Inactivation of coxsackieviruses B3 and B5 in water by chlorine

The inactivation rates of coxsackievirus B3 (CB3) and B5 (CB5) by chlorine in dilute buffer at pH 6 were very nearly the same and about half that of poliovirus (Mahoney) under similar conditions. Purified CB3, like the poliovirus, aggregated in the acid range but not at pH 7 and above. Purified CB5 aggregated rapidly at all pH values; still, the graph of log surviving infectivity versus time was a straight line. No chlorine inactivation data were obtained with dispersed CB5, for it could be dispersed only by addition of diethylaminoethyl dextran, which would react with the chlorine. Addition of 0.1 M NaCl to the buffer at pH 6 did not influence the aggregation of CB5 or the rate of chlorine action on either of the coxsackie-viruses, but at pH 10 it increased the disinfection activity of OCl- for both viruses roughly 20-fold. Cesium chloride had a similar but smaller effect. KCl was the most active of the three in this respect, making the inactivating effect of OCl- at pH 10 about equal to that of HOCl at pH 6.     

Inactivation of polioviruses and coxsackieviruses in surface water.

Inactivation rates of polioviruses 1 and 3 and coxsackieviruses A-13 and B-1 were determined in situ in the Rio Grande in southern New Mexico, using membrane dialysis chambers. Inactivation of the viruses was exponential, and the rates of inactivation were apparently affected principally by the water temperature. Stability of the viruses in river water differed, with poliovirus 1 and coxsackie B-1 being most stable. Typically 1-log reductions of infectivity at water temperatures ranging between 23 and 27 degrees C required 25 h for poliovirus 1, 19 h for poliovirus 3, and 7 h for coxsackie virus A-13. At water temperatures of 4 to 8 degrees C, the log reduction times for poliovirus 1 and coxsackievirus B-1 were 46 and 58 h, respectively. Results obtained with labeled poliovirus 1 and coxsackievirus B-1 and with infectious ribonucleic acid indicate that inactivation was due to damage to viral ribonucleic acid. Virus-inactivation rates were also affected by heat sterilization of river water, indicating the presence of a heat-labile or volatile inactivating factor. The inactivating factor in Rio Grande water was apparently present at a constant concentration over a 1-year period.     

Inactivation of polioviruses and coxsackieviruses in surface water.

Inactivation rates of polioviruses 1 and 3 and coxsackieviruses A-13 and B-1 were determined in situ in the Rio Grande in southern New Mexico, using membrane dialysis chambers. Inactivation of the viruses was exponential, and the rates of inactivation were apparently affected principally by the water temperature. Stability of the viruses in river water differed, with poliovirus 1 and coxsackie B-1 being most stable. Typically 1-log reductions of infectivity at water temperatures ranging between 23 and 27 degrees C required 25 h for poliovirus 1, 19 h for poliovirus 3, and 7 h for coxsackie virus A-13. At water temperatures of 4 to 8 degrees C, the log reduction times for poliovirus 1 and coxsackievirus B-1 were 46 and 58 h, respectively. Results obtained with labeled poliovirus 1 and coxsackievirus B-1 and with infectious ribonucleic acid indicate that inactivation was due to damage to viral ribonucleic acid. Virus-inactivation rates were also affected by heat sterilization of river water, indicating the presence of a heat-labile or volatile inactivating factor. The inactivating factor in Rio Grande water was apparently present at a constant concentration over a 1-year period.     

Inactivation of Norwalk virus in drinking water by chlorine.

Norwalk virus in water was found to be more resistant to chlorine inactivation than poliovirus type 1 (LSc2Ab), human rotavirus (Wa), simian rotavirus (SA11), or f2 bacteriophage. A 3.75 mg/liter dose of chlorine was found to be effective against other viruses but failed to inactivate Norwalk virus. The Norwalk virus inoculum remained infectious for five of eight volunteers, despite the initial presence of free residual chlorine. Infectivity in volunteers was demonstrated by seroconversion to Norwalk virus. Fourteen of 16 subjects receiving untreated inoculum seroconverted to Norwalk virus. Illness was produced in four of the eight volunteers and in 11 of 16 control subjects. A similar Norwalk virus inoculum treated with a 10 mg/liter dose of chlorine produced illness in only one and failed to induce seroconversion in any of eight volunteers. Free chlorine (5 to 6 mg/liter) was measured in the reaction vessel after a 30-minute contact period. Norwalk virus appears to be very resistant to chlorine which may explain its importance in outbreaks of waterborne disease.     

