Peroxygens and Other Forms of Oxygen - American Chemical Society

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Chapter 13

Peroxygens and Other Forms of Oxygen: Their Use for Effective Cleaning, Disinfection, and Sterilization

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Gerald McDonnell STERIS Corporation, Basingstoke, Hampshire, United Kingdom

Oxidation may be defined as the process of electron removal, where oxidising agents are substances which cause the loss of electrons from other molecules that are thereby accepted by the agent. As well as their many chemical uses, many oxidizing agents have potent antimicrobial activity. These include various halogens (like chlorine and iodine) and the peroxygens and other forms of oxygen, which include hydrogen peroxide, peracetic acid, chlorine dioxide and ozone. This group of biocides has become widely used for cleaning, antisepsis, disinfection and sterilization applications. This review will discuss the various applications and uses of peroxygens and other forms of oxygen, along with their various advantages and disadvantages. Applications include the use of these biocides in various liquid and gaseous forms, and include food and water disinfection, low temperature surface sterilization and large area remediation, as recently shown with the successful fumigation of buildings following recent bioterrorism attacks in the United States. For liquid applications, the formulation of these biocides plays an important role in optimizing their safety and efficacy. Equally, process controls including the effects of temperature can play an important role in the application of both liquid and gaseous processes. The current understanding of the mode of action of these biocides will be reviewed and recent investigations have identified new applications for these agents in the control of emerging and reemerging pathogens.

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© 2007 American Chemical Society

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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293 Oxidation may be defined as the loss of an electron by a molecule, atom, or ion and oxidizing agents as agents that remove electrons from the other substances, and are thus reduced themselves. Due to this activity, these agents will have dramatic effects on the structure and function of microorganisms that culminate to give potent biocidal activity. Many oxidizing agents are among the most widely used biocides for medical, dental, industrial and agricultural applications. They can be further classified into the halogens and the peroxygens/other forms of oxygen. The halogens include biocides based on chlorine (like sodium hypochlorite, widely used in bleach solutions), iodine (including iodine-releasing agents such as iodophors) and bromine. The chemical applications and biocidal activities of the halogens have been reviewed elsewhere (1, 2, 3). The peroxygens and other forms of oxygen include relatively simple biocides such as hydrogen peroxide, peracetic acid, chlorine dioxide and ozone. Some of these biocides, in particular hydrogen peroxide, have been used for various antiseptic, preservation and disinfectant applications for over 100 years; yet despite this, many recent advances have been made in the optimization of the antimicrobial activity, surface compatibility and application of these agents both in liquid and gaseous form. The types, uses, and modes of action of the peroxygens and other forms of oxygen are considered further in this review.

General Mode of Action The basic mode of action of the peroxygens is to react with the essential macromolecules that make up microbial life, including the oxidation of various proteins, carbohydrates, lipids and nucleic acids (4). These reactions will lead to the loss of structure and function of these molecules, including unfolding, fragmentation and cross-reaction with oxidized groups. Proteins, carbohydrates and lipids on the surface of microorganisms are the initial accessible targets. The various structures on these surfaces are essential to the survival, pathogenicity and basic structure of microorganisms; therefore the loss of structure and function at this stage alone is sufficient to observe loss of viability, in particular as observed in bacterial and viral studies. Initial damage to surface structures is followed by further interactions with various intercellular components, including proteins and nucleic acids, as the structure of the microorganism breaks down. Specific effects of different peroxygen chemicals have been reported as being particularly targeted by these biocides (4). For example, the effects of chlorine dioxide against certain amino acids (tryptophan, cysteine and tyrosine) have been reported. Further, peracetic acid and hydrogen peroxide have been shown to disrupt sulfhydryl (-SH) groups and sulphur bonds (S-S) in proteins, and fatty acid double-bonds; however, it is expected that these groups/bonds will be particularly sensitive to oxidation.

