Environ. Sci. Techno/. 1985, 19, 1192-1195

Interference in the Bactericidal Properties of Inorganic Chloramines by Organic Nitrogen Compounds Roy L. Wolfe, N. Robert Ward, and Betty H. Olson”

Environmental Analysis, Program in Social Ecology, University of California, Irvlne, California 927 17

The interference of nitrogenous organic compounds on the bactericidal activity of chloramines w a ~ examined. The amino acid glycine (0.1-0.5 mg/L as nitrogen) was sup- plemented into water samples obtained from a finished water reservoir to artificially increase the levels of organic nitrogen. Inactivation rates of total count bacteria were markedly slower with chloramines (1.6 mg/L; Cl,-N, 3:l w/w; pH 8) when preammoniation and concurrent ap- plication methods were compared to preformed chlor- amines. Levels of glycine as low as 0.1 mg (as nitrogen)/L reduced the bactericidal activity with the preammoniation procedure. No decrease in the bactericidal properties was observed when preformed chloramines were assayed in the presence of glycine. Significantly, the inorganic chloramine residuals (ammonia derived) and organic chloramine re- siduals (glycine derived) could not be distinguished by amperometric or ferrous ammonium sulfate-diethyl-p- phenylenediamine titration techniques. Hence, residual measurement by conventional analytical techniques may not indicate the true Ievel of disinfection potential of chloramines.

The presence of nitrogenous organic compounds in water may significantly reduce the microbicidal activity of in- organic monochloramines by interfering with the formation and stability of this disinfectant. This interference results primarily from two sets of chemical reactions that lead to a progressive increase in the levels of organic N-chloro compounds and a corresponding decline in inorganic mo- nochloramine. The initial distribution of organic and in- organic chloramines is determined by the concentrations and relative chlorination rates of organic nitrogenous compounds and ammonia (1). If the organic N compounds bind more rapidly with chlorine than ammonia, higher levels of organic chloramines will initially be formed. This kinetically determined distribution is followed by reaction toward equilibrium which frequently favors the formation of organic chloramines (2). As such, the expected level of inorganic chloramine residual may not be achieved during chloramine formation or the level of inorganic chloramine may decrease with extended residence time in the distri- bution system.

Significantly, many organic chloramine compounds, particularly the chlorinated amino acids, have little or no bactericidal activity ( 3 , 4 ) . Compounding this problem is the inability of current analytical techniques that measure combined chlorine residuals to distinguish between inor- ganic and many forms of organic chloramines (2 ,5 ) .

To our knowledge, there is no experimental evidence to date to verify that nitrogenous organic compounds reduce the disinfectant activity of inorganic chloramine as a direct result of preferential binding and exchange reactions. We report on a series of experiments designed to demonstrate interference in the bactericidal properties of inorganic chloramines by glycine when preammoniation and con- current addition application techniques are used. This information is particularly pertinent for water treatment facilities using source water with elevated levels of inter- fering nitrogenous organic compounds because the amount of “active” inorganic chloramine in water cannot be quantified.

Unfortunately, knowledge of the types and concentra- tions of nitrogenous organic compounds in surface waters is limited (2,s). However, the presence of amino acids and other primary amines such as peptides are thought to be among the most environmentally significant forms of or- ganic nitrogen in water supplies due to their ubiquity and their interference with inorganic chloramine formation. In general, the concentrations of free amino acids in fresh water varies from a few to several hundred micrograms per liter (7). Gardner and Lee (8) found approximately 20 pg of several amino acids/L in moderately eutrophic lake water whereas Zygmuntowa (7) observed 50-200 pg of free amino acids/L in highly eutrophic pond water. Hutchin- son (9) reported that free amino N, peptides, and non- amino N averaged 74,171, and 194 pg/L, respectively, in 15 Wisconsin lakes. The concentrations of free amino N in these lakes ranged from 20 to 1 2 1 pg/L, whereas the levels of peptide N ranged from 70 to 300 pg/L. Although not stated, the nutrient status of the lakes most likely ranged from moderately oligotrophic to highly eutrophic. The degree of eutrophication in lake water is important because it will influence the amount of nitrogenous organic compounds present (10). By comparison, the concentra- tion of free amino nitrogen in sewage effluents can be expected to be an order of magnitude greater than the levels found in oligotrophic fresh waters. Isaac and Morris (11) estimate that free amino nitrogen ranges from 200 to 750 pg/L in domestic sewage effluent.

