_—._-. K". - ; tun! w.;\'.'!‘. 8 {‘39 ‘— f'"‘:‘.‘, 33310 n 5 'b u!- \ ‘1 --.»...L.. Date This is to certify that the thesis entitled Studies on the Bactericidal Activity of Chloramine—T. presented by Jack M. Tadman has been accepted towards fulfillment of the requirements for Ph.D. Microbiology degree in 0h £Mkhv‘w‘v‘ V Majir professor October 17, 1955 STUDES ON THE BACTLRICIDAL ACTIVITY OF CI'EOMIINE-T BY Jack M. Tadman A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHIIDSOPHY Departinent of Microbiologr and Public Health 1955 an / '1 I25“ 3:" ' A e- ‘ j \5‘ ‘2) ' , TABLE OF CONTENTS 3. l Page 1 ACWOWENTS O O O O O O O O O O O O O O O O O O O O O C O l ABS'I'RACT O O O O O O O O O O 0 O O O O O O O O O O O O O O O O 2 A INTRODUCTION.........................3 HIS‘IOR ICAL REVIEW 0 O O O O O O I O O O O O O O O O O O O O O 6 CHEMICAL EQUILIBRIA IN CHLORAMINE—T SOLUTIONS . . . . . . . . l3 MAW AND MTHODS O O O O O O O O O O O O O O O O O O O O 19 THE RELATIONSHIP OF CONCENTRATION AND pH TO THE BACTERICIDAL ACTIVITY OF CHLORAMINE-T SOLUTIONS . . . . . . . . . . . . . 21 RELATIONSHIP OF IONIC STRENGTH TO BACTERICIDAL EFFICIENCY OF 0mm T O O O O O O O O O O O O C O O C O I O C O O O I 26 STABILITY OF CHLORANINE-T . . . . . . . . . . . . . . . . . . 27 A TECHNIQUE FOR RAPID SANITIZATION OF FOMITES USING cmRAl‘flNE-T C O O O O C O O O O C O O O O O O O C O O O O . 29 D13 CUSSION e o e e e e e e e e e e e e e e e o e o e e e e o e 3‘} CONCLUSIONS 0 e e e e e e e e o e e e e o o o o e e o 0 e o O ‘01 WHY e e 0 o o e e e e e e e e e o e e e o e e e e e 0 [+2 TABLE 10 LIST OF TABLES Effect of pH on bactericidal activity of Halazone andChloramine-Teeeeeeeoooeeeoeeo Dissociation of hypochlorous acid . . . . . . . . Effect of concentration and pH on bactericidal property of chloramine-T solutions . . . . . . . . Effect of ionic strength on bactericidal activity of chloramine-T Percent reduction of bacteria by chloramine-T and sodium hypochlorite after standing at room. temperature......o..........o. Percent reduction of bacteria by unbuffered chloramine-T at pH 8 with and without "activating" pre‘rmseoeeeeeeeeoeeeeeeeeoe Preliminary test of the effect of pH of pro-rinse Relationship of buffer concentrations in pre-rinse and chloramine-T . . . . . e . . . . . . Effect of chloramine-T concentration on sanitizer aCt iflty O O O O O O O O O O O O O O O O O O O 0 Effect of pH upon activity of sodium hypochlorite in 11/200 buffer C O O O O 0 O O O O O O O O O O O Page 10 26 28 30 31 32 33 33 LIST OF FIGURES FIGURE PAGE 1 N-chloro-p-toluenesulfonamide Concentration in Chloramine-T Solutions . . . . . . . . . . . . . . l7 2 Dichloramine-T Concentrations in Chloramine-T saluti ons O O O O O O O O O O O O O O O O O O O O 18 3 Chloramine-T, 50 p.p.m. as 012, 30 Second Contact Period, Oxidation-Reduction Potential, Bactericidal Activity and pH . . . . . . . . . . . 23 A Chloramine-T, 200 p.p.m, as 012, 15 Second Contact Period, Oxidation-Reduction Potential, Bactericidal Activity and pH . . . . . . . . . . . 25 5 Chloramine-T, 200 p.p.ms as 012, Dissociation and OXidation'RedUCtion POtential e e e e e e o e e e 36 6 Chloramine-T, 13,200 p.p.m~ as 012, Dissociation and Oxidation-Reduction Potential . . . . . . . . 37 7 Sodium.Hypochlorite, 200 p.p.m. as 012, Dissociation and Oxidation-Reduction Potential . . 38 ACKi‘tOnTLDGEIIENTS The author wishes to express his deep appreciation to Professor'w..L..Mallmann for his patient guidance and counseling during the course of this investigation. The author is indebted to Professor J. Carrell.Morris of the Department of Sanitary Chemistry at Harvard University for his assistance in determining concentration of species in chloramine-T solutions. These investigations were supported, in part, by a grant from the George Stearns Chemical Company, Inc., of Madison,'Wisconsin. ABSTRACT A study was made on the inter-relationship of pH, ionic strength, and concentration of chloramine-T. It was determined that a change in pH produced the most profound change in bactericidal activity with ionic strength and concentration of chloramine-T being of lesser importance. A direct relationship was found to exist between the con- centration of species present in the equilibria and the oxidation-reduction potential, thereby giving a.measure of bactericidal activity as determined by oxidation-reduction potential. A technique for the use of chloramine-T as a restaurant sanitizing agent is also given. This technique can be used in any eating or drinking establishment that utilizes a three tank wash and sanitizing system. After the utensil is cleaned with a detergent in the first tank, it is dipped in the second tank containing an acid rinse. The acid rinse neutralizes the alkalinity of the detergent and places an acid film on the utensil. When the utensil with the acid film is placed in the third tank containing chloramine-T, the chloramine-T is "activated" at the surface of the utensil by the lowering in pH, producing rapid bactericidal activity. The acid rinse is substantially buffered with citric acid at a pH of A and the chloramine-T solution is buffered at a pH.of 8 with KHQPO . It is possible with this method to have a rapid acting sanitizer that is stable and odorless. INTRODUCTION Chloramine-T is a sodium salt of N-chloro-p-toluenesulfonamide and its structure is represented as - 1‘ CH3 $02N\ Na Cl Although chloramine-T has been known for at least 50 years and its possible use as a disinfectant investigated often, the compound has never been widely used. Chloramine-T solution possesses certain advantages over Wehlorite solutions, the most important of which is stability, it being more resistant to decomposition by light and heat. It is relatively non-toxic and non-corrosive and can be used even on open wounds as a direct disinfectant