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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294 Despite the basic oxidation mode of action of these biocides, the overall effects observed in a microbial population will vary depending on the formulation and delivery process of the biocide. An important aspect in the optimization of the mode of action is access of the oxidizing agent to the microorganism. It is important to note that microorganisms are generally associated with various extraneous materials (or soils) including various proteins, carbohydrates and lipids. If preparations of these microbes in their natural state are added directly to a given concentration of the biocide, clumping may occur which limits the penetration to each microorganism. A clear example is with peracetic acid, simple solutions of which in water have an acidic pH; if a microbial population is exposed to this alone, it will lead to clumping and may underestimate the true activity of the biocide. In contrast, when peracetic acid is provided in a formulation (generally closer to a neutral pH) it can allow greater penetration of the biocide and access to. the microorganisms. Overall, unlike anti-infective (including antibiotic) investigations, the formulation and/or process control of a biocide will play a dramatic role in the overall mode of action and optimal activity in microbial inactivation. This is an important, yet often underestimated concept to appreciate. Examples of the impact of these effects will be discussed further.

Biocidal Applications Peroxygens are widely used for a range of biocidal applications (Table I). These applications can be considered as being based on the liquid or gaseous biocide at the point of use. Liquid applications can simply use various concentrations of the biocide in water; a notable example is the traditional use of hydrogen peroxide at 3-6% concentrations for skin and particularly wound treatment. In general, most liquid applications use the biocide in various formulations, being in combination with other chemicals that improve the activity, stability, or indeed the surface compatibility of the biocide alone. These formulation chemicals include stabilizing agents, anti-corrosives, surfactants, and chelating agents. For example, peracetic acid is a relatively unstable biocide and is therefore supplied in equilibrium with water, hydrogen peroxide and acetic acid (5). Commercial 35% PAA is provided with 7% hydrogen peroxide, 40% acetic acid and 17% water, which may also contain a stabilizer to improve shelf life. These solutions of peracetic acid can not generally be used directly for disinfection applications as they can be corrosive; however, stable and more compatible formulations based on peracetic acid as the major biocidal agent are widely used for general surface and medical device applications. Similar formulation optimization for antimicrobial efficacy, safety and surface compatibility has been described for hydrogen peroxide, chlorine dioxide, and other mixed oxidants.

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

Limited applications due to reactive and shortlived nature of the biocides. Hydrogen peroxide releasing agents, including benzoyl peroxide Liquid hydrogen peroxide, peracetic acid and chlorine dioxide

Control of bacterial/fungal growth in a product by inhibition

Removal of contamination ('soil') from a surface; can be combined with disinfection Microbial reduction or inhibition on living tissues (e.g., skin) Antimicrobial reduction of viable microorganisms to a safe or defined level in drinking water

Preservation

Cleaning

Antimicrobial reduction of viable microorganisms to a safe or defined level in air Defined process to render a surface of productfreefrom viable microorganisms

Air disinfection

Sterilization

Antimicrobial reduction of viable microorganisms to a safe or defined level on a surface

Surface Disinfection (including device and food surfaces)

Water Disinfection

Antisepsis

Hydrogen peroxide (including wound treatment) Chlorine dioxide (common replacement for chlorine disinfection) Ozone Electrolyzed water Liquid or gaseous hydrogen peroxide Liquid peracetic acid Liquid or gaseous chlorine dioxide Ozone Electrolyzed water Gaseous hydrogen peroxide and chlorine dioxide Ozone Gaseous or liquid hydrogen peroxide (including generation as a plasma gas) Liquid peracetic acid (at 50°C) Ozone

Examples

Applications

Description

Table I. Biocidal Application with Peroxygens and Other Forms of Oxygen

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296 Ozone was one of the earlier appreciated gaseous biocides, particularly used for the deodorization and disinfection of air, rooms and some industrial applications. More recently, ozone sterilization systems have been successfully developed for medical device and other surface applications, highlighting advances in ozone generation systems. During the 1980's and 1990's, various applications were developed using gaseous phase peroxygens, including peracetic acid, chlorine dioxide and particularly hydrogen peroxide. These processes have been used for area disinfection, surface (including device) sterilization and other industrial sterilization applications. They include the direct use of the biocide (usually with humidity control, depending on the process) and in combination with an energy source, e.g. in the presence of UV light or the generation of a plasma in the presence of the gas. Plasmas are generated by energizing the molecules of a gas to give a highly excited mixture of charged, reactive nuclei and free electrons. Plasma-based disinfection and sterilization systems have been described with peracetic acid, oxygen (for ozone generation) and notably hydrogen peroxide.