Our research findings during May and June 1982 in a finished water reservoir suggested that nitrogenous organic compounds interfered with disinfection when preammo- niation and concurrent application techniques were utilized to form inorganic chloramine. In these studies, we noted that the total count bacteria were inactivated significantly ( p < 0.05, df = 16, using linear regression analysis) more slowly at pH 8 when preammoniation and concurrent ap- plication techniques were used than when preformed in- organic chloramines were employed. Some of these data are presented in Table I. Organic amine containing compounds present in the San Joaquin Reservoir water were implicated as the interfering agents because the re- sidual chloramine concentrations, determined by amper- ometric and ferrous ammonium sulfate-diethyl-p- phenylenediamine (FAS-DPD) titration procedures, were identical regardless of disinfection application techniques. To test indirectly that organic amine compounds were interfering with inorganic chloramine formation and dis- infection, two experiments were conducted in which the amino acid glycine was supplemented into the San Joaquin Reservoir water to artifically increase the level of organic nitrogen.

These studies were conducted at 22 “C in sterile, 1-L beakers. Water samples were collected from the San Joaquin Reservoir between May and July 1982. The San Joaquin Reservoir (capacity 3 000 acre-ft) is a finished water reservoir maintained by the Metropolitan Water District (MWD) of southern California and is utilized for short-term storage. Generally, the water received by the reservoir is a blend of California State Project Water and Colorado River water, treated at the Robert B. Diemer treatment plant. This water has received full treatment

1192 Environ. Sci. Technol., Vol. 19, No. 12, 1985 0013-936X/85/0919-1192$01.50/0 0 1985 American Chemical Society

Table I. Evidence of Interference in the Inactivation of Total Bacterial Populations from the San Joaquin Reservoir Using Different Chloramine Application Techniques

sample date treatmenta X o b XI2 X Z S b 5/6/82

5/17/82d

5/20/82

preform preamm conc preform preamm conc preform preamm conc

4.02 f 0.03 (100)' 4.02 f 0.03 (100) 4.02 f 0.03 (100) 3.66 f 0.06 (100) 3.66 f 0.06 (100) 3.66 f 0.06 (100) 3.98 f 0.03 (100) 3.98 f 0.03 (100) 3.98 f 0.03 (100)

2.62e (4.0) 3.55 f 0.13 (34.0) 3.42 f 0.19 (25.1) 2.48e (6.0) 2.73 f 0.19 (1117) 2.83 f 0.20 (14.7) 2.55 f 0.31 (3.7) 3.19 f 0.15 (15.9) 3.09 f 0.09 (12.9)

1.45 f 0.17 (10.27) 3.44 f 0.21 (24.3) 3.28 f 0.34 (18.3) 0 (<0.44) 2.52 f 0.62 (7.2) 2.70 f 0.17 (11.0) 2.19 f 0.31 (11.6) 2.76 f 0.25 (5.9) 2.74 f 0.25 (5.7)

residual

::7 0 (<0.19) 3.06 f 0.11 (11.0) 3.00 f 0.08 (9.4) 0 (K0.44) 2.35 f 0.87 (4.9) 2.18 f 0.14 (3.3) 1.80 f 0.29 (0.67) 2.72 f 0.18 (5.5) 2.61 f 0.24 (4.2)

1.55 1.60 1.60 1.58 1.60 1.62 1.64 1.60 1.60

Preform = preformed chloramines; preamm = preammoniation technique; conc = concurrent application technique. Mean colony- forming units (log cfu/mL) and standard deviations after contact times of 0 (control), 15, 25, and 35 min. Each mean represents four observations. 'Numbers in parentheses represent mean percent survival. dContact times at 0, 11, 21, and 31 min. eMean represents duplicate analysis. 'Determined by ferrous ammonium sulfate-diethyl-p-phenylenediamine (FAS-DP) titrimetric procedure. Concentration measured immediately following assay.

