€Sm
REACTIONS OF CHLORINE, MONOCHLORAMINE
IN
ACT
The most important aspect of the treatment of water for drinking is disinfection. However, disinfectants are reactive oxidants and, upon ingestion, can undergo chemical reactions with components of saliva, food, and gastric fluid. Because aqueous chlorine and inorganic monochloramine are widely used disinfectants, it is important to determine what reactions these oxidants can undergo in the body, whether their products exhibit adverse health effects, and whether they are detoxified. Toxicological studies of aqueous chlorine and monochloramine have been reviewed ( I ) . However, the health effects associated with these disinfectants are probably not caused by the oxidants themselves, but by the products of their reactions with oreanic or inorganic compounds pres;nt in saliva or gastric fluid. Very little is known about the reactions of disinfectants with biomolecules in these fluids. Because the ch1orine:carbon ratio of the reaction mixture is comparatively small, the products are not likely
to be extensively chlorinated or oxidized. This means t h a t reaction products are not likely to be small fragments of larger molecules and their identification may r e q u i r e degradative techniques. This study will focus on the major reactive functional units of biomolecules present in saliva and stomach fluid and, using known reaction rate constants, predict the types of transformations expected for those functional groups which are most responsible for the dissipation of chlorine or monochloramine in the body.
E “”.
m
820 Envimn. Sci. Technol., Vol. 25, No. 5, 1991
Frank E. Scully, Jr. Old Dominion University Norfolk, VA 23529-0126 William N. White University of Vermont Burlington, VT 05405
Nature of the reactants The human body secretes between 1,000 and 1,500 mL of saliva i n a 24-hour period. MacDougall ( 2 ) d e scribes saliva as “ a clear, thin fluid: varying in pH from 5.45 to 7.4: and containing water (97-99%), electrolytes, organic materials . . . ” The major organic and inorganic comuonents of saliva have been idenhfied (3-11).The organic components include digestive enzymes as well as a complex mixture of glycoproteins called mucin (3)which is
.
0013-936x19110925-820502.5~0Q 1991 American Chemical Society
responsible for the slippery, viscous nature of saliva. The flow of saliva stimulated in normal humans by chewing wax is 6.7mL/5 min. (7).If a person takes 15 mL of chlorinated water in his mouth, it will mix with 0.5 mL saliva and dilute the average concentration of substrates 30-fold. (This 15mL estimate of a “swallow” is a conservative one personally measured by one of the authors.) Using this correction for dilution and the approximate concentrations of chemical components found in saliva (3-121, free amino acids in huM) would be man saliva (3 x diluted 30-fold to 1x M. Using these approximations, the molar concentrations of the maior components of saliva diluted by drinking water can be estimated. These are listed in Table 1. The human stomach typically secretes 2 to 3 liters of gastric fluid daily (121, of which 9197% is water. Stomach fluid is a muck more variable reaction medium than saliva. Following in. gestion of food and depending on the diet, the nature and concentrations 01 substrates for reac. tions of disinfec. tants can changc dramatically. Thc sight or smell oi food induces secretion of a copious amount of gastric fluid which contains inorganic salts, digestive enzymes, and mucus (12). In addition, the contents of the stomach are composed of fluid and particulate matter resulting from partially digested food. Therefore, the organic compounds present in the stomach depend on diet and include all major classes of biomolecules such as proteins, polysaccharides, lipids, and vitamins. Gastric fluid of the stomach also contains hydrochloric acid which can eive the stomach a DH as low as 1.3 (72). Gastric fluid is diluted upon ingestion of water. In a search of the literature, reference concentrations of organic substrates in human stomach fluid were not found. Therefore the average concentrations of substrates found in stomach fluid from rats administered 3 mL deionized water (13-15) will be used to determine re-
the nature of substrates for reactions with disinfectants. It would be reasonable to assume that the addition of food would increase the reaction rates of organics with disinfectants in saliva and stomach fluid because the concentrations of reactant substrates would increase. Therefore, the half-lives of disinfectants in saliva or stomach fluid would be expected to be shorter in the presence of food than the half-lives calculated below. However, the variables of diet and the proximity of eating to drinking disinfected water lead the discussion of the impact of diet beyond the scope of this paper.
action rates for different substrates. Although substrate concentrations in human gastric fluid would be preferred, concentrations in rat stomach fluid may correlate better with toxicological observations in the rat. Because the gastric fluid has already been diluted, no adjustment of these concentrations is necessary. In fact, these concentrations are probably lower than would be encountered in undiluted stomach fluid and actually present during animal feeding studies, because a volume of 3 mL is probably much more than the volume a rat drinks at one time. The total lipid concentration in stomach fluid is unknown. The concentration of protein in stomach flu.nd
L
‘ ’ I
Reactive forms of chlorine, chloramine In a survev of 80 localities most ofthe drinking waters tested contained residual aqueous chlorine concentrations between 0.4 and 2.8 mglL (C12, an average of appmximately 2 x M) (16). To simplify the calculations i n this study, a residual oxidant concentration (either a free or a combined residual) of 0.71 mg/L (as Cl,, 1 x io-’ M) will be assumed. Because the pH of the gastrointestinal tract can vary from a high of 7-8 down to a low of about 1 and because chloride concentrations can be greater than 0.1M. the reactive forms of the disinfectants also can vary accordingly. HOCl is a weak acid with a pK. of 7.5.At pH 7.0 the fraction of free available chlorine present as HOCl is 0.76 and the fraction present as hypochlorite (ClO-1 is 0.24. At low pH and in the presence of high concentrations of chloride ion, the fol-
associated with these disinfectants are probably not caused by the oxidants themselves
...
