Mechanisms of inactivation of poliovirus by chlorine dioxide and iodine.

Chlorine dioxide and iodine inactivated poliovirus more efficiently at pH 10.0 than at pH 6.0. Sedimentation analyses of viruses inactivated by chlorine dioxide and iodine at pH 10.9 showed that viral RNA separated from the capsids, resulting in the conversion of virions from 156S structures to 80S particles. The RNAs release from both chlorine dioxide- and iodine-inactivated viruses cosedimented with intact 35S viral RNA. Both chlorine dioxide and iodine reacted with the capsid proteins of poliovirus and changed the pI from pH 7.0 to pH 5.8. However, the mechanisms of inactivation of poliovirus by chlorine dioxide and iodine were found to differ. Iodine inactivated viruses by impairing their ability to adsorb to HeLa cells, whereas chlorine dioxide-inactivated viruses showed a reduced incorporation of [14C]uridine into new viral RNA. We concluded, then, that chlorine dioxide inactivated poliovirus by reacting with the viral RNA and impairing the ability of the viral genome to act as a template for RNA synthesis.     

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 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.     

Chlorine dioxide inactivation of Cryptosporidium parvum oocysts and bacterial spore indicators.

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number-cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21 degrees C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg. min/liter were needed to inactivate approximately 0.5 log(10) and 2.0 log(10) units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg. min/liter were required to achieve approximately 2.0 log(10) units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity.     

Peracetic acid as an alternative wastewater disinfectant to chlorine dioxide

AIMS: The aim of this study was to compare the efficiency of peracetic acid with that of chlorine dioxide in the disinfection of wastewater from a sewage treatment plant (serving about 650 000 inhabitants) that has been using peracetic acid as a disinfectant since 1998. METHODS AND RESULTS: A total of 23 samplings were made, each consisting of three samples: from secondary effluent, effluent disinfected with 2 mg l(-1) of peracetic acid and effluent disinfected with 2.2 mg l(-1) of chlorine dioxide (contact time 20 min). For each sample, measurements were made of the heterotrophic plate count at 36 degrees C, total and faecal coliforms, Escherichia coli, enterococci, pH, suspended solids and chemical oxygen demand (COD). During the first phase of the experiment the peracetic acid was seen to be less efficient than chlorine dioxide. To improve the disinfectant action a system of mechanical agitation was added which led to a greater efficiency in the inactivation of bacteria of faecal origin. CONCLUSIONS: Both products were found to be influenced by the level of microbial contamination, the amount of suspended solids and COD but not by the pH of the effluent before disinfection. The immediate mixing of the wastewater and disinfectant caused a greater reduction in enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Since peracetic acid was seen to produce a high abatement of micro-organisms, it can be considered as a valid alternative to chlorine dioxide in the disinfection of wastewaters.     

Disinfecting capabilities of oxychlorine compounds.

The bacterial virus f2 was inactivated by chlorine dioxide at acidic, neutral, and alkaline pH values. The rate of inactivation increased with increasing pH. Chlorine dioxide disproportionation products, chlorite and chlorate, were not active disinfectants. As chlorine dioxide solutions were degraded under alkaline conditions, they displayed reduced viricidal effectiveness, thereby confirming the chlorine dioxide free radical as the active disinfecting species.     

Disinfecting capabilities of oxychlorine compounds

The bacterial virus f2 was inactivated by chlorine dioxide at acidic, neutral, and alkaline pH values. The rate of inactivation increased with increasing pH. Chlorine dioxide disproportionation products, chlorite and chlorate, were not active disinfectants. As chlorine dioxide solutions were degraded under alkaline conditions, they displayed reduced viricidal effectiveness, thereby confirming the chlorine dioxide free radical as the active disinfecting species.     

Effect of disinfectants on pathogenic free-living amoebae: in axenic conditions.

