WO2017019685A1 - Methods of using chlorine dioxide for decontaminating biological contaminants - Google Patents

Abstract

Provided herein are methods of using a gas containing chlorine dioxide, or a liquid solution of chlorine dioxide, for reducing or eliminating a biological contaminant (e.g., an infectious biological contaminant, e.g., influenza, e.g., avian flu or swine flu) from a contaminated material (e.g., an agricultural material, e.g., a carcass, feed, bedding, and/or soil).

Description

METHODS OF USING CHLORINE DIOXIDE FOR DECONTAMINATING

BIOLOGICAL CONTAMINANTS

RELATED APPLICATIONS

This application claims priority to U.S. Application No. 62/197,225, filed on July 27, 2015, the entire content of which is hereby incorporated herein by reference.

FIELD OF INVENTION

The present disclosure relates to decontaminating material (e.g., an agricultural material, e.g., a carcass, feed, bedding, and/or soil) infected with a biological contaminant (e.g., influenza, e.g., avian influenza) using chlorine dioxide, e.g., gaseous chlorine dioxide or a liquid solution of chlorine dioxide.

BACKGROUND

Within recent years, there have been lethal outbreaks of emerging infectious diseases and growing concern regarding the transmission of virulent pathogens, such as, e.g., avian influenza ("avian flu") or swine flu. Millions of birds have been affected by avian influenza virus in the United States. This is a major threat to food safety that has potentially serious impacts on human wellbeing. There is a need to identify approaches that are effective for remediation of contaminated carcasses (e.g., bird carcasses) and other materials.

SUMMARY

Provided herein are methods of using a gas containing chlorine dioxide, or a liquid solution of a gas containing chlorine dioxide, for reducing or eliminating a biological contaminant (e.g., an infectious biological contaminant, e.g., influenza, e.g., avian flu or swine flu) from a contaminated material (e.g., an agricultural material, e.g., a carcass, feed, bedding, and/or soil). Experiments provided herein exemplify these methods and show that chlorine dioxide can effectively penetrate and/or decontaminate contaminated materials to treatment depths extending beyond the surface of the material.

In one aspect provided herein is a method of treating soil to a soil treatment depth, the method comprising applying to the soil an aqueous solution comprising a gas containing chlorine dioxide and allowing the solution to percolate through the soil, wherein a percolate formed by the solution, after it has percolated through at least the soil treatment depth of said soil, has a residual chlorine dioxide concentration of at least 15 mg/L and the method reduces the level of a contaminant in the soil.

In another aspect provided herein is a method of treating soil to a soil treatment depth, the method comprising applying to the soil an aqueous solution comprising chlorine dioxide and allowing the solution to percolate through the soil, wherein a percolate formed by the solution, after it has percolated through at least the soil treatment depth of said soil, has a residual chlorine dioxide concentration of at least 15 mg/L and the method reduces the level of a contaminant in the soil, wherein said soil treatment depth is 1 cm.

In some embodiments, the soil treatment depth is 1 inch (2.5 cm). In some embodiments, the aqueous solution is applied to the soil at a volume: surface area rate of at least about 0.18 L/ft² ( 1.9 L/m²) . In some embodiments, the aqueous solution is applied to the soil at a volume: surface area rate of at least about 0.5 L/ft². In some embodiments, the aqueous solution is applied to the soil at a volume: surface area rate of at least about 1 L/ft².

In some embodiments, the aqueous solution comprises 50 mg/L to 3,000 mg/L chlorine dioxide.

In some embodiments, the aqueous solution comprises at least 500 mg/L chlorine dioxide.

In another aspect provided herein is a method of treating soil, the method comprising applying an aqueous solution of at least 500 mg/L chlorine dioxide to the soil at a volume: surface area rate of at least about 0.5 L/ft², thereby eliminating a contaminant or reducing the level of a contaminant in the soil.

In some embodiments, the volume: surface area rate is about 0.5 L/ft² to about 2.0 L/ft² (about 5.4 L/m² to about 21.5 L/m²). In some embodiments, the volume: surface area rate is about 0.5 L/ft² to about 1.0 L/ft² (about 5.4 L/m² to about 10.8 L/m²) of soil.

In some embodiments, the volume: surface area rate is about 0.5 L/ft² to about 4 L/ft² (about 5.4 L/m² to about 43.1 L/m²). In some embodiments, the volume: surface area rate is about 1 L/ft² to about 4 L/ft² (about 10.8 L/m² to about 43.1 L/m²). In some embodiments, the volume: surface area rate is about 1 L/ft² to about 3 L/ft² (about 10.8 L/m² to about 32.2 L/m²). In some embodiments, the volume: surface area rate is about 1 L/ft² to about 2 L/ft² (about 10.8 L/m² to about 21.5 L/m²).

In some embodiments, the density of the soil is less than about 1.8 g/cm³. In some embodiments, the density of the soil is about 0.9 g/cm³ to 1.7 g/cm³.

In some embodiments, the solution is applied at a rate of 0.1 to 0.3 L/minute.

In some embodiments, applying the solution comprises spraying the solution over the soil.

In some embodiments, the percolate can be collected within less than 10 minutes after the applying. In some embodiments, the method is effective to inactivate a spore strip containing at least

10⁶ B. atrophaeus spores when the spore strip is placed at the treatment depth prior to the applying . In a further aspect provided herein is a method of fumigating a feed, a bedding, or a carcass, the method comprising exposing the feed, the bedding, or the carcass to a gas comprising chlorine dioxide at a concentration x time (CT) value sufficient to reduce the level of a contaminant in the feed, the bedding or the carcass.

In some embodiments, the CT value is at least 9000 ppm_v-hours.

In some embodiments, the CT value is at least 30,000 ppm_v-hours.

In some embodiments, the CT value is 9000 to 200,000 ppm_v-hours.

In some embodiments, the contaminant is an influenza virus.

In some embodiments, the influenza virus is an avian influenza virus. In some embodiments, the influenza virus is a highly pathogenic avian influenza virus.

In some embodiments, RT-PCR testing of a post-treatment sample taken from the soil, the feed, the bedding, or the carcass after application of the method indicates that the post-treatment sample is negative for the contaminant.

In some embodiments, the method results in at least a specified reduction in the level of (a) the contaminant or (b) a biological indicator, wherein the specified reduction is at least a 3 log reduction.

In some embodiments, the specified reduction is at least a 6 log reduction.

In some embodiments, the specified reduction is no detectable growth.

In some embodiments, the level of the contaminant or the biological indicator is assayed using a post-treatment sample taken from the surface of the soil, the feed, the bedding, or the carcass after application of the method.

In some embodiments, the level of the contaminant or the biological indicator is assayed using a post-treatment sample of the soil, the feed, the bedding or the carcass after application of the method, wherein the post-treatment sample is taken at a depth of at least about 1 cm.

In some embodiments, the post-treatment sample is taken at a depth of at least about 1 inch within the soil, the feed, the bedding, or the carcass.

In some embodiments, the post-treatment sample is taken from a depth of about 1 cm to about

16 cm.

In some embodiments, the chlorine dioxide is at least 95% pure. In some embodiments, the chlorine dioxide is at least 99% pure. Also provided herein is a method of fumigating a feed or a bedding, the method comprising exposing the feed or the bedding to a gas comprising chlorine dioxide at a chlorine dioxide concentration x time (CT) value sufficient to penetrate the feed or the bedding to at least a treatment depth. The treatment depth extends beyond the surface of the feed or the bedding and can be a treatment depth disclosed herein. In some embodiments, the treatment depth is 0.25 inch.

Also provided herein is a method of fumigating a feed or a bedding, the method comprising exposing the feed or the bedding to a gas comprising chlorine dioxide at a chlorine dioxide concentration x time (CT) value sufficient to penetrate the feed or the bedding to at least a treatment depth and to reduce the level of a contaminant in the feed or the bedding, wherein the treatment depth is 0.25 inch.

In some embodiments, the treatment depth is 1 inch (2.5 cm). In some embodiments, the treatment depth is 2 inches (5 cm).

In some embodiments, the CT value is at least 9000 ppm_v-hours.

In some embodiments, the CT value is 9000 to 200,000 ppm_v-hours. In some embodiments, the CT value is at least 30,000 ppm_v-hours.

In some embodiments, the contaminant is an influenza virus.

In some embodiments, the contaminant is an avian influenza virus. In some embodiments, the contaminant is a highly pathogenic avian influenza virus.

In some embodiments, RT-PCR testing of a post-treatment sample taken from the feed or the bedding after the exposing indicates that the post-treatment sample is negative for the contaminant. In some embodiments, the post-treatment sample is taken from the treatment depth of the feed or the bedding.

In some embodiments, the method is effective to produce at least a specified reduction in the level of a biological indicator (e.g., an E. coli biological indicator) placed at the treatment depth within the feed or bedding and comprising >10⁶ CFU prior to the exposing. In some embodiments, the specified reduction is at least a 3 log reduction in the number of E. coli CFU. In some embodiments, the specified reduction is at least a 6 log reduction in the number of E. coli CFU. In some embodiments, the specified reduction is a reduction to no detectable growth of E. coli.

Also provided herein is a method of fumigating a carcass, the method comprising exposing the carcass to a gas comprising chlorine dioxide at a chlorine dioxide concentration x time (CT) value sufficient to penetrate the carcass to at least a treatment depth and to reduce the level of a contaminant in the carcass, wherein the treatment depth is 0.1 inch (0.25 cm). In some embodiments, the CT value is at least 9,000 ppm_v-hours. In some embodiments, the CT value is at least 30,000 ppm_v-hours. In some embodiments, the CT value is 30,000 to 200,000 ppm_v-hours.

In some embodiments, the method is effective to produce at least a specified reduction in a dermally placed E. coli biological indicator that comprises at least 10⁶ colony forming units (CFU) prior to the exposing, wherein the specified reduction is at least a 3 log reduction in the number of E. coli CFU. In some embodiments, the specified reduction is at least a 6 log reduction in the number of E. coli CFU. In some embodiments, the specified reduction is no growth of E. coli CFU.

In some embodiments, the method is effective to produce at least a specified reduction in an intramuscularly placed E. coli biological indicator that comprises at least 10⁶ colony forming units

(CFU) prior to the exposing, wherein the specified reduction is at least a 2 log reduction in the number of E. coli CFU. In some embodiments, the biological indicator is placed at a depth of 1 inch (2.5 cm) within the carcass.

