Inactivation of Mycobacterium avium with Monochloramine

Sep 25, 2008 - University of Illinois at Urbana-Champaign, Urbana, Illinois. 61801, Department of Civil and Environmental Engineering,. University of ...
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Environ. Sci. Technol. 2008, 42, 8051–8056

Inactivation of Mycobacterium avium with Monochloramine J E A N N E L U H , †,§ N I N G T O N G , † L U T G A R D E R A S K I N , ‡,§ A N D B E N I T O J . M A R I Ñ A S * ,†,§ Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, Department of Civil and Environmental Engineering, University of Michigan at Ann Arbor, Ann Arbor, Michigan 48109, Center of Advanced Materials for the Purification of Water with Systems, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received April 24, 2008. Revised manuscript received July 10, 2008. Accepted August 8, 2008.

Batch experiments were performed to study the inactivation kinetics of Mycobacterium avium in the presence of monochloramine at 5-30 °C, pH 6-10, and 0.30-42.3 mg Cl2/ L. For each temperature and pH investigated, limiting high and low inactivation rates were observed for high and low disinfectant concentrations, respectively, within the range investigated. The rate of inactivation transitioned from high to low over a relatively narrow range of intermediate monochloramine concentrations. The observed temperature dependence of inactivation was consistent with an Arrhenius expression with activation energies of 58.0 and 71.7 kJ/mol for the high and low concentration ranges, respectively. The rate of inactivation increased with decreasing pH, consistent with trends reported for the reaction of monochloramine with protein thiols. Experiments performed at pH ∼3.5 showed that dichloramine was a weaker disinfectant than monochloramine, and that its contribution to the overall inactivation of M. avium with combined chlorine was negligible at pH 6-10. A kinetic model incorporating disinfectant concentration, temperature, and pH effects was used to illustrate that monochloramine efficiency to inactivate M. avium in water could vary broadly from adequate (e.g., 99.9% inactivation efficiency in 32 min at 5 mg Cl2/L, pH 6, 30 °C) to impractical (e.g., 99.9% inactivation efficiency in 9 d at 1 mg Cl2/L, pH 9, 5 °C).

Introduction To provide protection against outbreaks of waterborne diseases, drinking water utilities in the United States use various chemical disinfectants to control the presence of infective pathogens in treatment plants and distribution systems. Free chlorine is currently the disinfectant most commonly used to provide a protective residual in distribution systems (1). However, with the promulgation of the Stage 2 Disinfectants and Disinfection Byproducts (DBPs) Rule (2), * Corresponding author phone: (217) 333-6961; fax: (217) 3336968; e-mail: [email protected]. † Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign. ‡ University of Michigan at Ann Arbor. § Center of Advanced Materials for the Purification of Water with Systems, University of Illinois at Urbana-Champaign. 10.1021/es801133q CCC: $40.75

Published on Web 09/25/2008

 2008 American Chemical Society

many utilities have switched or will be switching from free chlorine to monochloramine with the goal of reducing the formation of regulated DBPs. There is therefore a need to assess the effectiveness of monochloramine for controlling emerging waterborne pathogens. One such pathogen is Mycobacterium avium, a waterborne opportunistic bacterium responsible for pulmonary infections in both immunocompetent and immunocompromised patients (3, 4), and the causative agent of lymphadenitis in children (5) and disseminated infections in HIV-positive patients (4). Person-to-person transmission of M. avium has not been observed (6), suggesting an environmental source for infection. Its isolation from drinking water systems (7, 8) and the identical DNA fingerprint patterns between M. avium isolates from AIDS patients and from their water supply points to drinking water as a potential source of M. avium infection (4, 8). Accordingly, the U.S. Environmental Protection Agency is evaluating M. avium for potential future regulation and has included it in its Preliminary Contaminant Candidate List (9). The presence of M. avium in drinking water containing a disinfectant residual has been credited to its known resistance to free chlorine and chloramines (10-14). However, while current literature demonstrates the resistance of M. avium to monochloramine (10, 11, 14), the need for a systematic study of the effects of water quality parameters such as pH, temperature, and disinfectant concentration on the inactivation kinetics of M. avium with monochloramine was identified and thus selected as the objective of this project. The results from this study will also provide information needed for assessing the potential development of future regulations for the control of M. avium in drinking water.

