APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1988, p. 649-654 Vol. 54, No. 30099-2240/88/030649-06$02.00/0Copyright C 1988, American Society for Microbiology

Factors Promoting Survival of Bacteria in ChlorinatedWater Supplies

MARK W. LECHEVALLIER,* CHERYL D. CAWTHON, AND RAMON G. LEE

American Water Works Service Co., Inc., Belleville Laboratory, 1115 South Illinois Street, Belleville, Illinois 62220

Received 14 September 1987/Accepted 21 December 1987

Results of our experiments showed that the attachment of bacteria to surfaces provided the greatest increasein disinfection resistance. Attachment of unencapsulated Klebsiella pneumoniae grown in medium with highlevels of nutrients to glass microscope slides afforded the microorganisms as much as a 150-fold increase indisinfection resistance. Other mechanisms which increased disinfection resistance included the age of thebiofilm, bacterial encapsulation, and previous growth conditions (e.g., growth medium and growth tempera-ture). These factors increased resistance to chlorine from 2- to 10-fold. The choice of disinfectant residual wasshown to influence the type of resistance mechanism observed. Disinfection by free chlorine was affected bysurfaces, age of the biofilm, encapsulation, and nutrient effects. Disinfection by monochloramine, however, wasonly affected by surfaces. Importantly, results showed that these resistance mechanisms were multiplicative(i.e., the resistance provided by one mechanism could be multiplied by the resistance provided by a secondmechanism).

The occurrence of coliform bacteria in otherwise high-quality drinking water has been a nemesis to the drinkingwater industry. Experience has shown that maintenance of achlorine residual cannot be relied upon to totally prevent theoccurrence of bacteria (13, 15, 20, 22, 29, 30, 38). Often, themechanisms responsible for the survival of coliform bacteriain drinking water supplies are unknown or poorly under-stood. As a result, solutions to the coliform occurrences areoften speculative and unsubstantiated.

Available research has shown that increased resistance todisinfection may result from the attachment of microorgan-isms to or the association of microorganisms with varioussurfaces, including macroinvertebrates (Crustacea, Nema-toda, Platyhelminthes, and Insecta) (26, 36), turbidity parti-cles (17-19, 23, 31), algae (34), carbon fines (7, 24), and evenglass microscope slides (29). Ridgway and Olson (31) haveshown that the majority of viable bacteria in chlorinateddrinking water are attached to particles. Presumably, mi-crobes entrapped in particles or adsorbed onto surfaces areshielded from disinfection and are not inactivated.

Several investigators have reported the isolation of encap-sulated bacteria from chlorinated drinking water (11, 30).They concluded that production of the extracellular capsulehelps to protect bacteria from chlorine. However, only cir-cumstantial evidence was given to support these conclusions.

Stewart and Olson (35) reported that the aggregation ofAcinetobacter sp. strain EB22 increased resistance to chlo-ramines by twofold. The researchers found that treatment ofthe strain with Tween 80 (a surfactant) eliminated theincreased disinfection resistance. Sloughing of cell aggre-gates from treatment filters or pipe walls has been suggestedas a possible mechanism by which coliform bacteria occur indrinking water supplies.Carson et al. (8) have reported that Pseudomonas aerugi-

nosa growing in distilled water was markedly more resistantto acetic acid, glutaraldehyde, chlorine dioxide, and a qua-ternary ammonium compound than were cells cultured ontryptic soy agar (TSA). In similar work by Berg et al. (3) and

* Corresponding author.

Harakeh et al. (16), it has been shown that bacteria grown ina chemostat at low temperatures and submaximal growthrates caused by nutrient limitation, conditions similar tothose in the natural aquatic environment, were resistant toseveral disinfectants. Legionella pneumophila grown in alow-nutrient, natural environment has been reported to be 6to 9 times more resistant than cells grown on agar (21). Berget al. (3) speculated that the increased resistance was due tochanges in the cell membrane permeability of slow-growingbacteria.Wolfe et al. (39) found that a number of bacterial genera

found in chlorinated water demonstrated a variety of disin-fection resistance patterns to free chlorine and monochlora-mine. Ward et al. (37) reported that a Flavobacterium strainwas more sensitive to monochloramine than to free chlorine.The fact that disinfection itself can select for a variety ofbacteria has been demonstrated by the results of work ofseveral researchers (2, 25, 28), who have indicated thatchlorination of water supplies select, for survivors which aremultiply antibiotic resistant. The results indicate that selec-tive pressures of water treatment can produce microorgan-isms with resistance mechanisms favoring survival in anotherwise restrictive environment.