without undue irritation. Chlorsmine-T solution in a suitable concentration for general restaurant sanitation has practically no odor and, therefore, would seem to be advantageous in those establishments where a chlorine odor might be offensive. Even with these manifold advantages chloramine-T has not been in widespread use. The main disadvantage with chloramine-T has been its relatively slow action on bacteria even in concentrations much higher than those employed for hypochlorite solutions. Much of the past work on the organic chlorine solutions has consisted of a direct comparison of these materials with hypochlorites which are generally accepted. The Public Health Service Ordinance Code for eating and drinking establistments and the Milk Ordinance and Code (1.1, 1.2) recommend that 50 p.p.m. available chlorine introduced as hypochlorite may be substituted for hot water (at 170 F.) for two minutes. Chloramine-T and other N-chloro compounds may be used in the place of hypochlorite in a concentration that is bactericidally equivalent to 50 p.p.m. available chlorine as hypochlorite. This problem was summarized by Weber in 1950 (39) in a study determining the effect of concentration and pH on the germicidal activity of chloramine-T. Working with con- centrations ranging from 50 to 1,000 p.p.m. available chlorine, he came to the following conclusions: (A) a thirty—fold increase (from so to 1,500 p.p.m.) does not sufficiently shorten the killing time to make chloramine-T equal in speed of reaction to the more alkaline mochlorites at 50 p.p.m. (B) it is necessary to adJust the pH of chloramine-T solutions in concentration of 500 to 1,000 p.p.m. available chlorine to a pH of between 7 and 7.5 in order to approach the bactericidal efficiency of hypochlorites, even more alkaline, at 50 p.p.m. These concentrations are economically impractical. Weber finally concluded, I'Although chloramine-T compounds would appear to have only limited usage where rapid germicidal action is required, they may be the germicide of choice under special conditions where long exposure periods are practicable." Inasmuch as a detailed study on the chemistry of hypochlorous acid and hypochlorites (ll, 16) has lent a great deal of information to the use of hypochlorites as sanitizing agaits, it was felt that a similar study of the chemistry of chloramine—T would give an insight into its mechanism and possibly show the way for more widespread use. The present work was conducted for the purpose of showing the relationship of the chemistry involved in chloramine-T solutions and its bacteri- cidal efficiency. Note should be made of the 1953 revision of the.Milk Ordinance and Code. Recommendations regarding the use of chemical bactericides have been somewhat modified. Hypochlorite in concentration between 50 and 100 p.p.ms available chlorine is suggested rather than 50 p.pam. as in the original (1939) Ordinance and.Code. Specific recommendations for chloramine-T generally follow those suggested by weber (39): "A 2~mdnute exposure is not sufficient for chloramine-T, unless the reaction is below pH 7.5 and the concentration of available chlorine is at least.750 ppm, or the reaction is below pH 7.0 and the concentration of available chlorine is at least 250 ppm. ‘When the reaction is below pH 8.5 and the concentration is at least 250 ppm of available chlorine, they may be employed satisfactorily where the exposure period is at least 20 mdnutes at a temperature of at least 75° F." The recommendations are a little.more realistic than those in the 1939 Code but are still lacking in full recognition of the pH- concentration relationship especially'with regard to chloramine-T. HISTORICAL.REVIEW Chloramine-T was first described by Chattaway in 1905 (9) and its uses as a disinfectant reported by Dakin, Cohen, and Kenyon (10) in 1916. It was one of the large group of similar compounds prepared in the course of a systematic investigation of germicidal preperties of N-chloro compounds. Chloramine—T is a white crystaline powder having a slight chlorous odor. It is freely soluble in water, a saturated solution at room temperature containing about 15% chlorine. It melts at 160 - 175 0. Solutions are stable to both heat and light (28). At the time of its discovery as a disinfectant in 1916, chloramine-T compared favorably with any other known disinfectant with regard to strength and efficiency. In 1923 Briscoe (3) and Feldhoff in 1929 (13) described the manufacture and antiseptic uses of chloramine-T. Because the inorganic hypochlorites are strongly alkaline in solution, they cannot be applied directly to wounds, cuts, sores, etc., without causing great irritation to the surrounding flesh and skin. Chloramine-T, however, is active and stable in a neutral solution and can be used even on the most delicate parts of the body without any after-effects. Compared with phenolic and cresolic disinfectants, chloramine-T has practically no odor, is non- poisonous, and can be taken internally without fear. Chloramine-T is without effect on metallic vessels and instruments and could, there- fore, be used for the sterilization of surgical instruments. According to Briscoe (3) chloramine-T would appear to be a sort of universal disinfectant: "It is absolutely non-toxic and can be used for treating wounds, sores, etc.; for sterilizing water and instruments; as a mouth wash; as an antiseptic wash; after shaving; as a gargle for the throat; in the bath; and in hygenic irrigation; as a dis- infectant wash for clothes and household utensils; etc., etc. Here it available in really large quantities in this country it would be a real boon to the public." Chloramine-T has never proved to be the boon that Briscoe expected it would be, primarily because its activity has been found to be considerably slower than