WO2005117914A2

WO2005117914A2 - Biocidal solution

Info

Publication number: WO2005117914A2
Application number: PCT/US2005/019665
Authority: WO
Inventors: Zsolt Kortvelyesi; Howard Mann; Gilbert Gordon
Original assignee: Sterilox Technologies, Inc.
Priority date: 2004-06-04
Filing date: 2005-06-03
Publication date: 2005-12-15
Also published as: WO2005117914A3

Abstract

A biocidal solution containing a mixture of free available chlorine and chlorine dioxide. A method of producing such a biocidal solution using electrolysis or acidification is also provided. The biocidal solution can be used to disinfect a surface such as sterilize a medical device, decontaminate a food item, treat water, or reduce or eliminate biofilm.

Description

A BIOCIDAL SOLUTION

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority to U.S. Provisional Application No. 60/576,606 filed on June 4, 2004.

FIELD OF THE INVENTION The present invention relates to a biocidal solution containing free available chlorine and chlorine dioxide.

BACKGROUND OF THE INVENTION Electrochemically activated brine solutions can be very effective biocides, as the activated species in such solutions act to oxidize biological material or micro-organisms. Such biocidal solutions have numerous commercial uses such as sterilizing or disinfecting drinking water, treating food and food preparation apparatus, and sterilizing or disinfecting medical apparatus. These solutions typically utilize a much lower salt concentration and are also sometimes referred to as "superoxidised water solutions". The free available chlorine (FAC) content of an electrochemically activated brine solution is seen as the primary indicator of its biocidal efficacy and solutions with high levels of FAC can be readily produced. In general, the FAC content of an electrochemically activated brine solution contains a combination of chlorine species, more specifically aqueous chlorine (Cl₂(_aq)), hypochlorous acid (HOC1), and hypochlorite ions (OCL^"). The pH of such a solution will have an effect on the proportion of these constituents in the solution. Chlorine dioxide does not contribute significantly to the FAC levels of an electrochemically activated brine solution. To produce electrochemically activated solutions having high levels of FAC, brine (or 'input solution') being passed into the electrolytic cell producing the solution needs to be of a high salt concentration. However, an input solution with a high salt concentration has a negative effect on equipment producing electrochemically activated solutions, due to the corrosive nature of the salt in the input solution. Further, the residual salt content of the electrochemically activated solution will also be high when the salt concentration of the input solution is high. This means that traditional electrochemically activated solutions with high FAC levels, because they have a high residual salt concentration, will also be corrosive against many devices that a user may wish to sterilize or disinfect. The FAC itself can also have a negative effect on materials requiring sterilization, for example, FAC will attack the polyurethane coatings on flexible endoscopes. FAC reacts rapidly with organic substances quickly consuming a substantial proportion of the active species (FAC). This is desirable when the organic substances are micro-organisms themselves. However, when the organic substances are, for example, residual protein material on an improperly cleaned or pre-washed medical device, the biocidal efficacy of the solution is quickly lost as the FAC reacts with the residual protein material, and is often lost before the device to be sterilized or disinfected has been sterilized or disinfected. Electrochemically activated solutions with relatively low FAC concentrations can still be effective broad spectrum biocides but, with respect to the materials being sterilized or disinfected, such solutions still suffer the negative effects, albeit to a lesser degree, associated with high level FAC solutions. However, lowering the FAC concentration (and hence also lowering the residual salt concentration) of the solutions to reduce their corrosive nature sacrifices biocidal efficacy. Chlorine dioxide (ClO₂) has a long history of use as a disinfectant for the preparation of drinking water, and a shorter history of use as a disinfectant/sterilizing agent for medical devices. ClO₂ is typically prepared for use in a healthcare setting by either the acidification or electrolysis of sodium chlorite solutions. In comparison to chlorine solutions, such as hypochlorous acid solutions, ClO₂ solutions are less sensitive to organic contamination, that is, ClO₂ tends to recycle itself to continue its biocidal activity. ClO₂ solutions are also less susceptible to changes in solution pH and are more oxidising than chlorine solutions. However, depending upon the concentration, ClO₂ solutions have potentially dangerous toxicological properties, possess greater hazards in storage and are more corrosive to metals. Because of such effects, use of ClO₂ as a disinfectant/sterilizing agent for medical devices and other surfaces has been of limited beneficial value. Accordingly, there is an ongoing need for a biocidal solution that is not only effective but that also has a high level of compatibility with the devices and other surfaces they are to sterilize or disinfect.