Inactivation of Norwalk virus in drinking water by chlorine

Norwalk virus in water was found to be more resistant to chlorine inactivation than poliovirus type 1 (LSc2Ab), human rotavirus (Wa), simian rotavirus (SA11), or f2 bacteriophage. A 3.75 mg/liter dose of chlorine was found to be effective against other viruses but failed to inactivate Norwalk virus. The Norwalk virus inoculum remained infectious for five of eight volunteers, despite the initial presence of free residual chlorine. Infectivity in volunteers was demonstrated by seroconversion to Norwalk virus. Fourteen of 16 subjects receiving untreated inoculum seroconverted to Norwalk virus. Illness was produced in four of the eight volunteers and in 11 of 16 control subjects. A similar Norwalk virus inoculum treated with a 10 mg/liter dose of chlorine produced illness in only one and failed to induce seroconversion in any of eight volunteers. Free chlorine (5 to 6 mg/liter) was measured in the reaction vessel after a 30-minute contact period. Norwalk virus appears to be very resistant to chlorine which may explain its importance in outbreaks of waterborne disease.     

Inactivation of hepatitis A virus and indicator organisms in water by free chlorine residuals.

Hepatitis A virus (HAV) and selected indicator organisms were mixed together in chlorine-demand-free buffers at pH 6, 8, or 10 and exposed to free chlorine residuals, and the survival kinetics of individual organisms were compared. HAV was enumerated by a most-probable-number dilution assay, using PLC/PRF/5 liver cells for propagation of the virus and radioimmunoassay for its detection. At all pH levels, HAV was more sensitive than Mycobacterium fortuitum, coliphage V1 (representing a type of phage common in some sewage-polluted waters), and poliovirus type 2. Under certain conditions, HAV was more resistant than Escherichia coli, Streptococcus faecalis, coliphage MS2, and reovirus type 3. It was always more resistant than SA-11 rotavirus. Evidence is presented that conditions generally specified for the chlorine disinfection of drinking-water supplies will also successfully inactivate HAV and that HAV inactivation by free chlorine residuals can reliably be monitored by practical indicator systems consisting of appropriate combinations of suitable indicators such as coliform and acid-fast bacteria, coliphages, the standard plate count, and fecal streptococci.     

Inactivation of poliovirus I (Brunhilde) single particles by chlorine in water.

Like the Mahoney strain, the Brunhilde strain of poliovirus aggregated slowly in dilute phosphate-carbonate buffer at pH 6 but not at all at or above pH 7. Infectivity decreased at rates approximately proportional to the concentration of free chlorine present at pH 6 over the entire range of 5 to 40 micrometer. The addition of 0.1 M NaCl to the buffer increased the rate about twofold, but this strain was still twice as resistant as the Mahoney strain. At pH 10, inactivation was much slower than at pH 6, but when 0.1 M NaCl was added, the rate was increased 31-fold, making the OCl- at pH 10 over three times more effective than HOCl at pH 6.     

Inactivation of particle-associated coliforms by chlorine and monochloramine.

Sieves and nylon screens were used to separate primary sewage effluent solids into particle fractions of less than 7- or greater than 7-micron size. The efficiency of separation was determined by using a particle counter. Indigenous coliforms associated with the particle fractions were tested for their resistance to chlorine and monochloramine. Coliforms associated with the less than 7-microns fraction were inactivated more rapidly by 0.5 mg of chlorine per liter at 5 degrees C and pH 7 than coliforms associated with the greater than 7-microns fraction. Homogenization of the greater than 7-microns fraction not only resulted in an increase in the number of less than 7-microns particles, but also increased the rate of inactivation to a rate similar to that of the less than 7-microns fraction. With 1 mg of monochloramine per liter at 5 degrees C and pH 7, particle size had no appreciable effect on the rate of inactivation. At pH 8, however, the less than 7-micron fraction was inactivated more rapidly than the greater than 7-micron fraction. The time required for 99% inactivation of the particle fractions with monochloramine at pH 7 or 8 was 20- to 50-fold greater than the time required for the same amount of inactivation with chlorine at pH 7. The results indicate that coliforms associated with sewage effluent particles are inactivated more rapidly with 0.5 mg of chlorine per liter than with 1.0 mg of monochloramine per liter. However, greater than 7-micron particles can have a protective effect against the disinfecting action of chlorine.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine.

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Inactivation of human and simian rotaviruses by chlorine.

The inactivation of simian rotavirus SA-11 and human rotavirus type 2 (Wa) by chlorine was compared at 4 degrees C by using single-particle virus stocks. Both virus types were usually more readily inactivated at pH 6.0 than at pH 8.0 when low chlorine concentrations (0.05 to 0.2 mg/liter) were used. A complete (5 log) reduction of both was obtained within 20 s at all pH levels when chlorine concentrations were increased to 0.3 mg/liter. Slight differences in the chlorine sensitivities of SA-11 and human rotavirus type 2 were noted but were not considered to be significant.     