Table 11. Interference in the Bactericidal Activity of Preammoniation and Concurrent Application Techniques by Glycine"

log mean cfu/mL bacteria surviving exposureb f SDf no glycine added 0.55 mg/L glycine added' contact

timer min preform preamm conc preform preamm conc

0 3.94 f 0.043 3.94 f 0.043 (100) 3.94 f 0.043 (100) 3.94 f 0.043 (100) 3.94 f 0.043 (100) 3.94 f 0.043 (100) 15 2.29 f 0.23 (2.24) 2.21 f 0.13 (1.86) 2.16 f 0.09 (1.66) 2.17 f 0.30 (1.70) 3.74e (63.0) 3.81e (74.1) 30 1.87 f 0.11 (0.85) 1.79 f 0.18 (0.71) 1.87 f 0.20 (0.85) 2.0 f 0.24 (1.15) 3.58 f 0.06 (43.7) 3.60 f 0.05 (45.7) 60 1.14 f 0.16 (0.16) 1.14 f 0.14 (0.16) 1.11 f 0.17 (0.15) 1.23 f 0.14 (0.19) 2.9P (11.0) 3.42e (30.2)

Inactivation of total count bacteria from the San Joaquin Reservoir (7/2/82 sample date) using preformed, preammoniation, and con- current addition techniques at 1.60 mg/L final chloramine concentration. Experiments were performed at pH 8 and 23 "C. bRepresents geometric mean of four observation after contact times of 0 (control), 15,30, and 60 min. cRepresents 0.55 mg/L nitrogen (as glycine) added prior to experiment initiation. Numbers in parentheses represent mean percents survived. e Means represent duplicate analysis. f SD = standard deviation.

consisting of coagulation, sedimentation, filtration, and chlorination. The water in the reservoir consistently ranges between pH 7.9 and pH 8.2 and carries no disinfectant residual.

Before analysis, the samples were allowed to equilibrate a t room temperature (22 "C) and mixed to disperse any aggregated bacteria and/or particulate material. Five hundred milliliters of the pooled sample was added into each sterile beaker. The amino acid glycine was then supplemented and completely mixed into the 500-mL samples. The final concentrations of glycine in the beakers ranged from 0.1 to 0.55 mg/L (nitrogen). Glycine was selected as the organic nitrogen source because it is the simplest amino acid and does not possess disinfectant interfering side chains other than the amine group. Moreover, glycine is one of the most frequently occurring amino acids isolated from aquatic environments (12,13). This amino acid has been purported to bind to chlorine approximately 13 times more rapidly than ammonia (14).

For the preammoniation studies, ammonium chloride (1.10 mg/L, nitrogen) was added and mixed 1 min prior to the addition of 500 mL of a double-strength chlorine solution (3.0-3.2 mg/L). Concurrent application studies were initiated by the simultaneous addition of ammonia and chlorine. For the preformed studies, water samples containing glycine were dosed with 500 mL of a double- strength chloramine solution (3.0-3.2 mg/L; Cl,-N, 3:l w/w). The solutions containing the disinfectant and samples were mixed at 100 rpm with a Phipps and Bird paddle stirrer for the duration of the assay. Disinfectant concentrations were measured before and after each in- activation experiment by using the amperometric and ferrous ammonium sulfate-diethyl-p-phenylenediamine

(FAS-DPD) titrimetric procedures (15). At preselected contact times, aliquots were removed and

added into 10 mL of 20 mM sodium thiosulfate (Baker) and 0.1 % Bacto-Peptone (Difco). Appropriate portions of the neutralized samples were then filtered through 0.45 pM cellulose acetate membranes (Gelman GN-6) and washed with at least 50 mL of sterile rinse water containing 10 mM phosphate buffer and 0.1 '3% Bacto-Peptone at pH 7.0. Membranes were placed on membrane Standard Plate Count Agar (16) and enumerated after a 7-day incubation at 24 "C. Data were statistically analyzed by linear re- gression and two-way analysis of variance (ANOVA) using packaged statistical programs (SPSS) contained on the Honeywell CP-6 at the University of California, Irvine.