that of hypochlorites when used under the same conditions. When used as a sanitizing agent in a manner similar to that of hypochlorites and without regard to some essential features, such as pH, ionic strength and concentration, its activity has been shown to be less than that of hypochlorite when in equal concentrations of available chlorine (7, 31, 39). The Public Health Service Milk Ordinance and Code (1.1) and the corresponding Public Health Service Ordinance and Code for Restaurants (42) recommend the use of hot water at 170° F. for a period of two minutes as a satisfactory bactericidal treatment for milk or food utensils. In lieu of hot water, hypochlorite (chlorine) compounds are permitted in a minimum concentration of 50 p.p.m. with an exposure period of two minutes. Other chlorine compounds, such as chloramine-T, are permitted to be used in a manner similar to that of hypochlorites. An exact concentration in terms of parts per million is not stipulated except for the modification previously noted in the 1953 revision of the Milk Ordinance and Code, but the recommendation is made that the other chlorine compound be bactericidally equivalent to 50 p.p.m. of available chlorine as hypochlorite. The problem of Just what concentration of available chlorine introduced as chloramine-T should be used in order to be bacteri- cidally equivalent to 50 p.p.m. of available chlorine introduced as hypochlorite is made more difficult by factors other than concentration which exert a tremendous influence on the killing time. In 1930 Johns (19) noted that chloramine-T was too slow when compared with hypochlorites. He also noted that trisodimn phosphate lowered its activity greatly, and he assmned that the reduction was due to an increase in pH. Again in 1931. Johns (20) noted that in regard to hypochlorites, pH exerted a greater influence than the concentration, particularly at the low end of the pH scale. Studying chloramine-T, Johns (21) noted that the pH decreases with a decrease in concentra- tion. The killing time decreases with a concentration decrease to approximately 200 p.p.m. available chlorine, at which point the killing time began to increase. In 1935 and later in 1937 Charlton and Levine (7, 8), in their studies of the germicidal properties of various chlorine compounds, considered pH to be the most important factor affecting the germicidal activity of chloramine-T. They found that dilute unbuffered chloramine-T solutions tend to become acid, probably from the absorption of 002. Later, in 1945 Marks, Eyes, and Strandskov (21;) found that pH was most important on the activity of chloramine-T and Halazone. Their results are shown in Table l. TABLE 1 Effect of pH on bactericidal activity of Halazone and chloramine-T Time (Minutes) for 99% m1 pH Dichloramine-T Halazone 3 87 35 A 72 33 5 60 26 6 A 21. 13 7 16 9 a 1.2 1.3 9 - 60 From Marks, Wyss, and Strandskov Because of their widespread use in water sanitation, the hypochlorites and inorganic chloramines (ammonia-chlorine combinations) have been the subject of study by many investigators, the study being much more intensive than that devoted to chloramine-T. Much of the information obtained with hypochlorites and inorganic chloramines has applied directly to the use of chloramine-T. It was early noted that the bactericidal efficiency of hypochlorites was profoundly affected by the pH. Andrewes and Orton in 1904 (2) were among the first to suggest that the active germicidal agent of hypochlorites is free hypochlorous acid. Holwerda (16), in 1928, studied the hydrolysis of hypochlorous acid. The results of his analysis are presented in Table 2. -10.. TABLE 2 Dissociation of WPOchlorous acid Percent turpochlorite present pH as undissociated HOCl Almost 100 99.6 95.8 69.7 18.7 2.2 ‘ \O (D \l 0‘ \It «P 10 0.2 From Holwerda (16) Since that time there have been similar studies relating pH and bactericidal activity of hypochlorites and inorganic chloramines (l, A, 5, ll, 12, 30, 31, 1.0). These studies all agree that the undissociated hypochlorous acid is the effective oxidizing agent whereas the oxidizing power of the salts and of the anion, 001’, is feeble. Fair gt a}, (11), in 191.8, correlated the effect of pH with the undissociated 8001 molecule and further determined that only if the pH is below 3 or if the concentration is greater than 1,000 p.p.m. is 012 present in measurable quantity. Butterfield (A), in 19%, com- pared inorganic chloramines and free chlorine against various gram negative bacteria. He determined that a residual free chlorine of 0.04 p.p.m. killed in one minute at a pH of 7 and temperature between 20 and 25 0. Inorganic chloramine under the same conditions requires concentration of 1.2 p.p.m. for 20 minutes. In order to kill in a one minute contact period, he required 25 times as much chloramine. The same amount of residual chloramine required 100 times the contact period for chlorine. It was also generally agreed that the bacteri- cidal efficiency of both hypochlorite and chloramines increased with an increase in temperature. The relationship between hypochlorous acid dissociation and its germicidal activity has led many investigators to the study of oxidation-reduction potentials and germicidal activity. Remington and Trimble (29), in 1929, studied the oxidation potential of sodium hypochlorite solutions by'bubbling oxygen or carbon dioxide through the solutions. It was noted that*when oxygen was bubbled through the solution there was no significant change. However, carbon dioxide raised the oxidation potential, simultaneously lowering the pH. Schmelkee (32), in 1933, determined that the oxidation potential of hypochlorous acid solutions at different pH values paralleled their bactericidal activity. He also noted that the addition of ammonia gas lowered the oxidation