APPLIED MICROBIOLOGY, Sept., 1965Copyright © 1965 American Society for Microbiology

Vol. 13, No. 5Printed in U.S.A.

Efficiency of Chlorine Dioxide as a Bactericide1MELAIN A. BENARDE, BERNARD M. ISRAEL, VINCENT P. OLIVIERI,

AND MARVIN L. GRANSTROM

Bio-Engineering Laboratory, Department of Civil Engineering, Rutgers, TheState University, New Brunswick, New Jersey

Received for publication 17 May 1965

ABSTRACT

BENARDE, MELVIN A. (Rutgers, The State University, New Brunswick, N.J.),BERNARD M. ISRAEL, VINCENT P. OLIVIERI, AND MARVIN L. GRANSTROM. Efficiency ofchlorine dioxide as a bactericide. Appl. Microbiol. 13:776-780. 1965-We found chlorinedioxide to be a more effective disinfectant than chlorine in sewage effluent at pH 8.5.Chlorine dioxide was also found to be a more stable bactericide in relation to pH in therange studied.

Chlorine dioxide, prepared early in the 19thcentury, was used in the treatment of water sup-plies in Europe after 1850. It was not until the1940's, however, that experimental data on itsbactericidal efficiency became available. Althoughchlorine and chlorine dioxide are similar in manyrespects, including the fact that both are power-ful oxidizing agents, Cl02 has 2.5 times the oxida-tion capacity of Cl2. It was this feature that rec-ommended C102 for the control of odors andtastes in water supplies.McCarthy (1944) reported C102 to be an effec-

tive germicide in water with low organic content.However, he found it to be less effective thanchlorine at equal concentrations when the organiccontent was high.

Shortly thereafter, Ridenour and Ingols (1947)reported C102 to be at least as effective as chlo-rine. Their conclusions were based upon the ab-sence of growth after 30 min of contact with thedisinfectant. Efficiency was based on residualvalues, and these did not represent equal initialdosages.Trakhtman (1946) and Bedulevich, Svetla-

kova, and Trakhtman (1953) found C102 to ex-ceed or at least equal chlorine in bactericidal ef-ficiency. They reported decreased effectiveness ofC102 under alkaline conditions.Because physicochemical characteristics and

kinetics of C102 were unavailable at the timethese studies were performed, the data obtainedare of questionable value.

Preparation of chlorine dioxide from chlorine,or by the action of acid on NaCl02, as is the gen-

lPresented in part at the 65th Annual Meetingof the American Society for Microbiology, AtlanticCity, N.J., April, 1965.

eral procedure, would of necessity introduce in-terfering substances which would not only hastenthe decomposition of C102, but would also yielderroneously high values on iodometric analysis.This would suggest that the initial concentrationsof C102 in the studies noted were probably lowerthan the reported values; the bactericidal effi-ciency of C102, when compared with chlorine,would suffer accordingly. Preparation of C102 byaddition of alkali results in major losses of C102almost instantaneously. Granstrom and Lee (un-published data) have shown the disproportiona-tion that occurs above pH 10. Additionally, theseolder methods probably did not account for thehigh degree of volatility exhibited by C102. De-pending upon the concentration and length of ex-posure, 7 to 30% loss can occur within 1 hr. Thismeans that dilutions prepared from stock solu-tions cannot be assumed to be quantitative. Spec-trophotometric analysis of each dilution imme-diately prior to use is required. Such a procedurehas only recently become available.The general analytical procedure for detection

of C102 residuals employed o-tolidine in a color-imetric determination. This procedure is subjectto similar interferences, as noted for the iodo-metric titration. Post and Moore (1959) reportedthat o-tolidine and o-tolidine arsenite methodsmade no distinction between Cl2 and C102.Consequently, it is nonspecific and allows only arange of values for any given concentration.Thus, critical comparison of chlorine versuschlorine dioxide was impossible. As a result,C102 is sparingly used in water and waste watertreatment.The study reported herein used as its point of

departure the detailed physicochemical findingsof Granstrom and Lee (1958) and Granstrom et

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CHLORINE DIOXIDE AS A BACTERICIDE

al. (uinpublished data) to avoid the pitfalls of thepast and to obtain more reliable and specific data.

MATERIALS AND METHODS

Organic-free distilled water. Organic-free waterwas prepared by distilling distilled water in thepresenice of an acid-permanganate solution (1%concentrated H2SO4 and 1% KMnO4). The dis-tillate traveled through a vertical column 6 ft(183 cm) long. It was necessary to use a heatingtape at the top of the column to break the film ofwater from the distillation flask; otherwise, creep-ing of the permanganate occurred and wouldappear in the distillate. Extremely small amountsof permanganate can interfere in the o-tolidinedeterminationi of chlorine by giving itself a posi-tive test (yellow color). This, however, provides ameans of checking the quality of the distillate(Israel, 1961).