in undiluted saliva; therefore the liDid concentration in undiluted sa&a will be used to estimate the concentration in stomach fluid. Because both saliva and gastric fluid are digestive fluids, this approximation is probably not unrealistic. Concentrations of substrates for reactions with disinfectants used in this study are listed in Table 2. Diet can have a dramatic effect on
-
-
TABLE 1
Concentrations of reactive substrates In saliva diluted by ingestion of 15 rnL drlnking water Subatrate
Chloride Protein-bound amino acids Free amino acids
Concentration
8xlWM 6xlWM 1x104M 4xt(r5M 3x104M 8x10dM 8 x lo* b”
~
Envimn. Sci. Technoi.. Voi. 25. No. 5, 1991 811
centrations of reactive substrates in stomach fluid used in Y
lowing equilibrium is established HOCl
kf
+ H+. +- TC
k. The rate constant for the forward reaction at 20 "C is 1.8 x lo4 M-' S (1 7)and the equilibrium constant is 2.58 x 103 M - ~( 1 8 ) . Equilibrium would be achieved rapidly in the acidic contents of the stomach as 20% of the HOCl is converted to Clzlaql.At pH 2 and in 0.01 M chloride the concentrations of reactive species are: [C1,(,,1] = 2.0 x lo-' M [HOC11 = 8.0 x
lo4
M
(2)
(3)
Whatever the species, it is clear that the active chlorinating/oxidizing agent in solutions at low pH and in the presence of high concentrations of chloride ion differs significantly from that at neutral pH. Other minor forms of reactive chlorine such as the hypochlorous a c i d i u m ion (H,OCl+I (19)will not be considered in this study. Inorganic monochloramine can also be protonated. The best estimates suggest that the pK, of NH,C1 is approximately 1 (20,21). NH,Cl+ H+
e
NH,Cl+
(4)
As the pH of a solution approaches 1 the concentration of NH,CI+ increases. For our purposes, it will be assumed that the reactions of disinfectants i n saliva take place at pH 7.0 and that the reactions of disinfectants in the stomach take place at pH 2 in the presence of 0.01 M chloride ion. Reactive functional groups in biom o1ecu 1es Based on the principal biomolecules present in saliva, in gastric fluid, and in a normal diet, it may be concluded that on ingestion a disin822 Environ. ai.Technol., Vol. 25, No. 5, 1991
fectant encounters three main classes of biomolecules with which it may react: proteins and amino acids, carbohydrates and sugars, and lipids or fats. Aqueous chlorine and monochloramine can react with organic molecules by three basic mechanisms: oxidation of a molecule, substitution of a chlorine atom for another atom, or addition to an unsaturated molecule (22).Fukayama et al. (23) have reviewed the reactions of cblorine with the major classes of biomolecules found in foods. Only proteins, amino acids, and unsaturated lipids react with aqueous chlorine to form chlorinated compounds, whereas carbohydrates form unchlorinated oxidation products. Disinfectants and reactive biom o1ecu 1es Reaction rate constants. Table 3 contains a summary of what i s known about the kinetic parameters for reactions of either aqueous chlorine or monochloramine with reagents or functional groups of importance in saliva or stomach fluid. In cases where kinetic data on the reactions of specific substrates or disinfectants are unavailable, information on the reaction rates of reasonably similar model compounds is reported. For example, the reaction of N-acetylglycine is presented as representative of the reaction of peptide nitrogen compounds. Because there are no known rate data for the reaction of aqueous chlorine with the double bonds in unsaturated lipids, the rate constant for the reaction of allyl alcohol is reported. A quick examination of Table 3 reveals the absence of available rate data for several biochemical substrates. However, the relative significance of several of these reactions can be estimated from other data in the table. For instance, because monochloramine is a much less reactive oxidant than aqueous chlorine, its reaction with peptide nitrogens in proteins probably proceeds with a rate constant much less than the reaction of C10- with proteins at
least at pH 7. Also, because the rate of ring substitution i n aromatic compounds is dependent on the electron-richness of the aromatic ring, the rate of chlorination of phenylalanine is probably much slower than the reaction of phenol. However, the absence of rate data for the reactions of Clzlaqlis a significant deficiency in the available data set for predicting the reactivity of aqueous chlorine with organic substrates in stomach fluid. Because the concentration of disinfectant in this modeling study is comparatively low by kinetic standards (1x M), it can be shown that only reactions with secondorder rate constants > l o 3 M-'s-' have much significance in the body. Therefore, the reactions of the nucleic acid bases (uracil and cytosine), peptide bonds, unsaturated lipids, and nitrite will not be given further consideration. Half-lives of disinfectants Using rate constants listed in Table 3 and concentrations listed in Tables 1and 2, the half-life of aqueous chlorine and monochloramine resulting from reaction with each functional group can be predicted. Amino nitrogen compounds. The mechanism for the chlorination of amino nitrogen requires the reaction of a free amino group with hypochlorous acid as illustrated below for the reaction of glycine: H,N-CH,-COO- + HOCl __c CINH-CHz-COO- + H,O (5) Consequently, the rate is dependent on the fraction of free available chlorine which is present as HOCl at a given pH and the fraction of free amino groups present at that pH. The rate expression can be written: rate = k aHocl [FACI, (1-1~1 IG~YIT (61 where k is the second-order reaction rate constant, and [FACIT and [Gly], are the total concentrations of free available chlorine and glycine, and respectively. The terms aHocl (l-ly) are the fractions of the protonated acid (HOCI) and unprotonated amine (glycine), respectively, which are constants at a given pH. Assuming that all the free amino acids in human saliva react at the same rate as glycine, the half-life of aqueous chlorine in the mouth can be estimated to be 0.7 seconds. For > 90% of the free available chlorine to react would require -11 s. The
in a manner similar to that outlined for the reaction of glycine: 2
NH,CI+ + H,N-CH,-COO- 7 NH: + ClNH-CH,-COO- (9)
