U.S. Patent for Methods of Eliminating Biofilms from Plumbing Systems (Patent No. 11,655,167, issued May 23, 2023) (2023)

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of US patent application serial no. No. 15/014,951, filed Feb. 3, 2016, claiming priority as a continuation-in-part of International Patent Application PCT/US2015/043331, filed Jul. 31, 2015, claiming priority from US Provisional Patent Application No. 614302, filed 1 August 2014, each of which is incorporated herein by reference in its entirety for all purposes.

FELD

The present disclosure relates to methods of treating sanitary systems to eliminate, remove and/or disinfect biofilm and biofilm-associated pathogens using treatment solutions comprising a mixture of chlorine and chlorine dioxide.

BACKGROUND

Sanitation-associated infections cause tens of thousands of illnesses and deaths each year. Clinically significant pathogens in plumbing include environmental gram-negative bacteria and free-living amoebae (FLA), which can invade plumbing systems in relatively small numbers, reproduce (proliferate) in large numbers, and release from plumbing into the environment as respirable bioaerosols. The only reported sanitary disease in the United States is Legionnaires' disease, a serious lung infection caused by the bacteriumLegionella. Domestic plumbing systems are now considered the main source of Legionnaires' disease. (Yoder et al., 2008) The US Center for Disease Control and Prevention (CDC) has estimated that there are up to 18,000 cases of Legionnaires' disease annually. The US Occupational Safety and Health Administration (OSHA) estimates that Legionnaires' disease causes approximately 4,000 deaths each year in the United States. Reported outbreaks of Legionnaires' disease have more than doubled in the last 10 years. Other sanitation-related pathogens, such asPseudomonasand non-tuberculous mycobacteria (NTM) can cause as many or more diseases asLegionella, but the lack of mandatory reporting and other factors make quantification difficult. The primary disease transmission vectors for these sanitary pathogens are inhalation and aspiration.

Since the early 20th century, water treatment and disinfection methods used in the United States and other developed countries have virtually eliminated the occurrence of waterborne enteric diseases such as typhoid and cholera resulting from fecal contamination of public water supplies. The focus of this historic, successful effort has been to combat "traditional pathogens," waterborne pathogens of fecal origin that contaminate source water and do not normally multiply in drinking water itself. The primary disease transmission vector for these traditional pathogens is ingestion.

E coliis a preferred reference organism in traditional water treatment; It is often used as a primary indicator of fecal pollution. Current data suggest soE coliobtained almost entirely from the feces of warm-blooded animals; Its presence in drinking water is considered to indicate significant faecal contamination after treatment or inadequate treatment.E coliis extremely sensitive to chemical disinfection such as chlorination. Its presence in a water sample is considered a sure sign of a serious deficiency in the treatment program or in the integrity of the distribution system. However, the lack ofE colialone does not provide sufficient assurance that the water is free of microbial contamination.

Components of water, pipe deposits and plumbing materials initially create a chemical need for oxidizing disinfectants such as chlorine. The amount of disinfectant remaining after the initial oxidant requirement has been met is referred to as "disinfectant residual". "Ct" - the concentration of residual disinfectant [C] times the contact time "t" - is a key concept used in the development of traditional disinfection protocols. For each drinking water disinfectant, Ct tables were developed for a range of challenge organisms, mainly suspended (planktonic), traditional (enteric) indicator pathogens such asE coliAndGiardien.

In general, public drinking water supplies in developed countries are subject to government standards that make the water safe for its intended use. In the United States, drinking water supplied by municipal water systems is treated in accordance with the National Primary Drinking Water Standards, a set of requirements established by the United States Environmental Protection Agency (USEPA) under the auspices of which the Safe Drinking Water Act (SDWA) was developed. Most drinking water regulations focus primarily on the quality of the water at the time it leaves the treatment plant.