The amoebicidal properties of chlorine, chlorine dioxide, ozone, and deciquam 222 were examined in axenic conditions. Naegleria spp. were found to be more sensitive to chlorine and chlorine dioxide than Acanthamoeba spp. No marked difference in sensitivity to ozone or deciquam 222 could be detected between the pathogenic (A-1) and nonpathogenic (1501) strains of Acanthamoeba and the pathogenic (MsT) and nonpathogenic (P1200f) strains of Naegleria. Methods of disinfection are discussed with reference to suitability of the disinfectants to real conditions.     

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 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.     

Use of acid-tolerant xylanase for bleaching of kraft pulps

A xylanase from Aspergillus kawachii, active at pH 2.0–2.5, was found to be suitable for improvement of bleachability. Due to the low working pH of the xylanase, the metal cations were also removed and thus the metal removal stage using chelating agents could be omitted. The use of acid-tolerant xylanase is especially beneficial prior to ozone or chlorine dioxide bleaching stages due to the minimization of pH adjustment steps during bleaching.     

Inactivation of bacteria by Purogene

The bacteriocidal efficacy of Purogene, a stabilized aqueous solution of chlorine dioxide (ClO2) was examined using bacteria of concern to public health. The organisms tested were: Escherichia coli, Pseudomonas aeruginosa, Yersinia enterocolitica, Klebsiella pneumoniae, Streptococcus pyogenes Group A, Salmonella typhimurium and Bacillus subtilis. The test organisms responded differently to inactivation by Purogene. At least a 4 log reduction in bacterial counts was noted when Purogene was applied at a concentration of 0.75 mg/l. Since Purogene is a stabilized complex, it was necessary to provide a chemical environment suitable for the release of ClO2 in this solution. This was done by varying the pH of Purogene from 3.5 to 8.6 (pH of Purogene is 8.6) while keeping the pH of the experimental medium constant (pH 7.0). The results showed that Purogene was most efficacious at the lowest pH tested (pH 3.5). This indicates that as chlorine dioxide solutions were reduced to chlorite (which predominates at pH 8.6), their bacteriocidal efficacy was reduced, suggesting free chlorine dioxide as the active disinfecting species.     

Inactivation of bacteria by Purogene

The bacteriocidal efficacy of Purogene, a stabilized aqueous solution of chlorine dioxide (ClO₂) was examined using bacteria of concern to public health. The organisms tested were: Escherichia coli, Pseudomonas aeruginosa, Yersinia enterocolitica, Klebsiella pneumoniae, Streptococcus pyogenes Group A, Salmonella typhimurium and Bacillus subtilis . The test organisms responded differently to inactivation by Purogene. At least a 4 log reduction in bacterial counts was noted when Purogene was applied at a concentration of 0.75 mg/l. Since Purogene is a stabilized complex, it was necessary to provide a chemical environment suitable for the release of ClO₂ in this solution. This was done by varying the pH of Purogene from 3.5 to 8.6 (pH of Purogene is 8.6) while keeping the pH of the experimental medium constant (pH 7.0). The results showed that Purogene was most efficacious at the lowest pH tested (pH 3.5). This indicates that as chlorine dioxide solutions were reduced to chlorite (which predominates at pH 8.6), their bacteriocidal efficacy was reduced, suggesting free chlorine dioxide as the active disinfecting species.     

Chlorine Dioxide Inactivation of Cryptosporidium parvum Oocysts and Bacterial Spore Indicators

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number–cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg · min/liter were needed to inactivate approximately 0.5 log₁₀ and 2.0 log₁₀ units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg · min/liter were required to achieve approximately 2.0 log₁₀ units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity.     

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.     

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability.

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.     

