and incubated in the dark for 30 min before the total residual chlorine concentration was measured. The results are plotted in Figure 1. Lysine exhibited a chloramine maximum with the addition of 3 equiv of aqueous chlorine. With 4 equiv an irreducible minimum was observed. The irreducible minimum after 30 min was a much larger percentage of the total chlorine maximum than with other amino acids studied (10-12). (B) Product Mixtures Formed at Cl2/Lys Molar Ratios of 1 and 2. The HPLC analysis of model solutions chlorinated

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with 1 and 2 equiv revealed the presence of unreacted lysine and two chlorinated products after 30 min. As each product eluted from the column, it was found to oxidize DPD in the presence of iodide, suggesting that the products contained oxidizing chlorine. Proof of the structure of the first compound was not possible because it could not be extracted from aqueous solution and analyzed by either GC/MS or NMR. However, it is believed to be N,N′-dichlorolysine (V) based on the chemistry of other amino acids and on the presence of VIII, an expected decomposition product of V. V decomposed fairly rapidly with a half-life of about 50 min. Decomposition of an R-N-chloramino acid to an aldehyde is observed in the decomposition of all other chlorinated amino acids studied (10-12). The second compound in the mixture has been identified as 5-chloraminopentanal (VIII) based on GC/MS analysis of extracts (see below). A third peak appeared in the chromatogram after 1 h and has been identified as 5-dichloraminopentanal (IX). When a lysine solution chlorinated for 30 min with 2 equiv is analyzed by GC/MS, two volatile products are detected. The mass spectrum of VIII showed major ions (relative intensity): m/z 137 (M + 2, 6), 135 (M+, 19), 118 (9), 99 (53), 83 (57), 70 (30), 66 (15), 64 (44), 55 (100), 41 (68). Exact mass measurements of the parent ion found m/z 135.0455 (calculated mass 135.0451). The one-chlorine isotope cluster at m/z 135 and 137 in the mass spectrum of the first peak in the chromatogram, its spectrum, and exact mass measurements of the parent ion are consistent with the structure of VIII. Cleavage of the single-bond β to the chloramino residue, a well-characterized fragmentation of amines, gives the [ClHNdCH2]+ ion with a characteristic cluster at m/z 64 + 66. Biemann has proposed that the molecular ion of lysine ethyl ester cyclizes to form an N-protonated 3,4,5,6-tetrahydropyridinium ion (m/z 84) with loss of ammonia and [COOC2H5]• (24). A similar cyclization of the enol form of the parent ion of VIII and loss of HOCl yields a 3,4,5,6tetrahydropyridinium radical cation (m/z 83, M - 52). A retro-Diels-Alder reaction results in loss of ethylene and formation of the m/z 55 fragment. The complete explanation of the mass spectrum of VIII is included in the Supporting Information (see paragraph at the end of the paper). The mass spectrum of IX contained the following major ions (relative intensity): m/z 173 (M + 4, 0.8), 171 (M + 2, 4.5), 169 (M+, 7), 152 (2), 154 (1), 135 (3), 133 (10), 117 (26), 98 (29), 89 (26), 82 (39), 70 (63), 64 (41), 62 (65), 58 (100). Exact mass measurements of the parent ion found m/z 169.0061 (calculated mass 169.0061). The two-chlorine isotopic cluster at m/z 169, 171, and 173 corresponded to the molecular weight of IX, and high-resolution mass spectral analysis was consistent with its formula. The low-resolution spectrum revealed fragmentation of IX similar to that of VIII. Loss of HCl produces the one-chlorine isotope cluster at m/z 133 + 135. Loss of the second chlorine atom or HCl produces a nitrenium ion (m/z 98) or an ionized nitrile (m/z 97). Cleavage of the single-bond β to the dichloramino residue would produce [Cl2N ) CH2]+ with m/z 98. However, this fragment would have a characteristic chlorine isotope cluster associated with it, and this is not observed. This observation suggests that the facile loss of HCl from this ion to form [ClsNtCH]+ (m/z 62 + 64), one of the more important fragments in the spectrum, suppresses its lifetime. The type of cyclization and fragmentation discussed above for VIII is also observed with IX. Loss of 17 (-OH) produces an ion with m/z 152 + 154 + 156, and subsequent loss of Cl produces a one-chlorine isotope cluster at m/z 117 + 119. A retro-Diels-Alder reaction of this fragment produces a one-chlorine isotope cluster at m/z 89 + 91. The complete explanation of the mass spectrum of IX is also provided in the Supporting Information (see paragraph at the end of the paper). The CHCl3 extract analyzed by GC/MS was shaken with 1 mL of water, and the aqueous layer was re-injected into the