Despite the research cited above, no comprehensive studyhas been conducted to evaluate the relative importance ofdisinfection resistance mechanisms. The current study wasdesigned to examine disinfection resistance mechanismswith respect to the survival of indicator bacteria in potablewater supplies. The results showed that the multiplicativeeffect of various resistance mechanisms can account for thepersistence of bacteria in chlorinated drinking water.

MATERIALS AND METHODSBacterial strains. Two strains of Klebsiella pneumoniae

were obtained from Ian W. Sutherland (University of Edin-burgh, Edinburgh, Scotland). The wild-type encapsulatedstrain was designated Kl, while a mutant lacking all capsuleproduction was labeled K3. Both strains were stored at-20°C in a 40% glycerol-2% peptone solution.For disinfection of suspended bacteria, cells were grown

on agar spread plates at 35°C for 24 h, 22°C for 48 h, or 10°C

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60.0

50.0

40.0

Relative 30.0Ratio

20.0.

IE] 35 CM 22 C

ML0I

tLz-LETSA EPS R2A .1 R2A

Medium

FIG. 1. Effect of growth medium and incubation temperature onthe production of extracellular polysaccharides. Relative ratio indi-cates the amount of capsule from the encapsulated strain comparedwith control values from the unencapsulated strain. Abbreviation: .1R2A, 0.1-strength R2A agar.

for 7 days. Various agar media was used, including TSA(Difco Laboratories, Detroit, Mich.), R2A agar (Difco),0.1-strength R2A agar (the medium was supplemented with13.5 g of Bacto-Agar [Difco] per liter), and extracellularpolysaccharide (EPS) agar. EPS agar was a medium de-signed to produce maximal capsule formation. EPS agarcontained the following (per liter of deionized water): 7.0 g ofK2HPO4, 3.0 g of KH2PO4, 0.1 g of MgSO4 7H20, 0.1 g of(NH4)2SO4, 0.01 g of CaC12, 0.001 g FeSO4, 0.1 g of NaCl,10.0 g of glucose, and 15.0 g of Bacto-Agar (final pH, 7.0).Bacteria were washed off the plates and suspended in 20 mMphosphate buffer (pH 7.0) at concentrations of 106 CFU/ml.

Biofilms of K. pneumoniae were grown on clean, sterile,glass microscope slides in EPS broth at 35°C. Slides wereheld in a vertical position by using cardboard inserts in themouths of the 125-ml flasks. For experiments under low-nutrient conditions, EPS broth was diluted 10,000-fold (finalconcentration of glucose, 1 mg/liter) in phosphate buffer.Biofilms of K. pneumoniae were grown on glass microscopeslides in the low-nutrient medium for 7 days at 35°C.

Preparation of the disinfectants. Stock solutions of thedisinfectants were prepared daily, and concentrations werechecked by using amperometric titration (1). A sodiumhypochlorite solution (5%) was obtained from the J. T.Baker Chemical Co. (Phillipsburgh, N.J.). Data from Fair etal. (14) indicate that over 85% of the free chlorine in water atpH 7.0 and 1 to 2°C is in the form of hypochlorous acid. AtpH 8.5 (1 to 2°C) over 90% of the free chlorine is in the formof the hypochlorite ion (14). A monochloramine solution wasprepared by using a 3:1 molar ratio of ammonia (ammoniumchloride; J. T. Baker Chemical Co.) to chlorine (pH 9.0).Amperometric titration indicated that no detectable freechlorine or dichloramines were present in the monochlora-mine stock solution.

Disinfection of suspended bacteria. K. pneumoniae (106CFU/ml) suspended in chlorine-demand-free phosphatebuffer (1) was treated with various disinfectants at 1 to 2°C.Portions were removed at designated times, dechlorinatedwith sodium thiosulfate, and plated in triplicate onto R2Aagar incubated at 35°C for 24 h.