reaction may be much faster than t h i s , if the free a m i n o groups present in saliva proteins and polypeptides are accounted for, but their concentration is not known. In any case, the reaction of free available chlorine with amino nitrogen compounds in the mouth is quite rapid and likely to be an important mechanism for the dissipation of free available chlorine in the body. The concentration of free amino acids in stomach fluid is higher than in saliva: but, because of the lower pH, the fraction of unprotonated amino groups is very low (1.7x 10"). At pH 2 both HOCl and Clzleqlcan react with amino acids. Two mechanisms for the chlorination of amines are possible, chlorination by HOCl and by Clzlaql.Each will be considered separately. Assuming that HOC1 is the sole reacting species, a calculation similar to that performed for saliva is possible. In this case the estimated half-life of HOCl in stomach fluid at 25 "C (pH 2) would be estimated as 9.4 min. Margerum (25)has shown that free amines react with C12(aqlat dif-
fusion-controlled rates according to the equation: R-NH,
-
+ C121aql
R-NHCl+ HC1 (7)
The rate expression is: rate = k
[FAC], (1-1~) [G~YIT (8)
where is the fraction of Clzla By the reaction of C1, e alone wi& amino acids, the h a k l i f e of free available chlorine in stomach fluid would be 2.6 min. The rate of chlorination of amino nitrogen compounds in the stomach is considerably slower than the same reaction in saliva, and Clz,eqlis a more important chlorinating agent than HOCl at low pH in the stomach. Whether the reactant is C121aql or HOC1, the reaction of aqueous chlorine with amino niixogen in stomach fluid is much slower than its reaction in saliva because of protonation of the amino nitrogen at this PH. In acid solution monochloramine will transfer its chlorine to the amino group of amino acids (21,26)
A calculation similar to that above can be carried out for the chlorine transfer reaction of monochloramine (1 x lo-' M) with amino acids (8.23x lo4 M). Instead of using the concentration of free available chlorine, [FAC], in the rate expression, the concentration of combined residMI, is ual chlorine, ([CRCI = 1x used. In saliva at pH 7.0 the half-life of monochloramine would be approximately 17.5hours. It is unlikely that the reaction of monochloramine with amino nitrogen compounds in saliva is significant. At pH 2 the half-life of monochloramine in the stomach is estimated to be 9.7 min. As mentioned previously, the half-life is probably less than this if the terminal amino groups of proteins and polypeptides are accounted for. The stability of N-chlorinated amino acids varies widely with structure ( 4 8 ) and with the pH of the solution in which they are formed (49).All appear to decarboxylate with loss of chloride to form aldehydes (49-52). Sulfur-containing amino acids. Methionine is one of eight amino acids that is not synthesized by humans and must be acquired in the diet. The recommended dietary allowance of total sulfur-containing amino acids (cysteine and methionine) in adults is 10 mg/kg body weight per day (700mg or approximately 5.2 mmol for an average adult)-(53). Neither of the free amino acids, cysteine or methionine, were detected in the study of saliva by Battistone and Burnett (6). A cystine concentration of 0.16-0.45 mglL (0.6-1.9@ andI a) methionine concentration of 0.005-0.010 mglL (30-60 nM) have been reported in saliva (11). These results should be confirmed by further analysis. Nevertheless, the significance of sulfurcontaining amino acids may not be limited to their concentration in the free amino acid pool, because they might effectively reduce oxidants when they are bound to proteins. Jacangelo and Olivieri (28) and lngols et al. (29) discuss the oxidation of cysteine residues in proteins as a critical part of the mechanism by which bacteria are inactivated by disinfectants. The main proteins in saliva are amylase and lysozyme Environ. Sci. Technol.. Vol. 25, No. 5, 1991 823
(31. An u-amylase derived from the rat pancreas contains four methionine residues and six cysteine residues that are oxidized and linked to each other in disulfide bridges ( 5 4 ) . A lysozyme derived from chicken egg white contains 6.2 mol percent cysteine and 1.55 mol percent methionine (54). The presence of cysteine, cystine, and methionine in the total pool of amino acids (free + protein-hound) in saliva should he determined. Rate constants for the reaction of HOC1 or monochloramine with the reduced sulfur moiety in sulfurcontaining amino acids are unknown. Therefore, in order to obtain an indication of the relative significance of the reactions of sulfurcontainingu amino acids and proteins in the dissipation of disinfectants in the body, the rates of reaction of other reduced sulfur compounds will be examined. Kice et al. ( 3 0 ) have studied the reduction of aqueous chlorine by henzenesulfinate (Ph-SO;) at pH values ranging from 5.2 to 9.0 at 25 'C. The pH d e p e n dence of the reaction suggests that t h e more a c t i v e form of chlorine is hypochlorite. HOCl is reduced with a rate constant 350 I times smaller. Because it is less highly oxidized tha.. the sulfinate ion and because it is an aliphatic sulfur compound instead of the aromatic sulfinate studied by Kice, cysteine probably reacts with hypochlorite at a much faster rate. In light of the rate of reduction of a model chloramine by dialkylsulfides discussed below, it would be reasonable to assume a second-order rate constant of 1 x 10" M-'s-' for the reduction of hypochlorite by sulfur-containing amino acids. If these amino acids represent two percent of the total amino acid and protein pool in saliva (2% of 6 x lo4 M is 1x M) and if their reaction with all free available chlorine species follows simple second-order kinetics (one sulfur reduces one oxidant molecule), then free available chlorine would he reduced in saliva with a half-life of approximately 0.42 s. In this case, the reaction of sulfur-containing amino acids would ef-
fectively compete with chlorination of amino nitrogen for dissipation of all disinfectant.More than 90% would he reduced in 6.3 s. If the rate constant were only 1 x lo4 M-' s-' (still a very fast rate), the half-life of free available chlorine would he 42 s and it would require 10.5 min for > 90% to react. If the reactions of cysteine, cystine, a n d methionine with free available chlorine are as pH-dependent as the reaction of benzenesulfinate is, then the rate constant for the reduction w o u l d be 350 times smaller or 2 . 8 x l o 3 M-' s-' (or 28.6 M-' s-' for the conservative estimate). Because the concentration of sulfur-containing amino acids would he higher in stomach fluid [an e:"--te of 2% of 1 x lo-' MI