Peracetic Acid Peracetic acid (CH COOOH; PAA) is a potent biocide at relatively low concentrations and is also considered environmentallyfriendlyas it rapidly breaks down to water, oxygen and a low concentration of acetic acid (5). Peracetic acid is also unique as an oxidizing agent in that it demonstrates somewhat stable activity even in the presence of organic and inorganic soils, which can interfere with optimal activity. Its main applications have been in liquid formulation disinfectants, in particular to optimize compatibility concerns with a broad spectrum of plastics, metals and other surface materials, as well as improved biocide stability. It should be noted that these formulations can vary significantly in antimicrobial efficacy and surface compatibility, independent of the concentration of peracetic used in the formulation. With optimized formulation, peracetic acid solutions can be successfully used on many sensitive materials, including those used in flexible endoscopes and dialysis equipment (5). Peracetic acid formulations are used for cleaning (to physically remove and breakdown soils), surface disinfection, waste-treatment and device disinfection/sterilization. Direct use of concentrated peracetic acid is limited to certain industrial applications, but formulations include stabilized solutions, two-component solutions (one containing peracetic acid and the other with formulation excipients) and generational type formulations. An example of a generational type reaction to make peracetic acid in formulation is shown in Figure 1. Gaseous peracetic acid disinfection (fumigation) and sterilization systems have also been described but have not seen widespread use due to the aggressive nature of the biocide in vapor form on various surfaces. 3

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

297 p ^ O

HO m*)2

B\ HO

/

N

H +

B

x

b —

O

X

2H20

0H

Sodium Per torate

H

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2

\ +

0—0^

2 HaCBQj) + 2 H 0 2

H

Hydrogen Peroside

Peracetic Acid (PAA)

Sodium M elaborate

Salicylic Acid

Figure 1. An example of a peracetic acid generational chemistry from sodium perborate and acetylsalicylic acid.

A unique low-temperature liquid chemical sterilization system, the STERIS S Y S T E M 1, was one of the first peracetic acid-based systems designed for the reprocessing of thermosensitive medical and dental devices including flexible endoscopes (6). The system consists of a processing unit that dilutes and flows a peracetic acid-based formulation. This is delivered as a two-component chemistry in a cup-within a-cup design (STERIS 20) consisting of - 3 5 % peracetic acid solution, separated from the dry formulation components that are dissolved, mixed and heated during the sterilization process. In the final sterilization process, the concentration of peracetic acid is -0.2%, at a p H of 6.4 and at 50-55°C. The total process time is ~30minutes, consisting of a 12 minute chemistry contact, followed by four sterile water rinses; this process has been shown to comply with the efficacy requirements of ISO 14937 as a true sterilization process (6, 7). The broad spectrum antimicrobial efficacy of the process is due to a combination of peracetic acid, the formulation and contact temperature to include bacteria, molds, yeasts, bacterial and fungal spores, viruses and protozoal dormant form (cysts/oocysts). Recent studies on the mode

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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of action of action of this process highlight the importance of formulation and process control in the activity of peracetic acid. For example, the process has been shown to cause the degradation of proteins, but only within the range of 45-60°C (Figure 2); at higher temperatures, for example, potential clumping of test proteins has been observed that may be expected due to the effects of temperature alone on protein structure.

30

35

40

45

50

60

Temperature (°C) Figure 2. SDS-PAGE analysis on protein (bovine serum albumin) samples exposed to the STERIS 20 formulation at various contact temperatures.