Materials and Methods Mycobacterium avium Stocks. Three shipments of M. avium strain ATCC 15769 (American type Culture Collection, Manassas, VA), designated as stocks 1, 2, and 3, were used for this study. Stock 1 was prepared in the following manner. The freeze-dried pellet from ATCC was aseptically rehydrated in liquid medium consisting of Middlebrook 7H9 broth (Difco 7H9) containing 10% (vol/vol) Middlebrook OADC Enrichment (Difco 0714) and dissolved in 0.2% (vol/vol) glycerol solution. A pure culture stock was obtained by performing the following procedure twice: streaking the bacterial suspension onto an agar plate, incubating at 37 °C for 18 d in an Isotemp Incubator (Fisher Scientific, Pittsburgh, PA), and picking a single opaque smooth colony for growth in liquid medium. The final culture was grown to stationary phase and centrifuged, and the pellet was resuspended in 20% vol/ vol of sterilized glycerol, before distributing the resulting suspension into vials and storing them at -80 °C. Preparation of stocks 2 and 3, corresponding to stocks 1 and 2 of Luh and Marin ˜ as (13), was similar to the preparation of stock 1, with the notable difference that the entire freeze-dried pellet from ATCC was used to prepare the stock bacterial suspension, as opposed to the use of a single colony. Preparation of Experimental Bacterial Suspension. For each disinfection experiment with stock 1, one vial was taken from the freezer and cells were recovered at 10,820g for 10 min in a refrigerated Sorvall model RC-5B centrifuge (Dupont, Hoffman States, IL). The pellet was resuspended in Middlebrook 7H9 liquid medium (prepared in 0.2% glycerol solution) containing 10% OADC Enrichment, and followed by two additional wash and recovery cycles. The resulting pellet was resuspended into 50 mL of liquid medium and incubated at VOL. 42, NO. 21, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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37 °C for 5 days to reach midlog phase. For each experiment, 5 mL of the resulting culture was centrifuged 3 times at 10,820g for 10 min, followed by resuspension in autoclaved 0.01 M phosphate buffer solution (PBS) of the desired pH. For disinfection experiments with stocks 2 and 3, preparation of the bacterial suspension was described previously (13), where 10 mL of the culture was concentrated to 5.5 mL using a Forma Centrifuge model 5528 (Thermo Electron Corporation, Marietta, OH) at 1,633g for 10 min. For stock 3, an additional centrifugation step at 170g for 5 min was performed to eliminate the possibility of clumping or large aggregates. Experimental Matrix. Experiments were performed to assess the effect of disinfectant concentration (0.30-42.3 mg Cl2/L) on the inactivation kinetics of M. avium with monochloramine at pH 8 and temperatures of 5, 20, and 30 °C (Table S1 in the Supporting Information). Experiments were also conducted at pH 6 and 10 to investigate the effect of pH on the inactivation kinetics of M. avium with monochloramine at 20 °C. The experimental pH values and temperatures were chosen to reflect the typical pH range of 6-9 for drinking water sources and winter to summer temperatures. Two experiments (51 and 52, Table S1) were performed at a target pH of 3.5 and 20 °C to assess the role of dichloramine on the inactivation kinetics of M. avium with combined chlorine. Test 51 contained dichloramine as the only measurable combined chlorine species, while Test 52 had both monochloramine and dichloramine present at comparable levels. Monochloramine Disinfection Experiments. Monochloramine disinfection experiments were performed in a 500 mL batch reactor immersed in a constant temperature water bath. Buffered monochloramine solution at target concentrations was prepared by combining sodium hypochlorite solution with excess ammonium chloride as described by Driedger et al. (15). A predetermined volume of M. avium stock suspension was added to 195 mL of monochloramine solution to start the experiment, and samples (5 mL) were subsequently taken at predetermined times and placed in 100 mL dilution bottles containing 90 mL of 0.01 M PBS (pH 7) and 5 mL of 0.12-1.0% sodium thiosulfate solution. The control sample (N0 sample), of ∼107 cfu/mL (Table S1), was taken immediately after the addition of the M. avium suspension, and the monochloramine concentration was measured at the beginning and end of each experiment (Table S1) using either the colorimetric or titrimetric DPD method (16). The CT value (exposure to disinfectant) was calculated using the assumption of firstorder decay of monochloramine over time. No free chlorine was detected in the samples. Control experiments were performed by maintaining M. avium in PBS buffer for the duration of the experiment to account for any natural decay of M. avium. Viability was determined following the method described in Luh and Marin ˜ as (13) using membrane filtration and colony enumeration. Dichloramine Disinfection Experiments. Disinfection experiments 51 and 52, designed to assess the potential role of dichloramine, were performed using the same experimental procedure as the one used for the monochloramine disinfection experiments. Dichloramine was prepared in accordance with the procedure of Hand and Margerum (17). In brief, a monochloramine solution of 10 mg of Cl2/L at pH 8 and 20 °C was first prepared. The pH of the solution was then gradually adjusted from pH 8 to 3.49-3.53 using perchloric acid and kept in the dark for 40 min (experiment 52) or 20 h (experiment 51). In experiment 51, the pH of the solution increased from 3.49 to 3.58 after 20 h.