Inactivation of hepatitis A virus and indicator organisms in water by free chlorine residuals

Hepatitis A virus (HAV) and selected indicator organisms were mixed together in chlorine-demand-free buffers at pH 6, 8, or 10 and exposed to free chlorine residuals, and the survival kinetics of individual organisms were compared. HAV was enumerated by a most-probable-number dilution assay, using PLC/PRF/5 liver cells for propagation of the virus and radioimmunoassay for its detection. At all pH levels, HAV was more sensitive than Mycobacterium fortuitum, coliphage V1 (representing a type of phage common in some sewage-polluted waters), and poliovirus type 2. Under certain conditions, HAV was more resistant than Escherichia coli, Streptococcus faecalis, coliphage MS2, and reovirus type 3. It was always more resistant than SA-11 rotavirus. Evidence is presented that conditions generally specified for the chlorine disinfection of drinking-water supplies will also successfully inactivate HAV and that HAV inactivation by free chlorine residuals can reliably be monitored by practical indicator systems consisting of appropriate combinations of suitable indicators such as coliform and acid-fast bacteria, coliphages, the standard plate count, and fecal streptococci.     

Inactivation of human and simian rotaviruses by chlorine dioxide.

The inactivation of single-particle stocks of human (type 2, Wa) and simian (SA-11) rotaviruses by chlorine dioxide was investigated. Experiments were conducted at 4 degrees C in a standard phosphate-carbonate buffer. Both virus types were rapidly inactivated, within 20 s under alkaline conditions, when chlorine dioxide concentrations ranging from 0.05 to 0.2 mg/liter were used. Similar reductions of 10(5)-fold in infectivity required additional exposure time of 120 s at 0.2 mg/liter for Wa and at 0.5 mg/liter for SA-11, respectively, at pH 6.0. The inactivation of both virus types was moderate at neutral pH, and the sensitivities to chlorine dioxide were similar. The observed enhancement of virucidal efficiency with increasing pH was contrary to earlier findings with chlorine- and ozone-treated rotavirus particles, where efficiencies decreased with increasing alkalinity. Comparison of 99.9% virus inactivation times revealed ozone to be the most effective virucidal agent among these three disinfectants.     

Inactivation of Giardia muris cysts by free chlorine.

The chlorine resistance of cysts of the flagellate protozoan Giardia muris was examined. This organism, which is pathogenic to mice, is being considered as a model for the inactivation of the human pathogen Giardia lamblia. Excystation was used as the criterion for cyst viability. Experiments were performed at pH 5, 7, and 9 at 25 degrees C and pH 7 at 5 degrees C. Survival curves were "stepladder"-shaped, but concentration-time data generally conformed to Watson's Law. Chlorine was most effective at neutral pH and was only slightly less so in acidic solutions. Comparison of inactivation data based on equivalent hypochlorous acid concentrations, which corrects for chlorine ionization, showed that the cysts have a pH-dependent resistance to inactivation. Concentration-time (C X t') products for free chlorine obtained at 25 degrees C ranged from a low of 50 mg min/liter at pH 5 to a high of 218 mg min/liter at pH 9 and were as high as 1,000 mg min/liter at 5 degrees C. It appears that G. muris cysts are somewhat more resistant to inactivation than G. lamblia cysts and rank among the microorganisms that are most resistant to inactivation by free chlorine.     

Inactivation of poliovirus I (Brunhilde) single particles by chlorine in water

Like the Mahoney strain, the Brunhilde strain of poliovirus aggregated slowly in dilute phosphate-carbonate buffer at pH 6 but not at all at or above pH 7. Infectivity decreased at rates approximately proportional to the concentration of free chlorine present at pH 6 over the entire range of 5 to 40 micrometer. The addition of 0.1 M NaCl to the buffer increased the rate about twofold, but this strain was still twice as resistant as the Mahoney strain. At pH 10, inactivation was much slower than at pH 6, but when 0.1 M NaCl was added, the rate was increased 31-fold, making the OCl- at pH 10 over three times more effective than HOCl at pH 6.     

Inactivation of clay-associated bacteriophage MS-2 by chlorine.

The model system consisted of bacteriophage MS-2, bentonite clay, and hypochlorous acid (HOC1). Factors that influenced association of the bacterial virus with bentonite were the titer of unadsorbed viruses, clay concentration, cation concentration, temperature, stirring rate, and the presence of soluble organics. Variation of the kinetic adsorption rate constant with stirring speed indicates that phage attachment is a diffusion-limited process; the attachment reaction has an apparent activation energy of 1 kcal/mol. About 18% of clay-associated bacteriophages was recovered by mixing the suspension with an organic eluent. Inactivation data were obtained from batch reactors operated under those conditions in which loss of HOC1 was minimal during the reaction. Bacteriophages attached to clay were more resistant to HOC1 than were freely suspended phages; for equivalent HOC1 concentrations, clay-associated phages required about twice the time that freely suspended phages required for loss of 99% of the initial virus titer.     