These effects on the structure of proteins may be significant in the control of 'infectious' proteins (known as prions) that have been implicated as the causative agents in a rare group of diseases known as transmissible spongiform encephalopathies (TSE's) such as Creutzfeld-Jakob disease (CJD) and Bovine Spongiform Encephalopathy (BSE). The prion proteins are proposed to transmit these diseases in the absence of any known nucleic acid and are known to be intrinsically resistant to disinfection and sterilization methods (8). Biocides that have a cross-linking mode of action such as glutaraldehyde and formaldehyde are not recommended to treat these agents, but some studies have shown promising efficacy with alternative oxidizing agent formulations and processes (9). Peracetic acid in the STERIS 20 formulation has demonstrated some activity to degrade prion proteins, but only in this formulation and when tested at 50°C. The mode of action would appear to be a combination of the exposure temperature

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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299 and formulation excipients that may allow for partial unfolding of the protein, and the ability of peracetic acid to degrade the protein structure. pH could also be an important variable, as when protein is added directly to a solution of peracetic acid it will cause clumping and, therefore, lack of biocide penentration; in contrast, peracetic acid in formulation under controlled pH conditions can show effective protein degradation.. Other formulations and uses of peracetic acid require further investigations and can not be assumed to have any efficacy against prions due to their unique structure and infectivity profile (e.g., as shown under experimental conditions, 10). Similar effects of formulation and process control in the activity of peracetic acid have been described in biofilm disinfection investigations. Biofilms are communities of microorganisms (either singular or multiple species) that develop on or are associated with surfaces (11). As biofilms grow and mature, they are found within an external matrix of protein, carbohydrate and other materials that can provide an excellent protection (and therefore resistance) mechanism to biocides and biocidal processes (4). The presence of biofilms has been implicated in microbial survival (e.g., in nosocomial infections with the use of inadequately reprocessed medical devices, product spoilage and crosscontamination) as well as surface damage over time. For these reasons, biofilm control is an important consideration and in particular in the disinfection of water and water-based systems. Peracetic acid-based formulations have often been recommended over aldehyde disinfectants as they have been proposed to better penetrate and remove biofilms from surfaces. This in fact may not always be the case, as exemplified by recent studies by Martiny et al (10). In this report, the activity of two peracetic acid-based disinfectants to decontaminate surfaces was compared to aldehyde-based disinfectants. Although the aldehydes showed an expected lack of activity, the peracetic acid-based products also showed clumping of the biofilm on surfaces, thereby limiting penetration, removal and disinfection. In contrast, the STERIS 20 process has been shown to degrade and remove Pseudomonas aeruginosa biofilm proteins and carbohydrates from contaminated surfaces, allowing adequate disinfection of intrinsic microorganisms (as shown in Figure 3). These results highlight the importance of formulation and process control in the activity of peracetic acid on surface disinfection and overall mode of action of the biocide.

Hydrogen Peroxide Hydrogen peroxide (H 0 ) is a simple yet powerful oxidizing agent and biocide (12). It may also be considered environmentally friendly, rapidly degrading into water and oxygen. Liquid applications include direct use on the skin (as described above) or for industrial applications, and in formulation, in particular to enhance the stability and antimicrobial activity at lower concentrations. It has also been widely used as an effective biocidal gas (13). 2

2

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

Figure 3. Results of a biofilm investigation with 4 disinfectants: 0.2% peracetic acid (STERIS 20 at 50°C), 0.5% orthophthaldehyde, 2%> glutaraldehyde and 10% bleach (~0.5% sodium hypochlorite). Pseudomonas aeruginosa biofilms were developed on test surfaces and exposed daily to individual disinfectant treatments. The levels of carbohydrate, protein (not shown) and viable bacteria were evaluated. The first point (at they-axis of both graphs) indicates initial, unexposed levels of bacteria or carbohydrate detected and subsequent points are following exposure (up to 5) to the test disinfectants. Resistant, stable biofilms developed over repeated applications of all biocides, with the exception of the exposure to the peracetic acidformulation.