Results and Discussion Effect of Disinfectant Concentration. Experimental results obtained to assess the effect of initial disinfectant concen8052

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FIGURE 1. Inactivation kinetics of M. avium with monochloramine at pH 8, 20 °C for (a) Stock 1, prepared from a single colony and (b) Stock 3, prepared from an entire freeze-dried vial of M. avium. tration, c0 ) 0.50-20.8 mg Cl2/L, on the inactivation kinetics of M. avium (stocks 1 and 3) with monochloramine at pH 8, 20 °C are presented in Figure 1. As depicted in Figure 1a for stock 1, different curves were generally obtained for different monochloramine concentrations tested. Each curve was characterized by a single phase of pseudofirst order kinetics without observable lag phase and thus was consistent with the Chick-Watson expression: N ) exp(-kiCT) N0

(1)

where N/N0 is the fraction of viable M. avium cells after time t of exposure to the disinfectant, ki is the second order inactivation rate constant in L/(mg × min), and CT is in mg × min/L. As illustrated in Figure 1a, the rate of inactivation was found to increase with increasing monochloramine concentration, with the occurrence of a maximum and minimum inactivation rate at the high and low ranges of disinfectant concentrations tested. This is in contrast to the single unique curve generally found in inactivation, regardless of disinfectant concentration used. Experimental data sets at the three lowest concentrations tested, c0 ) 0.50, 1.00, and 1.96 mg Cl2/L, produced a unique curve with inactivation rate constant kl that will be referred to as the low concentration range or low limiting rate curve. Similarly, data sets at the two highest concentrations c0 ) 5.00 and 10.0 (average of 13 individual experiments) mg Cl2/L, i.e., high concentration range, produced a high limiting rate curve with inactivation rate constant kh. Data sets at intermediate monochloramine concentrations, c0 ) 2.50 and 3.00 mg Cl2/L, produced kinetic curves with inactivation rate constants ki between the two limiting rate constants, or kl < ki < kh. The fact that these inactivation curves fell between the low and high rate limiting curves

TABLE 1. Summary of Rate Constants for the Inactivation of M. avium with Monochloramine temp (°C)

pH

stock no. c0 (mg/L as CL2) ki ( × 103) (L/mg × min)

5

8

1

0.98-5.60 7.00 8.33 11.2-42.3

0.55(0.01 1.69(0.05 2.20(0.04 2.85(0.04

20

8

1

0.50-2.00 2.50 3.00 5.00-10.0

3.16(0.08 4.43(0.05 7.58(0.11 12.3 (0.17

20

8

2

0.58-1.04 4.59-11.5

3.59(0.05 11.6 (0.23

20

8

3

0.53-2.01 2.53-3.05 4.07-5.12 7.66-20.8

3.35(0.11 4.38(0.08 7.02(0.17 10.3 (0.30

30

8

1

0.30-0.50 1.07 2.14 4.20-8.10

6.93(0.24 10.7 (0.67 16.4 (0.90 21.9 (0.17

20

3.58a 3.53b

1 1

N.D.a 5.94

–a 19.8b

20

6

1

0.50-1.00 2.50 3.00 5.00-10.0

4.54(0.11 9.68(0.13 14.2 (0.28 19.3 (0.59

20

10

1

0.50-1.00 2.50 3.00 5.60-11.0

2.55(0.05 3.51(0.04 4.37(0.12 7.68(0.13

a Dichloramine was the only measurable combined chlorine species and so no rate constant for monochloramine was obtained. The inactivation rate constant for dichloramine obtained from fitting data in Figure 5 with eq 1 was kd ) 0.0067 L/(mg × min). b Monochloramine and dichloramine were both present at measurable levels. The rate constant for monochloramine was obtained with eq 3 using kd ) 0.0067 L/(mg × min) for dichloramine.