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HPLC. Products VIII and IX were observed in the HPLC chromatogram. This strongly suggested that the compounds analyzed by GC/MS were not altered by extraction into an organic solvent and, in fact, were those observed in the HPLC chromatogram. This was not true for XII discussed below. (C) Product Mixtures Formed at Cl2/Lys Mole Ratios of 4 and 5. HPLC analyses of model solutions of lysine reacted with 4 and 5 equiv of aqueous chlorine suggested the formation of a mixture of two nonpolar products: 5-dichloramino-N-chloropentanimine (XII) and 5-dichloraminopentanenitrile (XV). The eluent of each peak oxidized DPD in the presence of iodide. The proton NMR spectrum of a CDCl3 extract of a chlorinated aqueous lysine solution exhibited the following (ppm): 8.2 (t, 1 H, HCdNCl), 3.7 (m, 4 H, CH2), 2.4 (m, 4 H, CH2), 1.93 (m, 2 H, CH2 of nitrile), 1.80 (m, 4 H, CH2 of both nitrile and chloroaldimine), 1.67 (m, 2 H, CH2 of chloroaldimine). The noise-decoupled 13C spectrum of the solution showed 10 singlets (ppm): 176.3 (CdNCl), 119.5 (CtN), 75.0 (CH2), 74.3 (CH2 of nitrile), 34.8 (CH2), 27.8 (CH2), 27.4 (CH2 of nitrile), 22.1 (CH2 of nitrile), 21.9 (CH2), and 17.0 (CH2 of nitrile). XV was identified in the mixture by comparison of the spectrum with that of a pure sample prepared by independent synthesis from an authentic sample of the parent aminonitrile. The proton spectrum of the authentic sample contained the following (ppm): 3.66 (t, 2 H, Cl2N-CH2-, J ) 6.6 Hz), 2.40 (t, 2 H, CH2sCtN, J ) 7.1 Hz), 1.88 (m, 2 H), 1.74 (m, 2 H). The noise-decoupled 13C spectrum of pure XV (CDCl3) contained 5 peaks (ppm): 119.2 (CtN), 74.4 (CH2), 27.5 (CH2), 22.2 (CH2), and 17.1 (CH2). Identification of XV in chlorinated lysine solutions was also confirmed by low-resolution GC/MS analysis of the CHCl3 extract of a model solution chlorinated with 4 equiv. The retention time and mass spectrum of XV in the total ion chromatogram correlated with those of the dichlorinated standard, 5-dichloraminopentanenitrile (XV). The mass spectrum of XV contained m/z (relative intensity) 170 (0.14, M + 4), 168 (0.75, M + 2), 166 (1.2, M+), 104 (15, [M+ - HCl - (CtN)]), 98 (20, M+ - 2HCl)), 82 (38), 64 (22) + 62 (11, [ClsNtCH]•+), 55 (100, [HNtCsCH2CH2]•+). Quantitative HPLC analysis using the dichlorinated standard showed that 42 ( 5% of the initial lysine is converted to XV within 30 min after chlorination. Extraction of the product mixture with chloroform and/ or decomposition in the GC apparently resulted in decomposition of XII. The total ion chromatogram revealed that two components, XV and another, had been extracted, but the mass spectrum of the second was not consistent with any suspected product. Furthermore, when the extract was reshaken with water and the aqueous layer analyzed by HPLC, XV was the only component remaining. The thermal lability of N-chloroaldimines as well as their instability in concentrated nonaqueous solution has been observed with the chloroaldimines derived from the amino acids isoleucine, valine, and phenylalanine (10-12). NMR analysis of a CDCl3 extract revealed a mixture of XII and XV. The characteristic downfield resonance of the chloroaldiminic proton (8.2 ppm) indicated the presence of both syn and anti isomers as seen in previous work (11, 12). The spectrum indicated that XV and XII were present in a 45/55 ratio. A 13C-NMR spectrum with an aldiminic carbon resonance at 175 ppm also supported the assignment of the structures. (D) Analyses of Product Mixtures at a Cl2/Lys Molar Ratio of 3. The HPLC analysis of a model solution chlorinated with 3 equiv yielded a mixture of the products previously described and one additional product (Figure 2). Co-injection of a chlorinated model solution and a solution of the 5-chloraminopentanenitrile (XIV), independently synthesized from the parent aminonitrile, showed the unidentified product was not XIV. Based on the most reasonable assignment of

FIGURE 2. High-performance liquid chromatogram with UV detection 30 min after chlorinating a model lysine solution (pH 7) to a Cl2/Lys molar ratio of 3.

