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 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 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.     

Long-term survival of hepatitis A virus and poliovirus type 1 in mineral water.

The survival in mineral water of hepatitis A virus (HAV) and poliovirus type 1 was compared, under controlled experimental conditions, at 4 degrees C and room temperature. Viral infectivity titers were determined by cell culture titration, while HAV antigenicity was monitored by radioimmunoassay-endpoint titration. Both viruses persisted longest at 4 degrees C. At this temperature, after 1 year of exposure, the inactivation of either HAV or poliovirus type 1 was not important. At room temperature, poliovirus type 1 was not detected after 300 days, whereas HAV was still infectious. For both temperatures, the computed regression coefficients of best-fit lines for inactivation rates for the two viruses were significantly different. The survival of HAV was also studied at 4 degrees C and room temperature in mineral water with 5- and 50-micrograms/ml protein concentrations (i.e., purity of the virus suspension) for 120 days. As shown by a comparison of the regression coefficients for the inactivation rates, the stability of HAV in mineral water depends on protein concentration and temperature. Radioimmunoassay-endpoint titration results showed inactivation patterns similar to those of cell culture titration, with the most significant reduction in HAV antigenicity at room temperature. At the two temperatures, the infectivity of HAV declined at a faster rate than the antigenicity.     

Measurement of the inactivation kinetics of poliovirus by ozone in a fast-flow mixer.

Inactivation kinetics of poliovirus type 1 in ozone demand-free water was investigated by utilizing a fast-flow mixing apparatus. Ozonated water and a solution of ozone demand-free water containing a known quantity of poliovirus type 1 were introduced simultaneously into a mixing chamber, both at a constant rate. This mixture was then passed through a narrow tube of known length and diameter into a neutralizing solution. By altering the rate of introduction and/or tube length, different contact periods between ozone and virus could be determined with an accuracy of 0.01 s. Inactivation of the poliovirus occurred in two steps. During the first step, which lasted for 0.2 to 1.0 s, 95 to 99% of the virus was inactivated, depending on the ozone concentration (which ranged from 0.1 to 2.0 mg/liter). The second step apparently continued for several minutes; in this period the remainder of the virus was inactivated. An obvious dose-response relationship was demonstrated during the first step of the inactivation curve. The pH of the water slightly affected the viral inactivation rate, but these small differences seem to have no practical value.     

Measurement of the inactivation kinetics of poliovirus by ozone in a fast-flow mixer.

Inactivation kinetics of poliovirus type 1 in ozone demand-free water was investigated by utilizing a fast-flow mixing apparatus. Ozonated water and a solution of ozone demand-free water containing a known quantity of poliovirus type 1 were introduced simultaneously into a mixing chamber, both at a constant rate. This mixture was then passed through a narrow tube of known length and diameter into a neutralizing solution. By altering the rate of introduction and/or tube length, different contact periods between ozone and virus could be determined with an accuracy of 0.01 s. Inactivation of the poliovirus occurred in two steps. During the first step, which lasted for 0.2 to 1.0 s, 95 to 99% of the virus was inactivated, depending on the ozone concentration (which ranged from 0.1 to 2.0 mg/liter). The second step apparently continued for several minutes; in this period the remainder of the virus was inactivated. An obvious dose-response relationship was demonstrated during the first step of the inactivation curve. The pH of the water slightly affected the viral inactivation rate, but these small differences seem to have no practical value.     

Magnitude of pollution indicator organisms in rural potable water.

A total of 460 water samples were randomly drawn from the potable water supply sources of rural communities in three counties of South Carolina. About 10% of the population, not incorporated in municipalities, was sampled. The samples were tested for total coliforms, Escherichia coli, and fecal streptococci. Significant levels of these pollution indicator organisms were detected in almost all the water supplies. Total coliforms were the most common, and only 7.5% of the water supplies were uncontaminated. E. coli, considered a reliable indicator of recent and dangerous pollution, was observed in 43% of the water supplies. Statistical analyses indicated that the bacterial populations, especially E. coli, were associated with the supply source depth and its distance from the septic tank. Total coliform counts were also weakly correlated to the pH of the water.     

Magnitude of pollution indicator organisms in rural potable water.

A total of 460 water samples were randomly drawn from the potable water supply sources of rural communities in three counties of South Carolina. About 10% of the population, not incorporated in municipalities, was sampled. The samples were tested for total coliforms, Escherichia coli, and fecal streptococci. Significant levels of these pollution indicator organisms were detected in almost all the water supplies. Total coliforms were the most common, and only 7.5% of the water supplies were uncontaminated. E. coli, considered a reliable indicator of recent and dangerous pollution, was observed in 43% of the water supplies. Statistical analyses indicated that the bacterial populations, especially E. coli, were associated with the supply source depth and its distance from the septic tank. Total coliform counts were also weakly correlated to the pH of the water.     

Chlorine inactivation of human norovirus,murine norovirus and poliovirus in drinking water

Aims: To evaluate the reduction of human norovirus (HuNoV) by chlorine disinfection under typical drinking water treatment conditions. Methods and Results: HuNoV, murine norovirus (MNV) and poliovirus type 1 (PV1) were inoculated into treated water before chlorination, collected from a drinking water treatment plant, and bench‐scale free chlorine disinfection experiments were performed for two initial free chlorine concentrations, 0·1 and 0·5 mg l^?1. Inactivation of MNV reached more than 4 log₁₀ after 120 and 0·5 min contact time to chlorine at the initial free chlorine concentrations of 0·1 and 0·5 mg l^?1, respectively. Conclusions: MNV was inactivated faster than PV1, and there was no significant difference in the viral RNA reduction rate between HuNoV and MNV. The results suggest that appropriate water treatment process with chlorination can manage the risk of HuNoV infection via drinking water supply systems. Significance and Impact of the Study: The data obtained in this study would be useful for assessing or managing the risk of HuNoV infections from drinking water exposure.     