revealed a transition in inactivation rate for monochloramine concentrations between 1.96 and 5.00 mg Cl2/L. Low and high rate limiting data sets and the two transition curves were fitted with eq 1. The resulting fitted lines are shown in Figure 1a, and the corresponding inactivation rate constants are listed in Table 1. To determine whether the observed effect of monochloramine concentration was stock-dependent, two additional stocks were tested at pH 8 and 20 °C. Results obtained with stock 3 are shown in Figure 1b, and those with stock 2 are presented in Figure S1 of the Supporting Information. The fitting lines from stock 1 (Figure 1a) are reproduced in Figures 1b and S1 for comparison. As depicted in Figure 1b, the resulting inactivation rates for stock 3 at c0 ) 0.53-2.01 mg Cl2/L and c0 ) 7.66-20.8 mg Cl2/L were generally consistent with the respective high rate and low rate limiting lines obtained for stock 1. Fitting these two limiting data sets of stock 3 with eq 1 resulted in kl ) 0.00335 L/(mg × min) and kh ) 0.0103 L/(mg × min), both of which were within 17% of the corresponding inactivation rate constants for stock 1. A more significant difference between stocks 1 and 3, however, was the concentration ranges at which the high and low limiting kinetic curves were observed, and the lack of a gradual transition in the transitional concentration range. In contrast to observation with stock 1, the two data sets of stock 3 at 5.01 and 5.12 mg Cl2/L did not fall into the high

rate limiting curve. Data sets at c0 ) 2.53-3.05 mg Cl2/L for stock 3 matched that obtained at c0 ) 2.50 mg Cl2/L for stock 1, while those obtained at c0 ) 4.07-5.12 mg Cl2/L for stock 3 were more consistent with that at c0 ) 3.00 mg Cl2/L with stock 1. The results obtained with stock 2 (see Figure S1 of the Supporting Information) were similar, and the corresponding low and high rate limiting constants (c0 ) 0.58-1.04 mg Cl2/L and c0 ) 4.59-11.5 mg Cl2/L, respectively, Table 1) were within 15% of the corresponding values for stock 1. Although in contrast to the stock 3 results, stock 2 data sets at c0 ) 4.59 and 4.68 mg Cl2/L were generally consistent with the high rate limiting curve of stock 1, tailing effects were observed at the highest inactivation levels investigated for stock 2 (Figure S1). This inactivation curve tailing effect, also observed for some of the curves with stock 3 (Figure 1b), was consistent with observations of inactivation of M. avium with free chlorine, for which a two-population model of susceptible and tolerant cells was used to represent the two-phase kinetics (13). The appearance of the second phase of slower kinetics in stocks 2 and 3 and its absence in stock 1 may be attributed to differences in stock preparation. The use of the entire freeze-dried pellet in stocks 2 and 3 may have resulted in a stock of mixed resistances, especially as M. avium is known to have colony types with different levels of resistance to antimicrobials (18). However, only the opaque dome-shaped smooth colonies, as determined by visual inspection, were consistently observed after the 15 day incubation period, with no other colony types present. The concentration effect observed in this study for M. avium has been reported for other mycobacteria. Pelletier et al. (11) observed a monochloramine concentration dependence at pH 7 and 17 °C within the first log of inactivation for M. avium (strains 723 and 743) and M. chelonei, where greater inactivation was achieved at 6.5 than at 1.0 mg Cl2/L. While the data available were limited, the two M. avium strains tested by Pelletier et al. showed more resistance at 1.0 mg Cl2/L, in the low limiting concentration range, than the strain used in this study. For a CT of 1440 mg × min/L, only ∼15% (less than 1-log) inactivation was obtained in the study of Pelletier et al., while 2-log (99%) inactivation was achieved at 1500 mg × min/L in this study. One hypothesis to explain the disinfectant concentration dependence at constant pH would be the occurrence of an adaptive response similar to that reported for E. coli (19). If so, it would be reasonable to assume that the adaptive response would be effective in repairing oxidative damage within the cell induced by monochloramine at relatively low concentrations, and would become ineffective at relatively high monochloramine concentrations when the rate of damage exceeds that of limiting reactions involved in oxidative damage repair. Effect of Temperature. The results for the inactivation of M. avium (stock 1) with monochloramine at pH 8 and temperatures of 5 and 30 °C are presented in Figure 2a and b, respectively. Consistent with the results of Figure 1a, two distinct limiting rate curves were achieved at relatively high and low concentration ranges. However, the concentration range for each of the kinetic curves obtained differed at each temperature; the concentration ranges for the low limiting curves were c0 ) 1.00-5.60 mg Cl2/L at 5 °C and c0 ) 0.30-0.50 mg Cl2/L at 30 °C, and the concentration ranges for the high limiting curves were c0 ) 11.2-42.3 mg Cl2/L at 5 °C and c0 ) 4.20-8.10 mg Cl2/L at 30 °C. The transition concentrations were also found to decrease with increasing temperature. Data sets at the low, high, and transition concentration ranges were fitted with eq 1 and the resulting fitting parameters are presented in Table 1. The second order rate constants obtained by fitting the inactivation curves at the low and high limiting concentration VOL. 42, NO. 21, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Effect of temperature on the inactivation kinetics of M. avium (Stock 1) with monochloramine at pH 8.