SUMMARY OF THE INVENTION In an embodiment, the present invention provides a biocidal solution comprising free available chlorine and chlorine dioxide. In another embodiment, the present invention provides a method of disinfecting a surface by exposing the surface to a biocidal solution comprising free available chlorine and chlorine dioxide.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a chart depicting the pH dependence of the rate of a FAC-ClO reaction. FIGURE 2 is a chart depicting the disinfection times as a function of disinfectant concentrations atpH 7.0. FIGURE 3 is a chart depicting the disinfection times as a function of disinfectant concentrations at pH 7.5 FIGURE 4 is a chart comparing sterile cylinders when different disinfectants and pretreatment methods are used. FIGURE 5 is a chart comparing the number of bacteria lost with different solutions and pretreatment methods.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term "FAC" refers to free available chlorine in a solution. It will of course be appreciated that other halides, when free in solution, also have biocidal efficacy and the present invention encompasses such other halides at the concentrations required to be equally effective as any stated concentration of FAC. A person skilled in the art will be readily able to determine such equivalent concentrations. Similarly, it will be appreciated that other halide dioxides, other than chlorine dioxide have biocidal efficacy and the present invention encompasses such other halide dioxides at the concentrations required to be equally effective as any stated concentration of FAC. A person skilled in the art will be readily able to determine such equivalent concentrations. The term "electrochemical cell" as used herein includes, unless the context otherwise requires, multiple electrochemical cells arranged either in series or in parallel, or a combination series-parallel arrangement. The invention has been described below primarily with reference to chlorine based acids and salts. It will of course be appreciated by those skilled in the art that the chlorine based acids and salts described herein could be replaced with other halide based acids and salts, and such other acids and salts are encompassed by the present invention. Similarly, although the invention has been described with reference to sodium salts, it should be understood that other metal salts can be used and a person skilled in the art will readily be able to determine which of such other salts are appropriate. In an embodiment, the present invention provides a biocidal solution comprising a mixture of ClO₂ and a FAC such as elemental chlorine (Cl₂), hypochlorous acid (HOC1) and/or hypochlorite ions (OCF). Without wishing to be bound by theory, it is believed that such mixtures form highly biocidal reactive intermediates which are stable for periods long enough to be useful to decontaminate medical devices, and other surfaces and items. Further, a biocidal solution according to the present invention can contain low concentrations of such reactive intermediates such that the solution is non-corrosive to many devices, surfaces, and items that may require sterilization, yet is still effective in biocidal terms. In a preferred embodiment, the present invention provides a biocidal solution having a chlorine dioxide concentration of about 0.1 to about 400 ppm and more preferably of about 80 to about 400 ppm and a FAC concentration of about 0.1 to about 350 ppm and more preferably of about 80 ppm to about 350 ppm. A biocidal solution containing a mixture of FAC and ClO₂, according to the present invention, can be made by electrolysis or acidification. With respect to electrolysis, a biocidal solution of the present invention can be produced by passing a mixture of a chloride salt solution and a chlorite salt solution through an electrolytic cell or by separately electrolyzing a chloride salt solution and a chlorite salt solution and then mixing the two solutions together. It would of course also be possible to pass the chloride and chlorite salt solutions as a mixture or individually through several electrochemical cells, and such cells may be arranged either in series or parallel. More specifically, in an embodiment, an electrochemical cell used to produce a biocidal solution of the present invention has an anode chamber and a cathode chamber which produces respective anolyte and catholyte solutions, hi this embodiment, the biocidal activity of the biocidal solution is conferred to the solution primarily in the anode chamber. The method includes supplying to the cell a