potential of chlorine solutions and simultaneous- ly lowered the bactericidal efficiency. Hallmann and Ardrey (23), in 19h0, studied the oxidation potential of hypochlorite at pH 5, 7, and 9. Comparisons were made on solutions prepared with chlorine demand- free distilled water and in the presence of suspended organic materials. They found that the oxidation potential gave a.more accurate measurement of relative germicidal activity, under these conditions, between different concentrations of chlorine than did the standard chemical tests. Trakhtmann (37), in 1949, determined.that the - 12 - bactericidal effect of calcium hypochlorite solutions, chlorine water, chloramine-T, and chlorine dioxide paralleled the oxidationereduction potential. Chang (6), in l9L5, made a detailed study of this problem as related to natural waters and concluded: "On the whole, it may be said that although potential measurements for relatively pure water treated with chlorine seemed to reflect the amount of H001, the complex.oxidation-reduction systems in the chlorinated water, the uncertainty of the reversibility of these systems, and the presence of the oxygen electrode.make the interpretation of oxidation potentials in terms of active chlorine extremely difficult. The presence of other interferring substances and the uncertain amount of dissolved oxygen in raw waters render the evaluation of potential.measurements almost impossible. The interpretation of potential measurements for chloramine or chlorimide compounds is even more difficult than that for chlorine compounds in view of the fact that each compound has its own characteristic potential and that the oxidation-reduction reaction is probably non-reversible. It is felt, therefore, that unless an electrode can be developed that is selective for the chlorine-chloride reaction, the measurement of oxidation potentials as evaluating genmicidally active chlorine is of practical value only in limited and specified circumstances and when the nature of the compound used is known." - 13 - Om-HCAL EQUEIBRIA IN CHLORAMINE—T SOLUTIONS In a solution of chloramine—T several equilibria involving the various molecules or ions containing active chlorine are simultaneously satisfied. The network of equilibria is represented by the following scheme: H20 1201‘ ,1 #:Rcwmiz ,1 H001‘—_*H7‘ ,1 001'“ 2 £101!!ng ,6 30123592 (solid) H20 acm ,l 1&0ch ,1 001- where R represents the divalent group CH3 O $02N= This network can be resolved into a series of simultaneous reactions each having its own individual equilibrium.constant. l. Hydrolysis Equilibrium KB 12012 ,1 ago-gnaw ,l HOCl 2. Disproportionation Reaction K0 2 Rcngmz ,1 RH2 3. Ionization of HDCl K1 H001;_A 001- ,1 H/ h. Ionization of RCIH K2 RClH:RCI“ ,1 Hz‘ The hydrolysis of RClH is included as a combination of processes (1.) and (2.) rather than as an independent step. Kfl = @ClXRClH) = 8 x 10-7 (35) (R012) KD : (RC12)(RH2) = 6.1 x 10'2 (26) RClH 6.5 x 10"2 (27) K1 : (H%)(0Cl’) : 2.95 x 10"8 - 6.8 2:10"3 depending upon temperature HOCl (15. 17, 18, 26, 33, 3h) K2 : (w‘Xacr) :- 2.8 f 0.2 x 10-5 (25) (30111) 3.5 x 10-5 (27) For an analysis of chloramine-T solutions the above general equilibrium equations with the equilibrium constants are operative. In addition to the general equilibrium equations, there are two stoichiometric equations applicable to these particular solutions. (HOCl) ,1 (001-) = (R32) - (R012) T .-. M11012) ,1 2 (RClH) ,1 2(RCl’) ,1 2(HCl) ,1 2 (001-) where T represents the total available chlorine of the solution in equivalents per liter. Soper (35) showed that for acid solutions, in which the ionization of RClH and H001 is negligible, the concentration of H001 is constant regardless of the total concentration of available chlorine in amounts to 0.014 p.p.m. In less acid or in basic solutions it will be seen that this represents an upper limit. Solving the previous equations with approximations which make no errors greater than 2% leads to the expression 1/2 (HOCI) = KH KD 4 1 1 ,1 2(1 ,1 Kymgfll 7IK2/(Hffi for the concentration of H001. In acid solutions or under other conditions where the second term.in the demoninator is small, this -15.. reduces to the equation developed by Soper (35), (HOCl) : KH JKD The concentration of the other active substances in these solutions is as follows: (3012) : T . K (ri- Ztl / Kz/(Wrfl- H D (R01) : T 2121 ,7 MEI (001') = K2 KH . (Kg re W ) (RCl') : K2T 2(H7‘)[2,L :1 Kz/(H’Zfl For the evaluations of these concentrations the following numerical values were chosen for the constants inasmuch as they more nearly approached the temperature conditions of the experimental work. KH .-. 8.0 x 10"7 KD : 0.061 x1 :: 3.7 x 10-8 x2 = 3.3 x 10-5 It should be noted that the constants K1 and K2 are ionization constants and they will depend upon the ionic strength of the solution. These solutions were calculated by Morris 33 _a_1_ (26) for an ionic strength of 0.02 - 0.03. Results of calculations are shown in Figures 1 and 2. The concentration of R01’ is not shown, for it varies only from 0.95‘1‘ at pH 6 to 1.00T at pH 7.5 and above. H001 and 001‘ concentrations are likewise not shown because 11001 is constant at -16-- maximum (0.01l. p.p.m. as 012) in solutions below pH 7; 0C1’ is at maximum.(0.13 p.p.m. as 012) at pH 9 decreasing by tenfold increments with each lower pH unit. -17.. Oil PRM.RCLH as etc 01 N PH l memo-p- vowceeeuueuwe 6mm m autonomy 1' 80“"! OI. TOTAL TITRABLE CHLORINE (88“.) .0 N 0.5. 