-dlFACl/dt = k,, IFACl[phenoll (101
I
824 Environ. Sci. Technol., Vol. 25.No. 5, 1991
ramine would have a second-order half-life in saliva of 3.7 s (55 s for > 90% to react) and a first-order half-life in stomach fluid of 0.13 s. The rate of reduction of chloramines by thiols such as cysteine may he sensitive to pH because of ionization of the sulfhydryl group. Tyrosine. Tyrosine, which contains a phenolic residue, is one of four aromatic amino acids. Because its rate of reaction with disinfectants has not been reported, the reaction of phenol will be examined as a model. Lee (32)and Soper and Smith (33) studied the rate of reaction of phenols with aqueous chlorine at pH 5-12. The reaction involved ring chlorination of the phenoxide ion by HOCl and apneared to follow the rate law:
where k,,, is the observed rate constant that is pH dependent. The reactive species appear to he HOCl and the phenoxide anion which account for the pH dependence. If the percentage of tyrosine in the total amino acid pool in saliva is the same as that in the free amino acid pool in gastric fluid ( -6% in the data from Reference 10) ...-..the total concentration of tyrosine in saliva would he 3.6 x lo-" M. This is similar to the concentration of free available chlorine assumed in this study ( 1 x M) and indicates second-order kinetic conditions. Assuming that the tyrosine ring will react as rapidly as phenol and equally rapidly whether it is the free amino acid or hound in a protein, then a half-life of - 9.4 min can be calculated. It w o u l d take almost 35 m i n for > 90% of the tyrosine to react. This suggests that, in saliva, chlorination of the aromatic ring of tyrosine is not significant. Because the concentration of ionized phenol decreases as the pH decreases, the observed rate of chlorination of phenol also decreases to < pH 5 where chlorination of phenoxide by HOC1 becomes too slow and another mechanism takes pre-
about the reactions of disinfectants with biomolecules in saliva or gastric fluid. ...-..in saliva, the half-life of free available chlorine would be estimated to be 1.23 s (or 2 min for the conservative ratel. Because monochloramine is a less potent oxidizing agent than free available chlorine, its reduction by sulfur-containing amino acids is likely to be considerably slower than reduction of free available chlorine. However, no measurements of the rate constants for reduction of monochloramine by thiols, disulfides, or dialkyl sulfides are known. Stanhro and Lenkovitch ( 5 5 ) have shown that bisulfite reduces organic N-chloramines at much slower rates than previously expected. Ruff and Kucsman ( 3 1 ) have determined the rate of reduction of chloramine-T (an N-chlorosulfonamide) by dialkyl sulfides. If chloramine-T is a good model, its reactivity suggests that monochlo-
cedence. Grimley and Gordon (34) have shown that at low pH and in high chloride concentrations the rate of chlorination of phenol dep e n d s on t h e concentration of Clz(aq), The mechanism involves formation of a chlorine-phenol complex that reacts further to form chlorophenol. It can be demonstrated that the reaction follows the rate law:
or more practically
As discussed above, C1z(aq)represents 20% of the free available chlorine at pH 2 in 0.01 M chloride. Because the concentration of free tyrosine in stomach fluid (3.6 x M [25])is comparable to that of free available chlorine assumed in this study, the second-order half-life of the reaction of aqueous chlorine with phenol is 4.7 s. It would require approximately 17.4 s for > 90% of the aqueous chlorine to react. These data suggest that chlorination of tyrosine could be a significant reaction in stomach fluid, but not in saliva. If tyrosine residues w h i c h are protein-bound react much more slowly than the free amino acids, then free tyrosine would be chlorinated at a significant rate, but it will not contribute to the dissipation of a significant amount of the total free available chlorine present. The same holds true if much higher concentrations of free available chlorine are used, whether protein-bound tyrosine residues react rapidly or not. For instance, if 7 1 mg/L free available chlorine is present in stomach fluid, there would be a 30-fold molar excess of chlorine over tyrosine. Burttschell et al. ( 3 5 ) have found that monochloramine will chlorinate phenol at a very slow rate at pH values encountered in drinking water treatment plants. Although there are no known rate constants, the reaction appears to be too slow to be of significance in saliva. The reaction at low pH is probably much faster, although no rate constants have been reported. When a model solution of N-acetyltyrosine (2.5 x M) was reacted with monochloramine ( 2 4 ) in 0.1M chloride, chlorination of the tyrosine ring was observed at low pH (pH 2), but not at high pH (pH 8). However,
-
even at low pH the reaction is slow. In 45 min at 37 "C one equivalent of monochloramine had converted 19% of the N-acetyltyrosine to the monochlorinated product (14). Tryptophan. Tryptophan reacts rapidly with solutions of aqueous chlorine to form an insoluble product (56).Although products have not been identified, chlorination of model compounds of similar structure has suggested that chlorination of the indole ring is involved. Burleson et al. (57)identified two oxindole products of the chlorination of tryptophan, one of which contained a chlorine atom. There are no rate data available on the reaction of tryptophan with aqueous chlorine. More work is needed to characterize the rates and products of this reaction. When one equivalent of monochloramine is reacted for 45 min at 37 "C with solutions of N-acetyltryptophan, 30% of the organic compound is destroyed at both pH 8 and pH 2 ( 2 4 ) . It requires a reaction with two equivalents of monochloramine to completely destroy all N-acetyltryptophan, suggesting that reactive intermediates also exhibited an oxidant demand. Reaction with NH;,Cl indicated that at least two products form that contain chlorine. Further work is needed to