Organic-free water was used in preparing thephosphate buffers needed for the various trials.

Glassware. To obtain chlorine demand-freeglassware, tubes, pipettes, reaction vessels, andsyringes were soaked overniight in chlorine waterof approximately 5,000 mg per liter.

Prior to use, these were rinsed several times indistilled water and heat-sterilized when necessary.

Neutralization of disinfectant. Tubes (16 by 150mm) containiing several crystals of Na2S203 weresterilized at 121 C for 15 min. These were used forquenchinig further disinfection action of bothchlorinie anid chlorine dioxide solutions.

Preparation of chlorine dioxide. A 4.0-g amountof NaCO1 was dissolved in 50 ml of distilled waterin a reaction vessel. To this was added a solutionof 2.0 g of K2S208 in 100 ml of distilled water.High-purity nitrogen gas (Linde Division, UnionCarbide Corp., New York, N.Y.) was used tosweep out the C102 formed; this N2-C102 gasmixture was passed through a dry NaCl02 columnto remove any traces of HOCI that might havebeen formed. The gases were then passed into atrap to remove any NaCl02 dust that might havebeen carried over. C102 was then collected in cool(O to 10 C) organic-free water.Preparation of chlorine. Aqueous chlorine stock

solutions were prepared by bubbling chlorine gasinto distilled water for 15 to 20 min. This yielded a10,000 mg per liter concentration.

Concentrations of C102 and Cl2 were preparedon an equivalent chlorine basis: 1 mg per liter ofchlorine is equal to 1.4 X 10-s moles per liter; 1 mgper liter of C102 is equal to 1.5 X 10-6 moles perliter. For this study, C102 was prepared to equal1.4 X 10-1 moles per liter. Thus, although equal inmolar concentration, C102 was low in per centconcentration. Since the high molar absorbtivityof C102 lends itself to spectral analysis, we wereable to measure the initial and residual concentra-tions of C102 directly at 357 m,u. This was not thecase with chlorine, whose molar absorbtivity isapproximately one-tenth that of C102. Althoughwe were able to prepare stock solutions of chlorine

of 20 ppm, using the spectrophotometer (238 m,u),the very low initial coincentrations used in thisstudy could not be so measured. For this reason,dilutions of chlorine were prepared in organic-freewater, since it had been shown in our laboratorythat quantitative dilutions of C12 could be made.For this study, initial dosages of both chlorine

and chlorine dioxide of 0.25, 0.5, 0.75, 2.0, and 5.0mg per liter were used.

Suspensions of washed cells (freashly isolatedfecal strain of Escherichia coli) were mixed bymagnetic stirrer in a reaction vessel with an equalvolume of disinfectant solution to yield the finaldesired concentrations of C12 or C102, or both, anda biacterial density of approximately 19,000 cellsper milliliter. The density of washed cells had beenstandardized spectrophotometrically by. optical-density measurement at 525 mp. Confiraation ofthis was obtained by spot-plate counts onr nutrientagar, using a Unopette (Becton, Dickinson andCo., Rutherford, N.J.) for delivery of 0.025 ml. Wefound this technique to be more accurate and re-producible, and a great deal easier, than standardplate counts. Figure 1 shows the type of spot-platecounts obtained.The sampling apparatus consisted of two auto-

matic hypodermic syringes, each with a two-wayflap valve assembly, permitting rapid drawing anddispensing of the solution from the reaction vessel.Figure 2 shows the apparatus ready for use. At15-sec intervals, samples were removed for estima-tion of remaining population. Sterile thiosulfatecrystals were used to quench further disinfectanteffects. In the case of chlorine, residuals weredetermined by the o-tolidine method. Chlorinedioxide residuals, however, were determinedspectrophotometrically. A 30-ml sample was with-drawn from the reaction vessel and placed in a10-cm cuvette. Absorbance values at 357 mi wereobtained at intervals, and were used to calculateconcentrations.

Plate counts were made on nutrient agar, after18 and 48 hr of incubation at 35.5 C. To preventclumping and spreading of colonies, it was neces-sary to add the sample to a layer of melted agarand swirl. Between trials, the sampling apparatuswas sterilized by drawing a strong chlorine solu-tion into it and allowing it to remain in contactfor 10 min. Rinsing with sterile organic-free dis-tilled water was repeated until negative o-tolidinetests were obtained. Sterility was determined byplate counts of the rinse water. This procedureadditionally provided a chlorine demand-freesystem.