solution including a chlorite salt or other salt that will release chlorine dioxide when subject to electrolysis and a chloride salt. The method further comprises applying current to the cell to produce a biocidal solution comprising chlorine dioxide and FAC. Preferably, the concentrations of the chlorite salt and chloride salt and amount of current supplied are sufficient to impart to the biocidal solution an FAC concentration of 0.1 to 350ppm, more preferably 80- 350ppm, and chlorine dioxide concentration of 0.1 to 400ppm, more preferably 80- 400ppm. In another embodiment, the present invention provides a method of producing a biocidal solution in an electrolytic cell that comprises the steps of: (a) providing first and second electrolytic cells, each cell having an anode chamber and a cathode chamber which produce respective anolyte and catholyte solutions; (b) supplying to the first cell a solution including a chlorite (or other halite) salt or other salt that will release chlorine dioxide upon electrolysis and supplying to the second cell a solution including a chloride salt; (c) supplying electric current to the first cell to produce a first electrolyzed solution including chlorine dioxide and supplying electric current to the second cell to produce a second electrolyzed solution including FAC; and (d) mixing said first and second solutions to provide a biocidal solution comprising FAC and ClO₂. Preferably, the biocidal solution has a FAC concentration of 0.1 to 350 ppm, more preferably 80- 350ppm, and a chlorine dioxide concentration of 0.1 to 400 ppm, more preferably 80- 400ppm. With respect to preparing a biocidal solution of the present invention by acidification, such a method involves acidifying a chlorite salt solution (or any other salt solution that produces chlorine dioxide when acidified) and mixing the acidified chlorite salt, which now includes ClO₂ in solution, with an electrochemically activated chloride solution. A chlorate salt could also be used, as could other salts that produce chlorine dioxide when acidified. Specifically, in an embodiment, a method of producing a biocidal solution of the present invention includes acidifying a first solution including a chlorite salt or other salt that will release chlorine dioxide when subject to electrolysis and adding the first solution to a second solution of an electrochemically activated chloride solution to produce a biocidal solution comprising FAC and ClO₂. Preferably, the chlorine dioxide concentration of the biocidal solution is 0.1 to 400pm and more preferably 80-350ppm and the FAC concentration of the biocidal solution is 0.1 to 350ppm and more preferably 80-300ppm. For all methods of the present invention, the person skilled in the art can readily determine appropriate concentrations for the chemical ingredients required to produce a biocidal solution of the present invention as well as the electrochemical conditions, for example appropriate current and pH, that should be applied to such ingredients. In a preferred embodiment of the invention, 80% of the biocidal active species is still present after 4 hours production. The pH of a biocidal solution of the present invention is preferably 6.1 to 6.3. In alternative embodiments of the invention, the pH may be any value between 5.8 to 7.5. The invention can also be effective outside these ranges. For example, pH values up to about 8 may also be used. When the pH is less than about 7.5, hypochlorous acid will be the primary contributor to the FAC component of the solution. At a pH of above about 7.5, hypochlorite ions will be the primary contributor. It is possible to characterize the solutions of the invention by, amongst other techniques, infrared analysis and high performance liquid chromatography (HPLC). A biocidal solution of the present invention provides improved biocidal performance in the presence of organic load, good materials compatibility with both plastics and metals, and acceptable toxicological profiles. A biocidal solution of the present invention can be used, for example, to sterilize and disinfect medical devices; treat food groups to make them safer for human consumption and improve shelf life of the commercial product by reducing microbial load naturally present or artificially introduced to the food groups; improve the shelf-life of non-food organic items such as flowers by reducing the microbial load on the subject that is to have its shelf-life extended; reduce or eliminate biofilm, such as biofilm within water lines by killing micro-organisms residing in the water lines; and otherwise decontaminate water.

