Preparation And Disinfection Efficiency Evaluation Of Pure Chlorine Dioxide Solution

The sterilization capacity of chlorine dioxide(ClO2)is 3-5 times higher than that of hypochlorous acid,showing a wide sterilization spectrum and no formation of organic chlorine,which is harmful to human health.The United Nations Health Organization has classified chlorine dioxide as the safest and healthiest disinfect product(Al level).However,the biggest problem of chlorine dioxide is storage instability and the need of activation prior to use.Domestic stable chlorine dioxide disinfectants release chlorine dioxide gas by reaction of chlorite with activation agent that may result in the decrease of antibacterial efficacy and the instable content of chlorine dioxide.Therefore,compared with pure chlorine dioxide,the present chlorine dioxide disinfectant products show poor antibacterial efficacy.In this study,we aim to establish a high-purity and stable chlorine dioxide solution preparation system,the traditional preparation method of chlorine dioxide solution was improved by using one-step chemical reaction method.To obtain minimum effective concentration of the prepared chlorine dioxide solution,the disinfection efficiency was evaluated via bacteria,spores and viruses killing tests.In addition,the influence factors of disinfection effect were also explored in the present study.Taken together,these findings indicate that the pure state chlorine dioxide solution with good disinfection efficacy was successfully prepared,which provides the scientific basis for further research and development of a safe and stable chlorine dioxide disinfectant.The details are as follows:1 Preparation of pure ClO2 solution and establishment of test methodThe purpose of the following experiments was to prepare pure ClO2 solution and to establish a simple and specific determination method of ClO2 solution.In this trial,sodium sulfite solution and sodium chlorate solution were used to generate ClO2 gas under the action of mineral acid.The gas was absorbed in ice water bath to prepare a pure ClO2 solution.By performing ultraviolet(UV)absorption scanning on standard ClO2 solution with different concentrations,and performing qualitative and quantitative detection,a standard curve was established.At the same time,the influence of common impurity ions ClO2-,ClO3-,ClO-,Cl-on the content determination was investigated,and its comparison of the accuracy of content determination by five-step iodometry.The results showed that ClO2 has a characteristic absorption peak at a wavelength of 359 nm,and each impurity ion didn’t affect the qualitative and quantitative detection of ClO2 solution at 359 nm.Compared with the traditional five-step iodometric method,the ultraviolet spectrophotometric detection method was much more accurate.The concentration of the prepared ClO2 solution was 135 mg· L-1 by using established UV detection.2 Screening test of the neutralization reagent for pure ClO2 solutionTo discovery the appropriate neutralization reagent and to understand the appropriate concentration of CO2 solution during disinfection tests,we performed the following experiments.Sodium thiosulfate recommended in the " Veterinary disinfection Technical Specifications " was selected as the neutralizing agent.According to the literature,The neutralization ratio was set to ClO2 solution:sodium thiosulfate solution=1:9,the neutralization time was set to 10 min;the neutralizer concentration is set to:0.1%,0.3%,0.5%,0.7%,1.0%,through the detection and comparison of the absorbance value of the neutralizer and ClO2 solution before and after neutralization,the most suitable concentration of neutralizer was established;E.coli 8099 was used as the test strain in the test,and the concentration of ClO2 solution used for the neutralization experiment was 135 mg·L-1，to determine whether the sodium thiosulfate solution as a neutralizing agent had an effect on the sterilization efficiency of ClO2 solution through the quantitative method of suspension.The results showed that 0.5%sodium thiosulfate could completely neutralize the ClO2 solution within 10 minutes without affecting the absorbance value and sterilization effect of the ClO2 solution.3 Killing tests of microorganism by exposure to pure ClO2 solutionTo investigate the bactericidal efficacy of ClO2 solution against Gram-positive bacteria,Gram-negative bacteria and spores.Standard strains E.coli 8099.Gluconococcus aureus 6538,and Bacillus cereus 8008 were selected as the test bacteria.A series of ClO2 solution concentrations were selected as 6 mg·L-1,3 mg·L-1,1.5 mg·L-1 in the preliminary test.The solution was applied to E.coli 8099,Staphylococcus aureus 6538 in the 30 s,1,2,3,4,5 min for qualitative and quantitative killing test,and the killing rate and killing index were determined by the plate