RESULTS AND DiscussioN

It is generally understood that the germicidalactivity of chlorine results from its hydrolysis inaqueous solutions to form hypochlorous acid, thedisinfectant constituent:

C12 + H20 HOCI + H+Cl- (1)

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BENARDE ET AL.

FIG. 1 (top). Spot-plate counts obtained with Unopette.FIG. 2 (bottom). Sampling apparatus.

Optimal activity occurs in acidic solutionswhere HOCI, the initial hydrolysis product, ex-

ists in undissociated form. As the pH of solutionsincreases, hypochlorous acid ionizes increasingly;the resulting hypochlorite ion, OC1-, has little

disinfectant value. Table 1 indicates the degreeof ionization at various pH levels.

HOCI OC1- + H+ (2)

Chlorine dioxide, on the other hand, does not

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CHLORINE DIOXIDE AS A BACTERICIDE

TABLE 1. Ionization of hypochlorous acid at variouspH levels

pH Per cent HOCI Per cent OCIh*(20 C

4.0 100.0 0.05.0 99.7 0.36.0 96.8 3.27.0 75.2 24.88.0 23.2 76.89.0 2.9 97.110.0 0.3 99.711.0 0.03 99.97

* Values calculated from the K values of equa-tions 1 and 2 (Moore, 1951).

E 0.75-

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50-

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i ---*- ~C102 pH 6.5 Buffer-0--- Cl2 pH 6.5 Buffer

-;- - -- C102 pH 8.5 Buffer-A---- Cl2 pH 8.5 Buffer

I \)Disinfection of E. coli inOrganic Free Buffer15,000 cells /ml 24° C

60 120 180 240MAXIMUM TIME FOR 99+% KILL, SECONDS

300

FIG. 3. Effect of pH on kill.

hydrolyze in aqueous solutions and, therefore,is not subject to dissociation in the manner ofHOCI (Mellor, 1922). A stock solution of 10.2mg per liter of C102 was diluted and its absorb-ance at 357 m,u was measured. At pH 4.0, 6.45,and 8.42, the C102 was unaltered (1.39 mg perliter, 100%). Thus, the intact C102 molecule ap-pears to be the bactericidal compound.

Figure 3 shows the effect of pH on the disin-fectant efficiency of chlorine and chlorine dioxidein organic-free buffer. It can be seen that at pH6.5 chlorine was somewhat more efficient thanC102. Based on our sampling times of 30 and 60sec, it would appear that half the time is neededfor equal kill at an initial dosage of 0.25 mg perliter. At 0.5 mg per liter, killing time was reduced,but chlorine remained more effective. Both com-pounds were equally efficient at an initial dose of0.75 mg per liter.

Increasing the pH to 8.5 altered the compara-tive disinfectant properties dramatically. It isseen that at 0.25 mg per liter C102 obtained99+% kill in 15 sec. Chlorine did not achievethis until 300 sec-20 times less effective. At 0.5

...\...-_.C102 .25 mg/I (doage)

Disinfection of E. coli inSewage Effluent

\ <>\ --_ C102 .5 _

Chlorine Dioxide vs. ChlorinepH 8.5 15,000 cells/ml

mU ~~~~~~~~24C30 60 _20 IOCI2 .75 3

30 60 120 180 240 300

CONTACT TIME, SECONDS

FIG. 4. Effect of contact time on organism survival

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60 120 10 240 300CONTACT TIME, SECONDS

Disinfection of E. coli inSewage Ef f luent

Chlorine Dioxide vs. ChlorinepH 8.5 15,000 cells/ml 241 C

C102 0.75 mg/i (dosage)

\\ < C12 ~~0.75Cl 02 0.5

C12 0.5

K __*C102 0.25

C12 0.25

30 60 120 180 240 300

CONTACT TIME, SECONDS

FIG. 5. Effect of contact time on disinfectantutilization.

mg per liter of C102, kill was also completed within15 sec, whereas chlorine requires 60 sec to achieveequal effects. Equal kill occured at 0.75 mg perliter for both compounds.