FIGURE 3. Arrhenius plot of the inactivation rate constants for the high (kh) and low (kl) concentration ranges. ranges are plotted against the corresponding temperatures in Figure 3 according to the Arrhenius expression:

( )

ki ) A × exp -

Ea RT

(2)

where ki is the inactivation rate constant of the low (kl) or high (kh) concentration range in L/(mg × min), A is the collision frequency parameter in L/(mg × min), Ea is the activation energy in J/mol, R ) 8.314 J/(mole × K) is the ideal gas constant, and T is absolute temperature in K. The parameters obtained by least-squares fitting of each set of rate constants with eq 2 are presented in Figure 3. The high concentration range was found to have a lower activation energy Ea) 58.0 kJ/mol compared to Ea) 71.7 kJ/mol for the low concentration range, although both were lower than the values reported for the inactivation of M. avium with free chlorine (96.5-100.3 kJ/mol) (13) and chlorine dioxide (74.1 kJ/mol) (20). Effect of pH. Experimental results for the inactivation of M. avium (stock 1) with monochloramine at 20 °C, and pH 6 and 10 are shown in Figure 4. Consistent with the pH 8 results in Figures 1a and 2, limiting kinetic curves at relatively high and low concentration ranges, and transitional curves at intermediate concentrations were observed at pH 6 and 10. Both high range and low range kinetics resulted in approximately the same monochloramine concentration ranges for all pH conditions, i.e., the low range limiting line occurred at c0 ) 0.50 and 1.00 mg Cl2/L and the high range limiting line occurred at c0 ) ∼5.00-10.0 mg Cl2/L for all three pH values. The experimental curves in Figure 4 were fitted with eq 1 and the resulting inactivation rate constants (Table 1) revealed that both limiting inactivation rates increased with decreasing pH. 8054

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FIGURE 4. Effect of pH on the inactivation kinetics of M. avium (Stock 1) with monochloramine at 20 °C. The possibility that dichloramine, expected to be present at higher levels at lower pH, may have contributed to the overall inactivation efficiency observed, was assessed in experiments 51 and 52. These experiments were performed at a pH ∼3.5 and 20 °C and results are shown in Figure 5. The CT values used to plot the data were calculated based on dichloramine (experiment 51) or monochloramine (experiment 52) concentrations. As depicted in the figure, the inactivation of M. avium (stock 1) with a mixture of monochloramine and dichloramine (experiment 52) was faster than that with dichloramine only (experiment 51). Furthermore, the inactivation rate constant for dichloramine obtained by fitting the corresponding data in Figure 5 with eq 1, k ) 0.00670 L/(mg × min), was too low for dichloramine to have an impact on the overall inactivation rate at pH 6-10 because the dichloramine concentrations in this pH range are much lower than those at pH ∼3.5. The inactivation rate constant obtained for dichloramine could be used to calculate that for monochloramine at pH 3.53 when the two combined chlorine species were present.