In the first experiment, glycine was supplemented into water, collected on June 25,1982, to a level of 1.10 mg/L as nitrogen. The concurrent, preamrnoniation, and pre- formed application methods were then employed. In each case, the concentration of ammonia nitrogen (3:l w/w) was equivalent to the concentration of organic nitrogen. For comparative purposes, inactivation curves were also de- termined without supplementation of glycine by using the three application methods. These data revealed that the bactericidal activity of chloramines was significantly (p < 0.05, df = 16) reduced by glycine when preammoniation and concurrent addition techniques were utilized (Table 11). No inhibition of the bactericidal activity, however, was noted in the presence of glycine when the preformed chloramines were tested. On this particular sampling date the rates of inactivation of the total count bacteria without glycine using preformed application were not significantly different (p < 0.05, df = 16) from preammoniation and concurrent addition techniques without glycine, suggesting

1193 Environ. Scl. Technol., Vol. 19, No. 12, 1985

Table 111. Combined Chlorine Determinations Using the Amperometric and FAS-DPD Procedures for San Joaquin Reservoir Samples Supplemented with Glycine

NH2Cl measuredn application technique glycine, mg/L N ammonia, mg/L N amperometric FAS-DPD theoretical NH2Cl concnb

preformed 0 0.55 1.54 1.63 1.60 preammoniation 0.1 0.55 1.50 1.60 1.39 preammoniation 0.25 0.55 1.50 1.63 0.79 preammoniation 0.55 0.55 1.52 1.63 0.32

Residual monochloramine concentration (inorganic + organic chloramine). *Theoretical distribution of NHzCl and N-chloroglycine on the basis (i) that nitrogen species bind a maximum of 5 X the weight of chlorine (5:l C12-N w/w) and (ii) that [NH2Cl] = k,[NH,][HOCl] and [NcGly] = k2[Gly-][HOCI] where [NH,] = concentration of un-ionized ammonia at pH 8, [Gly-] = concentration of glycine anion at pH 8, [HOCl] = concentration of unionized free chlorine, kl = fundamental rate constant for reaction of NH, and HOCl at 20 OC = 3.85 X lo6 (14) , and kz = fundamental rate constant for reaction of Gly- and HOCl a t 20 OC = 4.94 X lo7 (14) .

that the levels of naturally occurring nitrogenous organic compounds in the water were low and did not interfere with chloramine formation and subsequent disinfection.

In the second experiment, die-off rates using the pre- ammoniation technique were determined with the San Joaquin Reservoir samples collected on July 16, 1982, supplemented with glycine to final concentrations of 0.1, 0.25, and 0.55 mg/L as nitrogen. For comparative pur- poses, inactivation rates were also determined by using preammoniation and preformed application without ad- dition of glycine. The final concentration of ammonia added to each solution was 0.55 mg/L as nitrogen. These data indicated that the bactericidal activity with pream- moniation was significantly reduced ( p < 0.05, df = 16) when a concentration of 0.1 mg/L organic nitrogen as glycine was tested (Figure 1). After 60 min, the counts in the preformed and preammoniation solutions that did not receive glycine supplementation were reduced from 7000 cfu/mL to 50 and 70 cfu/mL, respectively, and were not significantly different at p < 0.05. This suggested that interfering nitrogenous organic compounds in the reservoir were low on this sample date also, and hence, limited disinfection impairment of chloramines was observed. In contrast, bacterial levels in the glycine-supplemented so- lutions exposed to preammoniation for 60 min were 500 (0.1 mg/L as nitrogen), 2000 (0.25 mg/L as nitrogen), and 4000 cfu/mL (0.55 mg/L as nitrogen). Regression analyses of the inactivation rates in this experiment indicated that the rates for preammoniation with glycine supplementation (at 0.1, 0.25, and 0.55 mg/L) were statistically different a t p < 0.05 (df = 16) from both preformed and pream- moniated solutions without glycine. Moreover, the am- perometric and FAS-DPD titrimetric procedures were unable to distinguish between organic and inorganic chloramines (Table 111). The impact of relatively small concentrations of amino acids on inorganic chloramine formation is shown by the theoretically calculated con- centrations of inorganic chloramine (Table 111). For ex- ample, the presence of 0.1,0.25, and 0.55 mg/L glycine (as nitrogen) reduces the active inorganic chloramine by 13%, 51 % , and 80%, respectively.