DETAILED DESCRIPTION The present application relates to methods for fumigating using a gas containing chlorine dioxide and/or applying a liquid solution comprising a gas containing chlorine dioxide to

decontaminate a material contaminated with a biological contaminant, e.g, a material used in agricultural or veterinary industry. Materials such as, e.g., feed, bedding, carcasses, and soil, can harbor biological contaminants, thereby contributing to the spread of infectious disease (e.g., an influenza, e.g., an avian influenza or swine flu). Such materials are difficult to permeate, particularly when the contamination extends beyond the surface. The present application provides new methods for effectively decontaminating such materials, e.g., by reducing or eliminating a contaminant. In embodiments, the methods are effective for decontaminating beyond the surface of the material. In embodiments, the methods are effective for eliminating a contaminant or reducing the level of a contaminant to at least a given depth (e.g., at least 0.5 inches, 1 inch, 2 inches, 3 inches, 4 inches, 5 inches, or 6 inches or at least 0.5 cm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm) within the material.

As used herein, singular terms such as "a," "an," or "the" include the plural, unless the context clearly indicates otherwise. As used herein, an "aqueous solution" is a solution that consists of more than 50% water by weight. In some embodiments, the aqueous solution comprises at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% water by weight.

As used herein, a "contaminant" refers to a biological contaminant such as a virus, a bacterium, or a fungus. Typically, the contaminant causes an infectious disease in an animal (e.g., a human and/or a non-human animal, such as a wild animal or an animal used in agriculture, e.g., poultry or livestock). In one embodiment, the contaminant is an influenza virus. In embodiments, the contaminant is an avian influenza virus. In embodiments, the contaminant is a highly pathogenic avian influenza virus. In embodiments, the contaminant is a swine flu virus. As used herein, a concentration-time value, also referred to as a "CT" or "CT value", is the time-weighted average of chlorine dioxide concentration in parts per million by volume (ppm_v) multiplied by the exposure time in hours. In a plot of chlorine dioxide concentration versus exposure time in hours, the CT would equal the area under the curve. For example, if the time weighted average chlorine dioxide concentration over a 12-hour exposure period were 750 ppm_v, the CT would be 9,000 ppm_v-hr. Similarly, if the time weighted average chlorine dioxide concentration over a 3 -hour exposure period were 3,000 ppm_v, the CT would still be 9,000 ppm_v-hr.

As used herein, "percent," "percentage" or "%"is intended to refer to the w/w% unless the context indicates otherwise.

As used herein and in the art, "ppm" refers to parts per million. In the describing liquid solutions comprising chlorine dioxide, the present specification employs the term "ppm" to refer to parts per million by weight. For solutions of chlorine dioxide in water, a concentration of 1 ppm chlorine dioxide is equivalent to 1 mg/L. As used herein, the term "ppm_v or ppmv" refers to parts per million by volume.

As used herein, to "reduce" or "reducing" the level of a contaminant includes eliminating or decreasing the number of the contaminant and/or inactivating the contaminant such that it is no longer viable (i.e., no longer capable of reproducing). Typically, a contaminant that is no longer viable is also no longer capable of causing disease. As disclosed herein, a reduction in a contaminant can be shown, for example, by tests of the contaminant itself or tests of a biological indicator. For example, tests of the contaminant or biological indicator can include cultures or viral isolation tests to detect growth.

As used herein and in the art, "soil" refers to a mixture that typically includes mineral matter, organic matter, air, and water. In some embodiments, the soil to be treated according to methods disclosed herein comprises 50% or less water, 40% or less water, 30%, or less water, 25% or less water, 20% or less water, 15% or less water, or 10% or less water. As used herein, "treatment depth" refers to the depth within a material to which chlorine dioxide penetrates and/or shows decontamination efficacy. Unless otherwise indicated, the treatment depth is measured from the surface of the material. Thus, for example, the treatment depth within a carcass is measured from the external surface of the carcass, and the treatment depth within feed or bedding is measured from the surface of a volume of feed or bedding. Methods of Treating Soil

In one aspect provided herein is a method of treating soil to a soil treatment depth, the method comprising applying to the soil a liquid solution (e.g., an aqueous solution) comprising chlorine dioxide and allowing the solution to percolate through the soil, wherein a percolate formed by the solution, after it has percolated through the soil treatment depth of said soil, has a residual chlorine dioxide concentration (e.g., a residual chlorine dioxide concentration disclosed herein, e.g., a residual chlorine dioxide concentration of at least 15 mg/L). Typically, the method is effective to reduce the level of a contaminant in the soil.

The "percolate formed by the solution, after it has percolated through the soil treatment depth of said soil" can be a percolate formed by the solution after it has percolated through at least the soil treatment depth of an appropriate sample of said soil. The residual chlorine dioxide concentration in the percolate can be determined by collecting at least a portion of the percolate and measuring the residual chlorine dioxide concentration in the percolate. Residual chlorine dioxide concentration is preferably assessed using Method 4500-C1O₂ E ("Amperometric Method II") in the "Standard Methods for the Examination of Water and Wastewater," 20th ed., 1998, or an equivalent method.

In some embodiments, the method further comprises collecting at least a portion of the percolate from the treatment depth of the soil. In some embodiments, the method further comprises determining the residual chlorine dioxide concentration in the percolate.

In some embodiments, the percolate can be collected from the treatment depth in less than 15 minutes from completion of the applying. In some embodiments, the percolate can be collected from the treatment depth in less than 10 minutes from completion of the applying. In some embodiments, the percolate can be collected from the treatment depth in 5 minutes or less from completion of the applying. In some embodiments, the percolate can be collected from the treatment depth in 2 minutes or less from completion of the applying. In some embodiments, the percolate can be collected from the treatment depth in 1 minute or less from completion of the applying.

In some embodiments, the percolate can be collected from the treatment depth in less than 15 minutes from commencement of the applying. In some embodiments, the percolate can be collected from the treatment depth in less than 10 minutes from commencement of the applying. In some embodiments, the percolate can be collected from the treatment depth in 5 minutes or less from commencement of the applying. In some embodiments, the percolate can be collected from the treatment depth in 2 minutes or less from commencement of the applying. In some embodiments, the percolate can be collected from the treatment depth in 1 minute or less from commencement of the applying. As used herein, "applying" a liquid solution to soil can include, e.g., dripping or spraying the solution over the soil or injecting the solution into the soil. In embodiments wherein the solution is injected into the soil, the treatment depth is measured downwards starting from the point of injection. In embodiments wherein the liquid solution is applied to the surface of the soil or over the surface of the soil, the treatment depth is measured downwards from the surface. The applying can be performed using liquid application methods known in the art, e.g., methods used in irrigation or for application of liquid fertilizers. For example, the liquid can be injected using a liquid fertilizer applicator, e.g., an injection harrow.

In embodiments, the residual chlorine dioxide concentration is a detectable concentration of chlorine dioxide. In embodiments, the residual chlorine dioxide concentration is at least 0.5 mg/L. In embodiments, the residual chlorine dioxide concentration is at least 1 mg/L. In embodiments, the residual chlorine dioxide concentration is 0.5 to 100 mg/L. In embodiments, the residual chlorine dioxide concentration is 1 to 100 mg/L.

In embodiments, the residual chlorine dioxide concentration is at least 15 mg/L. In embodiments, the residual chlorine dioxide concentration is 15 to 500 mg/L. In embodiments, the residual chlorine dioxide concentration is 15 to 200 mg/L. In embodiments, the residual chlorine dioxide concentration is 15 to 100 mg/L. In embodiments, the residual chlorine dioxide concentration is 15 to 50 mg/L.

In embodiments, the residual chlorine dioxide concentration is at least 5 mg/L. In embodiments, the residual chlorine dioxide concentration is 5 to 500 mg/L. In embodiments, the residual chlorine dioxide concentration is 5 to 200 mg/L. In embodiments, the residual chlorine dioxide concentration is 5 to 100 mg/L. In embodiments, the residual chlorine dioxide concentration is 5 to 50 mg/L.

In embodiments, the residual chlorine dioxide concentration is at least 10 mg/L In embodiments, the residual chlorine dioxide concentration is 10 to 500 mg/L. In embodiments, the residual chlorine dioxide concentration is 10 to 200 mg/L. In embodiments, the residual chlorine dioxide concentration is 10 to 100 mg/L. In embodiments, the residual chlorine dioxide concentration is 10 to 50 mg/L.

In embodiments, the residual chlorine dioxide concentration is at least 20 mg/L. In embodiments, the residual chlorine dioxide concentration is 20 to 500 mg/L. In embodiments, the residual chlorine dioxide concentration is 20 to 200 mg/L. In embodiments, the residual chlorine dioxide concentration is 20 to 100 mg/L. In embodiments, the residual chlorine dioxide concentration is 20 to 50 mg/L. In embodiments, the soil treatment depth is 0.5 cm. In embodiments, the soil treatment depth is 1 cm. In embodiments, the soil treatment depth is 1 inch. In embodiments, the soil treatment depth is 1 inch, 2 inches, 3 inches, 4 inches, 5 inches, or 6 inches. In embodiments, the soil treatment depth is 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, or 12 cm. In embodiments, the aqueous solution is applied at a volume: surface area rate of at least about

0.18 L/ft² (1.9 L/m²). In embodiments, the aqueous solution is applied at a volume: surface area rate of about 0.18 L/ft² (1.9 L/m²) to 2 L/ft² (21.5 L/m²). In embodiments, the aqueous solution is applied at a volume: surface area rate of at least about 0.5 L/ft² (at least about 5.4 L/m²). In embodiments, the aqueous solution is applied at a volume: surface area rate of about 0.5 L/ft² to about 2.0 L/ft² (about 5.4 L/m² to about 21.5 L/m²). In embodiments, the aqueous solution is applied at a volume: surface area rate of at least about 1 L/ft² (at least about 10.8 L/m²). In embodiments, the aqueous solution is applied at a volume: surface area rate of about 0.5 L/ft² to about 1.0 L/ft² (at least about 5.4 L/m² to about 10.8 L/m²).

In embodiments, the solution is applied at a rate of 0.05 to 1 L/minute. In embodiments, the solution is applied at a rate of 0.1 to 0.5 L/minute. In embodiments, the solution is applied at a rate of 0.1 to 0.3 L/minute.

In embodiments, applying the solution comprises spraying the solution over the soil. In embodiments, applying the solution comprises injecting the solution into the soil.

In some embodiments, the applying comprises applying the solution to the soil in two or more separate applications (e.g., two or more applications separated by at least 30 seconds, 1 minute, 2 minutes, 5 minutes, or 10 minutes) of the solution.

In embodiments, the aqueous solution comprises 50 to 3000 mg/L of chlorine dioxide. In embodiments, the aqueous solution comprises 100 to 3000 mg/L of chlorine dioxide. In

embodiments, the aqueous solution comprises at least 500 mg/L of chlorine dioxide. In embodiments, the aqueous solution comprises at least 1000 mg/L of chlorine dioxide. In embodiments, the aqueous solution comprises 500 mg/L to 2,000 mg/L chlorine dioxide. In embodiments, the aqueous solution comprises 500 mg/L to 3000 mg/L of chlorine dioxide.

