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 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 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.     

Comparison of ozone inactivation, in flowing water, of hepatitis A virus, poliovirus 1, and indicator organisms

In steadily flowing water at 20 degrees C and pH 7, five organisms had the following order of resistance to ozone (at constant levels of ozone): poliovirus 1 (PV1) less than Escherichia coli less than hepatitis A virus (HAV) less than Legionella pneumophila serogroup 6 less than Bacillus subtilis spores. The tests were repeated at 10 degrees C with HAV, PV1, and E. coli. Ozone inactivation of HAV and E. coli was faster at 10 degrees C than at 20 degrees C. At 20 degrees C, 0.25 to 0.38 mg of O3 per liter was required for complete inactivation of HAV but only 0.13 mg of O3 per liter was required for complete inactivation of PV1.     

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 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.     

Comparison of ozone inactivation, in flowing water, of hepatitis A virus, poliovirus 1, and indicator organisms.

In steadily flowing water at 20 degrees C and pH 7, five organisms had the following order of resistance to ozone (at constant levels of ozone): poliovirus 1 (PV1) less than Escherichia coli less than hepatitis A virus (HAV) less than Legionella pneumophila serogroup 6 less than Bacillus subtilis spores. The tests were repeated at 10 degrees C with HAV, PV1, and E. coli. Ozone inactivation of HAV and E. coli was faster at 10 degrees C than at 20 degrees C. At 20 degrees C, 0.25 to 0.38 mg of O3 per liter was required for complete inactivation of HAV but only 0.13 mg of O3 per liter was required for complete inactivation of PV1.     

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 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 Giardia lamblia and Giardia canis cysts by combined and free chlorine

Free chlorine and a combined organic N-chloramine (3-chloro-4,4-dimethyl-2-oxazolidinone, compound 1) were compared for efficacy as disinfectants against an admixture of cysts of Giardia lamblia and Giardia canis in water solution under a variety of test conditions; variables were pH, temperature, and water quality. In general, compound 1 was found to reduce the giardial excystation in the solutions at lower concentration or shorter contact time at a given total chlorine concentration than did free chlorine.     

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 Giardia lamblia and Giardia canis cysts by combined and free chlorine.

Free chlorine and a combined organic N-chloramine (3-chloro-4,4-dimethyl-2-oxazolidinone, compound 1) were compared for efficacy as disinfectants against an admixture of cysts of Giardia lamblia and Giardia canis in water solution under a variety of test conditions; variables were pH, temperature, and water quality. In general, compound 1 was found to reduce the giardial excystation in the solutions at lower concentration or shorter contact time at a given total chlorine concentration than did 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.     

Effect of chlorine treatment on infectivity of hepatitis A virus

This study examined the effect of chlorine treatment on the infectivity of hepatitis A virus (HAV). Prodromal chimpanzee feces, shown to induce hepatitis in marmosets (Saguinus sp.), was clarified, and the virus was precipitated with 7% polyethylene glycol 6000, harvested, and resuspended. The suspension was layered onto 5 to 30% linear sucrose gradients and centrifuged; the fractions containing HAV were dialyzed, and a 1:500,000 dilution of this preparation induced hepatitis and seroconversion in 2 of 4 marmosets. A 1:50 dilution of this preparation served as inoculum. Untreated inoculum induced overt hepatitis and seroconversion in 100% (5 of 5) of marmosets inoculated intramuscularly. Inoculum treated for various periods (15, 30, or 60 min) with 0.5, 1.0, or 1.5 mg of free residual chlorine per liter induced hepatitis in 14% (2 of 14), 8% (1 of 12), and 10% (1 of 10) of marmosets, respectively, and induced seroconversion in 29, 33, and 10% of the animals. Inoculum treated with 2.0 or 2.5 mg of free residual chlorine per liter was not infectious in marmosets as determined by absence of hepatitis and seroconversion in the 13 animals tested. Thus, treatment levels of 0.5 to 1.5 mg of free residual chlorine per liter inactivated most but not all HAV in the preparation, whereas concentrations of 2.0 and 2.5 mg of free residual chlorine per liter destroyed the infectivity completely. These results suggest that HAV is somewhat more resistant to chlorine than are other enteroviruses.     

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.