genation. Additionally, the reaction of thiols with organic chloramines is known to be pH dependent, with protonated chloramines reacting with the thiolate groups (22), which would be consistent with the pH effect observed in Figure 6. If the reaction with thiols is assumed to be primarily responsible for the pH-dependent component of the overall inactivation of M. avium, the observed inactivation rate constants kl and kh could be expressed as: kl ) kl,1 + kl,2

[H+] [H+] + Ka

kh ) kh,1 + kh,2

FIGURE 5. Comparison of the inactivation kinetics of M. avium (Stock 1) with dichloramine (pH 3.58, 20 °C) and a mixture of monochloramine and dichloramine (pH 3.53, 20 °C) to the high concentration range inactivation curves at pH 6, 8, and 10 (20 °C).

FIGURE 6. Effect of pH on the rate constant for the inactivation of M. avium (Stock 1) with monochloramine at 20 °C. Assuming that the two disinfectants acted simultaneously but without interfering with each other, the following expression equivalent to eq 1 could be used: N ) exp(-kmCTm - kdCTd) N0

(3)

in which the subscripts “m” and “d” are used to represent monochloramine and dichloramine, respectively. The resulting inactivation rate constant for monochloramine at pH 3.53 (experiment 52) is 0.0198 L/(mg × min), or within 3% of kh ) 0.0193 L/(mg × min) at pH 6. The overall pH dependence of kh and kl values is depicted in Figure 6. Assuming that the rate constant obtained for monochloramine at pH 3.53 represents the kinetics at the high concentration range, then one hypothesis for the pH dependence of kh would be the occurrence of two reaction terms: a pH independent term and a pH dependent one. From the literature, it is known that monochloramine can react with certain functional groups present in amino acids. Chlorine can transfer from monochloramine to the amine (R-NH2) groups (21), and some of the resulting organic chloramines as well as monochloramine can oxidize thiol (R-SH) groups (22, 23). Thiols are thought to play a key role in cell metabolism (22), with their oxidation possibly resulting in inactivation. This would be consistent with the results of Jacangelo et al. (24), who observed that the inactivation of E. coli by monochloramine corresponded to an inhibition of protein-associated processes such as substrate dehydro-

(4)

[H+] [H+] + Ka

(5)

in which kl,1 and kh,1 are pH independent components of the overall second order inactivation rate constant, kl,2 and kh,2 are second order inactivation rate constants associated with the overall reaction of protonated monochloramine with thiolate groups, and Ka is an effective acid-base dissociation constant for the reactive thiol groups. Least-square fitting of the experimental kl and kh values in Figure 6 with eqs 4 and 5 resulted in kl,1 ) 0.00254 L/(mg × min), kl,2 ) 0.00205 L/(mg × min), pKa,l ) 7.64, and kh,1 ) 0.00761 L/(mg × min), kh,2 ) 0.0120 L/(mg × min), pKa,h ) 7.80. As depicted in Figure 6, eqs 4 and 5 provided a good representation for the pH dependence of the observed low and high limiting rate constants. The approximately 3-fold increase of the pHindependent rate constants, from kl,1 ) 0.00254 L/(mg × min) to kh,1 ) 0.00761 L/(mg × min), would be consistent with M. avium losing the ability to repair DNA damaged by monochloramine oxidation, as discussed previously. In contrast, the nearly 6-fold increase in the pH-dependent rate constant, from kl,2 ) 0.00205 L/(mg × min) to kh,2 ) 0.0120 L/(mg × min), could not be explained by a loss of repair ability because the pH effect suggests that inactivation is the result of damage of vital external biomolecules, assumed to be proteins herein, exposed to the outer aqueous solution and not an intracellular process. Instead, this increase in reaction rate with increasing monochloramine concentration could be explained by changes of the protein structure as a result of the increased number of amine groups that react with monochloramine. Protein backbone fragmentation, which has been reported to be a possible outcome of chloramine formation at lysine residues (25), would provide increased access of aqueous solution and chloramines to the thiols located in less accessible areas, thus resulting in higher inactivation rates. Model Application. Equations 1, 2, 4, and 5 and corresponding parameters can be used to predict the inactivation kinetics of M. avium with monochloramine as a function of disinfectant concentration (except for the concentration range at which the transitional inactivation kinetics takes place), temperature, and pH. In addition, these equations can be used to determine the treatment requirements for a certain level of inactivation assuming that the activation energy value (Ea,h ) 58,000 J/mol) determined for kh at pH 8 could be applied to represent the temperature dependence of both kh,1 and kh,2, and similarly Ea,l ) 71,700 J/mol obtained for kl at pH 8 could be used for the temperature dependence of both kl,1 and kl,2. The corresponding collision frequency parameters can then be calculated as Ah,1 ) 1.64 × 108, Ah,2 ) 2.13 × 108, Al,1 ) 1.52 × 1010, and Al,2 ) 1.22 × 1010 L/(mg × min). The resulting inactivation model could be used to compare treatment requirements under different conditions as illustrated with the following example. The contact time that would be required for 99.9% inactivation of M. avium with 5 mg Cl2/L of monochloramine at pH 6 and 30 °C would be 32 min (CT ) 160 mg × min/L), as opposed to 9 d (CT ) 12,900 mg × min/L) for 1 mg Cl2/L of monochloramine VOL. 42, NO. 21, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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at pH 9 and 5 °C. Another interesting comparison would be the treatment requirements for the alternative disinfectant free chlorine which can be estimated with a previously developed model (13). The contact times that would be required for 99.9% inactivation of M. avium would be 12 min when using 5 mg Cl2/L of free chlorine at pH 6 and 30 °C (CT ) 60 mg × min/L), and 132 d when using 1 mg Cl2/L of free chlorine at pH 9 and 5 °C (CT ) 190,000 mg × min/L). Notice that while free chlorine is approximately three times more effective than monochloramine at 5 mg Cl2/L, pH 6 and 30 °C, monochloramine is 15 times more effective than free chlorine at 1 mg Cl2/L, pH 9 and 5 °C, although neither of the two disinfectants would provide protection under practical conditions against M. avium contamination at the latter set of conditions.