In one embodiment, the aqueous solution comprises about 3000 mg/L of chlorine dioxide and is applied to the soil at a volume: surface area rate of about 0.18 L/ft² (1.9 L/m²). In some embodiments, the method further comprises determining the estimated chlorine dioxide demand of the soil. The estimated chlorine dioxide demand of the soil can be determined, e.g., by applying a test solution (typically an aqueous test solution) comprising a known concentration of chlorine dioxide (e.g., a known concentration between 100 and 5000 mg/L or between 500 and 3000 mg/L chlorine dioxide) to a test sample of the soil, allowing the test solution to percolate through the soil treatment depth of the test sample to form a sample percolate, and determining the chlorine dioxide concentration in the sample percolate. The decrease in the chlorine dioxide concentration in the sample percolate compared with the test solution is the estimated chlorine dioxide demand of the soil. In some embodiments, method comprises applying to the soil a liquid solution comprising a concentration of chlorine dioxide that is greater than the estimated chlorine dioxide demand of the soil.

In another aspect provided herein is a method of treating soil, the method comprising applying an aqueous solution of at least 500 mg/L chlorine dioxide to the soil at a volume: surface area rate of at least about 0.5 L/ft² (or at least about 5.4 L/m²), thereby eliminating a contaminant or reducing the level of a contaminant in the soil.

In embodiments, the aqueous solution is applied at a rate of about 0.5 L/ft² to about 2.0 L/ft² (about 5.4 L/m² to about 21.5 L/m²).

In embodiments, the density of the soil is less than about 1.8 g/cm³. In embodiments, the density of the soil is about 0.9 g/cm³ to 1.7 g/cm³.

In some embodiments, the method comprises placing a spore strip (e.g., a B. atrophaeus spore strip) in the soil at the treatment depth before applying the aqueous solution. In typical embodiments, the spore strip initially comprises at least 10⁶ B. atrophaeus spores. In some embodiments, the method further comprises verifying inactivation of the spores on the strip after applying the aqueous solution.

Fumigation Methods

In a further aspect provided herein is a method of fumigating a material (e.g., feed, bedding, or a carcass), the method comprising exposing the material (e.g., the feed, the bedding, or the carcass) to a gas containing chlorine dioxide (e.g., a gas containing at least 95% chlorine dioxide, e.g., a gas containing at least 99% chlorine dioxide) at a concentration x time (CT) value sufficient to reduce the level of a contaminant in the feed, the bedding or the carcass.

In embodiments, the material is feed, bedding, dust, or a carcass.

In embodiments, the material (e.g., the feed) is kept in motion while it is being exposed to the gas containing chlorine dioxide. In embodiments, a conveyor is used to keep the material in motion. In embodiments, a fluidized bed produced by an air-lift column is used to keep the material in motion. In embodiments, a rotating basket or drum is used to keep the material in motion. In embodiments, the method further comprises keeping the material in motion while it is being exposed to the gas containing chlorine dioxide, e.g., by placing the material (e.g., causing the material to be placed) in an apparatus that serves to keep the material in motion. In embodiments, the apparatus is selected from the group consisting of a conveyor, a fluidized bed (e.g., a fluidized bed produced by an air-lift column), a rotating basket, and a rotating drum. In embodiments, the apparatus is a conveyor. In embodiments, the apparatus is a fluidized bed produced by an air-lift column. In embodiments, the apparatus is a rotating basket or drum.

In embodiments, the CT value is at least 9000 ppm_v-hours. In embodiments, the CT value is at least 30,000 ppm_v-hours. In embodiments, the CT value is 9000 ppm_v-hours to 200,000 ppm_v- hours. In embodiments, the CT value is 30,000 ppm_v-hours to 200,000 ppm_v-hours.

In embodiments, the contaminant is a virus. In some such embodiments, the contaminant is an influenza virus. In embodiments, the influenza virus is a swine flu virus or an avian influenza virus (avian flu virus). In embodiments, the influenza virus is a highly pathogenic avian influenza virus. In embodiments, RT-PCR testing of a post-treatment sample obtained from the material (e.g., the soil, the feed, the bedding, or the carcass) after the applying or exposing indicates that the post- treatment sample is negative for the contaminant.

In embodiments, a pre-treatment sample (e.g., a plurality of pre-treatment samples) is obtained from the material (e.g., the soil, the feed, the bedding, or the carcass) before the applying or exposing and analyzed to determine the presence or initial level of the contaminant. In embodiments, the method comprises obtaining a pre-treatment sample (e.g., a plurality of pre-treatment samples) from the material (e.g., the soil, the feed, the bedding, or the carcass) before the applying or exposing and/or analyzing a pre-treatment sample (e.g., a plurality of pre-treatment samples) obtained from the material (e.g., the soil, the feed, the bedding, or the carcass) before the applying or exposing to determine the presence or initial level of the contaminant.

In embodiments, a post-treatment sample (e.g., a plurality of posttreatment samples) is obtained from the material (e.g., the soil, the feed, the bedding, or the carcass) after the applying or exposing and analyzed to verify the efficacy of the method in reducing the level of the contaminant (e.g., to verify the absence of the contaminant or to verify that the level of the contaminant is reduced, e.g., relative to the level in a pre-treatment sample). In embodiments, the contaminant is absent from the post-treatment sample, or the level of the contaminant in the post-treatment sample is reduced, e.g., compared with the initial level of the contaminant (e.g., as assessed in a pre-treatment sample).

In embodiments, the method comprises obtaining a post-treatment sample from the material (e.g., the soil, the feed, the bedding, or the carcass) after the applying or exposing and/or analyzing a post-treatment sample obtained from the material (e.g., the soil, the feed, the bedding, or the carcass) after the applying or exposing, e.g., to verify absence of the contaminant in the post-treatment sample or a reduction in the level of the contaminant in the post-treatment sample, e.g., compared with the initial level of the contaminant (e.g., as assessed in a pre-treatment sample). In embodiments, the method results in at least a 1 log, 2 log, 3 log, 4 log, 5 log or 6 log reduction in the level of the contaminant. In embodiments, analysis of the post-treatment sample shows absence of the contaminant, or no growth of the contaminant.

In embodiments, the analysis is a PCR-based test, e.g., PCR or RT-PCR. Other PCR-based tests can demonstrate inactivation of a biological contaminant. In embodiments, analysis of a post- treatment sample taken from the material (e.g., the soil, the feed, the bedding, or the carcass) after application of the method indicates that the post-treatment sample is negative for the contaminant. In embodiments, the pre-treatment sample and/or post-treatment sample is obtained from the surface of the material or at a treatment depth within the material, e.g., at a depth of 1 inch, 2 inches, 3 inches, 4 inches, 5 inches, or 6 inches or 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, or 12 cm.

In one embodiment, the material is a carcass. In embodiments, a pre-treatment and/or post- treatment sample is obtained as described herein. In embodiments, a pre-treatment and/or post- treatment sample is obtained from the skin of the carcass (e.g., from the feathered or unfeathered skin of a bird carcass). In embodiments, a pre-treatment and/or post-treatment sample is a subcutaneous sample from the carcass. In embodiments, a pre-treatment and/or post-treatment sample is a muscle sample from the carcass (e.g., a sample obtained intramuscularly).

Treatment Depth and Efficacy

Prior to application of chlorine dioxide according to the methods disclosed herein, a biological or chemical indicator can be placed on or within the material to be treated (e.g., the soil, the bedding, the feed, or the carcass). For example, the biological or chemical indicator can be placed on the surface of the material or at a treatment depth within the material. In some embodiments, the treatment depth is a depth of 1 inch, 2 inches, 3 inches, 4 inches, 5 inches, or 6 inches or 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, or 12 cm. In some embodiments, the treatment depth is 0.1 inch (0.25 cm). In some embodiments, the treatment depth is 0.2 inch (0.5 cm). In some embodiments, the treatment depth is 0.25 inch (0.6 cm).

In embodiments, a biological indicator (e.g., a plurality of biological indicators) is placed on (e.g., on the surface) or within the material to be treated (e.g., within the soil, the bedding, the feed, or the carcass) before the material is treated using chlorine dioxide according to the methods disclosed herein. In embodiments, the biological indicator is placed at the surface of the material, or at a treatment depth within the material, e.g., at a depth of 1 inch, 2 inches, 3 inches, 4 inches, 5 inches, or 6 inches or 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, or 12 cm. The biological indicator can be, e.g., a biological indicator described herein, e.g., E. coli or B. atrophaeus.

In some embodiments, the reduction in the level of the contaminant (e.g., the elimination of the contaminant) is shown using a biological indicator. In some embodiments, the biological indicator is a B. atrophaeus spore strip. In embodiments, the spore strip is placed at a treatment depth (e.g., a treatment depth of 0.25 cm, 0.5 cm or another treatment depth disclosed herein) within the material. In some embodiments, the method comprises placing a biological indicator at the treatment depth within the material. In one embodiment, the material is a carcass. In embodiments, the biological indicator is placed on the skin (e.g., on the feathered or unfeathered skin of a bird carcass). In embodiments, the biological indicator is placed subcutaneously. In embodiments, the biological indicator is placed intramuscularly.

In embodiments, the method results in elimination of, or at least a specified reduction in the level of (a) the contaminant and/or (b) a biological indicator. In embodiments, the specified reduction is at least a 1 log, 2 log, 3 log, 4 log, 5 log or 6 log reduction. In embodiments, the specified reduction is at least a 3 log reduction. In embodiments, the specified reduction is at least a 6 log reduction. In embodiments, the method results in absence of the contaminant and/or the biological indicator (e.g., as assessed using PCR or RT-PCR). In embodiments, the method results no growth of the contaminant and/or the biological indicator.

In embodiments, the level of the contaminant or the biological indicator is assayed using a post-treatment sample taken from the surface of the soil, the feed, the bedding, or the carcass after application of the method. In embodiments, the level of the contaminant or the biological indicator is assayed using a post-treatment sample of the soil, the feed, the bedding or the carcass after application of the method, wherein the post-treatment sample is taken at a depth of at least about 1 cm. In embodiments, the post-treatment sample is taken at a depth of at least about 1 inch within the soil, the feed, the bedding, or the carcass. In embodiments, the post-treatment sample is taken from a depth of about 1 cm to about 16 cm.

In embodiments, the material is a carcass (e.g., a mammal carcass, e.g., a human or non- human carcass). In embodiments, the carcass is a bird carcass. In embodiments, the bird carcass is a wild bird carcass or a poultry carcass (e.g., a chicken carcass, turkey carcass, duck carcass, or goose carcass). In embodiments, the carcass is a carcass of an animal used in agriculture, e.g., a poultry carcass, a swine carcass, a cattle carcass (e.g., beef cattle carcass or dairy cattle carcass), a goat carcass, a bison carcass, a horse carcass, a rabbit carcass, an alpaca carcass, or an elk carcass. In embodiments, the carcass is a wild animal carcass.