2

Bacterial Count (log, cfu/cm Pseudomonas aeruginosa)

Carbohydrate Concentration (|4g/cm ) 2

Product Exposure

Product Exposure

0

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301 As for other oxidizing agents, the overall surface compatibility and antimicrobial efficacy will be formulation dependant (in the case of liquid applications) and process-dependant. An example is in the use of gaseous hydrogen peroxide; in its true gaseous or vapor form, peroxide demonstrates more potent antimicrobial activity that at similar concentrations in liquid form (e.g., ~350mg/L of liquid to give similar activity to l-2mg/L of the gas), but also greater compatibility with various surface materials including electronics and other sensitive materials. However, with consideration to the physical chemistry of the biocide, if the concentration of gaseous peroxide is increased above a given saturation point (dependant on the temperature) it will condense out on surfaces at high liquid concentrations (-70-80%) and pose additional safety and compatibility concerns than the gas. Hydrogen peroxide is used as an effective antiseptic, wound cleaner, hard surface cleaner, surface disinfectant and sterilization applications (both in liquid and gaseous forms; 4, 12). As an example, various gaseous applications with hydrogen peroxide are discussed in further detail. Hydrogen peroxide gas is readily generated by vaporization of liquid peroxide solutions (generally 30-60%) at 100°C (or at lower temperatures under vacuum), to give a colorless, odorless gas. The broad spectrum activity of the gaseous form has been well studied and published including bactericidal, fungicidal, viricidal, sporicidal, cysticidal, oocidal and, more recently priocidal activity (9, 13). The antimicrobial activity of gaseous peroxide is unique in that it is effective at both high and low humidity levels levels, where in contrast >60% humidity is required for other biocides like ozone, chlorine dioxide, formaldehyde and ethylene oxide. Gaseous processes include the fumigation of enclosed spaces such as isolators, laminar flow cabinets, rooms, vehicles and buildings. For example, peroxide gas was successfully used in the remediation of buildings contaminated during the anthrax spore bioterrorism events in the United States during 2001. Successful fumigation applications have been subsequently described in other general, industrial, and medical (hospital) applications (13, 14). The overall process is similar in all these applications using gas generator systems to dehumidify the given area to -40% relative humidity, conditioning the area to a given concentration of peroxide (~0.1-2mg/L), decontamination (by maintaining the biocide concentration) and aeration to remove peroxide gas to a safe level (-lpprn; 13). In addition to antimicrobial activity, gaseous peroxide has been shown to effectively neutralize bacterial exotoxins (including the protein-based anthrax and botulinum bacterial toxins), some chemical weapons and some cytotoxic drugs (15) for other applications. Sterilization processes have been developed for industrial, food packaging, medical and dental applications. These generally are performed under vacuum to optimize gas delivery, penetration and safe removal during a given process. Examples of gaseous sterilization apparatus are shown in Figure 4 and include processes with and without plasma generation as part of the sterilization process. Other sterilization processes control the temperature and flow of the gas to ensure adequate surface contact for the required exposure conditions.

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302

Figure 4. Hydrogen peroxide gas sterilization equipment On the left is the STERIS VHP MD system for medical device sterilization and on the right are the STERRAD NX (middle) and 100S systems for reusable medical device sterilization in hospitals. The STERIS system uses VHP under vacuum and the STERRAD process uses condensed peroxide gas in combination with plasma generation.

The mode of action of hydrogen peroxide is based on both the peroxide molecule itself, as an effective oxidizing agent, as well as other short-lived radicals and ions (e.g., "HO, O*, 0 , HOO') that form during the degradation to water and oxygen (4). It may be expected that the increased reactivity of the peroxide molecule and greater presence of break-down species may be responsible for the increased activity of gaseous over liquid peroxide. These various species will rapidly react and oxidize various groups on external/internal microbial constituents including lipids, proteins and nucleic acids. Some of these effects at a molecular level have been described in detail, as hydrogen peroxide is a natural by-product of the respiration of eukaryotic and prokaryotic cells, and unless controlled can cause internal damage to cell components by structure disruption and loss of function (16). Hydrogen peroxide gas has been shown to react with and disrupt peptide bonds, leading to protein fragmentation. This attribute was subsequently investigated for activity against prion proteins and was shown to be an effective priocidal biocide (9); interestingly, in contrast it was also shown that the activity of liquid (or condensed) peroxide against proteins and prions was dramatically limited (Figure 5). In studies with prion protein preparations, gaseous hydrogen peroxide was shown to degrade the prion proteins, while exposure to liquid peroxide caused clumping, lack of biocide penetration and little to no activity. It may be expected that similar clumping may also occur and prevent biocidal penetration when microorganisms are surrounded by contaminating soils, limiting the activity of liquid peroxide. It is certainly conceivable that these effects may be limited or activity enhanced by liquid formulation effects similar to those described for peracetic acid. !