characterize these products. Phenylalanine. Because the aromatic ring of phenylalanine is much less electron-rich than that of tyrosine, chlorination probably takes place at a much slower rate. The observations of Burleson et al. ( 5 7 )and the model studies of Carlson and Caple (58) suggest that chlorination of the aromatic ring of phenylalanine is too slow to compete effectively with other reactions. Histidine. No studies of the kinetics of the reaction of aqueous chlorine with histidine or suitable model compounds have been published. Further, there is no information on the reaction of monochloramine with histidine or with suitable model compounds. Iodide. For males and females over the age of 11 yr the recommended daily dietary allowance of iodide is 150 pg (1pmol). Saliva is known to contain about 0.4 pM iodide ( 2 2 ) which would be diluted 30-fold to 13 nM on ingestion of water. Kumar, Day, and Margerum (40) have studied the reaction of hypochlorous acid and iodide ion. With the iodide ion concentration present i n saliva, a half-life of 0.0005 s and a 90% reaction time of 0.0016 s are estimated. Although
this reaction is extremely fast, there is only enough iodide ion to consume about 0.13% of the total aqueous chlorine. The reaction results in the formation of chloride and hypoiodite ions. The latter, like hypochlorite ion, is an oxidant but is less reactive. Thus, it would be expected to react further in the digestive tract. Under conditions designed to simulate the gastrointestinal tract, Bercz and Bawa (59) demonstrated that monochloramine caused binding of radiolabeled iodide to nutrient biochemicals. The amount of binding varied with pH, but was generally less at neutral pH than at higher pH values. The rate of iodination of biomolecules is unknown and the average concentrations of iodide in saliva or stomach fluid are unknown. Thiocyanate. Definitive studies of the reaction of thiocyanate ion with aqueous chlorine have not been performed. The chlorine demand of thiocyanate in aqueous solution at low pH has been determined by Kobayashi and Okuda (60) and is 4 mol of chlorine for each mol of thiocyanate. This is sufficient to convert thiocyanate to sulfate and cyanide ions. Several studies indicate that the rate-determining step in the oxidation is the combination of the first mol of oxidant with the thiocyanate ion and that the subsequent oxidation steps are rapid (4245). Although the rate constant for the reaction of hypochlorous acid with thiocyanate ion has not been obtained, the reaction of this ion with other oxidants and with other substrates (with which it functions as a nucleophile) has been investigated quantitatively. Hydrogen peroxide reacts with thiocyanate about twice as fast as it does with triethylamine and one-quarter as fast as it does with thiodiethanol (46). As a nucleophile, thiocyanate ion is more reactive than dialkyl sulfides are toward methyl iodide, but slightly less reactive than dialkylamines are toward the same substrate ( 4 7 ) . On the other hand, this ion is considerably more reactive than either sulfides or amines toward substitution on platinum complexes (47). On the basis of these reactivity comparisons, it is reasonable to assume that thiocyanate will react with hypochlorous acid as readily as it does with ammonia and amines (10, to lo* M-ls-l at 25 "C [251). Using the smaller (and thus, more conservative)of these values, the half-life for the reaction of thiocyanate ion with aqueous chlorine at pH 7 in saliva will be 0.009 s and the time reEnviron. Sci. Technol., Vol. 25,No. 5, 1991 825
quired for 90% reaction will be 0.032 s. Not only is this reaction very fast, but there is sufficient thiocyanate ion in saliva to consume approximately 20 mglL aqueous chlorine. The rate of reaction of monochloramine with thiocyanate is not known. Relative significance of reactions Table 4 is a summary of the estimated half-livesof aqueous chlorine in saliva and in stomach fluid due to reaction of different biomolecules in Table 3. Table 5 is the corresponding summary of the half-lives for the reactions of monochloramine. The chemistry of the disinfectants in stomach fluid appears to differ significantly from that in saliva. Three factors appear to control this: the pH, the difference in chloride ion concentration, and substantially higher concentrations of organic substrates in the stomach. In saliva the reactions of thiocyanate ion, of amino nitrogen, and of sulfurcontaining amino acids probably dominate the chemistry of aqueous chlorine. Calculationsof reaction rates suggest that the reduction of aqueous chlorine by thiocyanate is probably the most important process in saliva.
Although aqueous chlorine also reacts very rapidly with amino nitrogen to form organic N-chloramines, it appears to depreciate the concentration of aqueous chlorine at a rate approximately two orders of magnitude slower than thiocyanate. Conservative estimates suggest that sulfur-containing amino acids may react as rapidly as amino nitrogen compounds to reduce aqueous chlorine at low concentrations. In any case, the reactions of aqueous chlorine appear to he so fast in saliva that at concentrations encountered in drinking water all free available chlorine is dissipated before water is swallowed. Monochloramine does not react as rapidly with organic amino nitrogen and probably is not reduced as rapidly by thiocyanate or by sulfur-containing amino acids. Because the residence time of aqueous disinfectants in the mouth is short, the chemistry of monochloramine may be slow enough to allow it to survive reactions in saliva and pass into the stomach unreacted. In stomach fluid, protonation of amino groups at low pH slows their reaction with any aqueous chlorine
ABLE 4
stimated half-lives of the reactions of aqueous chlorine with iomolecules in saliva and in stomach fluid Saliva
Half-life
1s
Stomach fluid
Thiocyanate
Amino nitrogen compounds
s-1 min min-I h h-100 h
lnknown
Suifur-containing amino acids Tryptophan (?)” Tyrosine, cytosine Uracil, lipids Peptide bonds Histidine, phenylalanine
Sulfur-containing amino acids Tyrosine Amino nitrogen compounds Uracil Histidine, phenylalanine Tryptophan, peptide bonds Lipids, cytosine
*The reaction of tryptophan is Significantly faster than the reaction of tyrosine (14.aithough lhe rate constant has not been measured.