EXAMPLES

Example 1 : Stability Measurement of CIO? and Cl₂ Solutions of pure aqueous chlorine dioxide (ClO₂) and free available chlorine (Cl₂) were prepared and analyzed by amperometric titration to measure the concentrations of ClO₂ and Cl₂ as a function of time. By using amperometric titration, the concentrations of both ClO₂ and Cl₂ and the individual concentrations of ClO₂ and Cl₂ were measured directly. Solutions were more than 95% pure, which was sufficient to carry out preliminary kinetic experiments on the differences in reactivity (i.e. chemical stability) as a function of time at pH 7 and pH 9 in order to determine the pH for the continuing, preliminary experiments. 200 mg/L of ClO solution and 200 mg/L of Cl₂ solution (1:1 ratio) were mixed in phosphate buffer. Samples from mixture were analyzed on the minutes and many hours time scale for starting components of ClO₂ and Cl₂. It was determined that at pH 7, the mixture is most reactive and at pH of 9, the mixture is least reactive.

Example 2: Effect of pH on Rate of Reaction of FAC^* and C1Q₂ 50 to 200 mg/L of FAC (Cl₂, HOC1, and OC1^") solution and 12.5 to 200 mg/L of ClO₂ solution (FAC/ClO₂ ratio of between 1:1 ^' and 8:1) are mixed in pH 6.5 and 7.5 phosphate buffers. Concentration of the reagents are measured by spectrophotometric analysis. TABLE 1 shows the wavelengths for the absorbance maxima and the molar absorbances (M^cm ) for the three reactants and the chlorite ion by-product.

FAC (Free Available Chlorine) represents the different chlorine species present in aqueous solution. The three species, that can be present in solution depending on the pH, are elemental chlorine TABLE 1 Molar absorbances of the reactants HOC1, OCT, ClO₂ and the chlorite ion product at different wavelengths

TABLE 2 shows the half-life and tγ^ of chlorine dioxide at pH 7.5 at 1:1 and 4:1 FAC/ClO ratio and TABLE 3 shows the same parameters at pH of 6.5. The reaction of FAC and ClO₂ is faster at pH of 7.5 than at 6.5 (at pH of 6.5, the reaction is about 5 to 6 times slower). Specifically, at pH 7.5 the slowest reaction (50 mg/L FAC & 12.5 mg/L ClO₂) has a half-life of ~28 minutes, whereas at pH 6.5 the fastest reaction (200 mg/L FAC & 200 mg/L ClO₂) has -39 minute half-life (t_1/2 for 50 mg/L FAC & 12.5 mg/L ClO₂ at pH 6.5 is ~113 minutes). As can also be seen from TABLE 2, the half-life of ClO₂ increases with decreasing chlorine concentration. At higher concentrations, the chlorite ion slows the reaction which would result in a longer lifetime for chlorine dioxide.

TABLE 2 Half-life (t Y₂) and the time required for 75% consumption (t 75%)of ClO₂ at pH 7.5

(Ck), hypochlorous acid (HOC1), and hypochlorite ion (OC1"). TABLE 3 Half life (t Vi) and the time required for 75% consumption (t 75%)of ClO at pH 6.5

Based on these measurements, the order of each of the species in the rate law can be determined. This results in the following equation:

The products of the FAC-ClO₂ reaction depend on the pH. With increasing pH, the amount of chlorite formed increases, which then further reacts with chlorine. For this reason, the chlorite concentration shows a maximum as a function of time — this is determined by the relative rates of the FAC-ClO₂ and the FAC-chlorite reactions — and then starts decreasing. This is clearly observed at pH 7.5, but at 6.5 the chlorite formation is lower and its concentration remains practically constant during the measurement.

Example 3: Measuring the effects of pH, reactant concentrations, and temperature on the chlorine (FAC) - chlorine dioxide (C1Q₂) reaction The FAC concentration is varied between 50 mg/L and 300 mg/L and the CIO2 concentration is changed between 12.5 mg/L and 200 mg/L. The ClO₂ to FAC ratios varies between 1:1 to 1:24. The measurements are taken at two pH values, 6.5 and 7.5, set by using phosphate buffers, and at two temperatures, 22 °C and 35 °C. The purpose of these kinetic studies is to predict the stability of the FAC-CIO2 mixtures to be produced using electrolysis or by direct mixing of stable reactive components. The temperature study is carried out in order to determine the role that the increase or decrease of temperature has on the reaction. By comparing the rates of the reaction at different reactant concentrations, it is possible to determine the order with respect to a species in the rate law. TABLE 6 shows the rate constants for two FAC species at two temperatures. The rate constant for the chlorine dioxide - hypochlorite ion pathway is about three orders of magnitude higher, than for the chlorine dioxide - hypochlorous acid reaction. This means that at pH values at which CIO^" is present, the reaction proceeds through this pathway almost exclusively. Thus, the rate of the overall reaction depends on the fraction of the FAC which is present as hypochlorite ion. The fraction of this species is increasing with increasing pH, resulting in faster reaction which is in agreement with our experimental findings.