FIGURE 4. Proposed scheme for the reactions of aqueous chlorine with lysine at low Cl2/Lys molar ratios and the rationale for the formation of V, VI, VIII, and IX. FIGURE 3. Total residual chlorine concentration and percent yields of products and unreacted lysine remaining 30 min after aliquots of primary wastewater containing 0.025 M phosphate buffer (pH 7) were inoculated with [3H]lysine and chlorinated to increasing Cl2/ Lys molar ratios. Polar products include all compounds that elute in the void volume with lysine. structure for products formed at a chlorination level this high, the product is suspected to be 5-dichloramino-N-chloropentanimine (XI). Analyses using radiolabeled-lysine revealed that XI is not a major chlorination product. Quantitative analyses using standard solutions of XV showed 21 ( 5% of the lysine in the model solution chlorinated with 3 equiv was converted to product XV 30 min after chlorination. (E) Quantitation of Chlorination Products in Model Solutions. By chlorinating an aqueous solution containing tritium-labeled lysine and measuring the amount of radioactivity associated with each product identified in the HPLC chromatogram, the yield of each lysine chlorination product was determined. Figure 1 summarizes the results of the quantitation of the various products at different Cl2/Lys ratios. Wastewater Solutions. The primary effluent used in this work had a total Kjeldahl nitrogen concentration of 22.9 mg/ L, an ammonia concentration of 21.9 mg/L, and a lysine concentration of 71 µg/L. Other amino acids present included β-alanine (85 µg/L), alanine (141 µg/L), valine (42 µg/L), tyrosine (120 µg/L), and phenylalanine (81 µg/L). In Figure 3, the distribution of lysine products formed when different levels of chlorine are added to the wastewater is shown. Also plotted is the total residual chlorine concentration at each point after 30 min.

Discussion Mechanism. Chlorination of lysine at molar ratios e2 produces V (suggested) and VIII (confirmed) as the major products. Because the first chlorination of an amino nitrogen is several orders of magnitude faster than the second

chlorination of that nitrogen (25), monochlorination of each of the two amino nitrogens is more likely to take place than dichlorination of only one of them. The HPLC analysis of chlorinated solutions demonstrated that V was the major product initially and that within 30 min VIII formed. VIII can form from V by the β-elimination of CO2 and chloride. The resulting imine will readily hydrolyze to the aldehyde. Over time there was an increase in VIII with a concurrent decrease of V. A reaction scheme for the formation of V and VIII is proposed in Figure 4. IX appeared to form at a later time, probably from the chlorine atom exchange between VIII and any of the chloramino species in solution (26, 27). In a wastewater this could include monochloramine. When 4 and 5 equiv are used to chlorinate the model solutions, XII and XV are the major products and account for at least 82% of the initial lysine. The dichlorinated nitrile (XV) also is observed when 3 equiv of chlorine are used, and this is suspected to be due to chlorine exchange between XIV and other chlorinated aminonitrogen compounds in solution. Figure 5 outlines a scheme to account for the formation of XII and XV from the exhaustive chlorination of lysine. As previously proposed (10-12), decomposition of the tetrachlorinated lysine can proceed by two pathways. Decarboxylation and dechlorination lead to the formation of XII. The initial loss of HCl prior to decarboxylation and a second dechlorination leads to formation of the nitrile. Model Solutions. Figure 1 summarizes the relative importance of the various lysine chlorination products at different Cl2/Lys molar ratios along with the observed amount of residual chlorine at each level. The amount of lysine present at each point is determined by the amount of radioactivity which elutes in the HPLC column void volume. Since all highly polar compounds elute here, lysine is grouped with other polar products. These probably include the highly ionized mono- and dichlorinated lysines II, III, and IV. No evidence for the formation of compounds such as VII, X, and XIII was observed, but formation of these compounds would