There is growing recognition that compliant drinking water can degrade after it enters the distribution system, the series of pipes that carry the water from the treatment plant to the consumer. In 2006, the National Academy of Sciences, at the request of the USEPA, published a study by the National Research Council's (NRC) Water Science Technology Board (WSTB): "Drinking water distribution systems: Assess and reduce risks". (NRC, 2006) The study highlighted the urgent need for new scientific knowledge that would enable cost-effective treatment of the distribution system to protect public health and minimize degradation of water quality after the water leaves the treatment plant. The distribution system is often categorized from largest to smallest components: transmission network (mains), distribution network, utility lines and building installations. The water treatment utility typically owns and is responsible for the infrastructure of the distribution system right up to the connection to the customer, which sometimes includes the service line. The customer is almost always responsible for the sanitary installation. The study highlights treatment challenges that are unique to plumbing fixtures.

The term "plumbing" refers to the piping in a building or home that distributes the water to the point of use. This also includes devices for treating the water, i.e. for softening, filtering, storing, heating and circulating the water before it leaves the tap. Home plumbing systems are made from a variety of materials, including copper, plastic, brass, lead, galvanized iron, and occasionally stainless steel. Many of these materials are typically not present in the main distribution system. Compared to other parts of the water distribution system, building sanitary facilities are characterized by longer water residence times, more stagnation, lower flow ratios, a higher surface area to volume ratio (due to relatively long sections of small diameter pipes), and lower (if any) disinfectant residuals and higher water temperatures. The special characteristics of the sanitary facilities create a unique ecological niche and contain a robust microbial ecology.

The microbial colonization of sanitary systems occurs mainly through the formation of natural biofilms on the inner surfaces of the pipes. (Declerck, 2010; Murga et al., 2001) Biofilms are complex heterogeneous aggregates of microorganisms and exogenous materials embedded in a highly hydrated matrix commonly referred to as extracellular polymeric substances (EPS). EPS consists of a number of components, including polysaccharides, proteins, lipids and nucleic acids. Biofilm development, chemical composition, microbial diversity, morphology and activity are affected by a number of factors, including water temperature, pH, hardness, disinfection history and the composition of the sanitary surface on which the biofilm forms. For example, biofilms that form on copper pipes in a hot water system differ from the biofilms that form on the interior surfaces of transmission lines, even in the same overall water system.

Biofilm formation on a plumbing surface can be triggered when a relatively small number of environmental microorganisms (typically found in high-quality, compliant drinking water) enter the plumbing system, attach to the interior surfaces of pipes and equipment, and excrete EPS and amplify very large numbers . Parts of the biofilm can detach or enter the environment as respirable droplets in infectious bioaerosols from plumbing, for example through shower heads, faucets and decorative fountains. Infection from these bioaerosols occurs mainly through inhalation and aspiration, sometimes through wound infection.

Clinically important biofilm-associated microorganisms that colonize the internal surfaces of sanitary systems in buildings include gram-negative environmental bacteria such asLegionella, Acinetobacter, Elizabethkingia(Flavobakterium),Stenotrophomonas, Klebsiella, Pseudomonasand NTM.

Legionella, the best-studied pathogen in sanitation, survives over a wide range of temperatures. It is acid tolerant up to pH 2.0 (Anand et al., 1983) and can survive temperatures up to 70 °C (158 °F) (Sheehan et al., 2005). Depending on the availability of essential nutrients (e.g. iron, L-cysteine),Legionellacan grow in water at 20-50°C.Legionellamultiply vigorously in water at 32–42 °C (89.6–107.6 °F) with small amounts of available nutrients, e.g. B. in unsterilized tap water (Yee and Wadowsky, 1982), especially in slow-flowing or stagnant water.Legionellais comparatively less susceptible to chlorination thanE coliand they can reportedly survive chlorine doses of up to 50 mg/L when contained in protozoan hosts.

Bacteria and other microorganisms living in the biofilm are often physiologically different from their free-floating (plankton) counterparts and have been shown to be far more resistant to conventional disinfectants such as chlorine. For example, biofilm bacteria growing on the surface of granular activated carbon particles, metal coupons, or glass slides were 150 to more than 3,000 times more resistant to hypochlorous acid (free chlorine, pH 7.0) than unattached cells. In contrast, the resistance of biofilm bacteria to monochloramine disinfection was 2- to 100-fold higher than that of unattached cells. (LeChevallier et al. 1988)