TABLE 6 Comparison of the Rate Constants For the Different Reaction Pathways and Temperatures

In TABLE 6, the rate constants at 35 ° C also are shown. From this table it can be seen that at this higher temperature the reaction takes place approximately three times faster, which is due to the temperature dependence of the rate of the main reaction, between OC1^" and CIO2. Higher disinfectant (FAC & CIO2) concentrations may be used which would result in higher initial disinfection, but the high concentration of these species may decrease rapidly due to the reaction between FAC & CIO2. This would mean that high concentration of the corrosive species would be present only for a short time, which would result in lower corrosion, thereby increasing disinfection efficiency and decreasing corrosion. In a preferred embodiment, 1-2 g/L ClO₂ and FAC are used and after 2-3 hours, a relatively high (500-600 mg/L) CIO2 concentration is maintained but a low FAC (~50 mg/L or less) concentration is maintained. The ClO₂ is less corrosive, thus, its high concentration would not contribute significantly to the corrosion but would provide sufficient disinfectant residual. Example 4: Preparing a Mixture of FAC and Chlorine Dioxide A 100 ml chlorite solution (a chlorine dioxide precursor solution) is prepared from solid sodium chlorite using distilled or deionized water. The chlorite solution contains approximately 24,000 mg/L of sodium chlorite. Equal volumes of the chlorite solution and a 1.0 M HC1 solution are mixed and the mixture is allowed to react for five minutes during which chlorine dioxide forms. The concentration of this chlorine dioxide solution, after the 5 minute reaction time with HC1, is approximately 6,000 mg/L ClO₂. After this reaction time, this solution is used for preparing the Working Stock Solutions of ClO₂. Working Stock Solutions are prepared from concentrated stock solutions of FAC (4,000 mg/L) and ClO₂ (-6000 mg/L). TABLE 7 and TABLE 8 shows the compositions of the Working Stock Solutions. Each solution is brought to 50 mL volume with distilled water.

TABLE 7

Four different synthetic mixtures are prepared from the Working Stock Solutions as follows:

TABLE 6 provides the time required for 95%> of ClO₂ to react with the FAC (t₉₅»_o).

TABLE 6

Example 5: Preparing a FAC-C1Q_?_ Mixture Five test solutions are prepared on the apparatus described in GB patent 2352728 (Sterilox Medical (Europe) Limited). The hypochlorous acid-ClO₂ mixture solution is prepared by passing a mixture of sodium chloride and sodium chlorite through one electrolytic cell. Final pH of the solution is 7.0. Example 6: Preparing FAC-C1Q?_ Mixture Separate solutions of hypochlorous acid and ClO₂ are each prepared on the apparatus described in GB patent 2352728. The solutions are then mixed to form 5 test solutions. Final pH of the solution is 7.0.

Example 7: Preparing FAC-ClOτ_ Mixture Five test solutions are prepared by acidifying a sodium chlorite solution and mixing the acidified sodium chlorite solution with an electrochemically activated chloride solution. Again, solutions are prepared such that the final pH is 7.0.

Examples 8. 9. and 10: Preparing FAC-CIO? Mixture

Examples 7, 8, and 9 are each respectively the same as Examples 4, 5 and 6 but the solutions were produced such that their final pH is 7.8. Because of the higher pH, hypochlorite ions are the primary contributor to FAC for these solutions.