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TABLE 2. Yields and Half-Lives of Lysine Chlorination Products in Model Solutions and in a Wastewater at pH 7.0 chlorination product

max 30-min yield (%) model soln wastewater

V VIII IX XI XII XV

FIGURE 5. Proposed scheme for the reactions of aqueous chlorine with lysine at high Cl2/Lys molar ratios and the rationale for the formation of XI, XII, and XV. require disproportionate chlorination of the R-amino moiety of the amino acid. Since primary amino nitrogens are much more rapidly substituted with one chlorine atom than with a second, it is unlikely that these compounds could have formed in significant yields. In a short communication, Franck and Randau (28) have reported a study of the reaction of lysine with 2 equiv of sodium hypochlorite at room temperature for 3 h in aqueous solution. Experimental details such as the pH or any buffering of the reaction were not reported. Of the total amount of lysine subjected to oxidation, 35% was recovered unreacted, 4% was converted to ∆1-piperideine, 4% was converted to tetrahydroanabasine, 9% was converted to 2-piperidone, and 3% was converted to N-chloro-2-iminopiperidine. ∆1-Piperideine, the cyclic imine formed by intramolecular condensation of 5-aminopentanal, would be formed from VIII or IX if the N-chloramino residues are reduced to amines. However, our results suggest that, in solutions chlorinated with 2 equiv, VIII and IX are far more important chlorination products than what is implied by Franck and Randau’s observations (28). The tetrahydroanabasine they observed could be formed by condensation of two ∆1-piperideines, a bimolecular process that is not likely to take place in dilute solution in a wastewater. The remaining products they observed require intramolecular cyclization and subsequent oxidation. Piperidone could have formed under our reaction conditions; but, because it is so polar, it would have eluted in the void volume and been labeled as other polar products in Figure 1. N-Chloro-2-iminopiperidine identified in Franck and Randau’s study may correspond to the product we suspected to be XI, a minor chlorination product in our studies. Significance of Lysine Chlorination Products in Chlorinated Wastewaters. To ensure adequate disinfection, many disinfected wastewaters are held for at least 30 min following chlorination before they are discharged. Consequently, the products formed within 30 min were of primary concern in this study, since these are the products that would affect receiving waters. All major chlorination products identified in the model studies after 30 min were also observed in our studies of a wastewater. At the chloramine maximum of the wastewater (120 mg/L applied chlorine) VIII, IX, and XV account for more than 50% of the lysine chlorination products

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9.2 44.9 10.5 4.4 41.9 40.5

12.5 27.8 8.9 2.7 41.9 42.3

half-life (h) (model soln) 0.8 >45 >45 1.5 23 210

(Figure 3). Based on the work of LeCloirec et al. (29, 30), which demonstrated that inorganic monochloramine can react with aldehydes to form nitriles, XV could form from reaction of IX with monochloramine. Table 2 lists the observed maximum yields and stabilities of all of the lysine chlorination products identified. Every one of the compounds listed contains an N-chlorinated amino residue and therefore responds to iodimetric analyses in the same way NH2Cl does. Because organic chloramino compounds are, in general, much less bactericidal than inorganic monochloramine, all of the compounds identified here could contribute to interference with the proper measurement of the bactericidal properties of a chlorinated wastewater. Over the past 15 years, it has been recognized that chlorinated municipal wastewaters can have adverse effects on various life stages of fish and other aquatic life. This has led to concern about the environmental impact of the discharge of chlorinated wastewater into otherwise clean receiving waters. Chloramines as a group have been particularly associated with toxic effects in fish (1-3). The longerlived these compounds are, the greater would be their expected environmental significance. While V and XI decompose fairly rapidly in aqueous solution, VIII, IX, XII, and XV all have significantly long lifetimes.

Acknowledgments We are grateful to Karl Wood of the Purdue University Mass Spectroscopy Center and Asoka Ranasinghe of the University of North Carolina at Chapel Hill for their help in obtaining and interpreting mass spectral data and to the Hampton Roads Sanitation District Commission for their cooperation in providing total Kjeldahl nitrogen and ammonia analyses on wastewater samples. This research was supported by the National Science Foundation, Grant BCS-9218565, Dr. Edward Bryan, project manager. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the National Science Foundation.