014‘ 18- new. not, as ct, U l I ”III” 0.l Fl G UR E 2 I encasement sustenance II ma setuueu _.SOLLBILITY 0F DIGHLDRAMIAE-T TOTAL TITRABLE MINE (RM) |000 2000 4000 -19- WtTERIAIS AND METHODS The organism used in these experiments was Micrococcus pyogenes var. m strain 209. The culture was prepared for testing by daily transfers for a minimum of three days prior to the test date. Immediately prior to the actual test a 24-hour culture containing appxoximately 20 ml. was placed in a sterile 125 ml. flask with six small glass beads and shaken for 15 minutes on a Burrell wrist action shaker. The glass beads were added to facilitate breaking of clumps. Nail-shaped glass rods 1" x.l/h", that had previously been sterilized, were dipped in the broth culture, removed, and allowed to dry for 30 minutes. The inoculated rods were placed in the chlorine or chloramine-T solution for the prescribed contact period, at the end of which they were transferred to a tube of physiological saline containing 0.01% sodimm thiosulphate. At the end of one minute in the thiosulphate- saline solution the rods were transferred to a tube containing 10 m1. of broth recommended by the Food and Drug Administration for disinfectant testing. The broth tubes containing the rods were shaken vigorously and samples of the broth were plated in tryptone— glucose-extract (plus 1% milk) agar. The tests were all performed at 20 C. and all cultures were incubated at 35 C. This testing procedure was adopted because (A) the test conditions simulated the action of a disinfectant on contaminated eating utensils, glasses, dishes, etc., and (B) it provided a distinct advantage in that there was no residual disinfectant carried over from the test to the final broth and agar -20- plates. The latter was checked periodically by adding potassium iodide, acid, and starch to the final saline tube. Buffers used were citrate (pH 1. - 6), phosphate (6 - 8), and borate (8 - 10). pH was determined with a glass electrode and a Beckman model H-Z meter. Oxidation potential was determined with a bright platinum electrode and a Beckman model C meter. Because of the confusion existing in use of the term, "available chlorine", the method for determining chlorine concentrations for the present investigation is noted here. Concentrations were determined by titrating 25.0 ml. of chlorine solution with standardized sodium thiosulphate after acidifying in the presence of an excess of potassium iodide. Soluble starch served as an indicator. p.p.m. as 012 : ml. 0.1 E Na28203 x 35.46 x t. This is accepted procedure as given in human (11.) and most analytical references . -21- THE RELATIONSHIP OF CONCENTRATION AND pH TO THE BACTEBICIDAL ACTIVITY OF CHLORAMINE-T SOLUTIONS Many early investigators of the problem regarding concentration and pH of chloramine-T solutions showed that concentration was rela- tively less important than the pH of the solutions. These experiments were performed without regard to the chemical equilibria that were established at various concentrations and various pH levels. Referring to Figures 1 and 2, it can be seen that two materials in the equilibria are affected profoundly by pH. These materials are the un-ionized N-chloro-p-toluenesulfonamide and dichloramine-T. Hypochlorous acid in the acid range is constant at a very low level and decreases as the pH rises above 7. Hypochlorite ion is at an even lower level than hypochlorous acid in the acid range but rises sharply to reach a maximum at approximately pH 8.5. Because of the decided effect of pH as shown in the past, it seems illogical to assume that the bactericidal factor in chloramine-T solutions is mochlorous acid or hypochlorite ion, and that the active ingredients would then be those that are increased with a change in pH. Referring to Figures 1 and 2, it will be seen that it is impossible to separate the activities of N—chloro-p-toluenesulfonamide and dichloramine—T inasmuch as they both are affected in a similar manner by pH. It can be seen that both un-ionized compounds increase tenfold with each tenfold increase in hydrogen ion concentration. Examination of Table 3 shows the killing activity of various chloramine-T solutions ranging in concentration from 50 p.p.m. as 012 to h,000 p.p.m. as C12 and the pH of each adjusted to give similar concentrations of the un-ionized species in - 22 _ each series. The reduction of bacterial numbers in each test was not significantly different even though concentration changed 80 fold (50 to h,000), thereby lending support to the hypothesis that the un— ionized.materials are the effective bactericidal agents in a chloramine-T solution. TABLE 3 Effect of concentration and pH on bactericidal property of chloramine-T solutions Chloramine-T Concentration- (p.p.m. as 012) 500 4,000 5O pH Percent Bacteria Killed Figure 3 indicates the bactericidal activity of chloramine-T solution at 50 p.p.m. as C12 and the pH varying from.L to 10. The oxidation potential was also recorded and is indicated in Figure 3. The killing curve generally parallels the oxidation-reduction potential curve. This is in accordance with earlier work relating oxidation potential to bactericidal activity. The oxidation potential in a chloramine—T solution indicates, then, the effective concentration of the active ingredients. In a solution of chloramine-T containing 50 p.p.m. as 012 it is not possible to reach the precipitation level for dichloramine—T because an equilibrium between the two un-ionized materials in solution is reached before the solubility point of -23- FIGURE 3 Wit-T, so can as at. so new contact Hence Gammon-Incomes Forearm. “enema“ mwm an m “ILLWOLYO dichloramine-T. This accounts for the plateau reached at approximately pH 6 as one goes down the pH scale. At the other end of the killing curve another plateau is reached as the concentration of active com- pounds become too low to be effective under the conditions of this test. Figure l. is similar to Figure 3 except the chloramine-T solution contained 200 p.p.m. as 012. This eXperiment was designed to show the relative importance of the two un-ionized ingredients. As the solubility maximum for dichloramine-T is approached, there is a tendency for the bactericidal efficiency to level off; however, it would not be expected that the bactericidal efficiency will reach a plateau if the un-ionized N-chloro-p—toluenesulfonamide in solution continues to increase and is assumed to be active. Limitations of the testing procedure forestalls indication of what may be the true picture. Because the oxidation-reduction potential continues to rise rapidly even after the precipitation point of dichloramine-T, a logical assummion would be that bactericidal activity will increase until the maximum un-ionized N-chloro-p-toluenesulfonamide is reached. new" BACTERIA KMO 70 O O 50‘— a O -25.. —-—-—--—-—-—--_-—-_-_-— Domains-1’ Mommas FIGURE 4 Gimme-Y. :00 m u a. I0 m 0.7“? m MW“ Ibiflflo‘NOYIOfl "Mil. I!