‘ABLE 5
lstimated half-lives of the reactions of monochloramine with lifferent biomolecules in saliva and in stomach fluid lalf.life
Saliva
Sulfur-containing amino acidsa
IS
s-I min 1 min-I h 1 h-100 h Unknown
Stomach fluid
Sulfur-containingamino acids Amino nitrogen compounds Thiocyanate Histidine, phenyialanine Tyrosine, tryptophan Lipids, purines, pyrimidines
* Asriiming no elfort of nH nn ieaCfion rate
826 Environ. Sci. Technol., Vol. 25. No. 5. 1991
Amino nitrogen compounds Tyrosine Histidine, phenylalanine Tryptophan. peptide bonds Lipids, purines, pyrimidines
I
that might be swallowed. Because the residence time of water in the stomach can be longer than in the mouth, the half-life for this reaction (several minutes) may still be significant. However, estimates suggest that the reduction of aqueous chlorine by sulfur-containing amino acids will be as fast or faster than the formation of N-chloramino acids in the stomach. The rate of chlorination of amino acids by aqueous monochloramine is not significantly different at low pH than at high pH. However, the higher concentration of organic amino nitrogen in the stomach shortens the halflife of monochloramine to 9 min because of reactions w i t h amino nitrogen compounds. Because the effect of pH on the rates of reduction of monochloramine by sulfur-containing amino acids is unknown, it is difficult to predict whether monochloramine will survive in the stomach long enough to be absorbed. Estimations of the rate suggest that low concentrations of monochloramine will not. There is a need to realistically evaluate the significance of the presence of sulfur-containing amino acids (cysteine, cystine, and methionine) in saliva and in stomach fluid. To do this, their concentrations must be measured and the stoichiometq and rate constants for their reaction with free available chlorine, with monochloramine, and with N-chloramino acids determined at various pH values. Sulfur-containing amino acids may reduce these oxidants at toxicologically significant rates. Effect of dose on reaction products The concentration of thiocyanate in human saliva is sufficient to reduce up to 15 mL of 20 mglL aqueous chlorine. With concentrations of aqueous chlorine above 20 mglL [Cl,) slower reactions become significant and the products of these reactions may have toxicological effects which are of no consequence at lower concentrations. Several reactions discussed above can form different products depending on the amount of disinfectant added to saliva or stomach fluid. For instance, N-chloramino acids react further (although at a much slower rate 1251) to form dichloramino acids which decompose to form other products (50-52). Because of the comparatively fast reaction of thiocyanate with aqueous chlorine, secondary by-products of the chlorination of amino nitrogen compounds could be more
significant in saliva than in gastric fluid in clinical studies involving concentrations of free available chlorine greater than 20 mg/L. Formation of N , N-dichloramino acids and their decomposition products are not likely to occur in the body except with high concentrations of aqueous chlorine. This suggests that there is a threshold level of chlorine above which more extensive oxidation will take place. Stomach fluid from rats fasted 48 hours and administered 4 mL deionized water has a chlorine demand of approximately 500 mglL (61). Le., more than 500 mglL free available chlorine (as Cl,) must be added to it before a non-chloramine residual can be detected. At lower dosages N-chlorinated compounds are formed, d e p e n d i n g o n t h e amount of chlorine added (48-52). Consequently, the highly chlorinated compounds, chloroform, chlorinated acetic acids, and chlorinated acetonitriles observed by Mink et al. (64)in the gut and plasma of rats administered 8000 mg/L free available chlorine, are not likely to be observed at much lower concentrations. The aromatic ring of tyrosine residues can become dichlorinated (14, 57, 63). yielding a product having different chemical and perhaps different toxicological properties than the monochlorinated derivative. However, the tyrosine ring cannot become dichlorinated until the first chlorine atom is substituted on the ring. Consequently, different specific products will form depending on the chlorine concentration used. Siissmuth has found that chlorinated solutions of methionine, tyrosine, phenylalanine, cysteine, and glycine are mutagenic with histidine-auxotrophic strains of Salmonella t y p h i m u r i u m ( 6 5 ) .However, the ch1orine:carbon ratio he used in the chlorination appears to be considerably higher than would be encountered in the reaction of aqueous chlorine with amino acids in saliva and stomach fluid under normal drinking water conditions. Conclusion This study finds five deficiencies in the data base needed to accurately model reactions of aqueous chlorine and monochloramine in the body: characterization of the composition of human stomach fluid; accurate measurements of the concentrations of sulfur-containing amino acids in saliva and in stomach fluid: reliable rate constants for the reactions of sulfur-containing
amino acids with aqueous chlorine and monochloramine; a good understanding of the reactions and rates of these disinfectants at low pH and in the presence of high chloride concentrations; and the rates and reactions of monochloramine with several organic and inorganic substrates found in food, saliva, and stomach fluid. C1?l,,, represents a significant fraction of the oxidant present in stomach fluid at low pH. However, its reactions with model compounds have received little attention. The reactions of monochloramine at low pH in the presence of high chloride concentrations are of particular interest. For instance, the following reaction could affect studies of monochloramine: NH3C1'
+ C1-
3 "
+ CL1aq, (13)
In this case, an otherwise mild oxidizing agent at neutral pH is converted to a potent oxidizing agent at low pH. The rate and equilibrium constant for this reaction should be determined. Knowledge of the reactions of aqueous chlorine and monochloramine with other reactive organic substrates is particularly lacking. Needed information is outlined in Tables 3-5. More information is needed on the concentrations of unsaturated fatty acids in saliva and in stomach fluid and on the rates of their reaction with free available chlorine. In light of the number of moles of unsaturated fats in the normal dailv diet. this is esDeciallv sianificant.. Finally, because different products can form at different dosage levels, a linear extrapolation of to& cological effects observed at high dosage levels to effects in humans at low dosages does not appear to be appropriate for aqueous chlorine and monochloramine. It is quite possible that more toxicologically significant products are formed at low dosages which are destroyed at higher dosages. Therefore, in order to determine the true toxicological significance of the ingestion of oxidizing disinfectants, it is important to identify products formed at low dosages of chlorine and monochloramine and evaluate their toxicological significance separately. I
I
Acknowledgment The study described in this document was supported by EPA under a work assignment to Life Systems, Inc. This docu-
ment has not been reviewed by the Agency for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. The study described herein was resented lo the Science Advisory Boar: of EPA, December 6, 1989, and at the 24th Annual Conference on Trace Substances i n Environmental Health, Cincinnati. OH. July 9-12,1990.