Protozoa play a crucial role in the microbial ecology of biofilms associated with sanitation systems. Protozoa graze on biofilm organisms. A number of biofilm-associated pathogens (e.g.Legionella, NTM,Pseudomonas) are able to parasitize and reproduce within FLA species commonly found in drinking water. Once these bacterial pathogens are consumed and phagocytosed by the protozoan host, they survive, multiply, and eventually spread to infect new hosts. In the host, the bacteria are protected from environmental influences such as disinfectants and high temperatures. Reportedly, this process can not only promote bacterial survival, but also result in the upregulation of the bacteria's virulence genes, thereby directly affecting their ability to infect humans and cause disease.L. pneumophilaIt has been shown to be able to parasitize and reproduce on more than twenty different species of protozoaAkanthamöben, Naegleria, AndMy Hartman(Donlan et al., 2005; Kuiper et al., 2004). Protozoa have been shown to be extremely resistant to chlorine and other common drinking water disinfectants.

While chlorine is still the dominant disinfectant used in public water supplies, alternative disinfectants such as chlorine dioxide, monochloramines, and ozone are also used to treat water for human consumption. (White, G.C. 1999) Chlorine, chlorine dioxide and ozone are used in the wastewater treatment plant, sometimes sequentially, as "primary" disinfectants to achieve water quality goals in the finished water - ie. H. at the point where the water leaves the plant. Chlorine and monochloramines are added to the water as "secondary" disinfectants to maintain the quality of the water that is distributed all the way to the customer. In general, the antimicrobial effectiveness of each of these disinfectants increases with temperature, approx. doubles for every 10°C increase in water temperature. This finding is consistent with the Arrhenius equation, a well-known formula for the temperature dependence of reaction rates.

Chlorine is the chemical most commonly used to disinfect public water supplies. The pH value of the water treated with chlorine has a great influence on its disinfection efficiency. Chlorine dissolved in water exists in three equilibrium species: Chlorine gas (Cl₂), Hypochlorination (00⁻) and hypochlorous acid (HOCI). The ratio between the three components depends on the pH of the water. If the pH is below 2, chlorine gas is important. When the pH is between 2 and 7, the balance strongly favors hypochlorous acid, a potent antimicrobial agent. When the pH rises above 7, hypochlorous acid dissociates to form hypochlorite ions, which have poor antimicrobial properties. At pH>8, hypochlorination dominates. Therefore, when chlorine is used to disinfect water, the pH must be controlled to a lower pH to ensure that hypochlorous acid, the antimicrobial species, predominates. The amount of chlorine that remains after the water's initial oxidant needs are met is called the "free residual concentration". EPA regulations allow a concentration of residual free chlorine in drinking water of up to 4 mg/L. Chlorine, in permitted doses, has been shown to be effective in inactivating a large number of traditional (faecal-borne) pathogens in drinking water.Cryptosporidium minor, an encapsulated protozoal intestinal parasite, is the notable exception.

Chlorine dioxide is a relatively strong, fast-acting disinfectant that inactivates pathogens over a wide pH range from about pH 5 to 9. Chlorine dioxide is sometimes used as an alternative to chlorine for primary disinfection; However, the ability of chlorine dioxide to remain in the distribution system is unclear. Chlorine dioxide is not typically used for secondary disinfection in the United States; However, it has been used as a secondary disinfectant in several European countries, including Italy, Germany, France and Switzerland.

The amount of chlorine dioxide that remains after the water's initial oxidant needs are met is referred to as the "free residual concentration". EPA regulations allow a residual concentration of free chlorine dioxide in drinking water of up to 0.8 mg/L. Chlorion, the EPA-regulated disinfection byproduct of chlorine dioxide, has a maximum permitted concentration in drinking water of 1.0 mg/L, effectively limiting the dose of chlorine dioxide that can be used to treat drinking water. The antimicrobial effectiveness of chlorine dioxide at pH 5-9 against a broad spectrum of common faecal-borne pathogens in drinking water is comparable to or better than chlorine at pH 5-7. Chlorine dioxide is more effective than chlorineKryptosporidium. Chlorine dioxide is readily soluble in water, but unlike chlorine, it does not react with water (hydrolyzes); Rather, it exists as a dissolved gas. At STP, chlorine dioxide is about 10 times more soluble in water than chlorine; The solubility of chlorine dioxide increases with decreasing water temperature.