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Literature Cited (1) Scully, F. E., Jr.; Hartman, A. C.; Rule, A.; LaBlanc, N. Environ. Sci. Technol. 1996, 30, 1465-1471. (2) Feng, T. H. J. Water Pollut. Control Fed. 1966, 38, 614-628. (3) Marks, H. C.; Strandskov, F. B. Ann. N.Y. Acad. Sci. 1950, 53, 163-171. (4) Wolfe, R. L.; Ward, N. R.; Olson, B. H. J. Am. Water. Works Assoc. 1984, 76 (5), 74-88. (5) Wolfe, R. L.; Olson, B. H. In Water Chlorination: Chemistry, Environmental Impact and Health Effects; Jolley, R. L., et al., Eds.; Lewis Publishers: Chelsea, MI, 1985; Vol. 5, pp 555-573. (6) Wolfe, R. L.; Ward, N. R.; Olson, B. H. Environ. Sci. Technol. 1985, 19, 1192-1195. (7) Johnson, J. D. In Water Chlorination: Environmental Impact and Health Effects; Jolley, R. L., Ed.; Ann Arbor Science: Ann Arbor, MI, 1978; Vol. 1, pp 37-63. (8) Jensen, J. N.; Johnson, J. D. Environ. Sci. Technol. 1990, 24 (7), 981-985. (9) Jensen, J. N.; Johnson, J. D. Environ. Sci. Technol. 1990, 24 (7), 985-990. (10) Nweke, A.; Scully, F. E., Jr. Environ. Sci. Technol. 1989, 23, 989994. (11) McCormick, E. F.; Conyers, B.; Scully, F. E., Jr. Environ. Sci. Technol. 1993, 27, 255-261. (12) Conyers, B.; Scully, F. E., Jr. Environ. Sci. Technol. 1993, 27, 261-266. (13) Scully, F. E., Jr.; Winters, D. S.; Conyers, B. Preprints of Papers, 204th National Meeting of the American Chemical Society, Washington, DC; American Chemical Society: Washington, DC, 1992; Vol. 32, No. 2, p 3. (14) Scully, F. E., Jr.; Howell, G. D.; Kravitz, R.; Jewell, J. T.; Hahn, V.; Speed, M. Environ. Sci. Technol. 1988, 22, 537-542. (15) Franson, M. A., Ed. Standard Methods for the Examination of Water and Wastewater, 18th ed.; American Public Health Association: Washington, DC, 1992.

(16) Conyers, B.; Walker, E.; Scully, F. E., Jr.; Marbury, G. D. Environ. Sci. Technol. 1993, 27, 720-724. (17) Blazevic, N.; Kolbah, D.; Sunjic, V.; Kakfez, F. Synthesis 1979, 161-175. (18) Besace, Y.; Marszak-Fleury, A. Bull. Soc. Chim. Fr. 1971, 4, 14681472. (19) Long, G. M.; Troutman, D. H. J. Am. Chem. Soc. 1948, 71, 24692473. (20) Scully, F. E., Jr.; Howell, G. D.; Penn, H. H.; Mazina, K. Environ. Sci. Technol. 1988, 22, 1186-1190. (21) Burdige, D. J.; Martens, S. C. Geochim. Cosmochim. Acta 1988, 52, 1571-1584. (22) Cowie, G. L.; Hedges, J. I. Mar. Chem. 1992, 37, 223-238. (23) Mopper, K.; Dawson, R. Sci. Total Environ. 1986, 49, 115-131. (24) Biemann, K. In Mass Spectrometry of Organic Ions; McLafferty, F. W., Ed.; Academic Press: New York, 1963; pp 529-596. (25) Morris, J. C. In Principles and Applications of Water Chemistry; Faust, S. D., Hunter, J. V., Eds.; John Wiley & Sons: New York, 1967; pp 23-53. (26) Gray, E.; Margerum, D.; Huffman, R. In Organometals and Organometalloids, Occurrence and Fate in the Environment; Brinkman, F. E., Bellama, J. M., Eds.; American Chemical Society: Washington, DC, 1979; pp 264-277. (27) Isaac, R. A.; Morris, J. C. Environ. Sci. Technol. 1985, 19, 810814. (28) Franck, B.; Randau, D. Angew. Chem. Int. 1966, 5, 131. (29) LeCloirec, C.; Martin, G. In Water Chlorination: Chemistry, Environmental Impact and Health Effects; Jolley, R. L., et al., Eds.; Lewis: Chelsea, MI, 1985; Vol 5, pp 821-834. (30) LeCloirec, C.; Benabdesselam, H.; Martin, G. Presented at the 5th International Conference: Chemistry and Protection of the Environment, Louvain, Belgium, September 1985.

Received for review June 19, 1996. Revised manuscript received January 8, 1997. Accepted January 14, 1997.X ES960534T X

Abstract published in Advance ACS Abstracts, March 15, 1997.

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