Example 11 : Preparing a Mixture of FAC and Chlorine Dioxide 1. Preparing Buffers and Ethanol Solution a. Buffer 1 (pH 6.5) 55.196g of NaH₂PO ^»H₂O and 170ml of NaOH solution (1.0M) are added to a 1L volumetric flask and the flask is filled to the mark with distilled water to produce a 6.5 pH buffer solution. b. Buffer 2 (pH 7.0) 55.196g of NaH₂PO₄ ^»H₂O and 277ml of NaOH solution (1.0M) are added to a 1L volumetric flask and the flask is filled to the mark with distilled water to produce a 7.0 pH buffer solution. c. Buffer 3 (pH 7.5) 55.196g of NaH₂PO₄*H₂O and 355ml of NaOH solution (1.0M) are added to a 1L volumetric flask and the flask is filled to the mark with distilled water to produce a 7.5 pH buffer solution. d. 5% Ethanol Solution 6.4 ml of 96% ethanol is diluted to 100 mL. 2. Preparing Chlorite Solution (chlorine dioxide precursor solution) A 100 ml chlorite solution (a chlorine dioxide precursor solution) is prepared from solid sodium chlorite using distilled or deionized water. The chlorite solution contains approximately 24,000 mg/L of sodium chlorite. 3. Preparing a Chlorine Dioxide (CIO2) Solution Equal volumes of the chlorite solution and a 1.0 M HCl solution are mixed and the mixture is allowed to react for five minutes during which chlorine dioxide forms. The concentration of this chlorine dioxide solution, after the 5 minute reaction time with HCl, is approximately 6,000 mg/L ClO₂. 4. Preparing a Diluted Chlorine Dioxide Solution (600 mg/L) 30 mL of the pH 7 buffer solution and 10ml of the CIO2 solution are added to a 100 ml volumetric flask and the resultant solution is diluted to the mark with distilled water to produce a diluted chlorine dioxide solution (600 mg L). 5. Preparing Diluted FAC Solutions a. Diluted FAC solution (650 mg/L) 19mL of a FAC solution (bleach, pH 11, 4,000 mg/1) is diluted to 100 mL with distilled water to produce a diluted FAC solution (650 mg/1). b. Diluted FAC solution (600 mg/L) 30 ml of the 7.0 pH buffer solution and 17 mL of the FAC solution are added to a 100 mL volumetric flask and the resultant solution is diluted to the mark with distilled water to produce a diluted FAC solution (600 mg/1). 6. Preparing a FAC/CIO2 Mixture 5ml of the diluted FAC solution (600 mg/1) and 5 ml of the diluted ClO₂ solution are mixed to produce a FAC/CIO2 mixture. The following different disinfectants are prepared and pretreatment methods are performed to assess biocidal efficacy:

Example 12: Efficacy of Solutions of Example 11 Against C. Sporosenes Inoculated Onto Porcelain Penicylinders 1. Preparation of Bacterial Culture Clostridium sporogenes (ATCC #3584, Manassas, Virginia) is maintained in 20 ml of soil extract nutrient broth containing 10% egg meet medium (SEEM) (Difco Laboratories) in a 25 x 150 mm screw-capped test tube incubated for 72±8 hours at 35±2°C. From this C. sporogenes bacterial stork, 0.8 ml is inoculated into 80 ml SEEM and incubated for 72±8 hours at 35±2°C. The suspension of C. sporogenes is decanted through moist sterile cotton in a glass funnel. This filtered suspension of C. sporogenes is used immediately to label cylindrical carriers (Fisher Scientific, Pittsburgh, PA). 2. Preparation of Cylindrical Carriers Cylindrical carriers are washed with Triton X-100 by swirling in a beaker and rinsing four times with tap water and twice with deionized water. These cylinders are then sterilized in a steam sterilizer at 121 °C for twenty minutes. The sterile cylinders are then dried in an oven at 60°C. When cool, these sterile cylinders are ready to be labeled with C. sporogenes. 3. Labeling and Drying of Cylindrical Carriers Approximately 30 ml of the cotton-filtered suspension of C. sporogenes is placed into a 100 ml glass beaker. Twenty-five (25) cylindrical carriers are added to the suspension. The cylinders are allowed to soak in the suspension for 60 seconds and then transferred to a double layer of filter paper in a petri plate and stood on end. The petri plates containing spore-labeled carriers are placed into a vacuum desiccator chamber above Drierite desiccant. A vacuum is drawn on the desiccator chamber for 20 minutes at 15 mm of Hg. The spore-labeled carriers are allowed to dry for 24±4 hours at ambient temperature. 4. Determining the Number of CFU/ml of Bacteria in the Suspension Used to Label Cylindrical Carriers Serial ten-fold dilutions of bacterial culture as used to label carriers are made as 1.0 ml into 9 ml portions of fluid thioglycollate medium (FTM) (Difco Laboratories) One (1.0) ml portions of several dilutions are placed into petri plates. Approximately 25 ml of molten nutrient agar (at 45 °C) are added to the petri plates and gently swirled to mix. The plates are incubated at 35±2°C for > 72 hours anaerobically. Colony counts are multiplied by the appropriate dilution factor to determine colony-forming units (CFU) per ml (CFU/ml) of bacteria in the culture suspensions used to label carriers. 5. Test Procedure Ten (10 ml) of the solutions of Example 11 are placed into sterile 25 x 150 mm capped test tubes and allowed to come to temperature in a 25±1°C water bath. One tube is prepared for each carrier. Using sterile flamed forceps, one labeled carrier is placed into each tube of disinfectant. After 15, 30, 60, 120, and 240 minutes of exposure, a sterile flamed nichrome wire hook is used to remove each spore-labeled carrier from the disinfectant and to place it into 10 ml of FTM contained in a plastic centrifuge tube with a rubber stopper. The tubes are agitated for 60 seconds on a vortex mixer and serial tenfold dilutions are made as 1.0 ml into 9 ml FTM. One (1.0) ml portions of several dilutions are placed into petri plates. Approximately 25 ml of molten nutrient agar (held at 45° C) are added to the petri plates and gently swirled to mix. The plates are allowed to solidify and then incubated anaerobically for > 72 hours at 35±2°C in a GasPak Anaerobic Chamber System (Becton Dickinson, Cockeysville, MD) Three C. sporogenes-labeled cylinders are tested per exposure time per test chemistry. All tubes are incubated for 3 days at 35±2°C and then scored for growth (+) or no growth (-). 6. Validation of Neutralization A sterile carrier is placed into 10 ml of a solution containing 200 mg/L of ClO₂ and 200 mg/L of FAC in a buffer having 7.0 pH. The carrier is transferred from the disinfectant, with an additional 0.1 ml of disinfectant, to a test tube containing 10 ml of FTM. One serial ten-fold dilution is made as 1.0 ml into 9 ml of FTM and approximately 200 CFU of C. sporogenes (in 1.0ml of broth) are added to each tube. This is done twice. These tubes are incubated for 3 days at 35±2°C and then scored for growth (+) or no growth (-) of bacteria. Growth validated that the recovery process neutralizes any inhibitory activity of test-strength disinfectant. 7. Measure of the Number of Spores Per Labeled Cylindrical Carrier One bacteria labeled carrier is placed into 10 ml of FTM in a 18 x 11 mm plastic tube with silicone rubber stopper and agitated on a vortex mixer for 60 seconds. This is followed with a serial of ten-fold dilutions as 1.0 ml into 9 ml portions of FTM. One (1.0) ml portions of several dilutions are placed into petri plates. Approximately 25 ml of molten nutrient agar (at 45° C) is added to the petri plates and gently swirled to mix. The plates are allowed to dry and then incubated anaerobically for > 72 hours at 35±2°C. The colonies are counted and multiplied by the appropriate dilution factor to determine the number of spores on a spore-labeled carrier. This assay procedure is repeated for 5 cylinders. 8. Results Figure 4 shows a comparison of the sterile cylinders when different disinfectants and pretreatment methods are used. Figure 5 shows a comparison of the number of bacteria lost with different solutions and pretreatment methods. As indicated by Figures 4 and 5, a high level oxidizing solution capable of rapid disinfection is created using a mixture of FAC and ClO₂. At pH of 7.0, a biocidal solution of 300 mg/1 of FAC and 300 mg/1 of ClO₂ achieves the same level of disinfection as a 650 mg/1 solution of FAC. Due to the lower FAC concentration, however, this biocidal solution is less corrosive than a 650 mg/1 solution of FAC.