In embodiments, the material is bedding. In embodiments, the bedding is a poultry bedding. In embodiments, the bedding comprises pine flakes. In embodiments, the bedding consists of, or consists essentially of, pine flakes. In embodiments, the bedding comprises straw.

In embodiments, the material is feed. In embodiments, the feed is poultry feed. In embodiments, the feed comprises grain (e.g., corn), stone grit, or oyster shells. In embodiments, the feed comprises mash, pellets, crumbles, or scratch grain.

Influenza

The methods described herein are useful in controlling and preventing further spread of influenza outbreaks. While influenza viruses typically remain infectious only within their host species, at times infections may spread to other species. As used herein, the term "influenza" includes all types and strains of influenza, as well as variants. Types of influenza include influenza types A, B, and C. In embodiments, the influenza virus is an influenza A virus.

Influenza viruses are classified into subtypes by based on antibody responses to the proteins hemagglutinin and neuraminidase that are expressed on viral particles. There are at least sixteen hemagglutinin antigens denoted as HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, HI 5, and H16 and at least eleven neuraminidase antigens, denoted as Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, and Nl 1 so at least 198 different subtypes are possible. Virus subtypes can mutate into a variety of strains, some of which are pathogenic to one species but not others and some of which are pathogenic to multiple species.

Avian Influenza

Wild birds (e.g., ducks, gulls, shorebirds) as well as domestic poultry (e.g., chickens, turkeys, ducks, geese) can be infected by the avian influenza virus. Depending on the ability of the virus to produce disease in poultry, avian influenza viruses are classified as low pathogenic avian influenza

(LPAI) or highly pathogenic avian influenza (HPAI). HPAI has a high death rate, e.g., in chickens and turkeys, and spreads rapidly. As used herein, the term "avian influenza virus" includes any classification and any strain of avian influenza virus. In one embodiment, the influenza virus is an HPAI H5-N2 virus. Swine Flu

There are also different subtypes and strains of influenza viruses in pigs (swine). Subtypes of influenza A virus that affect swine include H1N1, H1N2, H3N1 and H3N2. Efficacy of Decontamination

In embodiments, the methods described herein are effective to reduce the level of a contaminant or eliminate a contaminant, as indicated either by measuring the contaminant itself, or by measuring an appropriate biological indicator. In embodiments, the methods reduce the level of the contaminant or biological indicator by at least a 1-log order reduction ("1 log reduction"), a 2-log order reduction ("2 log reduction"), a 3-log order reduction ("3 log reduction"), a 4-log order reduction ("4 log reduction"), a 5-log order reduction ("5 log reduction"), or a 6-log order reduction ("6 log reduction").

In embodiments, the methods described herein are effective to achieve sterilization. As used herein, "sterilizing" or "sterilization" requires at least a 6-log order reduction ("6-log reduction") of an enumerable biological material (such as, e.g., spores, bacterial colony forming units (CFU), viral titers). The reduction can be determined using an analytically quantified biological indicator.

In some embodiments, a method described herein results in more than a 6 log reduction in the level of a contaminant. In embodiments, a method described herein results in at least a 7-log order reduction ("7 log reduction"), an 8-log order reduction ("8 log reduction"), a 9-log order reduction ("9 log reduction"), or a 10-log order reduction ("10 log reduction") in the level of the contaminant.

In some embodiments, a method described herein results in no detectable growth of the contaminant. As used herein, a method "eliminates" or results in "elimination" of a contaminant when the method results in no detectable growth of the contaminant. Biological Indicators

A biological indicator is an organism other than the contaminant that is being targeted by the method that is used as a surrogate for the contaminant. The biological indicator is used to assess or verify the efficacy of the method in reducing or eliminating the contaminant. Typically, the biological indicator is placed on (e.g., on the surface) or within (e.g., at a treatment depth) the material (e.g., the soil, the feed, the bedding, or the carcass) being treated with the method before the material is treated, e.g., by exposing the material to a gas comprising chlorine dioxide or by applying a liquid solution (e.g. aqueous) comprising chlorine dioxide.

Because the viral envelope of influenza viruses is susceptible to disinfectants and detergents, vegetative bacteria may be more difficult to devitalize than enveloped viruses (e.g., influenza viruses) and can serve as an appropriate biological indicator of the efficacy of decontamination of a virus, e.g., an influenza virus, e.g., an avian influenza virus. General aerobic bacteria (e.g., Escherichia coli) can be quantified based on growth in laboratory culture. Such bacteria are naturally present in the poultry production environment; accordingly, they can be collected from poultry for use in laboratory experiments, e.g., as described herein, or obtained from other sources. For example, the bacteria can be serially diluted in (i) microbiological media bottles or (ii) on agar plates for enumeration of colony forming units (CFU). The media bottles or agar plates can be placed in experimental materials (e.g., carcasses, feed, bedding, or soil) that are subjected to treatment using a method disclosed herein.

In one embodiment, the biological indicator is a spore strip. In one embodiment, the biological indicator is Bacillus atrophaeus (B. atrophaeus, formerly known as Bacillus subitilis var. niger). B. atrophaeus has been used previously as the primary indicator organism during large-scale chlorine dioxide fumigation operations to eliminate anthrax contamination and is expected to function as an appropriate biological indicator for other

contaminants, e.g., avian influenza. B. atrophaeus spore strips are commercially available, e.g., from Mesa Labs, Inc. A spore strip is produced to contain a known number of spores, e.g., from lxlO⁶ to 4xl0⁶ spores. After exposure to fumigation and/or liquid chlorine dioxide treatment, spore strips can be analyzed to determine viability of spores as an indicator of the efficacy of the treatment.

Chemical Indicators

Penetration of the gaseous or liquid chlorine dioxide can be assessed using a chemical indicator. For example, a potassium iodide (KI) chemical indicator can be used. This colorimetric indicator can elucidate the extent of chlorine dioxide penetration into a material of interest.

Penetration to a treatment depth can be verified using a chemical indicator. For example, as disclosed herein, a potassium iodide indicator can be placed at the treatment depth within the material to be treated (e.g., the feed, the bedding, the carcass, or the soil) prior to treatment with chlorine dioxide. Testing for Avian Influenza Virus

Any method known in the art can be used for detection of avian flu virus. For example, PCR- based methods, such as PCR or RT-PCR, can be used. In one embodiment, an RT-PCR method is used. In a specific embodiment, the presence or absence of avian flu virus is determined using the VetMAX™-Gold avian influenza virus detection kit (Life Technologies, Inc.). Chlorine Dioxide

Chlorine dioxide gas or liquid (e.g., aqueous) solutions can be produced using any means known in the art. A chlorine dioxide generator, such as, e.g., the chlorine dioxide generator described in U.S. Pat. No. 6,468,479 can be used. In some embodiments, the chlorine dioxide is generated as disclosed in U.S. Patent or Patent Publication Nos. US 6,645,457; US 6,468,479; US 7,807, 101; US 7,678,388; US 8, 192,684; US 8,741,223; and/or US 2009/0081310. In one embodiment, a chlorine dioxide generator is used to generate chlorine dioxide either as a gas, or as an aqueous (or other suitable liquid carrier) chlorine dioxide solution. For fumigation methods, an emitter can be used to remove chlorine dioxide from solution and deliver it in air. Water recovered from the emitter can be recycled and reused. Methods and devices for generating chlorine dioxide are disclosed in, for example, U.S. Patent or Patent Publication Nos. US 6,645,457; US 6,468,479; US 7,807, 101; US 7,678,388; US 8, 192,684; US 8,741,223; US 2009/0081310; US 5,290,524, and US 5,234,678.

In some embodiments, the gas comprising chlorine dioxide that is used in the methods disclosed herein comprises chlorine dioxide and air. In some embodiments, the gas consists essentially of chlorine dioxide and air.

When chlorine dioxide is produced, a small percentage of impurities (such as, e.g., chlorite and/or molecular chlorine) may also be present as byproducts. In embodiments, when the chlorine dioxide is generated, it is at least 95% pure (i.e., comprises 5% or less impurities relative to chlorine dioxide). In some embodiments, when the chlorine dioxide is generated, it is at least 96, 97, 98, or 99% pure (i.e., comprises 4% or less, 3% or less, 2% or less, or 1% or less impurities relative to chlorine dioxide). In some embodiments, the gas comprises less than 5% molecular chlorine relative to chlorine dioxide. In some embodiments, the gas comprises less than 1% molecular chlorine relative to chlorine dioxide. In embodiments, the gas is dissolved in a liquid solution, e.g., an aqueous solution. In some embodiments, the solution comprises less than 5% molecular chlorine relative to chlorine dioxide. In some embodiments, the solution comprises less than 1% molecular chlorine relative to chlorine dioxide.

Fumigation with Chlorine Dioxide Gas

In embodiments of the methods described herein, the methods comprise exposing a material (e.g., a feed, bedding, carcass, or soil) to a gas comprising chlorine dioxide at a CT value. In embodiments, the CT value is sufficient to reduce the level of a contaminant in the material. In embodiments, the CT value is sufficient to reduce the level of a biological indicator (e.g., a biological indicator disclosed herein) in the material. In embodiments, the CT value is sufficient to result in a negative RT-PCR result for the contaminant in a post-treatment sample taken from the material after application of the method.

In embodiments, the CT value is at least 5000 ppm_v-hours. In embodiments, the CT value is 5,000 to 200,000 ppm_v-hours. In embodiments, the CT value is at least 9000 ppm_v-hours. In embodiments, the CT value is 9000 to 200,000 ppm_v-hours. In embodiments, the CT value is at least 4,000; 5,000;, 6,000; 7,000; 8,000; 9,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; 100,000; 110,000; 120,000; 130,000; 140,000; or 150,000 ppm_v-hours. In embodiments, the CT value is at least 30,000 ppm_v-hours. In embodiments, the CT value is 30,000 to 200,000 ppm_v-hours.

Typically, the fumigation methods disclosed herein are carried out in an enclosed volume. In some embodiments, the enclosed volume is a structure used for raising agricultural animals, e.g., a barn. In embodiments, the gas is introduced into the enclosed volume to achieve a desired minimum chlorine dioxide concentration during the exposing, e.g., a concentration in the range of 500 to 3000 parts per million by volume (ppm_v). In embodiments, the gas is introduced into the enclosed volume to achieve a minimum concentration of chlorine dioxide in the range of 200 to 15,000 ppm_v. In embodiments, the gas is introduced such that the concentration of chlorine dioxide does not exceed about 20,000 ppm_v.

In embodiments, the gas is introduced into the enclosed volume to achieve a peak

concentration of chlorine dioxide in the range of 200 ppm_v to 20,000 ppm_v. In embodiments, the gas is introduced into the enclosed volume to achieve a peak concentration of chlorine dioxide in the range of 3,000 ppm_v to 20,000 ppm_v. In embodiments, the gas is introduced into the enclosed volume to achieve a peak concentration of chlorine dioxide in the range of 5,000 ppm_v to 20,000 ppm_v. In embodiments, the gas is introduced into the enclosed volume to achieve a peak concentration of chlorine dioxide in the range of 10,000 ppm_v to 20,000 ppm_v.