Monochloramine is an oxidizer that is sometimes used as a secondary disinfectant to maintain a relatively low but persistent disinfectant residual throughout the distribution system. Monochloramine reacts with organic matter much more slowly than chlorine; As such, it is often part of a strategy to minimize the formation of regulated disinfection byproducts associated with chlorine. The antimicrobial effect of monochloramines against a broad spectrum of traditional faecal-borne pathogens in drinking water is far less than that of chlorine or chlorine dioxide. (Van der Wende and Characklis, 1990)

The relative effectiveness of chlorine, chlorine dioxide, and monochloramines against biofilms and biofilm-associated organisms differs from that against common pathogens. Information on the effectiveness of chlorine dioxide against biofilms is conflicting, but generally appears to be superior to chlorine. Chlorine has a limited ability to penetrate biofilms or inactivate bacteria living in biofilms, while monochloramine is reported to have the ability to penetrate biofilms and inactivate organisms in biofilms.M. avium, an NTM species, is more resistant to chlorine than indicator bacteria and survives in distribution systems despite ambient residual chlorine concentrations; Most strains appear to be more resistant to monochloramine than to free chlorine. All NTM species are believed to be at least 100 times more resistant to chlorine and other disinfectants compared toE coli(Taylor et al., 2000).

Microbial control treatments applied to sanitary systems fall into two general categories: (1) acute and (2) continuous. Acute treatments are usually short-term interventions to eliminate biocontamination. Continuous treatments are usually part of routine operations and are used to control biocontamination. For plumbing in buildings, traditional acute treatment options are thermal and chemical methods. Because of the associated health and safety risks and damage to the physical plant (eg, severe corrosion), emergency treatment has generally been limited to emergency decontamination of building plumbing systems in connection with disease outbreaks. (White, G.C. 1999)

High temperature water (e.g. 170°F/77°C) is sometimes used for emergency treatment of domestic hot water systems in a process called "thermal shock" or "superheat and flush". Thermal shock poses significant scalding hazards, is difficult to implement and can cause serious damage to plumbing systems. The high temperatures required to kill sanitation-related pathogens such asLegionellaare difficult to achieve and maintain consistently for a sufficient period of time in all parts of a building's plumbing system. Even when target temperatures are reached, biofilm established by the thermal shock is not removed.

Chemical disinfectants are sometimes used in higher than usual doses to acutely treat pathogen-infested drinking water systems in a process known as "chemical shock". The most commonly used form of chemical shock is "hyperchlorination" with chlorine. The relatively high levels of chlorine used are reported to cause corrosion, lead to leaks, and otherwise degrade plumbing materials. Drinking water systems are likely to be recolonized within a few weeks of hyperchlorination. (Williams et al., 2011) Even when pH and chlorine concentration targets are met, hyperchlorination has been reported to be ineffective in removing established biofilms. Most hyperchlorination protocols use chlorine in doses sufficient to develop a free chlorine residual of at least 5 mg/L (up to 50 mg/L or more) lasting up to 24 hours. Since the effectiveness of chlorine depends on the pH, the pH of the water should be below 8 and preferably below 7.2. The use of such high concentrations of chlorine is likely to result in corrosion of pipes and damage to plumbing system components, especially at the preferred pH levels where hypochlorous acid predominates. When flushed through faucets, chlorine at the concentrations used in hyperchlorination can significantly outgas and release chlorine vapors well above OSHA limits.

In a study of the acute treatment of a hospital sanitary system, a shock dose of 50-80 mg/ml chlorine dioxide was administered over a period of 8 hours under acidic (low pH) conditions. The protocol involved flushing all outlets with 50-80 mg/ml for about an hour. According to reports, the biofilm in the cold and hot water faucets was significantly reduced, but not eliminated; Treatment of the shower heads was reportedly unsuccessful, and >3000 CFU/mL recovered. (Walker et al., 1997) When flushed through faucets, chlorine dioxide can significantly outgas at the concentrations used in the study, releasing chlorine dioxide vapors well above OSHA limits.