Disinfection of biofilms. Bacteria attached to glass micro-scope slides were rinsed with sterile phosphate buffer toremove unattached cells. Biofilms were disinfected in 100 mlof chlorine-demand-free phosphate buffer (pH 7.0) at 4°C ina 250-ml beaker agitated with a magnetic stirring bar. Bio-

films on the glass surface exhibited no measurable freechlorine demand (M. W. LeChevallier, C. D. Cawthon, andR. G. Lee, submitted for publication). At the end of thereaction time, sodium thiosulfate (0.01%) was added to stopthe reaction. Biofilms on the glass slides were scraped off byusing a sterile rubber policeman and homogenized by theprocedure of Camper et al. (6). Enumerations were made intriplicate with R2A agar, as described above.

Determination of EPS. EPS were isolated from the Kleb-siella strains by using high-speed centrifugation (5). Cellswere centrifuged at 33,000 x g by using a Sorvall RC-5Brefrigerated superspeed centrifuge (DuPont Instruments,Wilmington, Del.), and then were suspended and centrifuged(33,000 x g) again. The supernatant was decanted and storedat -20°C until analysis. Hexose sugar concentrations andtotal dry weight were used as measures of extracellularcapsular material. The protein concentration was used as ameasure of the degree of cellular disruption. Hexose sugarswere measured by the phenol-sulfuric acid method of Duboiset al. (12). Hexose determinations were performed with aspectrophotometer (optical density at 490 nm; Spectronic1001; Bausch & Lomb, Inc., Rochester, N.Y.) and areexpressed as glucose equivalents. Total protein determina-tions were made by using the biuret reagent method (re-search kit 690; Sigma Chemical Co., St. Louis, Mo.). Resultsof control experiments showed that small amounts of glu-cose and protein could be extracted from the growth me-dium. By calculating the ratios of extracellular polymersfrom the encapsulated and unencapsulated strains of K.pneumoniae, the effect of materials extracted from thegrowth medium could be negated. All determinations wereperformed in triplicate.

Quality control and statistical comparisons. A quality as-surance program, as outlined previously (1, 4), was usedthroughout the course of the study. The materials usedduring each experiment were checked for sterility. Thetemperatures of autoclaves and incubators were monitoredon a per-use basis. Spectrophotometers, pH meters, andanalytical balances were calibrated on a regular basis.

Statistical comparisons were made by using the Stat-Pacstatistics program (Northwest Analytical, Portland, Oreg.)on a personal computer (Kaypro).

RESULTSImpact of encapsulation. Two strains of K. pneumnoniae (an

encapsulated strain and a mutant which was incapable ofcapsule production) were used to examine the impact ofcapsule production on disinfection efficiency. The amount ofencapsulation was determined by two methods. The capsulematerial was extracted by centrifugation and measured astotal dry weight and as hexose sugars. While these assaysmeasured different components of the capsular material,regression statistics showed that the two measurementswere in good agreement (r = 0.96). No measurable proteinwas found in the extracted capsular material, indicating thatlittle cellular disruption occurred during the centrifugationprocedure.Growth of the K. pneumoniae strains on various media

(TSA, R2A, EPS, and 0.1-strength R2A agar) at 10, 22, and35°C produced various amounts of capsular material (Fig. 1).The results, expressed as the ratio of carbohydrate materialfrom the encapsulated strain to that from the unencapsulatedstrain, showed that the greatest amount of capsular material(a 55-fold increase) is produced at 10°C on EPS agar.

Despite large differences in the amount of capsular mate-rial produced by growth on different media at various

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temperatures, no change in the susceptibility to free chlorineor monochloramine disinfection (pH 7.0, 1 to 2°C) wasobserved between the two strains (Tables 1 and 2). Althoughthe results shown in Fig. 1 indicate that the encapsulatedstrain grown on EPS agar at 10°C produced 55 times moreextracellular material than did the unencapsulated strain, thesusceptibilities of the two strains to the disinfectants werenot significantly different.Experiments showed that free chlorine (pH 7.0, 22°C)

reacted with extracellular capsular material (material equiv-alent to 107 bacteria per ml), but that the chlorine reactionrate was relatively slow, producing a demand for freechlorine of 0.3 mg/liter in 30 min. No reduction in mono-chloramine residuals was detected during the 60-min contacttime with the capsular material. Extracellular extracts fromthe unencapsulated strain and the deionized water controlshowed no measurable chlorine demand.Impact of growth conditions. To examine the impact of