F r a n k E. Scully, Jr.,11) is professor a n d chairperson ofthe Department of Chemistry a n d Biochemistry at Old Dominion University. He received h i s Ph.D. i n physical organic chemistry from Purdue University. His research focuses on the reactions of aqueous chlorine a n d chloramines with organic components of waters a n d wastewaters. William N. White IrJ is professor of chemistry a t the University of Vermont. His research h a s been centered on the mechanisms of organic reactions, a n d includes kinetic investigations of the reactions of chloramines a n d chloramides. He received his doctorate in organic chemistly from Harvard University.
References (1) National Research Council. Drinking
(2) (3)
(4)
(5)
Water a n d Health, Vol. 7; National Academy Press: Washington. DC. 1987, pp. 81-83, 90-99. MacDougall, J. A,. In Gostroenterology; Bogoch. A,. Ed.; McGraw-Hill: New York. 1973, pp. 153-171. Woman. S.; Mandel. 1. D. In Diseases of the Salivary Glands; Rankow, R. M.; Polayes. I. M., Eds.; W. B. Saunders: Philadelphia, 1976, pp. 32-53. Jenkins. G. N. The Physiology a n d Biochemistry of the Mouth, 4th ed.; Blackwell Scientific Publications: Oxford, 1978. pp. 284-357. Jenzano, J. W. et al. Anal. Biochem. 1986.159.370-76.
(6) Battistone, G. C.; Burnett. G. W. Arch. Oral Biol. 1961, 3.161-70. (7) Syqanen. S.; Piironen. P.; Markkanen.
H. Arch. OmlBiol. 1987.32(9). 607-10. (8) Tannenbaum, S. R. et al. 1. Not. Cancerrnst. 1974, 53, 79-84. (9) Green. L. C. et 81. Anal. Biochem. 1983,131,24245. (10)
Barsotti. D. J.; Pylypiw. H. W.; Harrington. G . W. Anal. Lett. 1982. 1 5 .
1411-22. (11) Altman, P.
L.; Dittmer, D. S. Metabolism; FASEB: Bethesda. MD, 1976. pp. 2 3 7 4 1 . (12) Bogoch, A. et al. In Gastroentero1og)s Boeoch. A,. Ed.; McGraw-Hill: New Yoyk. 1973. chapter 11. Environ. Sci. Technol.. Vol. 25. No. 5. 1991 027
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Forstner, G. G.; Mullinger. M. A,; Bogoch, A. In Gastroenterology; Bogoch, A.. Ed.; McGraw-Hill: New York. NY, 1973, chapter4. 1141 Scully. F. E., Jr.; "Characterization of Chlorinated Organic Compounds Formed on Ingestion of Chlorinated Water"; U S Environmental Protection Agency. Final Report, project #CR813092-01, Ringhand, H. P., project officer; Health Effects Research Laboratory: Cincinnati, OH. 1151 Scully. F. E., Jr. et al. Chem. Res. Toxicol. 1990.. 3141. 301-06. 1161 Symons, J. M. et al. 1. Am. Water Works Assoc. 1975.67.634-47, 1171 Eiaen. M.; Kustin. K. 1. Am. Chem. I131
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ter a n d Wastewater: Johnson, J. D., Ed.; Ann Arbor Science Publishers: Ann Arbor. MI, 1975, pp. 249-76. I201 Gray, E.; M q e N m , D.; Huffman. R. In Orgonometals ond Orgonometalloids, Occurrence and Fate in the Environment Brinkman, F. E.; Bellama, J. M., Eds.; American Chemical Society: Washington, DC,1979, pp. 264-77. 121) Isaac, R. A,; Morris, J. C. Environ. Sci. Technol. 1985, 19,810-14. I221 National Research Council. Drinking Water a n d Health, Vol. 2; National Academy Press: Washington, DC, 1980. pp. 144-82. (231 Fukayama, M. Y. Environ. Health Perspec!. 1986,69,267-74. 1241 Morris, J. G. In Principles a n d Applications of Woter Chemistry; Faust. S.D.; Hunter, J. V., Eds.; John Wiley: New York, 1967, pp. 23-53. 1251 Margerum, D. W.; Gray, E. T.; Huffman, R. P. In Organometols and Organometalloids, Occurrence a n d Fate in the Environment: Brinckman, F. E.; Belloma, J. M., Eds.: American Chemical Society: Washington, DC, 1979, pp. 278-91. I261 Snyder, M. P.; Margerum, D. W. Inorg. Chem. 1982,Zl. 2545-50. I271 Mauger, R. P.; Soper, F. G. I. Chem. Soc. 1946,71-75. I281 Jacangelo. J. G.; Oliuieri, V. P. In Water Chlorination: Chemistry, Environmental Impact a n d Health Effects, Vol. 5; Jolley, R. L. et al., Eds.; Lewis Publishers: Chelsea, MI, 1985; Chapter 45, pp. 575-86. 129) Ingols, R. S. et al. Ind. Eng. Chem. 1953.45.9961000. I301 Kice, I. L.; PUIS, A. R. I. Am. Chem. SOC.1977,99,3455-60. I311 Ruff, F.; Kucsman. A. I. Chem. SOC., Perkin 111975, 509-19. I321 Lee. C. F. In Principles a n d Applica-
tions of Water Chemistry; Faust, S. D.; Hunter, J. V., Eds.; John Wiley: New York, 1967, pp. 55-74. I331 Soper. F. G.; Smith, G.F. 1. Chem. SOC.1926,1582. I341 Grimley, E.; Gordon, G. I. Phys. Chem. 1973.77.973-78,
Burttschell, R. H. et al. 1.Am. Water Works Assoc. 1959, 51, 205-14. 1361 Gould, J. P.: Richards, J. T.; Miles, M. G. WaterRes. 1984, 18121. 205-12. 1371 Gould, J. P.; Hay, T. R. Water Sci. Technol. 1982,14,629-40. 1351
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828 Environ. Sci. Technol., Vol. 25, No. 5 , 1991
M. G. WaterRes. 1984. 28181. , ,, 991-99. ~~~
~~
(39)Israel, G. C.; Martin, J.K.; Soper, F.G.