Inactivation of clay-associated bacteriophage MS-2 by chlorine.

The model system consisted of bacteriophage MS-2, bentonite clay, and hypochlorous acid (HOC1). Factors that influenced association of the bacterial virus with bentonite were the titer of unadsorbed viruses, clay concentration, cation concentration, temperature, stirring rate, and the presence of soluble organics. Variation of the kinetic adsorption rate constant with stirring speed indicates that phage attachment is a diffusion-limited process; the attachment reaction has an apparent activation energy of 1 kcal/mol. About 18% of clay-associated bacteriophages was recovered by mixing the suspension with an organic eluent. Inactivation data were obtained from batch reactors operated under those conditions in which loss of HOC1 was minimal during the reaction. Bacteriophages attached to clay were more resistant to HOC1 than were freely suspended phages; for equivalent HOC1 concentrations, clay-associated phages required about twice the time that freely suspended phages required for loss of 99% of the initial virus titer.     

Inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine.

The kinetics of inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine were studied at 5 degrees C with a purified preparation of single virions and a preparation of cell-associated virions. Inactivation of the virus preparations with chlorine and chlorine dioxide was studied at pH 6 and 10. The monochloramine studies were done at pH 8. With 0.5 mg of chlorine per liter at pH 6, more than 4 logs (99.99%) of the single virions were inactivated in less than 15 s. Both virus preparations were inactivated more rapidly at pH 6 than at pH 10. With chlorine dioxide, however, the opposite was true. Both virus preparations were inactivated more rapidly at pH 10 than at pH 6. With 0.5 mg of chlorine dioxide per liter at pH 10, more than 4 logs of the single-virus preparation were inactivated in less than 15 s. The cell-associated virus was more resistant to inactivation by the three disinfectants than was the preparation of single virions. Chlorine and chlorine dioxide, each at a concentration of 0.5 mg/liter and at pH 6 and 10, respectively, inactivated 99% of both virus preparations within 4 min. Monochloramine at a concentration of 10 mg/liter and at pH 8 required more than 6 h for the same amount of inactivation.     

Inactivation of gerbil-cultured Giardia lamblia cysts by free chlorine.

Giardia lamblia cysts were harvested from Mongolian gerbils and exposed to free chlorine in buffered water at pH 5, 7, and 9 at 15 degrees C. The contact times required to obtain a 2-log reduction in cyst survival (i.e., a 99% kill) were interpolated from survival curves generated at fixed concentrations of chlorine in the range of 0.25 to about 16 mg/liter. Concentration-time (C.t') products for 99% inactivation ranged from about 120 to nearly 1,500 mg.min/liter. These values are higher than those reported previously for free chlorine using G. lamblia cysts from infected humans. The cysts isolated from gerbils, as with other Giardia cysts, were unusually sensitive to chlorine in alkaline solutions.     

Inactivation of particle-associated coliforms by chlorine and monochloramine

Sieves and nylon screens were used to separate primary sewage effluent solids into particle fractions of less than 7- or greater than 7-micron size. The efficiency of separation was determined by using a particle counter. Indigenous coliforms associated with the particle fractions were tested for their resistance to chlorine and monochloramine. Coliforms associated with the less than 7-microns fraction were inactivated more rapidly by 0.5 mg of chlorine per liter at 5 degrees C and pH 7 than coliforms associated with the greater than 7-microns fraction. Homogenization of the greater than 7-microns fraction not only resulted in an increase in the number of less than 7-microns particles, but also increased the rate of inactivation to a rate similar to that of the less than 7-microns fraction. With 1 mg of monochloramine per liter at 5 degrees C and pH 7, particle size had no appreciable effect on the rate of inactivation. At pH 8, however, the less than 7-micron fraction was inactivated more rapidly than the greater than 7-micron fraction. The time required for 99% inactivation of the particle fractions with monochloramine at pH 7 or 8 was 20- to 50-fold greater than the time required for the same amount of inactivation with chlorine at pH 7. The results indicate that coliforms associated with sewage effluent particles are inactivated more rapidly with 0.5 mg of chlorine per liter than with 1.0 mg of monochloramine per liter. However, greater than 7-micron particles can have a protective effect against the disinfecting action of chlorine.     

Inactivation of human and simian rotaviruses by chlorine

The inactivation of simian rotavirus SA-11 and human rotavirus type 2 (Wa) by chlorine was compared at 4 degrees C by using single-particle virus stocks. Both virus types were usually more readily inactivated at pH 6.0 than at pH 8.0 when low chlorine concentrations (0.05 to 0.2 mg/liter) were used. A complete (5 log) reduction of both was obtained within 20 s at all pH levels when chlorine concentrations were increased to 0.3 mg/liter. Slight differences in the chlorine sensitivities of SA-11 and human rotavirus type 2 were noted but were not considered to be significant.