growth conditions, the two Klebsiella strains were grown onhigh-nutrient medium (EPS agar) and in low-nutrient me-dium (1/10,000-strength EPS broth). Growth of the bacteriain the low-nutrient (1 mg of glucose per liter) solutionsignificantly increased the resistance of the microorganismsto free chlorine disinfection (Table 3). The encapsulatedstrain of K. pneumoniae grown in the low-nutrient solutionwas nearly three times more resistant to free chlorine thanwas the same strain grown in high-nutrient solution. Thelow-nutrient-grown unencapsulated strain was nearly twotimes more resistant than the same strain grown in high-nutrient solution. Growth in low-nutrient solution made theencapsulated strain significantly (P < 0.05) more resistantthan the unencapsulated strain to free chlorine. However,neither growth medium nor capsule production affecteddisinfection by monochloramine (Table 3).Impact of surfaces. Data from Table 3 indicate that con-

centration and time (C x 7) values to achieve 99% inactiva-tion of unattached K. pneumoniae grown on EPS agar(high-nutrient solution) were 0.065 ± 0.02 mg- min/liter forhypochlorous acid and 33 + 18 mg. mim/liter for monochlo-ramine (pH 7.0, 1 to 2°C). These values represent units ofactivity for which 99% of the test organisms are killed.Multiples of the concentration multiplied by time unitsrequired to inactivate K. pneumoniae attached to glassmicroscope slides are presented in Fig. 2. Because of theprotection provided by attachment of the bacteria to theglass surfaces, higher multiples were needed to achieve a99% (2 log unit) reduction. Data from Fig. 2 indicate thatattachment of K. pneumoniae to glass microscope slidesincreased resistance to hypochlorous acid nearly 150-fold,

TABLE 1. Free chlorine concentration multiplied by time valuesfor 99% inactivation of unattached K. pneumoniae

Free chlorine C x T value for cells grown on the

Growth following mediuma:temp (°C) R2A

EPS TSA R2A (0. -strength)

35 0.08 (0.06) 0.06 (0.07) 0.07 (0.06) 0.0722 0.07 (0.06)10 0.06 (0.07) 0.07 (0.05)

a Strains were scraped off the high-nutrient growth medium, diluted to 106CFU/ml, and disinfected at 1 to 2°C (pH 7.0). Values of free chlorineconcentration multiplied by time (C x T) are in milligrams * minutes per literand are for unattached encapsulated K. pneumoniae. Values in parenthesesindicate the free chlorine concentration values (in milligram - minutes perliter) for the unattached unencapsulated strain.

TABLE 2. Monochloramine concentration multiplied by timevalues for 99% inactivation of unattached K. pneumoniae

Monochloramine C x T value for cells grown on theGrowth following medium':temp (C) R2A

EPS TSA R2A (0.1-strength)

35 65 (40) 23 (22) 44 (52) 47 (30)22 20 (25)10 18 (15) 15 (18)

a Strains were scraped off the high-nutrient growth medium, diluted to 106CFU/ml, and disinfected at 1 to 2°C (pH 7.0). Values of monochloramineconcentration multiplied by time (C x T) are in milligrams * minutes per literand are for unattached encapsulated K. pneumoniae. Values in parenthesesindicate monochloramine concentration multiplied by time values (inmilligrams * minutes per liter) for the unattached unencapsulated strain.

while resistance of attached organisms to monochloramineincreased only 2-fold.Impact of incubation time. The data presented in Fig. 3

demonstrate the impact of incubation time on disinfectionefficiency. The unencapsulated strain of K. pneumoniaegrown on glass microscope slides (in EPS broth at 35°C) for7 days was more resistant to disinfection (1 mg of freechlorine per liter for 10 min at pH 7.0 and 4°C) by hypo-chlorous acid than were cells cultured for 2 days. Noincrease in disinfection resistance was observed when thebacteria were treated with monochloramine (5 mg of NH2Clper liter for 10 min at pH 7.0 and 4°C). Although celldensities increased threefold during the prolonged incuba-tion period (Fig. 3), microscopic examination showed thatthe glass slides were sparsely colonized. Because levels ofbacteria were similar in both experiments, it is unlikely thatthe increased disinfection resistance to hypochlorous acidwas due to the higher cell densities.