“ _ IMLLIVOLTO t RELATIONSHIP OF IONIC STRENGTH TO BACTERICIDAL EFFICIENCY OF CHLORAMINE—T As stated previously, the values of constants K1 and K2 are dependent upon the ionic strength of the solution. It is important, therefore, to take into account ionic strength.when using chloramine-T. Table A illustrates the effect of changing ionic strength, keeping concentration of chloramine-T and pH constant. The chloramine-T concentration was at 200 p.p.m. as 012 and pH at 7. The ionic strength was changed by changing the concentration of the buffer, ranging between.M/lO to M/l,000. Table A illustrates the effect of that change. As the ionic strength increases, the bactericidal . activity of chloramine-T likewise increases. TABLE 4 Effect of ionic strength on bactericidal activity of chloramine-T Concentration of Phosphate Buffer (pH 7) ‘ 14/10 14/100 M/1,000 Percent Bacteria 92.6 8h.2 83.6 Killed 200 p.p.m. as 012, 30 seconds contact period -27... STABILITY OF CHLORAMINE-T That chloramine-T is stable in solution has been amply demonstrated in the past (3). It is sufficiently stable to be recomended as a reagent in chemical analyses where chlorine is required (22, 36, 38) and has been adopted as a standard reagent for the determination of the chlorine demand of water for the estimation of the extent of contamination with certain CW agents (27 ). In an acid solution chloramine-T has the properties of hypochlorous acid. Table 5 illustrates the bactericidal stability of chloramine-T and sodium hypochlorite when maintained at a pH of 7. In addition to those chlorine solutions made in distilled water, similar solutions were prepared and immediately prior to the first test a suspension of washed, heat-killed bacterial cells was placed therein. A parallel chemical titration showed 0.8% loss of chlorine from the chloramine-T and 20% loss by sodium hypochlorite in 21. hours. Addition of killed bacterial cells had somewhat more effect upon the chloramine-T, but even then the bactericidal activity was maintained higher than the hypochlorite. -28- TABLE 5 Percent reduction of bacteria by chloramine-T and sodium hypochlorite after standing at room temperature Time (Hours) Compound 0 9 2h morm3-T 78 e 5 72 e l 70e 7 Chloramine-T with cells 74.7 66.9 63.8 N300]. 77e3 56e2 55e8 NaOCl with cells 75.9 54.1 52.6 50 p.p.m. as 012 in 11/100 phosphate buffer, pH 7 -29.. A TECHNIQUE FOR RAPID SANITIZATION OF FOMITES USING CHLORAMINE-T During the course of the investigationS'with chloramine-T, improvements were made on an existing.method that used chloramine-T as a sanitizing agent in eating and drinking establishments. The existing technique was designed for those establishments that maintained a three tank system for cleaning and sanitizing of eating utensils. This method involved two steps in the sanitizing process. After thorough cleaning in the first tank containing a detergent, the utensils were rinsed in the second tank which contained a dilute acid, and from there dipped directly into the third tank containing chloramine-T. The theory behind the sanitizing operation revolved about placing an acid film on the utensil in the second tank, which was carried over to the third tank, lowering the pH of the chloramine-T at the mrface of the utensil, thereby making the chloramine-T more bactericidal. In this manner it was assumed that the chloramine-T would remain at a fairly high pH level and would not emit chlorine odor, which is undesirable in those establisments where the cleaning was performed in close proximity to the patrons. The materials described in that procedure were used under condi- tions previously described herein. The bacterial-coated glass rods were first dipped in the acid solution for 15 seconds and then transferred to the chloramine-T solution, allowed to remain there for 30 seconds, and then transferred to thiosulphate-saline solution. After one minute in the thiosulphate-saline solution the rods were transferred to F. D. A. broth. The broth tubes were shaken vigorously - 3o - and samples of the broth.were plated out in TGE(M) agar. The results from this experiment are shown in Table 6. As one control, the rods were dipped first in saline solution before the chloramine-T to determine the difference in bactericidal activity induced by the acid rinse. There is no significant difference between the number of survivors in the control and acid pro-rinse. TABLE 6 Percent reduction of bacteria by unbuffered chloramine-T at pH 8 with and without "activating" pre-rinse Chloramine-T Concentration Pro-rinse (p.p.m. as C12) 50 100 200 M/ZO citrate buffer at 91.8 97.5 99.9 pH 6.0 Commercial PradUCt 64e7 72e8 86e8 None 5h.l 68.0 80.2 Analysis of the pre-rinse demonstrated a relatively high pH (6.3) when tap water was used and little buffering capacity. The pH in the chloramine-T solution decreased significantly after transferring four glass rods from.the acid pre-rinse to the chloramine-T solution. Because the reasoning seemed logical for a procedure such as the one