Chlorine, chlorine dioxide and monochloramines are used for continuous treatment of potable water inside buildings, especially hot water. Studies of continuous use of chlorine dioxide in a hospital drinking water system showed that a longer time (>12 months) was required to achieve a significant reductionLegionellaPositivity in the hot water system. (Srinivasan et al., 2003)

The current net replacement value of plumbing in buildings is over $0.6 trillion (NRC, 2006). In addition, the costs associated with corrosion-related plumbing failures in buildings are unpredictable and include property damage and mold costs. Corrosion of copper pipe, an important plumbing material, depends on a number of complex variables and is not yet fully understood. However, chlorine is known to attack copper pipes. At low pH, where hypochlorous acid predominates, chlorine corrosion can be severe.

Mixtures of chlorine and chlorine dioxide have been described in the literature, but only in the context of enabling the use of inexpensive processes for the production of chlorine dioxide or minimizing the formation of toxic disinfection byproducts in groundwater. For example, Rosenblatt et al. describes the production of a mixture of chlorine and chlorine dioxide using a relatively inexpensive process based on sodium chlorate with subsequent conversion of the chlorine component to chloramines by the addition of ammonia or alternatively separating chlorine and chlorine dioxide to remove chlorine and thus avoid undesirable downstream effects (f .eg bad smell) associated with chlorine-contaminated chlorine dioxide in distribution systems. (Rosenblatt et al., 1994) Rittman et al. allows for the use of mixtures of chlorine and chlorine dioxide to minimize the formation of regulated disinfection by-products associated with the chlorination of drinking water at a water treatment plant. (Rittmann et al., 2002) In no case is the use of a mixture of chlorine and chlorine dioxide to treat biofilms taught, just as the use of the mixture is also not taught in building plumbing. cat one. al applied an equal dose of chlorine dioxide and chlorine at pH < 7.2, conditions where hypochlorous acid predominates, to disinfect wastewater from a municipal sewage treatment plant. (Katz et al., 1994) The results of Katz et al. showed that the combination provided relatively stable residues of both disinfectants and reduced the concentration of an unwanted disinfection byproduct. cat one. However, al al does not teach the use of the mixture for biofilm treatment or the use of the mixture for building plumbing. In a study on the inactivation ofLegionellaIn a model piping system, a combination of chlorine and chlorine dioxide showed no significant synergistic effect. (Zhang, 2007) Norgaard describes the use of chlorine dioxide to treat biofilm, but never suggests combining it with chlorine. In fact, Norgaard states that biofilm is unaffected by chlorination and points to the disadvantages of using chlorine to treat building plumbing due to its corrosive properties at low pH. (Norgard, 2012).

To avoid excessive system noise and the possibility of erosion-corrosion, generally accepted limits for process water flow rates are 8 feet per second for cold water and 5 feet per second for cold water. second for hot water, up to a temperature of approx. 140°F in systems where the water temperature regularly exceeds 140°F should not exceed lower flow rates such as 2 to 3 feet per hour. second.

RESUMÉ

Sanitation-related diseases are a recognized, significant and growing public health problem. There is a lack of effective, practical means and methods for treating plumbing systems in buildings and preventing diseases associated with plumbing. Requirements for a viable method and process include (1) removal of biofilms and (2) inactivation of biofilm-associated pathogens while (3) minimizing corrosion of the physical facility and (4) reducing release of toxic chemical vapors to the environment. It is a surprising finding of the methods of the present disclosure that the use of a mixture of chlorine and chlorine dioxide can remove biofilm and/or inactivate biofilm-associated pathogens. It is a further surprising finding that such mixtures can be effective under conditions that are far less corrosive to the material of the piping system than traditional methods of treating pipes in buildings that do not use mixtures of chlorine and chlorine dioxide and that use Prescribe the use of ​​lower chlorine dioxide pH for chlorine-containing solutions. Since the effectiveness of chlorine decreases with increasing pH, it is particularly surprising that the mixtures according to the present method show better biofilm removal than chlorine or chlorine dioxide alone, even at pH > 7.2. As used herein, "biofilm elimination" or "elimination of a biofilm" refers to the partial or complete destruction of a biofilm, resulting in the partial or complete inactivation of biofilm-associated pathogens and/or the partial or complete removal and/or removal of the biofilm from the surface to which it is held.