Interaction of resistance mechanisms. In the studies de-scribed above, we examined individual mechanisms of dis-infection resistance. It was of interest to know whethercombined resistance mechanisms were additive or multipli-cative. If the resistance of attached K. pneumoniae (150-fold) was additive to the resistance conferred by growth onlow-nutrient solution (3- to 4-fold), then the combined resis-tance of attached bacteria grown in a low-nutrient mediumwould be 153- to 154-fold. The data shown in Fig. 4 indicatethat the resistance of attached bacteria grown in a low-nutrient medium was multiplicative. Bacteria grown underthese conditions were approximately 600-fold (150 x 4) moreresistant than unattached bacteria grown in rich media.

DISCUSSION

Results of this study indicate that attachment of bacteriato surfaces provides the primary means for bacteria tosurvive disinfection. Attachment of unencapsulated K.pneumoniae grown in high-nutrient medium to glass micro-scope slides afforded the microorganisms a 150-fold increasein disinfection resistance (Fig. 2). In a previous study(LeChevallier et al., submitted), heterotrophic plate countbacteria grown on metal coupons were 2,400 times moreresistant to free chlorine than were unattached cells, whilebiofilms grown on granular activated carbon particlesshowed more than a 3,000-fold increase in chlorine resis-tance. These results are in agreement with those of Ridgwayand Olson (31), who found that bacteria in chlorinateddrinking water were primarily associated with particle sur-faces.

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TABLE 3. Concentration multiplied by time values for 99% inactivation of unattached K. pneumoniaegrown at high and low nutrient levels

C x T for"

Nutrient level Encapsulated Unencapsulated

Free chlorine Monochloramine Free chlorine Monochloramine

High (10 g/liter) 0.065 ± 0.02b 32.4 ± 18 0.06 ± 0.01'C 31.2 ± 20Low (1 mg/liter) 0.17 + 0.056" 29.3 ± 20 0.11 ± 0.04c 37.5 ± 1.0

a Values are expressed as the concentration multiplied by time (C x T; in milligrams * minutes per liter) for 99% bacterial inactivation ± standard deviation.Values within a column followed by the same superscript letter are significantly different.

b p < 0.001.c P < 0.05.

The results of the present study demonstrate that attach-ment of bacteria to relatively inert surfaces (such as glass)can significantly increase resistance to disinfection. Attach-ment to a surface alters the way a disinfectant interacts witha bacterium. Theoretically, the physical hindrances of asurface could affect the ability of a disinfectant to approachthe cell membrane. A freely suspended organism, for exam-ple, is susceptible to a disinfectant from all sides and at allangles, while an organism attached to a surface is susceptibleonly from one side (excluding lateral diffusion, which, in abiofilm, is probably negligible [9, 27]). In addition, theconcentration of organic solutes at a solid surface mayincrease the free chlorine demand at the interface. Transportof the disinfectant to the biofilm surface, then, becomes animportant rate-limiting step. Characklis (10), using knownrate constants, calculated that the total chlorine consump-tion rate is determined by the diffusion rate of the disinfec-tant through a biofilm rather than the reaction with thepipeline wall material. The importance of mass transfer fromthe bulk fluid and the diffusion of the compounds within thebiofilms has been modeled for several nutrients (9, 27).Additional research is needed to understand the interactionof disinfectants with microorganisms growing on pipelinesurfaces.Data from Fig. 3 indicate that the age of the biofilm

influences the disinfection efficiency by free chlorine. Theresults showed that biofilms grown for 7 days are morechlorine resistant than biofilms grown under identical condi-tions for 2 days. The exact reason for this increased resis-

LogReductionViableCount

3.0 Fa HOC7 |

2.502.0-

1

0.0.1 1.5 2 2.5 75 100 125 150

Disinfectant Dose(CxT Multiple)

FIG. 2. Disinfection of K. pneumoniae attached to glass micro-scope slides. The log reduction in viable counts is calculated as acomparison with counts for undisinfected controls. Disinfectiondose is calculated as multiples of the activity (concentration multi-plied by time [C x Ti, in milligrams minutes per liter) necessary toinactivate 2 log units (99%) of unattached bacteria.

tance is not known, but it probably was not due to the highercell density at 7 days. Microscopic examination of thebiofilms showed that cells were sparsely distributed on theglass surface even after 7 days of growth. It is possible thatphysiological changes in the biofilm population, such asstarvation effects, or a coalescence of the biofilm may havemade the cells more resistant. Nutrient limitation has beenshown to increase bacterial resistance to various disinfec-tants (3, 8, 16).