In embodiments, the material is exposed to the gas comprising chlorine dioxide for an exposure time of about 1 to 48 hours. In embodiments, the material is exposed to the gas comprising chlorine dioxide for an exposure time of about 1 to 24 hours. In embodiments, the material is exposed to the gas comprising chlorine dioxide for an exposure time of about 1 to 12 hours. In embodiments, the exposure time is about 3 to 12 hours.

Typically, the fumigation methods disclosed herein are carried out at a relative humidity (RH) in the range of 5% to 80%. In embodiments, the RH is in the range of 10 to 80%. In embodiments, the RH is at least 5%. In embodiments, the RH is at least 70%. In embodiments, the RH is between 5 and 56%. In embodiments, the RH is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%, or is in a range or subrange between these values, e.g., 5-55%, 35-55%, 40-55%, 45-50%, 45-48%, 50%-80%, etc.. In embodiments, the fumigation methods disclosed herein are carried out at a temperature in the range of about 50°F to about 175°F (about 10°C to 80°C). In embodiments, the temperature is in the range of about 50°F to about 100°F (about 10°C to about 38°C). In embodiments, the temperature is in the range of about 60°F to about 95°F (about 15°C to about 35°C). In embodiments, the temperature is at least about 70°F (at least about 21°C). In embodiments, the temperature is about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175°F (or the corresponding temperature in °C, which can be calculated using the formula T(°C) = (T(°F) - 32) / 1.8).

In embodiments, the methods comprise climatizing the enclosed volume in which the fumigation is carried out, e.g., to achieve a desired RH or RH range (e.g., an RH or RH range disclosed herein) and/or a desired temperature or temperature range (e.g., a temperature or temperature range disclosed herein).

In embodiments, the air flow rate in the enclosed volume is at least about 3 feet per second (ft/sec) (0.9 m/s), for example, at least about 3 ft/sec (0.9 m/s), 5 feet/sec (1.5 m/s), 10 ft/sec (3 m/s), 15 ft/sec (4.5 m/s), or 20 ft/sec (6 m/s). In embodiments, the air flow rate is 3 to 20 ft/sec (0.9 to 6 m/s). In embodiments, the air flow rate is 5 to 20 ft/sec (1.5 to 6 m/s). In some embodiments, the velocity of the gas stream at or in the vicinity of the material being treated increases due to the circulation of air in the enclosed volume.

In some embodiments, the method comprises regulating the air flow rate in the enclosed volume, e.g., such that it is at least about 3 ft/sec (0.9 m/s), 5 feet/sec ( 1.5 m/s), 10 ft/sec (3 m/s), 15 ft/sec (4.5 m/s), or 20 ft/sec (6 m/s). In embodiments, the method comprises regulating the air flow rate in the enclosed volume such that it is 3 to 20 ft/sec (0.9 to 6 m/s). In embodiments, the air flow rate is 5 to 20 ft/sec (1.5 to 6 m/s).

Liquid Solutions of Chlorine Dioxide

In some embodiments, a liquid solution of chlorine dioxide used in the methods described herein is prepared by combining a concentrated liquid solution of chlorine dioxide (e.g., a liquid chlorine dioxide solution comprising at least 500, 1000, 2000, or 3000 mg/L chlorine dioxide) with dilution water. In some embodiments, the method comprises combining a concentrated liquid chlorine dioxide solution with dilution water. The dilution water can be any locally available water source, such as, e.g., tap water, well water, pond water, lake water, river water, etc. Dilution water can be added in an amount so as to achieve a desired chlorine dioxide concentration (e.g., a concentration disclosed herein) in the liquid solution.

In preferred embodiments, the concentration of chlorine dioxide in a solution is determined by Method 4500-ClO₂ E ("Amperometric Method II") in the "Standard Methods for the Examination of Water and Wastewater," 20th ed., 1998, or an equivalent method. Iodometric titration, which is described in Aieta, E. M. et al. (1984) Journal - American Water works Association, 76 (l):64-70, can also be used to determine the concentration of chlorine dioxide, chlorite, and chlorine in aqueous solutions.

In some embodiments, the liquid chlorine dioxide solution (e.g., the concentrated liquid chlorine dioxide solution) is made using a chlorine dioxide generator (e.g., a generator as disclosed in U.S. Patent Nos. 6,486,479 and/or 6,645,457). In some embodiments, the liquid chlorine dioxide solution (e.g., the concentrated liquid chlorine dioxide solution) is a solution as described in U.S. Patent No. 7,678,388. In some embodiments, the liquid chlorine dioxide solution is an aqueous solution that comprises chlorine dioxide and a chlorine scavenging means (e.g., sodium chlorite) for converting dissolved chlorine to chlorine dioxide. In some embodiments, the chlorine scavenging means comprises chlorite. In some embodiments, the chlorine scavenging means comprises sodium chlorite. In some embodiments, the chlorine scavenging means is sodium chlorite. In some embodiments, the liquid chlorine dioxide solution (e.g., in the concentrated liquid chlorine dioxide solution) is an aqueous solution that is prepared to initially have a chlorine dioxide concentration of 1000 to 3000 mg/L (e.g., 2000 to 3000 mg/L), a pH of 1 to 6 (e.g., about 5 to 6), and a ratio of chlorine scavenging means: chlorine dioxide in the range of about 1 :4 to 1: 15 (w/w) (e.g., about 1 : 10 tol : 15, e.g., about 1 : 13) based on a sodium chlorite to chlorine dioxide system. In some

embodiments, the chlorine scavenging means comprises sodium chlorite. In some embodiments, the chlorine scavenging means is sodium chlorite. In some embodiments, the initial concentration of chlorine dioxide in the liquid chlorine dioxide solution decreases by less than 10% after two days of storage at room temperature and at normal atmospheric pressure. In some embodiments, the initial concentration of chlorine dioxide in the liquid chlorine dioxide solution decreases by less than 10% after 45 days of storage at room temperature and at normal atmospheric pressure. In some embodiments, the initial concentration of chlorine dioxide in the liquid chlorine dioxide solution (e.g., in the concentrated liquid chlorine dioxide solution) decreases by less than 10% after 90 days of storage at room temperature and at normal atmospheric pressure.

In some embodiments, the liquid chlorine dioxide solution (e.g., the concentrated liquid chlorine dioxide solution) is an aqueous solution that is prepared to initially have a chlorine dioxide concentration of about 2000 to 3000 mg/L, a pH of about 5 to 6, and a ratio of sodium

chlorite: chlorine dioxide in the range of about 1 :4 to 1 : 15 (w/w).

In some embodiments, the liquid chlorine dioxide solution (e.g., the concentrated liquid chlorine dioxide solutionis an aqueous solution that is prepared to initially have a chlorine dioxide concentration of 10 to 3000 mg/L of water (e.g., from 1000 to 2500 mg/L), a chlorite ion

concentration of 1 to 3000 mg/L of water (e.g., from 100 to 1000 mg/L), and optionally, a pH of 1 to 6.5 (e.g., a pH of 5 to 6).

Typically, the liquid chlorine dioxide solution (e.g., the concentrated liquid chlorine dioxide solution) is an aqueous solution. In some embodiments, the aqueous solution comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% water by weight. In some embodiments, the liquid chlorine dioxide solution (e.g., the concentrated liquid chlorine dioxide solution) has an initial chlorine dioxide concentration of 500-3500 mg/L, 1000 to 3500 mg/L, 1000 to 3000 mg/L, 2800 to 3200 mg/L, or about 3000 mg/L. In some embodiments, the liquid chlorine dioxide solution (e.g., the concentrated liquid chlorine dioxide solution) is an aqueous solution having an initial chlorine dioxide

concentration of about 3000 mg/L. Optionally, the liquid chlorine dioxide solution (e.g., the concentrated liquid chlorine dioxide solution) can further comprise chlorite (e.g., sodium chlorite). The chlorite can act as a chlorine scavenger. In some embodiments, the initial chlorite concentration is 1 to 3000 mg/L of water (e.g., 100 to 1000 mg/L). In some embodiments, the initial

chlorite: chlorine dioxide ratio (w/w) in the solution is 1 :4 to 1 : 15 (e.g., about 1 : 10 to 1 : 15). In some embodiments, the initial chlorite concentration in the solution is 200 mg/L to 750 mg/L. In some embodiments, the liquid chlorine dioxide solution (e.g., the concentrated liquid chlorine dioxide solution) comprises at least 90% by weight of chlorine dioxide with respect to all chlorine species. In some embodiments, the liquid chlorine dioxide solution (e.g., the concentrated liquid chlorine dioxide solution) comprises at least 95% by weight of chlorine dioxide with respect to all chlorine species. In some embodiments, the liquid chlorine dioxide solution is an aqueous solution comprising

500-3500 mg/L chlorine dioxide and 100 to 1000 mg/L chlorite. In some embodiments, the liquid chlorine dioxide solution has a pH of 1 to 6 (e.g., 4 to 6, e.g., 5 to 6).

In some embodiments, the liquid chlorine dioxide solution is an aqueous solution that comprises a chlorine dioxide concentration of 200 to 10,000 mg/L (e.g., 500 to 10,000 mg/L) and has a pH of 1 to 8 (e.g., about 5 to 8, e.g., about 6 to 8). In some embodiments, the liquid chlorine dioxide solution is prepared to include a chlorine scavenging means (e.g., chlorite, e.g., sodium chlorite). In some embodiments, the liquid chlorine dioxide solution comprises sodium chlorite, wherein the solution is prepared such that the ratio of sodium chlorite: chlorine dioxide is initially in the range of about 1 :4 to 1 : 15 (w/w) (e.g., about 1 : 10 to 1 : 15 , e.g., about 1 : 13). In some embodiments, the liquid chlorine dioxide solution is refrigerated or otherwise stored at a temperature less than 25°C to prevent thermochemical degradation of chlorine dioxide.

Soil

There are various types and densities of soil. The average density of soil found in the natural environment is roughly 1.3 grams (g) per cubic centimeter (cm³), with a range from approximately 0.9 to 1.8 g/cm³. Soil of various types will begin to experience root penetration resistance when compacted beyond a certain density (the critical bulk density for soil resistance, which differs for different soil textures, ranges from 1.60 to 1.85 g/cm³ for sandy soil, 1.40 to 1.80 g/cm³ for coarse- loamy soil, from 1.40 to 1.80 g/cm³ for coarse-fine-silty soil, and for clayey soil it varies depending on the clay percent and structure. See, e.g., Sumner, M.E. (1999) Handbook of Soil Science, CRC Press).