colony counting method;On basis of the pretests,1.5 mg·L-1,0.75 mg·L-1,0.375 mg·L-1 ClO2 solution were exposed to E,coli 8099,Gluconococcus aureus 6538,and Bacillus cereus 8008 for 5,10,15,30,60 min,respectively,during qualitative and quantitative killing efficiency evaluation.The killing rate and killing index of ClO2 were also calculated at the same time.The findings showed that the killing rates of 0.75 mg·L-1 ClO2 solution on Escherichia coli(2.2 × 108 cfu·mL-1,5 min),Staphylococcus aureus(6.7 × 107 cfu·mL-1 10 min)and Bacillus cereus(L6 × 107 cfu·mL-1 30 min)were up to 99.99%,99.99%and 99.99%,respectively.4 Killing tests of viruses by exposure to pure ClO2 solutionIn this chapter,we aim to investigate the disinfection efficiency of ClO2 solution,the Newcastle disease virus(NDV)La Sota strain and the swine fever rabbit attenuated strain were used as test viruses.Based on the results of ClO2 solution to kill bacteria and spores,the ClO2 solutions(1.5 mg·L-1,0.75 mg-L-1 and 0.375 mg· L-1)were selected for qualitative and quantitative killing tests of NDV.Quantitative killing test:2.5 mL ClO2 solution and 2.5 mL NDV solution were took respectively,then inoculation the allantoic cavity of the chicken embryo to calculate the EID50 of the chicken embryo by observing the survival of the chicken embryo within 120 hours and performing hemagglutination titer(HA)determination of the dead chicken embryo.Qualitative killing test:2.5 mL each concentration of ClO2 solution were taken to react with 2.5 mL NDV respectively.After treatment for 5,10,15,30,and 60 min,0.5 mL of the mixed solution were drawn into 4.5 mL of neutralizer,and 0.1 mL chicken embryos were taken to inject into the allantoic cavity,and the 120-hour chicken embryo survival was measured to determine EID50,and the dead chicken embryos were tested for HA.1.5 mg·L-1 0.75 mg-L-1,0.375 mg·L-1 ClO2 solution were exposed to swine fever virus,the reaction mixture was injected into the rabbit’s body via ear vein injection.To evaluate the killing efficacy of ClO2 solution on classical swine fever virus,the rectal body temperature was measured with 7 days after administration ClO2 solution.The results showed that the killing rate of NDV after 5 minutes of 1.5 mg·L-1 and 0.75 mg-L-1 dose groups reached to 99.99%,but the qualitative test results showed that they could not achieve 100%killing effect.At the same time,the results of killing rate of hog cholera rabbit attenuated virus showed that 0.375 mg·L-1 ClO2 solution could kill all viruses in a 20℃ water bath for 5 min.These findings showed that 0.75 mg· L-1 ClO2 solution has a good killing effect on both viruses.5 Influencing factors of pure ClO2 solution disinfection effectsBased on the "Veterinary disinfection Technical Specifications ",E.coli 8099,Gluconococcus aureus 6538,and Bacillus cereus 8008 were used as test bacteria,the concentration,disinfection time,temperature,organic matter concentration,pH value during sterilization and disinfection disinfection of ClO2 solution were investigated.Four concentration of ClO2 solution groups(0.75 mg·L-1,0.66 mg·L-1,0.56 mg·L-1)for 5,10,15,30,60 min were set and exposed to bacterial suspension(1×107～5×107 cfu·mL-1)respectively.The average killing index and killing rate of bacterial suspension were counted to determine the influence of concentration and disinfection time on the sterilization and disinfection effect of ClO2 solution;At 5 ℃±1℃,10 ℃ ± 1℃,20 ℃±1 ℃,30 ℃ ± 1 ℃,the five bacterial suspensions(1×107～5×107 cfu·mL-1)and 0.75 mg·L-1 ClO2 were mixed to react.After the reaction,the average kill index and kill rate were measured to investigate the effect of temperature on the sterilization and disinfection effect of ClO2 solution.5%,10%,and 20%fetal bovine serum were configured as organic interferents and then an organic interfering substance was added to a mixture of bacterial suspension of 1×107～5×107 cfu·mL-1 and 0.75 mg·L-1 ClO2 solution,and the average killing index and killing were measured after the reaction.To evaluate the effect of organic matter concentration on the sterilization and disinfection effect of ClO2 solution;configure ClO2 solution with pH value of 2.0,4.0,6.0,8.0,10.0 and bacterial suspension with concentration of 1×107～5×107 cfu·mL-1.After the reaction,the influence of pH value on the solution ClO2 sterilization effect was investigated by measuring the average kill rates and kill index.The results showed that the sterilization and disinfection effect of the ClO2 solution was elevated with the increase of ClO2 solution concentration,the extension of the action time,and the temperature increase within 10 ℃～30 ℃.When fetal bovine serum was higher than 5%,the sterilization efficacy of ClO2 solution could be reduced,the change of pH of ClO2 solution in the range of pH<10 had little effect on its bactericidal efficacy.