Susceptibility of chemostat-grown Yersinia enterocolitica and Klebsiella pneumoniae to chlorine dioxide

The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two enteric bacteria, Yersinia enterocolitica and Klebsiella pneumoniae, to chlorine dioxide was investigated. These organisms were grown in a defined medium in a chemostat and the influence of growth rate, temperature, and cell density on the susceptibility was studied. All inactivation experiments were conducted with a dose of 0.25 mg of chlorine dioxide per liter in phosphate-buffered saline at pH 7.0 and 23 degrees C. The results indicated that populations grown under conditions that more closely approximate natural aquatic environments, e.g., low temperatures and growth at submaximal rates caused by nutrient limitation, were most resistant. The conclusion from this study is that antecedent growth conditions have a profound effect on the susceptibility of bacteria to disinfectants, and it is more appropriate to use the chemostat-grown bacteria as test organisms to evaluate the efficacy of a certain disinfectant.     

Susceptibility of chemostat-grown Yersinia enterocolitica and Klebsiella pneumoniae to chlorine dioxide.

The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two enteric bacteria, Yersinia enterocolitica and Klebsiella pneumoniae, to chlorine dioxide was investigated. These organisms were grown in a defined medium in a chemostat and the influence of growth rate, temperature, and cell density on the susceptibility was studied. All inactivation experiments were conducted with a dose of 0.25 mg of chlorine dioxide per liter in phosphate-buffered saline at pH 7.0 and 23 degrees C. The results indicated that populations grown under conditions that more closely approximate natural aquatic environments, e.g., low temperatures and growth at submaximal rates caused by nutrient limitation, were most resistant. The conclusion from this study is that antecedent growth conditions have a profound effect on the susceptibility of bacteria to disinfectants, and it is more appropriate to use the chemostat-grown bacteria as test organisms to evaluate the efficacy of a certain disinfectant.     

Susceptibility of the brine shrimp Artemia and its pathogen Vibrio parahaemolyticus to chlorine dioxide in contaminated sea-water.

Adults and nauplii of the brine shrimp, Artemia, together with Vibrio parahaemolyticus, were placed in sewage-contaminated sea-water which had been treated with chlorine dioxide (Hallox E-100TM) to test its potential as a disinfectant for salt water aquaculture. The nauplii were very susceptible to low concentrations of chlorine dioxide (47 micrograms/l Cl-), but the adults were slightly more resistant. Sterile sea-water treated with lower concentrations of chlorine dioxide (less than 47 micrograms/l Cl-) had no effect on the shrimp, but inhibited the growth of V. parahaemolyticus. In sewage-contaminated sea-water, chlorine dioxide levels of 285-2850 micrograms/l, necessary for the inactivation of V. parahaemolyticus and any native bacteria, destroyed the Artemia culture. Hallox E-100TM persisted in sea-water for 18 h, but later decayed. We conclude that: (i) Artemia nauplii are a sensitive and convenient test-organism to determine low concentrations of chlorine dioxide in sea-water; (ii) chlorine dioxide is efficient for controlling V. parahaemolyticus in sea-water; and (iii) chlorine dioxide should be further evaluated as a potential disinfectant for aquaculture, but, for higher organisms than Artemia.     

Chlorine dioxide disinfection of single and dual species biofilms, detached biofilm and planktonic cells

Disinfection efficacy testing is usually done with planktonic cells or more recently, biofilms. While disinfectants are much less effective against biofilms compared to planktonic cells, questions regarding the disinfection tolerance of detached biofilm clusters remain largely unanswered. Burkholderia cepacia and Pseudomonas aeruginosa were grown in chemostats and biofilm tubing reactors, with the tubing reactor serving as a source of detached biofilm clusters. Chlorine dioxide susceptibility was assessed for B. cepacia and P. aeruginosa in these three sample types as monocultures and binary cultures. Similar doses of chlorine dioxide inactivated samples of chemostat and tubing reactor effluent and no statistically significant difference between the log(10) reductions was found. This contrasts with chlorine, shown previously to be generally less effective against detached biofilm particles. Biofilms were more tolerant and required chlorine dioxide doses ten times higher than chemostat and tubing reactor effluent samples. A second species was advantageous in all sample types and resulted in lower log(10) reductions when compared to the single species cultures, suggesting a beneficial interaction of the species.