2

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303

Liquid IL^

SC

P«"P

VHP

Figure 5. The activity of hydrogen peroxide against prion proteins. This Western blot demonstrates dilutions of a prion protein (PrP ) preparation on the left and the effect of immersion in liquid hydrogen peroxide at 5 and 50% v/v in water and on exposure to vaporized hydrogen peroxide (VHP). 5

Although the subtle effects of low concentrations of hydrogen peroxide on nucleic acids have been described to include reactions with nucleotide bases and the sugar-phosphate backbone, the effects at truly biocidal concentrations have only recently been investigated. Exposures to gaseous hydrogen peroxide have shown significant nucleic acid unfolding and fragmentation, depending on the gas concentration and exposure time (McDonnell, unpublished results). These effects will obviously initially disrupt the essential replication, transcription and translation activities of nucleic acids, but will also eventually culminate in enough damage that any natural repair mechanisms of the cell will not allow for recovery. The inactivation of nucleic acids in a important attribute for any biocide or biocidal process, in particular in consideration of viricidal activity, where the viral nucleic acid has in some cases been shown to be able to infect cells despite loss of viral structure (17).

Chlorine Dioxide Chlorine dioxide (C10 ) is an unstable, water soluble gas (18). It is used as an effective biocide in various liquid and gaseous applications; however, due to its intrinsic instability, it needs to be generated at its site of use by various different chemical and electrochemical methods. Examples of various chemical reactions used in the generation of chlorine dioxide are shown in Figure 6. 2

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

304

Chlorine Gas

2 NaCI0

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Sodium chlorite

2

+ Cl

2

Chlorine

2CI0

2

Chlorine Dioxide

+ 2NaCI Sodium Chloride

HCI + NaOCI Hydrochloric Sodium Acid Hypochlorite

Figure 6. Examples of the generation of chlorine dioxide by various chemical reactions, via the reaction of chlorine with sodium chlorite.

The biocide is also considered environmentally friendly, as it rapidly breaks down into innocuous components. Chlorine dioxide is directly used in liquids (including water), in formulation (usually as two-components which are mixed to generate the biocide in the presence of other formulation excipients) and in gaseous form (in the presence of >70% relative humidity; 18). These applications all demonstrate broad spectrum antimicrobial activity over recommended product/process contact times and conditions. Surface compatibility is generally recorded as being less than that experienced with hydrogen peroxide (depending on the process control, formulation and biocide concentration); despite this, gaseous and liquid applications have been successfully developed for disinfection of even sensitive plastics used in reusuable, thermosensitive devices or other surfaces. In gaseous applications, incompatibility observed with surfaces has been linked to the production of chlorine-intermediates that can particularly form on degradation on exposure to light (thereby fumigation applications are best applied under darkness). Chlorine dioxide has been particularly widely used for water disinfection (as a safer alternative to the use of direct chlorination) and food surface disinfection, with other applications including general surface disinfection (in liquid and gas phases) and, in gaseous applications, for use in medical/dental sterilization processes (although many of these systems are not currently commercially available). Antiseptic applications, at lower concentrations have also been used in limited situations. Various two-component formulations are currently used for device disinfection and gaseous chlorine dioxide has been successfully

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

305 applied for large area fumigation, notably for building decontamination in the remediation of anthrax spore contamination in the United States.