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From Fig. 3 it would seem that pH affects C102efficiency. Our data and the data of Mellor (1922)and Granstrom et al. (unpublished data) indicatethat C102 is not directly affected by pH in aque-ous solutions; other factors are probably contrib-uting to the loss of efficiency. Although the chem-ical reactions of C102 with many materials areyet to be studied, it may well be that the rates ofreaction of C102 with substances found in oursystem are pH-dependent.To establish the comparative bactericidal ef-

ficiencies that would more nearly resemble in-plant conditions, tests were conducted in sterile,unchlorinated sewage effluent to which a knowncell density of E. coli was added. Since there werematerials in the effluent that absorbed at 357 m,u,spectral analysis of C102 residuals, in this in-stance, was impractical. Thus, both chlorine andC102 residuals were determined by o-tolidine.Figure 4 presents the results of the effluent study.

It can be seen that the combination of organicmatter and pH sharply curtails disinfectant ac-tivity. For initial C12 dosages under 1 ppm, lessthan 30% of the initial population is removed. Atthe same initial dosages, chlorine dioxide achievesup to 70% removal. An even more dramatic ex-ample of the relative efficiencies of the two com-pounds is seen with chlorine at 5.0 ppm, whichobtains 90%/ reduction after 5 min, comparedwith chlorine dioxide at 2.0 ppm, which obtainsapproximately 100% kill in 30 see.

Generally, reports of chlorine efficiency arestated in terms of residual chlorine values. Wefound that residuals are not in themselves indic-ative of the concentration required for kill. Sim-ilarly, the initial concentration is not the killingdose. We feel that the actual value lies betweenthe two. The amount of material used is probablycloser to the true killing value.

Figure 5 presents residual values obtained forchlorine and chlorine dioxide in the effluent stud-ies. For complete removal of organisms in 30 see,about 0.9 ppm of C102 was used when dosed withan initial concentration of 2.0 ppm. In the case ofC12, 2.25 ppm were actually used of an initial doseof 5.0 ppm to obtain 90%/o removal in 5 min.Our data indicate that, even though residuals

are present after 5 min, major reductions occurwithin the first minute of contact and do not oc-cur appreciably thereafter.

This would suggest that residuals actually havelittle disinfectant value. Thus, reports of killingconcentrations based on residual values seem un-realistic.

It should be borne in mind that the plateau ef-fects in the case of chlorine are probably due tothe formation of monochloramines (pH 8.5) andother chloro compounds which are of little valueas disinfectants. In the case of chlorine dioxide,however, reaction with ammonia to form chlor-amines has not been reported.The fact that 100%kill has not been obtained with residuals up to0.5 mg per liter, as measured by o-tolidine, whichis nonspecific, would suggest that CIO2 undergoesreaction to form organo-chloro compounds thathave little disinfectant ability.

Additionally, Fig. 5 shows that less chlorinedioxide was used to obtain greater bacterial re-ductions. This may be due to fewer side-reactionsat pH 8.5 that tie up the essential disinfectingmaterial. As fewer molecules of chlorine dioxidewere present initially, 1.4 X 10-5 as opposed toits usual 1.5 X 10-s moles per liter, the resultswould tend to support even more strongly theconclusion that C102 exhibits greater bactericidalactivity than chlorine.

ACKNOWLEDGMENT

This investigation was supported by PublicHealth Service grant PHT 1-61A-61.

LITERATURE CITED

BEDULEVICH, T. S., M. N. SVETLAKOVA, AND N. N.TRAKHTMAN. 1953. New data on the use of chlo-rine dioxide in water purification. Gigiena iSanit. 19:14-17.

GRANSTROM, M. L., AND G. F. LEE. 1958. Genera-tion and use of chlorine dioxide in water treat-ment. J. Am. Water Works Assoc. 50:1453-1466.

ISRAEL, B. M. 1961. Hydrolysis of organo-halo-genating agents. Ph.D. Thesis, University ofWisconsin, Madison.

MCCARTHY, J. A. 1944. Bromine and chlorine aswater disinfectants. J. New Engl. Water WorksAssoc. 58:55-68.

MELLOR, J. W. 1922. Comprehensive treatise oninorganic and theoretical chemistry. Longmans,Green & Co., New York.

MOORE, E. W. 1951. Fundamentals of chlorinationof sewage and waste. Water Sewage Works98:No. 3.

POST, M. A., AND W. A. MOORE. 1959. Determina-tion of chlorine dioxide in treated surfacewaters. Anal. Chem. 31:1872-1874.

RIDENOUR, G. M., AND R. S. INGOLS. 1947. Bac-tericidal properties of chlorine dioxide. J. Am.Water Works Assoc. 39:561-567.

TRAKHTMAN, N. N. 1946. Chlorine dioxide inwater disinfection. Giegiena i Sanit. 11:10-13.

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