In one embodiment, the present disclosure provides a method for removing a biofilm from a plumbing system, which method comprises contacting the biofilm on interior surfaces of the plumbing system with a treatment solution comprising a mixture of chlorine and chlorine dioxide. In some embodiments, the method may be performed where the plumbing system is a building's plumbing system. In some embodiments, the method may be used in a plumbing system that includes a building water system connected to and supplied by a building piping system. In some embodiments, the building water system supplied from an on-site plumbing system includes a cooling tower.

In other embodiments of the method for removing biofilm from a piping system, the treatment solution comprises a mixture of chlorine and chlorine dioxide in a 50:50 weight ratio. In some embodiments, the treatment solution has a pH of from about 6.5 to approx. 9.0 and possibly a pH of approx. 7.2, approx. 7.5, approx. 8.0 or even approx. 8.5. In some embodiments, the treatment solution has a pH of about 6.5 or higher, approx. 7.2 or higher, approx. 7.5 or higher, approx. 8.0 or higher, or even approx. 8.5 or higher.

In one aspect of the present invention, methods are described for treating plumbing systems in buildings, the methods comprising contacting biofilm on the interior surfaces of the plumbing system with a treatment solution, the treatment solution comprising a mixture of chlorine and chlorine dioxide with a pH value ≥ 6.5, a pH ≥ 7.2, a pH ≥ 7.5, a pH ≥ 8.0, a pH ≥ 8.5 or a pH ≥ 9.0.

In some embodiments of the biofilm removal method, the concentrations of chlorine and chlorine dioxide in the treatment solution are each at least 1.5 mg/L (ie, 3 mg/L total disinfectant). In some embodiments, the concentrations of chlorine and chlorine dioxide in the treatment solution are at least 1.5 mg/L, at least 3.0 mg/L, at least 6.0 mg/L, at least 12.0 mg/L, or at least 50.0 mg/L, respectively L L. In some embodiments, in addition to the concentration of chlorine and chlorine dioxide described above, the treatment solution may also optionally have a pH of about 6.5, approx. 7.2, approx. 7.5, approx. 8.0, approx. 8.5 or approx. 9.0.

As used herein, "treatment solution" is the solution containing the desired mixture of disinfecting reagents (e.g., a 50:50 mixture of chlorine and chlorine dioxide) that is present in the piping system and thereby in contact with the biofilm on the internal surfaces that get in touch. In some embodiments of the biofilm removal method, the treatment solution is contacted with the interior surfaces of the plumbing system by introducing a mixture of chlorine and chlorine dioxide in water into the plumbing system and circulating the building's plumbing system. In some embodiments, the treatment solution can be contacted with the internal surfaces of the piping system by separately applying chlorine and chlorine dioxide to the piping system such that the treatment solution mixture is formed in situ. As described elsewhere herein, residual water may be present in the plumbing system and thereby become part of the treatment solution and serve as a diluent for the component disinfectant reagent solutions added to the system.

In some embodiments, the treatment solution should be prepared so that the pH of the plumbing system is approximately pH 7.2 or higher. In some embodiments of the treatment method, the treatment solution has a pH between 7.2 and 9.0. In some embodiments, the treatment solution has a pH >8.0. In some embodiments, the treatment solution has a pH between 8.0 and 9.0.

In some embodiments, the biofilm removal treatment methods of the present disclosure may be used in plumbing systems where the interior surfaces of the plumbing system are colonized with a biofilm comprising a microorganism selected from the group consisting of:Acinetobacter, Elizabethkingia (Flavobakterium),Escherichia coli, Klebsiella, Legionella, non-tuberculous mycobacteria (NTM),Pseudomonas, Stenotrophomonasprotozoa and combinations thereof.

Due to the low corrosivity of the treatment solutions when used at high pH (e.g. pH 7.2 and higher), in some embodiments the treatment process can be carried out where the inner surfaces of the piping system comprise a material selected from the following being : list consisting of copper, brass, iron, galvanized steel, stainless steel, PVC, HDPE and combinations thereof.