Other Oxygenated Species Ozone (0 ) is probably the most reactive and short-lived oxidizing agent used for biocidal applications (19). It may be produced by passing oxygen (or the oxygen in air) through a high energy source (e.g., U V light, electrochemical cells or corona discharge). Ozone itself is a simple molecule, which rapidly degrades on reaction with surfaces, contaminants etc. into water and oxygen. The reactivity of the molecule makes it a very effective antimicrobial in both liquid and gaseous applications (the latter requiring humidity control of generally >70% relative humidity). In addition, during the generation and degradation of ozone, many other oxygenated species are produced including ions (e.g., the superoxide O " and peroxide 0 " ions) and radicals (hydroxyl "OH and hydroperoxyl H 0 radicals); these are equally reactive and will culminate in the overall antimicrobial activity associated with ozone. However, it is also the reactivity of ozone and other species that can cause some of the disadvantages in the use of the biocide, in particular surface compatibility (e.g., various plastics and metals) and maintenance of microbiocidal concentrations over the required exposure times (especially for endosporicidal activity). Ozone is used for water disinfection, deodorization, surface disinfection (in particular in gaseous form) and as a gaseous sterilization process. The use of ozone as a biocide has been advanced by the development of more efficient and reproducible ozone generation systems. For example, although various patents and processes have been described, it is only recently that ozone sterilization for devices has become more widely available (Figure 7; 20).

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3

2

2

2

2

Figure 7. The TS03 125L ozone sterilizer.

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306 This new sterilization process uses a >3 hour exposure time including humidification of the load (at 85-95% relative humidity), ozone exposure and ventilation. Although being a simple, economic and effective sterilization method, the process is currently limited due to some material restrictions and compatibility concerns over multiple exposure times. Ozone itself has been well studied as an antimicrobial, but initial claims of some priocidal activity have yet to be substantiated and are under further investigation. Electrolyzed water generators use water and a low concentration of sodium chloride (or another salt) to generate a solution of mixed oxygenated species by passing solutions through an electrolysis device, subjecting it to a voltage across a membrane and collection of the resulting anolyte for direct use as an antimicrobial solution (Figure 8). The parallel collected catholyte solution has also been used as an effective cleaning solution.

Anode

Figure 8. Electrolyzed water generators. The theory of operation is shown on the left, by passing the water sample through an electrolysis unit and separation of the anolyte which is usedfor biocidal purposes. An example of a STERILOX generator is shown on the right.

The biocidal activity of the anolyte is primarily due to the generation of hypochlorous acid (HOCI), but also other oxygenated species such as ozone and superoxide radicals (21). A variety of systems have been developed, some of which control the pH of the resulting anolyte (which is acidic) by mixing with a

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307 given proportion of the basic catholyte portion also separated during generation. The anolyte is rapidly antimicrobial, but is also short-lived (thereby requiring to be generated at site of use) and environmentally friendly. Similar to ozone applications, although having the advantage of potent biocidal activity, the active species are quickly neutralized in the presence of contaminating soils and can also be rather aggressive to sensitive surface materials. For example, even in pH adjusted systems, a specialized protective coating is recommended to be applied on flexible endoscopes being reprocessed with electrolysed water to prevent damage on repeated uses. Overall, successful applications of these systems have included odor control, water disinfection and low temperature device disinfection.

Conclusions Peroxygens and other forms of oxygen are widely used for cleaning, antiseptic, disinfection and sterilization applications. They have been shown to be potent biocidal agents, including activity against those standard investigated microorganisms such as bacteria, fungi and viruses, as well as less studied pathogens such as parasites and prions. Biocidal activity can be successfully and safely applied on a variety of surfaces with little overall environmental impact. However, it is clear, yet often underestimated, that the success of these requirements will depend of the formulation of the biocide (in liquid applications) and in the control of liquid/gaseous processes (e.g., temperature, delivery, humidity etc.). What may appear to be similar formulations and processes may indeed pose varying antimicrobial activities, surface compatibility profiles and safety considerations. These variables should not be underestimated in the application and use of peroxygens for biocidal purposes. Further, these effects will also play a role in the overall mechanisms of action of these biocides. Peroxygens, as oxidizing agents, clearly have non-specific modes of action against the various macromolecules that make up microbial life. The oxidation of these molecules causes a loss of essential structure and function, which culminate in a loss of viability; however, the various formulation and process effects associated with these biocides have been shown to play an important role in the optimization and even restriction of these effects. It is clear that in the future that further improved formulations, delivery/control processes and optimized applications will be developed with peroxygens and other oxygenated species, as well as a greater understanding of their spectrum and mechanisms of biocidal activity.

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