described involving a pro-rinse and a stable chloramine-T solution, a series of experiments was performed to determdne: (A) the minimum pH of the pro-rinse necessary to produce high bactericidal activity, (B) maximum pH of the chloramine-T solution necessary to retain - 31 _ stability, and (C) the minimum effective concentration of chloramine-T. It was soon found that varying the pH of the chloramine-T solution between 7 and 9 did not produce any significant difference in bactericidal activity if the pre-rinse was maintained constant. However, a change in the pH of the pre-rinse produced quite a pronounced change in bactericidal activity (see Table 7). TABLE 7 Preliminary test of the effect of pH of pre-rinse M/ZO Citrate Buffer Physiological ¥_pH Saline 4 5 6 Chloramine-T ( l 2 376 57, 500 Saline 80,500 66,500 112,300 219,500 Number of survivors in unbuffered chloramine-T During the course of the experimentation cited above, it was - observed that the pH of the chloramine-T solutions decreased signifi- cantly with just a small amount of acid rinse carried over on the test rods. To overcome this difficulty and secondly, because it would also be advantageous to raise the ionic strength, buffer systems were incorporated in both the acid rinse and the chloramine-T solution. After considerable experimentation with various buffers and buffer concentrations (see Table 8), it was determined that citrate buffer, M/20 at a pH of L'was an active pre-rinse. Phosphate buffer, M/ZOO, at a pH of 8.0 gave sufficient buffering capacity in the chloramine-T to resist any pronounced change in pH of the chloramine-T. In regard to - 32 - the latter point, it was determined that the chloramine-T solution, buffered as described, could take up approximately a 10% addition of the pre-rinse while having the pH lowered to 7. As has been shown earlier, this pH could even go lower and the chloramine-T would still remain stable; however, at decided acid pH (below pH 5) chloramine-T in a concentration of 200 p.p.m. has a definite chlorine odor. TABLE 8 Relationship of buffer concentrations in pre-rinse and chloramine-T Pro-rinse Buffer Chloramine-T Buffer Concentration Concentration 4: 3:120 I 147/200 I 152200 1 W20 69.6 91+.l 99.6 M/2oo 56.6 87.1 84.3 11/500 45.7 68.6 88.3 Percent reduction of bacteria in chloramine-T (50 p.p.m. as 012, pH 8.0 phosphate buffer). Pre-rinse is citrate buffer at pH 4.0 The final study of this procedure involved a determination of the concentration of chloramine-T necessary to produce sufficient bacteri- cidal activity to be favorably compared with sodium hypochlorite. This comparison presents another problem inasmuch as regulations for the use of sodium.hypochlorite or other hypochlorites do not take into account the effect of pH, merely stating that a minimum concentration of 50 p.p.ms satisfies the United States Public Health Service regulations. Because of its basic nature, sodium.hypochlorite will produce a basic - 33 - solution in.mest kinds of water. Tables 9 and 10 illustrate the relative bactericidal activities of hypochlorite and chloramine-T. It can be seen from.this comparison that this technique using a suitably buffered pre-rinse and buffered chloramine-T solution produces equal, if not better, bactericidal activity than hypochlorite even when the available chlorine concentrations in both are maintained at 50 p.p.m. as C12. TABLE 9 Effect of chloramine-T concentration on sanitizer activity .Method Chloramine-T Concentration (p.p.m. as 012) 50 100 200 Chloramine-T 39.3 48.8 62.h Pro-rinse and Chloramine-T 96.h 99.2 99.8 Procedure as recommended fer use. Pro-rinse: M720 citrate buffer at pH A; 15 seconds contact chloramine-T: M/200 phosphate buffer at pH 8; 30 seconds contact. TABLE 10 Effect of pH upon activity of sodium hypochlorite in.M/200 buffer Contact pHWBP of NaOCl Soluti n Period p.m. (Seconds) 7 9 10 ll 15 73.0 30 91.8 57.5 h2.5 22.9 22.fi 45 93.9 96.5 54.9 60 99.2 75.5 - BL - DISCUSSION The fact that it is possible to calculate efficiencies of disinfectant mixtures by consideration of equilibria in the solution has important consequences with respect to the mechanism of the dis- infecting process. It has been amply demonstrated in the past with hypochlorites and the inorganic chloramines that the disinfectant efficiency of chlorine does not depend upon concentration. This fact is again proven in the present studies. It was not the purpose of this study to determine a satisfactory standard of disinfection involving chlorine compounds. It can readily be seen that the chemical determination of chlorine residual without consideration of pH is of little value as an indication of sanitization. The usefulness of the oxidationsreduction potential is limited to experimental procedures because, as pointed out by Chang (6), in a complex:system involving a number of compounds, each having its own oxidation potential, the significance of any one of the compounds cannot be seen. In the present study the oxidation potential of chloramine-T solution parallels to a certain extent the bactericidal activity, and the bactericidal activity parallels the concentration of un-ionized molecules involved in the equilibria.network. For additional informa- tion on the relationship of the oxidation-reduction potential in chemical equilibria refer to Figures 5, 6, and 7. Figures 5 and 6 illustrate the dissociation of chloramine-T in the presence of N/10 H01. The pH curves indicate the degree of dissociation and the Eh ourves indicate the corresponding oxidation potential. Figure 7 -35- is a similar study with sodium hypochlorite. Again, it can be seen that the oxidation-reduction potential increases with the acidity. There is a direct relationship between the oxidation potential of sodium hypochlorite solution and the concentration of undissociated