Example 13: Disinfection Times of a FAC-C1Q₂ Mixture FIGURE 1 shows how the rate of the reaction of FAC and CIO2 changes with pH of 7.0 and 7.5. FIGURES 2 and 3 show the dependence of the disinfection time on the disinfectant concentrations at both pH values. At pH 7.0, the disinfection time is linearly dependent on the reciprocal of the bleach (FAC) concentration. However, in contrast to the role of bleach, the rate of disinfection is independent of the chlorine dioxide concentration. The absence of chlorine dioxide dependence can be described based on the previously determined rate law. The reaction between FAC and CIO2 takes place slower at pH 7.0, meaning that the amounts of intermediates are low as compared to pH 7.5 and their concentrations are controlled by the amount of OCl^" present. Although not wishing to be bound by theory, from the concentration dependence at pH 7.5, the intermediates are thought to play role in the disinfection. These two facts together result in that the CIO2 concentration has no significant effect on the disinfection time. At pH 7.5, the concentration dependence for disinfection is different than at the pH 7.0. At the higher pH, the disinfection time depends on the concentration of both reactants. Although not wishing to be bound by theory, the unexpected effectiveness of the synthetic mixtures and the complex pH-concentration dependence of the disinfection time is thought to be due to the role of intermediates which are formed in these solutions. At higher pH (7.5), the concentration of hypochlorite ion is higher, resulting in an increase in the concentration of this intermediate. Although not wishing to be bound by theory, this increased amount of intermediate is thought to be responsible for the more effective disinfection at this pH value. By taking into account the previously measured concentration dependence, to achieve a 5 minute disinfection time at pH 7.0, the present invention provides a biocidal solution comprising 1,200 mg/L FAC and 200 mg/L CIO2. To achieve a 5 minute disinfection time at pH 7.5, the present invention also provides a biocidal solution comprising 300 mg/L of each disinfectant. This lower concentration would result in less toxic and less corrosive solutions. TABLE 8 provides additional biocidal solutions according to the present invention and their disinfection times. Solutions 1 and 2 have a slightly longer disinfection time than Solution 3. In each case, one of the sterilants is in lower concentration than in Solution 3.

TABLE 8 Composition of the Proposed Synthetic Mixtures And Their Estimated Disinfection Time

As shown from this Example, the efficacy of a FAC/CIO2 solutions at pH 7.5 increases more rapidly with increasing disinfectant concentration than at pH 7.0. For this reason if rapid disinfection is needed, a FAC/ClO₂ solution at pH 7.5 may be used. But if the toxicity of the disinfectant solutions is more important, a FAC/CIO2 solution at pH 7.0 may be used, because the disinfectant concentrations can be decreased without a significant loss of their efficiency. However, the disinfection time is about 30 to 60 minutes when using a FAC/CIO2 solution at pH 7. Based on the test results, the time required for total disinfection (disinfection time), shows a different dependence on either ClO₂ and FAC concentration at the two tested pH values (7.0 and 7.5). At pH 7.5 (the pH value which was tested in both cases) , the disinfection time depends on the square of both the FAC and ClO concentrations. It is thought that the pH changes the relative amount of intermediates and synergistic effects of the disinfectant. The foregoing description and examples have been set forth merely to illustrate the invention and are not intended as bemg limiting. Each of the disclosed aspects and embodiments of the present invention may be considered individually or in combination with other aspects, embodiments, and variations of the invention. In addition, unless otherwise specified, none of the steps of the methods of the present invention are confined to any particular order of performance. Modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art and such modifications are within the scope of the present invention. Furthermore, all references cited herein are incorporated by reference in their entirety.

Claims

We claim:

1. A biocidal solution comprising a free available chlorine and a chlorine dioxide.

2. The biocidal solution of claim 1, wherein the free available chlorine is an elemental chlorine, a hypochlorous acid, a hypochlorite ion, or any combination thereof.

3. The biocidal solution of claim 1, wherein the concentration of the chlorine dioxide is about 0.1 to about 400 ppm.

4. The biocidal solution of claim 3, wherein the concentration of the chlorine dioxide is about 80 to about 400 ppm.

5. The biocidal solution of claim 1, wherein the concentration of the free available chlorine is about 0.1 to about 350 ppm.

6. The biocidal solution of claim 5, wherein the concentration of the free available chlorine is about 80 to about 350 ppm.

7. A method of producing the biocidal solution of claim 1, comprising: passing a mixture of a chloride salt solution and a chlorite salt solution through an electrolytic cell.

8. A method of producing the biocidal solution of claim 1 , comprising: separately electrolyzing a chloride salt solution and a chlorite salt solution; and mixing the chloride salt solution and the chlorite salt solution together.

9. A method of disinfecting a surface comprising: providing the biocidal solution of claim 1; and exposing the surface to the biocidal solution of claim 1 to disinfect the surface.

10. A method of sterilizing a medical device comprising: providing the biocidal solution of claim 1; and exposing the medical device to the biocidal solution of claim 1 to sterilize the medical device.

11. A method of reducing or eliminating biofilm comprising: providing the biocidal solution of claim 1; and exposing the biofilm to the biocidal solution of claim 1 to eliminate or reduce the biofilm.