In one embodiment, a method disclosed herein is for treatment of soil. The soil can be any soil type, e.g., a soil type disclosed herein. Soil textures can be determined using the USDA classification provided in the USDA soil texture triangle chart (See Soil Survey Division Staff. 1993. Soil survey manual. Soil Conservation Service. U.S. Department of Agriculture Handbook 18). In one embodiment, the soil is sandy clay, silty clay, clay loam, sandy clay loam, silty clay loam, loam, silt loam, sandy loam, loamy sand, sand, or silt. In another embodiment, the soil is loamy sand, sandy loam, sandy clay loam, clay loam, loam, silt loam, or silty clay loam. In another embodiment, the soil is loamy sand, sandy loam, sandy clay loam, loam, or clay loam. In one embodiment, the soil is loamy sand, sandy loam, loam, or clay loam.

In one embodiment, the soil comprises 10-30% silt, 40-80% sand, and 10-30% clay.

In another embodiment, the soil comprises 10-30% silt, 40-80% fine sand, and 10-30% clay.

In one embodiment, the soil has a density of less than about 1.85 g/cm³. In one embodiment, the soil has a density of in the range of about 0.9 g/cm³ to 1.85 g/cm³, e.g., a density in the range of about 0.9 g/cm³ to 1.7 g/cm³, e.g., a density in the range of 0.9 g/cm³ to 1.65 g/cm³. In embodiments, the soil has a density below the critical bulk density for soil resistance.

Incorporation by Reference

All patent documents and other references referred to herein are hereby incorporated by reference in their entirety.

EXAMPLES

Example 1 : Fumigation of carcasses and other materials at 9.000 ppnty-hr and at 30.000 ppnty-hr

Experimental Design

Chemical indicators and biological surrogates were employed to gauge C10₂ penetration and concomitant microbial kill in materials from the poultry industry including bedding, feed, soil, litter (used bedding including feces), and carcasses. This study investigated the requisite C10₂ dosage concentration and exposure time (concentration ^χ time or CT value) required to achieve biocidal activity at various penetration depths within bird carcasses (feathered skin surface, subcutaneous, and intra-muscular) as well as in the other materials.

Methods

Chemical indicators comprised of 5% potassium iodide (KI, rehydrated crystals in silicone) were used to qualitatively measure C10₂ mass transfer throughout the candidate poultry materials. These solid KI indicators produce a brown/black colorimetric chemical reaction in the presence of C10₂ and were placed at various points within chicken carcasses (surface, skin, and muscle), poultry feed, and bedding. Preliminary tests were also performed with skinless chicken breasts that were brined in a 5% KI aqueous solution overnight. This whole-tissue indicator served as a visual determinant throughout the entire muscle.

Biological indicators were employed concurrently to correlate CIO₂ penetration gradients with discretely quantifiable microbial kill. Vegetative bacteria may be more difficult to devitalize than enveloped viruses; therefore, Escherichia coli served as a biological surrogate for avian influenza in the present study. A CT value that can effectively kill E. coli is anticipated to inactivate avian influenza viral particles. As such, biological indicators (Bis) containing liquid bacterial broth (>10⁶ CFU/ml) in glass screw-cap vials were placed at similar sites within the chickens, feed, and bedding. Caps were opened slightly to facilitate gas transfer. In all BI locations, control vials containing liquid media only (no bacterial inoculum) were placed by identical methods and subsequently cultured to demonstrate 'no growth,' thereby discounting any cross-contamination.

In addition to using Bis in glass vials, as another method of using a BI, certain experimental materials were directly inoculated with E. coli (see Table 1). The materials were soaked directly in overnight culture of E. coli on the order of 10¹⁰ CFU/ml to saturate the porous materials with the BI. For these trials, DuMor® brand pellets and grain mixtures (scratch) were steam sterilized and then submerged in bacterial broth for a 30 second contact time. The inoculated feed materials were then allowed to dry at 37° F for 30 minutes before use. In order to recover viable bacteria after exposure to experimental fumigation conditions, individual pellets or pieces of grain were incubated in 1 ml of LB media for 30 minutes, centrifuged at 10,000 rpm for 5 minutes to pellet debris, and the supernatant was used for serial dilutions on agar plates. Bacterial colonies were enumerated to determine the log- fold reduction in viable cells (CFU). Control samples inoculated with 10⁸ CFU/ml culture broth absorbed and sustained the viability of roughly 10⁶ CFU/ml as a baseline.

All experimental materials were subjected to CIO₂ fumigation at CT values set forth below in a fumigation chamber maintained at -72° F and 75-80% relative humidity. Chlorine dioxide gas concentrations were analytically determined in 15-minute intervals by drawing samples of the fumigation chamber atmosphere with calibrated impingers ( 1 L/min for 2 min) and then performing a standard iodometric titration with sodium thiosulfate. At the termination of each fumigation cycle, the CIO₂ atmosphere was removed by scrubbing before KI and BI samples were retrieved and evaluated by visual inspection or subsequent growth in laboratory culture. Positive controls for both KI and BI were kept unprotected in the fumigation chamber to experience full contact with CIO₂ gas;

corresponding negative controls remained outside of the fumigation chamber.

KI indicators produce a qualitative result in response to C10₂ presence. Quantification of bacterial populations was performed by cultivating the Bis on agar plates to allow enumeration of individual colony forming units (CFUs). In brief, Bis containing a starting concentration >10⁶ CFU/ml were serially diluted by 10^"1 to 10^"4, spread evenly on a set of 100 mm ^χ 15 mm plates containing standard LB broth (Lennox, 1.5% agar), and incubated overnight at 37° C. After outgrowth was observed (12-hr), distinct bacterial colonies were counted and used to calculate the relative abundance and log-order reduction of CFUs as a result of fumigation. Positive and negative controls respectively confirmed the CFU inoculum concentration and discounted any bacterial contaminants.

Materials

Live chickens were sacrificed 1 hour prior to fumigation. Store-bought whole chickens and breasts (both skin-on and skinless) served as comparators. Core samples of muscle were removed with a 17-inch, stainless steel tuna grader (Hi -Liner Fishing Gear & Tackle Inc.) in order to place KI indicators and biological indicators. Placement of subcutaneous indicators was performed though small incisions in the skin and resealed.

Poultry bedding and feed materials were acquired from Tractor Supply Co. and prepared in 2- ft diameter baskets. The bedding consisted of 4 inches of compacted pine shavings (2.5 lbs) on top of 6 inches of soil (sandy loam, 64 lbs). The feed was a mixture of DuMor® brand pellets, cracked corn, stone grit, and oyster shells (20 lbs, ~4 inches). Core samples were removed in order to place KI and BI samples at various depths within each material and at the interface of soil and bedding. Pellets and grain samples directly inoculated with E. coli were prepared in Pyrex Petri dishes that remained opened during fumigation for maximal CIO₂ exposure.

Results of CT 9, 000 ppm_v-hours Fumigation The initial fumigations of chickens, feed, and bedding were performed at a CT value of 9,000 ppm_v-hours. These fumigations proceeded for just over 3 hours and reached a peak CIO2 concentration of roughly 5,000 ppm_v.

Following the fumigation, KI-brined skinless breasts revealed about 1-2 mm penetration into the muscle tissue. Surface biological indicators exposed to 9,000 ppm_v-hrs confirmed at least a 6-log reduction in viable E. coli cells, fulfilling the defined criterion of sterilization (data not shown). Despite complete kill on the surface, this CT value did not achieve a significant reduction in bacteria under the skin (data not shown).

The results of microbiological assessments of E. coli in various other materials following CT 9000 ppm_v-hours fumigation are provided in Table 1. The results from controls are also provided in Table 1.

The 4-inch packed, porous bedding material was readily penetrated at CT 9,000 ppm_v-hrs as evidenced by the complete colorimetric change of chemical indicators. In the bedding, the biological indicator showed that at a depth of 1 inch, the fumigation was effective in achieving no growth (quantitative result of 0 CFU/ml, which is more than a 7 log reduction compared with the

corresponding control inoculum from test fume 1 that was not fumed and showed a quantitative result of 1.6 x 10⁷ CFU/ml as set forth in Table 1). At a depth of 2 inches in the bedding (at the interface of pine flakes and sandy loam), the quantitative result for the biological indicator was 8.9 x 10⁶ CFU/ml, which was about a 1-log reduction compared with the corresponding control inoculum from test fume 2 that was not fumed and showed a quantitative result of 3.4 ^χ 10⁷ CFU/ml as set forth in Table 1).

The feed mixture with 2-inch of overburden was partially permeable to CIO2 and resulted in incomplete color change of KI indicators. In the feed mixture at a depth of 2 inches, the quantitative result from the fumigation was 3.6 x 10⁴ CFU/ml. which was about a 3 -log reduction compared with the corresponding control (the control inoculum from test fume 1 that was not fumed showed a quantitative result of 1.6 ^χ 10⁷ CFU/ml as set forth in Table 1). For the pellets and feed grains that were directly inoculated with E. coli, the fumigation achieved no growth, which was a 6 log reduction or more compared with the corresponding controls.

Table 1: Results of CT 9,000 ppm_v-hr fumigation: Viability of biological indicator (E. coli)

E. coli survival (CFU/ml)

Sample Sample Z² Description Qualitative Quantitative Test Fume Type¹ No. Result Result number³

Bedding material: pine

B Basket A 1 flakes No Growth 0 1

Interface of pine flakes

B/S Box A 2 and sandy loam Growth 8.0 x 10⁶ 2

Feed mixture (pellets,

F Basket 1 2 corn, oyster, grit) Growth 3.6 x 10" 1

F Dish l 0.25 Pellets directly inoculated No Growth 0 4 w/ E. coli

Feed grains directly

F Dish 2 0.1 inoculated w/ E. coli No Growth 0 4

0 CFU/ml, liq. media;

C Dish 1 0.25 pellets contaminant No Growth 0 4

0 CFU/ml, liq. media;

C Dish 2 0.1 grain mix contaminant No Growth 0 4

10⁶ CFU/ml inoculum,

pellets

c Dish 1 e6+ N/A not fumed Growth ·/ 1.4 10⁶ 4

10⁶ CFU/ml inoculum,

c Dish 2 e6+ N/A grain mix not fumed Growth ·/ 3.2 10⁶ 4

10⁶ CFU/ml inoculum,

c e6+ N/A not fumed Growth ·/ 1.6 x 10⁷ 1

10⁶ CFU/ml inoculum,

c e6+ N/A not fumed Growth ·/ 3.4 x 10⁷ 2 c e6- 0 10⁶ CFU/ml inoculum No Growth 0 2 c e7- 0 10⁷ CFU/ml inoculum No Growth 0 2 c e8- 0 10⁸ CFU/ml inoculum No Growth 0 2 c e9- 0 10⁹ CFU/ml inoculum No Growth 0 2

B = Bedding, F = Feed, S = Soil, D=Dermal, M=Muscle, C=Control

²Z: Depth from surface (inches)

3 Fume 1: 10⁶ BI inoculum, Chamber

Fume 2: 10⁶ BI inoculum, Tube

Fume 3: 10⁶ BI inoculum, Chamber

Fume 4: 10⁶ BI inoculum, Chamber

Sandy loam proved to be a significant barrier to mass transfer of CIO₂ with no discernible soil penetration by chemical detection.