In general, the presence of residual water in the plumbing system or additional water added during the process when using the methods of the present disclosure can affect the disinfectant concentrations of the treatment solution and the pH. Additionally, as discussed elsewhere herein, residual water in the system can affect the remaining free concentration of chlorine and chlorine dioxide disinfectants. Accordingly, the presence and chemistry of water in the system should be considered when applying the treatment methods described herein.

In some embodiments, the concentration of each component in the treatment solution may be at or below acceptable levels in drinking water or may exceed acceptable levels in drinking water. Thus, in some embodiments of the treatment method, the weight ratio of chlorine to chlorine dioxide in the treatment solution may be from 80:20 to 20:80, and in some preferred embodiments, it may be a weight ratio of 50:50. In general, the higher the concentration of the treatment solution, the shorter the contact time required to treat the building's plumbing.

In some embodiments of the building pipe treatment methods described herein, the chlorine and chlorine dioxide concentrations in the treatment solution are 0.8 mg/l or less residual chlorine dioxide and 0.4 mg/l or less residual chlorine. In other embodiments, the concentrations of chlorine and chlorine dioxide in the treatment solution are greater than 0.8 mg/L residual chlorine dioxide and greater than 0.4 mg/L residual chlorine.

In another aspect of the present invention, the treatment solution is effective over a wide range of temperatures, including the entire temperature range characteristic of domestic water (0-60°C; 32-140°F). Accordingly, the building installation treatment methods described herein can be practiced over a wide temperature range and even at cold water temperatures. In some embodiments, the temperature of the treatment solution is between 55 and 80°C. In other embodiments, the temperature of the treatment solution is between 20 and 55 °C, and in some embodiments, the temperature of the treatment solution is between 20 and 55 °C 0 to 20 °C. In fact, application at cold water temperatures provides the benefits of increased chlorine dioxide solubility. A colder treatment solution can also circulate faster than a warmer treatment solution and provides more clearance to achieve the desired turbulent flow, which can improve biofilm removal due to increased shear forces and better mixing at the treatment solution-biofilm interface. Applying the treatment solution at warmer temperatures provides additional effectiveness due to increased reaction rates, but increases the release of chemical vapors (outgassing) and the rate at which the treatment solution reacts with organics in the water and with plumbing materials. Another consideration is that aqueous solutions at temperatures >43.3°C (110°F) can cause scalding.

In another aspect of the present invention, the use of the treatment solution can advantageously be achieved by circulating through the building's plumbing at a flow rate with a Reynolds value of at least 4,000.

In contact with plumbing system surfaces, the treatment solution may have temperatures between 0 and 80°C (32-176°F) and pH values ​​between pH 6.5 and 9, preferably pH 7.2 and 8. The treatment solution may also contain a chelating agent , such as sodium silicate, to further mitigate corrosion, especially when the treatment solution is applied at higher temperatures and higher concentration. In another aspect, the present invention is directed to methods of containing open faucets during acute chemical treatment of building plumbing systems when the treatment solution is flushed through faucets. The method includes attaching a channel to an outlet (e.g. a faucet), the channel walls being partially or completely impermeable to chlorine and chlorine dioxide gas, thereby providing a partial or total barrier to the transmission of these gas vapors. The line can be a pipe, a hose, a hose, a channel or the like. The opposite end of the line is directed to a physical or chemical wash that prevents the release of chemical vapors into the environment; Example embodiments include, without limitation, the opposite end of the conduit (a) terminates directly above a drain with a small air gap; (b) built into a drain; or (c) attached to the conical end of a funnel where the contact between the channel and the funnel is sealed and the funnel forms a barrier against chlorine dioxide gas and (i) attached to a drain, (ii) attached to a drain, (iii) sealed to a drain, or (iv) in the immediate vicinity of a drain. As another example, the opposite end of the line can also be passed through a plug placed in the drain, with the plug closing off the vapor path from the drain. In one variant, the plug may be a sponge soaked in a cleaning solution, for example an aqueous solution containing sodium thiosulphate (a reducing agent), which inactivates the chlorine and chlorine dioxide vapours. The sponge is placed in a drain and may contain a chemical cleaning agent. The water source is (a) a faucet, (b) a shower head, or (c) a faucet. The line may contain (a) check valves, (b) remote valves, (c) temperature sensors, (d) pH sensors, (e) chemical sensors, (f) data acquisition devices, (g) data storage devices and/or (h) data transmission devices . The treatment solution is passed through the line while maintaining isolation between potable water and wastewater systems as plumbing codes may require.