Although the types and concentrations of organic N compounds in the San Joaquin Reservoir have not been quantified, determinations of the total Kjeldahl nitrogen and ammonia levels were determined from four locations within the Metropolitan Water District of southern Cal- ifornia (MWD) system. The levels of total Kjeldahl ni- trogen ranged from 0.19 to 0.32 mg/L. Ammonia con- centrations were on the order of 0.1 mg/L, indicating that the levels of total organic nitrogen were approximately 0.1-0.2 mg/L. The concentration of total organic nitrogen for these locations appear low compared to values reported by others (6), but it is likely that these levels fluctuate

1194 Environ. Scl. Technoi., Vol. 19, No. 12, 1985

41

TOTAL COUNT INACTIVATION

Conc. I C12:N I pH

1 BO I 3: I I 8.2

3-

2- - PREAMM 0 5 5 - PREAMM 0 25 - PREAMM 0 10

-PREAMM o -PREFORM 0

1'0 20 30 40 50 6b

Contact Time (Min) Figure 1. Inactivation of total count bacteria from the San Joaquin Reservoir using preammoniation and preformed application techniques (7/16/82 sample date). Glycine was supplemented into samples at levels of 0.1, 0.25, and 0.55 mg/L (final concentration as nitrogen) prior to preammoniation treatment. Experiments performed at pH 8.0 and 22 O C . Each datum represents the mean of two observations.

considerably during the year and are markedly influenced by the nutrient status of the reservoir and by seasonal events such as algal blooms. Morris et al. (6) observed that the concentration of total organic nitrogen in a New England municipal water supply increased from 2.2 to 21.7 mg/L during a summer algal bloom. Although not de- termined, the levels of amino acids and other primary amines in their samples most certainly increased in pro- portion to the concentration of total organic nitrogen. This suggests that interference by organic amine compounds is most likely to occur in systems that obtain source water from eutrophic lakes and rivers or during seasonal algal blooms.

Friend (17) and Morris (1) have studied the rate of reaction of chlorine with amino acids and other nitrogenous compounds. Some of Morris' revised data are presented in Table IV. It can be seen that the reaction of chlorine with certain nitrogenous compounds (alanine, glycine, methylamine, dimethylamine, and diethylamine) is more rapid than with others (ethyl aminoacetate, glycylglycyl- glycine). From these data the type of organic amine com-

Table IV. Chlorination Rates for Organic Nitrogen Compounds Relative to Ammonia

relative reaction rate

nitrogenous compound (~RNHCII~NH~C? amino acids

glycine alanine 1 e u c i n e serine glutamic acid

peptides glycylglycine glycylglycylglycine

me thylamine dimethylamine diethylamine morpholine diethanolamine ethyl aminoacetate chloramide N-chloromethylamine

others

13 16 14 - 7.6 13

1.9 1.7

77 86 16 9 9 2 5.5 x 10-6 1.8 x 10-4

kRNHCl = chlorination rate of organic nitrogen compound (I, 14); kNHzCl = chlorination rate of ammonia (14) = 3.85 x lo8 8-I at 20 OC.

pounds in the water will be more important in terms of their relative rates of N-chlorination and interference with inorganic chloramine formation than merely the amount of organic N present.

In addition, recent studies by Snyder and Margarum (18) and Isaac and Morris (2) have shown that chlorine (CP) can be transferred from inorganic chloramines to amino acids and peptides via a direct-transfer mechanism. The rate and extent to which this reaction proceeds are largely dependent upon the type and concentration of the N substrate. Isaac and Morris (2) observed that with equimolar concentrations of NH&l (pH 7, 25 “C) and several organic nitrogen compounds, including glycine (at

M), 50% of NH&l was converted to organic chlor- amine in 1-2 days. With concentrations of individual nitrogenous compbunds and 10“ M, 50% conversion re- quired only a few hours. The concentrations of nitrogenous compounds in their work were chosen to reflect the levels in oligotrophic freshwater and wastewater systems.