(8)

(9) (10)

(11)

(12)

Acknowledgments We thank Dr. Benito Corona-Vasquez and Dr. Chuanwu Xi for training provided in the early stages of this study, and Orlando Coronell for helpful discussions and suggestions. The Illinois Distinguished Fellowship program of the University of Illinois at Urbana-Champaign, the United States Environmental Protection Agency (cooperative agreement CR-826461010), and the WaterCAMPWS, a Science and Technology Center of Advanced Materials for the Purification of Water with Systems (NSF agreement CTS-0120978) are gratefully acknowledged for their support. The opinions of this paper do not necessarily reflect those of the sponsors.

(13) (14) (15) (16)

Supporting Information Available

(17)

Additional experimental data. This information is available free of charge via the Internet at http://pubs.acs.org.

(18)

Literature Cited (1) U.S. EPA. 40 CFR Parts 9, 141, and 142 National Primary Drinking Water Regulations: Long Term 2 Enhanced Surface Water Treatment Rule; Final Rule. Fed. Regist. 2006, 71 (3), 653–702. (2) U.S. EPA. 40 CFR Parts 9, 141, and 142 National Primary Drinking Water Regulations: Stage 2 Disinfectants and Disinfection Byproducts Rule; Final Rule. Federal Register 2006, 71 (2), 388– 493. (3) Huang, J. H.; Kao, P. N.; Adi, V.; Ruoss, S. J. Mycobacterium avium-intracellulare Pulmonary Infection in HIV-Negative Patients without Preexisting Lung Disease: Diagnostic and Management Limitations. Chest 1999, 115, 1033–1040. (4) von Reyn, C. F.; Maslow, J. N.; Barber, T. W.; Falkinham, J. O., III.; D’Arbelt, R. Persistent Colonisation of Potable Water as a Source of Mycobacterium avium Infection in AIDS. Lancet 1994, 343, 1137–1141. (5) Wolinsky, E. Mycobacterial Lymphadenitis in Children: A Prospective Study of 105 Nontuberculous Cases with LongTerm Follow-Up. Clin. Infect. Dis. 1995, 20, 954–963. (6) Falkinham, J. O., III. Epidemiology of Infection of Nontuberculous Mycobacteria. Clin. Microbiol. Rev. 1996, 9, 177–215. (7) Falkinham, J. O., III.; Norton, C. D.; LeChevallier, M. W. Factors Influencing Numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in Drinking Water

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