High-pressure inactivation of hepatitis A virus within oysters

Previous results demonstrated that hepatitis A virus (HAV) could be inactivated by high hydrostatic pressure (HHP) (D. H. Kingsley, D. Hoover, E. Papafragkou, and G. P. Richards, J. Food Prot. 65:1605-1609, 2002); however, direct evaluation of HAV inactivation within contaminated oysters was not performed. In this study, we report confirmation that HAV within contaminated shellfish is inactivated by HHP. Shellfish were initially contaminated with HAV by using a flowthrough system. PFU reductions of >1, >2, and >3 log(10) were observed for 1-min treatments at 350, 375, and 400 megapascals, respectively, within a temperature range of 8.7 to 10.3 degrees C. Bioconcentration of nearly 6 log(10) PFU of HAV per oyster was achieved under simulated natural conditions. These results suggest that HHP treatment of raw shellfish will be a viable strategy for the reduction of infectious HAV.     

Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone.

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.     

Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone.

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.     

Liquid-phase study of ozone inactivation of Venezuelan equine encephalomyelitis virus.

Ozone, in a liquid-phase application, was evaluated as a residue-free viral inactivant that may be suitable for use in an arboviral research laboratory. Commonly used sterilizing agents may leave trace residues, be flammable or explosive, and require lengthy periods for gases or residues to dissipate after decontamination of equipment such as biological safety cabinets. Complete liquid-phase inactivation of Venezuelan equine encephalomyelitis virus was attained at 0.025 mg of ozone per liter within 45 min of exposure. The inactivation of 10(6.5) median cell culture infective doses (CCID50 of Venezuelan equine encephalomyelitis virus per milliliter represented a reduction of 99.99997% of the viral particles from the control levels of 10(7.25-7.5) CCID50/ml. A dose-response relationship was demonstrated. Analysis by polynomial regression of the logarithmic values for both ozone concentrations and percent reduction of viral titers had a highly significant r2 of 0.8 (F = 63.6; df = 1, 16). These results, together with those of Akey (J. Econ. Entomol. 75:387-392, 1982) on the use of ozone to kill a winged arboviral vector, indicate that ozone is a promising candidate as a sterilizing agent in some applications for biological safety cabinets and other equipment used in vector studies with arboviruses.     

Comparison of Giardia lamblia and Giardia muris cyst inactivation by ozone.

Inactivation of Giardia lamblia and Giardia muris cysts was compared by using an ozone demand-free 0.05 M phosphate buffer in bench-scale batch reactors at 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times of 2 and 5 min. The viability of the control and treated cysts was evaluated by using the C3H/HeN mouse and Mongolian gerbil models for G. muris and G. lamblia, respectively. The resistance of G. lamblia to ozone was not significantly different from that of G. muris under the study conditions, contrary to previously reported data that suggested G. lamblia was significantly more sensitive to ozone than G. muris was. The simple Ct value for 2 log unit inactivation of G. lamblia was 2.4 times higher than the Ct value recommended by the Surface Water Treatment Rule.     

Effect of ionic environment on the inactivation of poliovirus in water by chlorine.

The rate of inactivation of poliovirus in water by chlorine is strongly influenced by the pH, which in turn influences the relative amounts of HOCl and OCl- that are present and acting on the virus in the region of pH 6 to 10. The distribution of HOCl and OCl- is influenced to a lesser extent by the addition of NaCl. The major part of the sharp increase in disinfection rate seen with this salt is thought to be due to its effect on the virus itself resulting in an increased chlorine sensitivity, especially at high pH.     

Heat inactivation of poliovirus in wastewater sludge.

The effect of raw and anaerobically digested sludge on heat inactivation of poliovirus was investigated. Raw sludge was found to be very protective of poliovirus plaque-forming ability at all temperatures studied, but digested sludge had variable effects that were highly dependent upon the experimental conditions. In low concentrations and at relatively low inactivation temperatures, digested sludge is nearly as protective of poliovirus as raw sludge. However, at higher tempeatures and concentrations, digested sludge caused a significant acceleration of poliovirus inactivation. The difference between the protective capability of raw and digested sludge is not due to loss of protective material, because this component is present in the solids of digested sludge as well as in those of raw sludge. Instead, the difference is due to a virucidal agent acquired during digestion. Addition of this agent to the solids of either raw or digested sludge reverses the protective potential of these solids during heat treatment of poliovirus.     

Heat inactivation of poliovirus in wastewater sludge.

The effect of raw and anaerobically digested sludge on heat inactivation of poliovirus was investigated. Raw sludge was found to be very protective of poliovirus plaque-forming ability at all temperatures studied, but digested sludge had variable effects that were highly dependent upon the experimental conditions. In low concentrations and at relatively low inactivation temperatures, digested sludge is nearly as protective of poliovirus as raw sludge. However, at higher tempeatures and concentrations, digested sludge caused a significant acceleration of poliovirus inactivation. The difference between the protective capability of raw and digested sludge is not due to loss of protective material, because this component is present in the solids of digested sludge as well as in those of raw sludge. Instead, the difference is due to a virucidal agent acquired during digestion. Addition of this agent to the solids of either raw or digested sludge reverses the protective potential of these solids during heat treatment of poliovirus.