In summary, the preferential binding of amine-con- taining compounds such as amino acids and peptides with chlorine could greatly reduce the disinfection potential of inorganic chloramines. In addition, long retention times in distribution systems may result in conversion from in- organic chloramines to the less microbicidal organic chloramines. This problem is compounded by the inability of any currently employed analytical technique to spe- cifically measure the levels of inorganic chloramine in drinking water. Current research efforts should be directed toward determining the types and concentrations of or-

ganic amine compounds in drinking water and their impact on disinfection with chloramines.

Acknowledgments

We thank Edward G. Means and Michael J. McGuire of the Metropolitan Water District of southern California (MWD) for their valuable comments on the manuscript.

Registry No. NH2C1, 10599-90-3; Gly, 56-40-6; NP, 7727-37-9.

Literature Cited (1) Morris, J. C. “Principles and Applications of Water

Chemistry”; Faust, S. D.; Hunter, J. V., Eds.; Wiley: New York, 1967; Chapter 2.

(2) Isaac, R. A.; Morris, J. C. Environ. Sei. Technol. 1983,17,

(3) Feng, T. H. J. Water Pollut. Control Fed. 1966, 38 (4),

(4) Krusg, C. W.; Hsu, Y.; Griffiths, A. C.; Stringer, R. “Halogen Action on Bacteria, Viruses and Protozoa”. Proc. Natl. Spec. Conf. Disinfect., 1970, 113-136.

(5) White, G. C. “Disinfection of Wastewater and Water for Reuse”; Van Nostrand Reinhold New York, 1978; Chapter 2.

(6) Morris, J. C.; Ram, N.; Baum, B.; Wajon, E. “Formation and Significance of N-chloro Compounds in Water Supplies”. 1980, EPA-600/2-80-031.

738-742.

614-628.

(7) Zygmuntowa, J. Acta Hydrobiol. 1972, 14, 317-325. (8) Gardner, W. S.; Lee, G. F. Environ. Sei. Technol. 1973, 7,

(9) Hutchinson, G. E. “A Treatise on Limnology”; Wiley: New 719-724.

York, 1957; p 1015. (10) Gocke, K. Arch. Hydrobiol. 1970, 67, 285-367. (11) Isaac, R. A.; Morris, J. C. in “Water Chlorination: Envi-

ronmental Impacts and Health Effects”; Jolley, R. L.; Brungs, W. A,; Cumming, R. B., Eds.; Ann Arbor Science: Ann Arbor, MI, 1980; Vol. 3, Chapter 17.

(12) Litchfield, C. D.; Prescott, J. M. Limnol. Oceanogr. 1978,

(13) Newell, I. L. J. N. Engl. Water Works Assoc. 1976, 90,

(14) Isaac, R. A.; Morris, J. C. “Water Chlorination: Environ- mental Impact and Health Effects”; Jolley, R. L.; Brungs, W. A.; Cotruvo, J. A.; Cumming, R. B.; Mattice, J. S.; Jacobs, V. A., Eds.; Ann Arbor Science: Ann Arbor, MI, 1983; Vol. 4, Chapter 3.

(15) “Standard Methods for the Examination of Water and Wastewater”, 14th ed.; American Public Health Association: Washington, DC, 1975.

(16) Taylor, R. H.; Geldreich, E. E. J.-Am. Water Works Assoc. 1979,44, 1110-1117.

(17) Friend, A. G. Ph.D. Dissertation, Harvard University, Cambridee. MA. 1956.

15, 250-256.

315-324.

(18) Snyder, M.’P.; Margarum, D. W. Inorg. Chem. 1982, 71, 2545-2550.

Received for review July 16, 1984. Revised manuscript received December 7, 1984. Accepted July 11, 1985. The research was supported by a grant (MWD 1373) from the Metropolitan Water District of Southern California.

Environ. Sci. Technol., Vol. 19, No. 12, 1985 1195