Results of CT 30, 000 ppm_v-hours Fumigation A subsequent experimental run utilized a CT value of 30,000 ppm_v-hrs. In this second fumigation trial, poultry meat, feed, and bedding were similarly exposed to gaseous CIO₂ over the course of 3 hours with a maximal concentration of 12,000 ppm_v. The results from the BI are shown in Table 2. BI sets spanning a range of 10⁷ to 10⁹ CFU/ml were used to quantify the extent of microbial devitalization.

C10₂ fumigation at CT 30,000 ppm_v-hrs resulted in at least a 6-log reduction in both surface and subcutaneously placed Bis in whole chicken carcasses. The intra-muscular BI showed more than a 2-log reduction in CFU.

Bedding and feed materials were fully permeated at CT 30,000 based on the colorimetric change of KI. In the bedding, the biological indicator showed that at a depth of 1 inch, the fumigation was effective in achieving no growth (quantitative result of 0 CFU/ml, which is more than an 8 log reduction compared with the corresponding control inoculum from test fume 3 that was not fumed and showed a quantitative result of 2.6 ^χ 10⁸ CFU/ml). At a depth of 4 inches in the bedding sample used in test fume 2, no growth was observed. However, at a depth of 4 inches in the bedding sample used in test fume 3, growth was observed. Also, in the bedding sample containing litter, growth was observed. The 6-inch soil layer again remained impermeable to gaseous CIO2.

Table 2: Results of CT 30,000 ppm_v-hr fumigation: Viability of biological indicator (E. coli)

¾ = Bedding, L = Litter, F = Feed, S = Soil, D=Dermal, M=Muscle, C=Control

2Z: Depth from surface (inches)

³Fume 2: 10⁶ BI inoculum, Chamber; Fume 3: 10⁷ BI inoculum, Chamber

General Conclusions

Taken together, the chemical and microbiological results from this example demonstrate that CIO₂ fumigation at 9,000 ppm_v-hrs rendered the surface of chicken carcasses safe for handling and disposal. Treatment at this CT value also served as a suitable sterilizing process for poultry feed, provided that the pellets or grain experience sufficient exposure to CIO₂ gas. The present study demonstrated the successful decontamination of these feed materials. In embodiments, particularly those involving processing mass quantities of feed, additional measures or apparatuses to achieve adequate contact time with the inherently high surface area can be used. For example, the feed could be kept in motion during the fumigation using a fluidized bed produced by air-lift column, by using conveyors during fumigation to move the feed, or by tumbling batches of feed in rotating baskets or drums. A CT value of 30,000 ppm_v-hrs achieved deeper penetration and decontamination of chicken tissues, feed, and bedding material.

Example 2: RT-PCR confirmed efficacy of fumigation of various materials at 9.000 ppnty-hr

The efficacy of fumigation was further verified using RT-PCR. Various field samples were collected from areas in and around contaminated egg layer barns affected by avian flu (an HPAI). The samples included soil samples, feed samples, dust samples, and litter samples collected from residual materials in locations in and around the barns (e.g., floors, steps, fans, feed, soil, conveyor belts). One of the barns had been dry cleaned barn (cleaned without liquid, e.g., with brooms, shovels, and/or a leaf blower) (Barn C) and one of the barns had been partially cleaned (Barn B). Samples from a feed bin and a grain conveyer were taken; these samples contained grains. Samples were subjected to fumigation at 9,000 ppm_v-hr chlorine dioxide gas or no fumigation (as a control).

The presence or absence of avian influenza virus in the samples was confirmed by a USDA- approved RT-PCR protocol employing the VetMAX™-Gold avian influenza virus detection kit (Life Technologies, Inc.). The results are shown in Table 3 below; the samples generally tested positive for avian influenza by RT-PCR prior to fumigation and tested negative for avian influenza following fumigation at 9,000 ppm_v-hr chlorine dioxide gas.

Table 3: RT-PCR Results of 9,000 ppm_v-hr fumigation

RT-PCR Results (Ct)

Sample Sample Z² Description Qualitative Quantitative Test Fume Type¹ No. Result Result number³

Feed from auger

outside of barns, not

F F0 0.5 fumed Positive 34.7 4

F EG16 0.25 Feed grains from egg Positive 36.8 4 conveyer (Barn B),

not fumed

Feed and dust from

floor (Barn B), not

F / Dust FL16 0.25 fumed Positive 37.8 4

Compacted chip of

litter from steps

L ST16 0.25 (Barn B), not fumed Positive 37.1 4

Dust and flakes from

egg conveyor (Barn

Dust EGC20 0.25 C), not fumed Positive 29.2 4

Litter from floor

L FL20-2 0.25 (Barn C), not fumed Positive 31.7 4

Litter from steps

L ST20 0.25 (Barn C), not fumed Positive 32.7 4

Feed from auger

F FOf 0.5 outside of barns Negative > 40 4

Feed grains from egg

F EG16f 0.25 conveyer (Barn B) Negative > 40 4

Feed and dust from

F / Dust FL16f 0.25 floor (Barn B) Negative > 40 4

Compacted chip of

litter from steps

L ST16f 0.25 (Barn B) Negative > 40 4

Dust and flakes from

egg conveyor (Barn

Dust EGC20f 0.25 C) Negative > 40 4

Litter from floor

L FL20-2f 0.25 (Barn C) Positive 36.7 4

Litter from steps

L ST20f 0.25 (Barn C) Positive 31.3 4

^:B = Bedding, L = Litter, F = Feed

2Z: Depth from surface (inches)

3Fume 4: AI Viral Load, Chamber

Example 3 : Soil treatment with liquid chlorine dioxide solution

Liquid C10₂ solutions of varying concentration were applied to different types of well- compacted soil in a series of tests to determine the approximate volumetric flow rate of each solution required to inactivate 10⁶ log concentrations of a biological indicator (surrogate bacterial spores) placed at varying levels within the soil. The objective of these tests was to establish appropriate application conditions for liquid CIO2 solution in a tightly controlled test environment designed to simulate natural soil conditions.

Materials and Methods

Test Bed Construction and Preparation

Soil "test beds" were constructed which incorporated a one-inch thick concrete block into the bottom of a wooden frame with internal dimensions of approximately 30 centimeters (cm) (length) x 30 cm (width) x 19 cm (height). The concrete block allowed for effective compaction of soil samples placed within the test beds. Following construction, the internal surfaces of each soil test bed were coated with an impervious liner to prevent the absorption of CIO2 solution into the test bed materials. Numerous small holes were drilled through the concrete block and test bed bottom in order to allow for collection of solution that had infiltrated down through the soil samples. A catchment basin was placed immediately beneath the test bed to collect residual percolated solution.

The bottom of each test bed was lined with a layer of "geogrid" material to promote effective collection of residual C10₂ solution that had percolated downward through the soil. Once the liner had been put in place, five 10⁶ log spore strips with unique sample identification numbers were placed in an even distribution across the top surface of the geogrid liner, along with one negative control spore strip. After the geogrid material and spore strips had been put in place, a second layer geogrid material was carefully placed over the spore strips to help protect their integrity during testing.

Biological Indicator

Bacillus atrophaeus (B. atrophaeus) spores were used as a surrogate organism to demonstrate effective CIO2 soil treatment. Bacterial indicator (BI) spore strips that contained approximately 1.5 x 10⁶ B. atrophaeus spores (i.e., 1,500,000 spores per strip) served as the biological indicator.

Soil

Three types of soil that represent a cross-section of the many variations in soil type that might be encountered in an actual field environment were placed into the test beds atop the geogrid liners and spore strips for purposes of CIO2 efficacy testing. These soil types were as follows: (1) loamy sand: 80% fine sand (all grains less than 1.0 millimeter in diameter), 10% silt and 10% clay; (2) loam: 50% fine sand, 35% silt and 15% clay; or (3) clay loam: 40% fine sand, 30% silt and 30% clay.

Two depths of each soil type were placed into test beds for the CIO2 efficacy testing. The soil depths were 1 cm (0.394 inches) or 1 inch (2.54 cm).

Once the soil samples had been placed within the test beds, they were compacted to dry bulk densities set forth in Table 4, to simulate densities found in the real world (e.g., in an urban setting). Soil compaction within the test beds was accomplished by means of a hammering device similar to that used to conduct soil compaction "Proctor" testing in the field environment. The three soil types were compacted in the test beds to the bulk density values shown in Table 4

Table 4: Dry Bulk Density of Test Soils

Chlorine Dioxide Liquid CIO₂ concentration levels were 500 mg/L and 1,000 mg/L. High purity aqueous solutions of chlorine dioxide were prepared in a laboratory environment using standard small-scale techniques. The actual concentration of each batch of CIO₂ solution was measured immediately before each soil test bed application by means of amperometric titration of a representative sample volume with a 0.1 normal sodium thiosulfate solution. This titration method is based on Method 4500-ClO₂ E ("Amperometric Method II") in the "Standard Methods for the Examination of Water and Wastewater," 20th ed., 1998.

Two different volumes of 500 and 1000 mg/L CIO₂ solution were applied to the soil test beds. These CIO₂ solution test volumes were as 1.075 liters (L) and 0.5375 L. Because the soil test beds contained approximately one square foot (ft²) of surface area, the equivalent CIO₂ solution application rates (volume/surface area) tested were about 1.075 L/ft² and about 0.5375 L/ft².

CIO 2 Application Process

The 500 and 1,000 mg/L CIO₂ solutions were applied to the soil test beds using a Flexflo® peristaltic pump, which pulled solution from a stock solution reservoir and applied it to the soil surface by means of spray nozzles affixed atop the test beds. The Flexflo® pump delivered each solution at a constant flow rate of 0.215 L per minute. An application regimen of one minute on and one minute off was utilized in the case of the 1.075 L volume, for a total application time of 10 minutes. In the case of the 0.5375 L volume, a regimen of 30 seconds on and 30 seconds off was used, for a total application time of five minutes. The CIO₂ solution was applied evenly over the entire compacted soil surface within each test bed until the desired volume had been applied.

Quality Control (QC)

Both positive and negative control spore strips were employed during the C10₂ soil treatment testing. Positive controls are untreated, impregnated spore strips of identical composition that are submitted for "blind" laboratory analysis along with treated spore strips for purposes of QC. Positive controls provide evidence of spore strip product quality as well as evidence that conditions for growth during analytical incubation were conducive. Negative controls are unimpregnated, treated spore strips that are submitted for laboratory analysis along with actual treated samples for purposes of QC. Negative controls provide evidence that sample spore strips have not been compromised by external sources of contamination.