HOC1 (6). Similarly, in the chloramine-T solutions there is a direct correlation between the oxidation potential and the concentration of un-ionized molecules. Relating oxidation-reduction potential, con- centration of un-ionized molecules, and bactericidal activity, it is assumed, therefore, that (A) the un-ionized molecules in chloramine-T are the bactericidal agents and (B) the bactericidal concentration of the un-ionized molecules in a chloramine-T solution can with some degree of accuracy be determined by the oxidation-reduction potential. The importance of ionic strength in a disinfecting solution has never been realized. It is Just briefly mentioned in the work of Morris at a_l_ (26). The importance of this factor is shown in the present work and must be considered if disinfectants which have an ionizing phase are being used. It is logical to assume that in disinfectants such as hypochlorous acid and chloramine-T, which ionize extensively, the ionic equilibrium will to some degree determine the activity of the compounds, especially so considering that it is the un-ionized molecule which is bactericidally active. Weber (39) was correct in his statement, as were Charlton and Levine (7), when they observed that there was relatively little difference in the bactericidal activity of chloramine-T even though the concentrations were varied from 1,000 to 4,000 p.p.m. available 6... 022 J: a _ M m . . . . 8"] ~ e 00 I :a 1 n A O 1 u 85’ oo \\INI -Illullla IIIIIIIIIIIIIII t lllll lll||\ \3.\ 2m \x . seizmhoa «.6 «zeo.:.<1.xo 0.: 2 ounce. wooofiwwm coo. 2%. com n. mozzéoszo m wiser. O- -33.. .—._.—. ._a e 00 o sus‘ \ ‘\ \ m \ \*| 8U m \\ m \ \ n X \ \ \ s u. .t x 32 a X . . .2352... 30.533.20.523 use 22:63.5 use 3.13 03.3.5452»: .533 N umber... -39- chlorine. These investigators also noticed that pH was a decidedly inportant factor in the activity of chloramine-T. A similar series of studies has been made on the hypochlorites. Johns (19) reported in 1934 that there was no difference in bactericidal activity between 25 and 10 p.p.m. available chlorine, but that 2 p.p.m. was distinctly more active than 10. The explanation for this was believed to lie in the effect of pH on the prOportion of the total "available chlorine" existing in the form of H001. In 1935 Charlton and Levine (7) reported that, " 'available chlorine' was not found to be a direct measure of the germicidal efficiency of the calcium hypochlorite studied. A solution containing 1,000 p.p.m. available chlorine was only slightly more germicidal than the same solution diluted with distilled water to 100 p.p.m. (the reaction was changed by diluting from pH 11.3 to pH 10.1.) and very much less efficient than 20 p.p.m. of the same disinfectant at a reaction of pH 8.3." This same type of study has been repeated with essentially the same results (5, 31, 1.0). It is obvious that the bactericidal activity of chlorine- containing canpounds cannot be treated in an empirical manner. It is necessary when using compounds such as these, if they are to be used most efficiently, that the chemistry underlying the bactericidal activity be investigated thoroughly. Even though at this time the physiology of chlorine sanitation is not understood, the chemistry is. With this knowledge, it is possible to use a compound such as chloramine-T as a rapid bactericidal agent. A technique for the use of chloramine-T in restaurant sanitation has been presented and possesses certain advantages over hypochlorite - 40 - sanitation. These advantages make chloramine-T a desirable restaurant sanitizer to be used in preference to hypochlorites. It is more stable, has less odor, is relatively non-toxic, and is, therefore, not irritating to the skin of the workers coming in contact with it. Contrary to the statement of‘Weber (39), chloramine-T compounds do appear to have a wider usage where rapid bactericidal action is required. CONCLUSIONS 1. The bactericidal activity of chloramine-T solutions is determined by (A) pH, (B) ionic strength, and (C) concentration, in that order of importance. 2. The oxidation-reduction potential of chloramine-T is an indication of the concentration of un-ionized molecules in solution. The un-ionized molecules have been shown to be the bactericidal agents, and the over-all bactericidal activity of a chloramine-T solution can, in some degree, be determined by the oxidation- reduction potential. 3. A technique has been presented which effectively utilizes chloramine-T as a restaurant sanitizer. 2. 3. 1.. 5. 7. 9. 10. BIBLIOGRAPHY Allen, L. A. and E. Brooks. 1952. Some Factors Affecting the Bactericidal Action of Chlorine. Proc. Soc. Ap. Bact., 15’ 155‘1650 Andrewes, F. W. and K. S. P. Orton. 1901.. Disinfectant Action of mpochlorous Acid. Centrabl. f. Bakt. K. 0rigina1e., 35: 6h5'65ls 811-8150 Briscoe, M. 1923. The Manufacture and Uses of Chloramine-T. Chem. Age (London), 9, 168-169. Butterfield, C. T. 191.8. Bactericidal Pr0perties of Chloramines and Free Chlorine in Water. Pub. H. Repts., 63(29), 934-9h0e Butterfield, C. T., E. Wattie, S. Megregian, and C. W. Chambers. 191.3. 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Effect of Concentration and Reaction (pH) on the Germicidal Activity of Chloramine-T. Pub. H. Repts., 65) 503-5120 Weber, G. R. and bi. Levine. 1941.. Factors Affecting Germicidal Efficiency of Chlorine and Chloramine. Am. Jr. P. H., 31., 719. Milk Ordinance and Code, 1953 Recommendations of the Public Health Service, Public Health Bulletin No. 229, Government Printing Office, Washington, D. C. Ordinance and Code Regulating Eating and Drinking Establishments, 1950 Recommendations of the Public Health Service, Sanitation Division of the State Services Bureau, Bulletin No. 37, Government Printing Office, Washington, D. C. r“. u. M. . Mu a d. P. a ’- [\1" 1..‘~ V-v