In a previous study (22) it has been shown that biofilms ofcoliform bacteria in distribution systems are sparsely distrib-uted along pipe surfaces. Thus, the term biofilm in thiscontext refers to even the sporadic accumulation of organicmaterial, microorganisms, and detritus at a surface (33). Thedata presented in this report indicate that thick films are notrequired to provide disinfection resistance; even sparselydistributed attached cells were hundreds of times moreresistant to free chlorine than were monodispersed, sus-pended bacteria.

Possession of an extracellular capsule per se did notincrease the disinfection resistance of K. pneumoniae. Re-sults presented in Tables 1 and 2 indicate that there was nosignificant difference in disinfection resistance between theencapsulated and unencapsulated strains of K. pneumoniae.Growth of the strains in a low-nutrient medium (1 mg ofglucose per liter), however, increased bacterial resistance tofree chlorine threefold (Table 3). Evidently, growth undernutrient-limiting conditions changed the capsule material.Rudd et al. (32) have found that the extracellular polymerproduction of Klebsiella aerogenes NCTC 8172 increased atlow growth rates. This finding is consistent with the resultspresented in Fig. 2. The highest capsule production was

FIG. 3. Effect of incubation time on disinfection of attachedbacteria. K. pneumoniae was grown on glass microscope slides inEPS broth at 35°C. Biofilms were treated with 1 mg of free chlorineper liter for 10 min or 5 mg of monochloramine per liter for 10 min.Bars indicate the log reduction of viable counts due to disinfection;lines show the cell density of biofilms on the glass microscope slides.

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Log 2.0 NutnReduction 1.5-

Viable

Count 1.0-

0.5-

15 75 100 125 150 300 450 600Concentration x Time

RatioFIG. 4. Multiplicative effect of resistance mechanisms. Data

represent the combination of resistance provided by growth of K.pneumoniae attached to glass microscope slides and resistanceprovided by growth under low-nutrient conditions. Disinfectantdose is calculated as multiples of the activity (in milli-grams minutes per liter) necessary to inactivate 2 log units (99%) ofunattached bacteria.

observed with growth at 10°C on EPS medium. Rudd et al.(32) also have found that the majority of the capsularmaterial was in a soluble phase at high growth rates, whilelow growth rates produced a colloidal form of the polymer.In this study, it is possible that the soluble type of capsularmaterial did little to protect the cells from disinfection, whilethe increased resistance of encapsulated bacteria grown inlow-nutrient medium may have been due to the colloidaltype of polymer.Growth of the unencapsulated K. pneumoniae in low-

nutrient media increased its resistance to free chlorinetwofold (Table 3). Berg et al. (3) have speculated thatchanges in the bacterial cell membrane may account forincreased resistance of microorganisms grown in low-nu-trient medium. Because the unencapsulated strains lackedother defense mechanisms, it is possible that cell membranechanges were responsible for the observed resistance; how-ever, further research is necessary to substantiate this point.The choice of disinfectant residual influenced the type of

resistance mechanism observed. Disinfection by free chlo-rine was affected by surfaces, age of the biofilm, encapsula-tion, and nutrient effects. Disinfection by monochloramine,however, was only affected by surfaces. Not only do thesedata support the hypothesis of Ward et al. (37), who sug-gested that monochloramine and free chlorine have differentmechanisms of action, they also show that the mechanismsof resistance differ. This information may be useful indeveloping a program of alternating disinfectants for thecontrol of biofilm bacteria.The data presented in Fig. 4 show that the resistance

afforded by one mechanism could be multiplied by theresistance provided by a second mechanism. This interac-tion of resistance mechanisms could account for the survivalof bacteria in highly chlorinated water supplies. In drinkingwater distribution systems, encapsulated bacteria attachedto pipe surfaces grow under low-nutrient conditions for longperiods of time and are usually disinfected with a freechlorine residual. Given this scenario, it is easy to under-stand how biofilms can survive in potable water pipe sys-tems.

ACKNOWLEDGMENTS

We thank Richard H. Moser for comments and suggestions. Theclerical assistance of Marsha Rohr and Kathy Waigand is alsoacknowledged.

This study was supported by funds from the American WaterWorks Co., Inc.

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