1.Chem. Soc.,
1950,1282-85.
Kumar, K.; Day. R. A.; Margerum, D. W. Inorg. Chem. 1986, 25,4344-50. 1411 Lister. M.W.; Rosenhlum. P. Can. 1. Chem. 1961,39,1645-51. I421 Wilson, 1. R.; Harris, G. M. 1. Am. Chem. SOC.1960.82.4515-17. 1431 Wilson. 1. R.; Harris, G. M. 1. Am. Chem. SOC.1961,83,286-89. 1441 Briot. G.T.; Smith, R. H. Austr. 1. Chem. 1973,26,1863-69 1451 Mahadevappa, D. 5.; Gowda. B. T.; Gowda, N.M.N. Z. Naturforsch. 8: Anorg. Chem. Org. Chem. 1979, 348, I401
~~
52-57.
1962. 84, 1355-61:
Connick. R. E.; Chia, Y. I. Am. Chem.
SOC.1959,81,1280-84. 119) Rosenhlatt. D.H. In Disinfection: Wa-
1
(38) Gould, J. P.; Richards, J. T.; Miles,
Curci, R.; Edwards, J. 0. In Organic Peroxides. Vol. I; Swern, D., Ed.; Wiley-lnterscience: New York, 1970, pp. 199-264. 1471 Pearson, D. G.; Sohel, H.; Songstad, J. I. Am. Chem. Soc. 1960,90,319-26. 1481 Isaac, R. A.; Morris, J. C. In Water Chlorination: Environmental Impact a n d Health Effects, Vol. 4; Jolley, R. L. et al., Eds.; Ann Arbor Science: Ann Arbor. MI, 1983, pp. 63-75. (49) Stanbro, W. D.; Smith, W. D. Environ. Sci. Technol. 1979, 13,446-51. 150) Nweke, A.; Scully, F. E., Jr. Environ. Sci. Technol. 1989,23,989-94. I511 Stelmaszynska, T.; Zgliczynski. J. M. Bur. 1.Biochem. 1978,92.301-8. 1521 Stanhro, W. D.; Lenkovitch, M. J. In!. I. Chem. Kinetics 1985, 17,401-11. I531 Committee on Dietary Allowances, National Academy of Sciences. Recommended Dietary Allowances, 9th ed.; National Academy of Sciences: Washington, DC, 1980,pp. 39-54. I541 Fasman, G. D., Ed. Handbook of Biochemistry and Molecular Biology, 3rd Ed., Vol. Ill; CRC Press: Cleveland, OH, 1976. 1551 Stanhro, W. D.; Lenkovitch, M. J. SciI461
ence 1982,215, 967-68. J. R.; Mitchell. S. K. In Water
I561 Kirk,
Chlorination: Environmentol Impact a n d Health Effects, Vol. 3; Jolley, R. L. et al., Eds.; Ann Arbor Science Publishers: Ann Arbor, MI, 1980, pp. 283-303. I571 Burleson, J. L.; Peyton, G. R.; Glaze, W. H. Environ. Sci. Technol. 1980. 14(11), 1354-59.
I581 Carlson. R. M.; Caple, R. In Water
Chlorination: Environmental Impact a n d Health Effects. Val. 1, Jolley, R. L.: et al.. Eds.: Ann Arbor Science Publishers: Ann Arbor, MI, 1978. pp. 65-75... 159) Bercz, J. P.; Bawa, R. Toxicol. Lett. 1986. 34(2-3),14147. 160) Kobayashi, T.; Okuda, T. Water Res. 1972, 6,197-209. I611 Scullv. F. E.. Ir. et al. In Water Chlori~~
nation: Chemistry, Environment ImpactandHealth Effects. Vol. 5, Jolley, R. L. et al., Eds.; Lewis Publishers: Chelsea, MI, 1985, pp. 175-84. I621 Scully, F.E.. Jr. et al. Environ. Heolth Perspec. 1986,69,259-65. I631 Nickelsen, M. G . et al. Chem. Res. Toxicol. 1991, 411). 94-101. I641 Mink, F. L. et al. Bull. Environ. Contam. Toxicol. 1983, 30, 394-99. 1651 Sussmuth, R. Mutat. Res. 1982, 105, 23-8.