Spore Strip Analysis

Within 24 hours of collection, spore strips were shipped to an outside laboratory for analysis. At the outside laboratory, they were aseptically placed in sterile tubes containing a soybean-casein digest broth and incubated at 30 to 35 °C degrees Celsius for a period of seven days. Tubes were monitored daily during the incubation period for a change in turbidity. A change in turbidity indicates metabolic activity by viable spores.

Results

CIO 2 Application and Residual Analyses Results of the amperometric titration analyses performed on CIO₂ stock solutions prior to soil test bed application and on residual solutions that percolated completely through the test beds, for the 1.075 L/ft² and 0.5375 L/ft² C10₂ application rates, are shown in Table 5 and Table 6, respectively. Table 5: C10₂ Stock Solution and Residual Liquid Concentration for 1.075 L/ft² Application Rate

Table 6: C10₂ Stock Solution and Residual Liquid Concentration for 0.5375 L/ft² Soil Application Rate

Soil Type Soil Depth 500 mg/L Target 1,000 mg/L Target

Stock Residual Stock Residual Solution Solution

Loamy Sand 1 cm 499 27 998 108

I inch 499 13 998 No Liquid

Loam 1 cm 499 0 998 0

1 inch 499 No Liquid 998 No Liquid

Clay Loam 1 cm 499 13 1,011 No Liquid

1 inch 499 No Liquid 1,011 No Liquid Spore Strip Analyses

Results of the spore strip analyses are presented in Tables 7 through 10. Tables 7 and 8 show results for the 1.075 L/ft² application rates of 500 mg/L and 1000 mg/L C10₂ solution, respectively. Tables 9 and 10 present results for the 0.5375 L/ft² application rates for the same two C10₂ solution test solution concentration levels.

Table 7: 500 mg/L C10₂ Concentration, 1.075 L/ft² Soil Application Rate

Table 8: 1,000 mg/L C10₂ Concentration, 1.075 L/ft² Soil Application Rate

Soil Depth Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Negative Positive

Control Control

Loamy 1 cm - - - - - - +

Sand

1 inch - - - - - - +

Loam 1 cm - - - - - - +

1 inch - - - - - - +

Clay 1 cm - - - - - - +

Loam

1 inch - - - - - - + Table 9: 500 mg/L C10₂ Concentration , 0.5375 L/ft² Soil Application Rate

Table 10: 1,000 mg L C10₂ Concentration, 0.5375 L/ft² Soil Application Rate

Conclusions

Results of the spore strip analyses demonstrated that the 1.075 L/ft² and 0.5375 L/ft² CIO2 solution application rates were both effective in inactivating high concentrations of surrogate spores placed in various types of soils at depths of up to one inch. With respect to the 1.075 L/ft² rate, each of 12 applications, including six at 500 mg/L and six at 1,000 mg/L, successfully inactivated all five 10⁶ log B. atrophaeus spore strips embedded beneath one-cm and one-inch layers of all three soil types tested, including loamy sand, loam and clay loam. Similar results were found with the 0.5375 L/ft² CIO2 solution application rate, although complete spore strip inactivation did not occur in every instance. All five spore strips embedded beneath one-cm and one-inch layers of all three soil types were inactivated in ten of 12 test applications, including five at 500 mg/L and five at 1,000 mg/L. The two exceptions to complete spore strip inactivation with the 0.5375 L/ft² solution application rate were the 500 mg/L application to a one-cm depth of clay loam soil and the 1,000 mg/L application to a one-cm depth of loam soil. In both instances, three of the five spore strips embedded beneath the soil layer were found to have survived the C10₂ treatment. Under identical treatment conditions applied to one-inch depths of each soil type, all five spore strips were inactivated. It was more difficult to achieve even surface coverage when the lower application rate was used; some unevenness in coverage at the lower application rate can explain these results.

Spore strip findings were generally consistent with the CIO₂ residual analyses results obtained during testing of percolated solutions. The 1.075 L/ft² CIO₂ solution application rate percolated completely through the soil test bed in all 12 test scenarios, including 500 mg/L and 1,000 mg/L applications to one-cm and one-inch depths of loamy sand, loam and clay loam soil types. A residual concentration of CIO₂ was measured in 10 of the 12 percolated liquids, with a range of 14 mg/L to 108 mg/L in the case of the 500 mg/L solution and 13 mg/L to 256 mg/L in the case of the 1,000 mg/L solution. No residual CIO₂ concentration was identified in the percolated liquid resulting from either the 500 mg/L or 1,000 mg/L application to the one-inch depth of loam soil.

The 0.5375 L/ft² CIO₂ solution application rate percolated completely through the soil test bed in five of six test scenarios using a one-cm depth of soil. The only soil type that absorbed the entire volume of solution at the one-cm depth was clay loam. The range of residual CIO₂ concentrations noted in percolated liquids ranged from 13 mg/L to 108 mg/L. No residual CIO₂ concentration was identified in the percolated liquid resulting from either the 500 mg/L or 1,000 mg/L application to the one-cm depth of loam soil. The 0.5375 L/ft² application rate percolated completely through the soil test bed in only one of six test scenarios using a one-inch depth of soil. The soil type through which this volume of solution did percolate was loamy sand.

Claims

CLAIMS What is claimed is:

1. A method of treating soil to a soil treatment depth, the method comprising applying to the soil an aqueous solution comprising chlorine dioxide and allowing the solution to percolate through the soil, wherein a percolate formed by the solution, after it has percolated through at least the soil treatment depth of said soil, has a residual chlorine dioxide concentration of at least 15 mg/L and the method reduces the level of a contaminant in the soil, wherein said soil treatment depth is 1 cm.

2. The method of claim 1, wherein the soil treatment depth is 1 inch (2.5 cm).

3. The method of claim 1 or 2, wherein the aqueous solution is applied to the soil at a

volume: surface area rate of at least about 0.5 L/ft² (5.4 L/m²).

4. The method any one of claims 1 to 3, wherein the aqueous solution comprises 50 mg/L to 3,000 mg/L chlorine dioxide.

5. The method any one claims 1 to 4, wherein the aqueous solution comprises at least 500 mg/L chlorine dioxide.

6. A method of treating soil, the method comprising applying an aqueous solution of at least 500 mg/L chlorine dioxide to the soil at a volume: surface area rate of at least about 0.5 L/ft² (5.4 L/m²), thereby eliminating a contaminant or reducing the level of a contaminant in the soil.

7. The method of any one of the preceding claims, wherein the rate is about 0.5 L/ft² to about 2.0 L/ft² (about 5.4 L/m² to about 21.5 L/m²).

8. The method of any one of the preceding claims, wherein the rate is about 0.5 L/ft² to about 1.0 L/ft² (about 5.4 L/m² to about 10.8 L/m²).

9. The method of any one of the preceding claims, wherein the density of the soil is less than about 1.8 g/cm³.

10. The method of any one of the preceding claims, wherein the density of the soil is about 0.9 g/cm³ to 1.7 g/cm³.

1 1. The method of any one of the preceding claims, wherein the solution is applied at a rate of 0.1 to 0.3 L/minute.

12. The method of any one of the preceding claims, wherein applying the solution comprises spraying the solution over the soil.

13. The method of any one of the preceding claims, wherein the percolate can be collected within less than 10 minutes after the applying.

14. The method of any one of the preceding claims, wherein the method is effective to inactivate a spore strip containing at least 10⁶ B. atrophaeus spores whein the spore strip is placed at the treatment depth prior to the applying .

15. A method of fumigating a feed or a bedding, the method comprising exposing the feed or the bedding to a gas comprising chlorine dioxide at a concentration x time (CT) value sufficient to penetrate the feed or the bedding to at least a treatment depth and to reduce the level of a contaminant in the feed or the bedding, wherein the treatment depth is 0.25 inch (0.6 cm).

16. The method of claim 15, wherein the treatment depth is 1 inch (2.5 cm).

17. The method of claim 15, wherein the treatment depth is 2 inches (5 cm).

18. The method of any one of claims 15 to 17, wherein the CT value is at least 9000 ppm_v-hours.

19. The method of claim 18, wherein the CT value is 9000 to 200,000 ppm_v-hours.

20. The method of claim 18 or 19, wherein the CT value is at least 30,000 ppm_v-hours.

21. The method of any of claims 15 to 20, wherein the contaminant is an influenza virus.

22. The method of claim 21, wherein the contaminant is an avian influenza virus.

23. The method of claim 21 , wherein RT-PCR testing of a post-treatment sample taken from the feed or the bedding after the exposing indicates that the post-treatment sample is negative for the contaminant.

24. The method of claim 23, wherein the post-treatment sample is taken from the treatment depth of the feed or the bedding.

25. The method any one of claims 15 to 24, wherein the method is effective to produce at least a specified reduction in an E. coli biological indicator placed at the treatment depth within the feed or bedding and comprising at least 10⁶ CFU prior to the exposing, wherein the specified reduction is at least a 3 log reduction in the number of E. coli CFU.

26. The method of claim 25, wherein the specified reduction is at least a 6 log reduction in the number of E. coli CFU.

27. The method of claim 25, wherein the specified reduction is no detectable growth of E. coli.

28. A method of fumigating a carcass, the method comprising exposing the carcass to a gas

comprising chlorine dioxide at a concentration x time (CT) value sufficient to penetrate the carcass to at least a treatment depth and to reduce the level of a contaminant in the carcass, wherein the treatment depth is 0.1 inch (0.25 cm).

29. The method of claim 28, wherein the CT value is at least 30,000 ppm_v-hours.

30. The method of claim 28, wherein the CT value is 30,000 to 200,000 ppm_v-hours.

31. The method of any one of claims 28 to 30, wherein the method is effective to produce at least a specified reduction in a dermally placed E. coli biological indicator that comprises at least 10⁶ colony forming units (CFU) prior to the exposing, wherein the specified reduction is at least a 6 log reduction in the number of E. coli CFU.

32. The method of any one of claims 28 to 30, wherein the method is effective to produce at least a specified reduction in a dermally placed E. coli biological indicator that comprises at least 10⁶ colony forming units (CFU) prior to the exposing, wherein the specified reduction is a reduction to no detectable growth of E. coli.

33. The method of any one of claims 28 to 30, wherein the method is effective to produce at least a specified reduction in an intramuscularly placed E. coli biological indicator that comprises at least 10⁶ colony forming units (CFU) prior to the exposing, wherein the specified reduction is at least a 2 log reduction in the number of E. coli CFU.

34. The method of any one of claims 28 to 30, wherein the intramuscularly placed E. coli

biological indicator is placed at a depth of 1 inch (2.5 cm) within the carcass.