DETAILED DESCRIPTION

For purposes of the descriptions herein and the appended claims, the singular forms "a" and "an" include plural references unless the context clearly dictates otherwise. The use of "comprises", "includes", "comprises", "includes", "includes" and "includes" are interchangeable and not intended to be limiting. It is further to be understood that when the term "comprising" is used in descriptions of various embodiments, those skilled in the art will understand that in some specific cases an embodiment may alternatively be described using the language "consisting essentially of" or "consisting of". ” Technical and scientific terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise expressly defined.

When a value range is specified, unless the context clearly dictates otherwise, each intermediate integer of the value and each tenth of each intermediate integer of the value, unless the context clearly dictates otherwise, is assumed to be between the upper and lower limits is within that range, and any other specified or intermediate value within this specified range is covered by the invention. The upper and lower limits of these smaller areas may independently be included within the smaller areas and are also covered by the invention subject to any expressly excluded limits in the specified area. When the specified area includes one or both boundaries, areas excluding (i) one or (ii) both of these included boundaries are also encompassed by the invention. For example, 1 to 50 includes 2 to 25, 5 to 20, 25 to 50, 1 to 10, and so on.

It is to be understood that both the foregoing general summary and the following detailed description, including drawings and examples, are exemplary and explanatory only and do not limit the inventions herein.

Mixtures of chlorine and chlorine dioxide in a treatment solution are surprisingly effective in eliminating biofilms on interior plumbing surfaces by killing (or inactivating) biofilm-associated pathogens and loosening and/or removing the biofilms, even at high pH (e.g., pH > 7.2), which results in minimal corrosion of the pipe system. This finding applies to chlorine and chlorine dioxide treatment solutions where the concentrations of these disinfectants are at the relatively low levels permitted in drinking water (eg, 0.8 mg/L chlorine dioxide; 4 mg/L chlorine). The method and treatment solutions are also effective at removing biofilm at much higher concentrations (eg, 25 mg/L chlorine dioxide; 25 mg/L chlorine). Additionally, it is surprising that this finding occurs at pH values ​​much higher than 7.2 (e.g. pH 8.5) and even in cold tap water (e.g. 0-20 °C/32-68 °F ) applies.

Co-application of a chelating agent such as sodium silicate with the treatment solution can provide improved protection of the metal components of building materials against corrosion, especially when the treatment solution is applied at higher concentrations and temperatures.

Penetration of biofilm on the surfaces of pipes is favored by an increased flow and especially by a turbulent flow at the interface between biofilm and treatment solution. Without wishing to be bound by theory, we believe this is likely due to increased contact between the treatment solution and the surface to be treated, eliminating a boundary layer associated with laminar flow and/or stagnant contact. The degree of turbulence that can be achieved depends on the flow rate and pipe diameter; For example, a 2 inch diameter pipe requires a flow velocity of about 2 feet per second to achieve turbulent flow (Reynolds number ~4,000).

The removal of biofilm on the treated surfaces is also favored by an increased flow. Without being bound by any theory, we hypothesize that this is due to the increased shear forces having an abrasive effect on the biofilm.

Based on these findings, certain embodiments of the invention provide new means and methods for treating plumbing systems in buildings.

When treating indoor plumbing systems, it is desirable to remove surface-adherent biofilms and kill biofilm-associated pathogens such as bacteria, viruses, and protozoa without causing significant physical damage to pipes and other components of indoor plumbing systems and without releasing these to environmentally harmful chemical fumes. Removal of surface-adherent biofilms and killing of biofilm-associated pathogens without damaging copper pipes and other system components can be achieved by flushing the building's plumbing system with a mixture of chlorine and chlorine dioxide in aqueous solution (treatment solution). The treatment solution may comprise a mixture of chlorine and chlorine dioxide in a ratio of 80:20 to 20:80 (weight basis) at a total concentration of up to 200 mg/L for up to 24 hours. The treatment described herein can advantageously be carried out at pH values ​​of the treatment solution above neutral, especially pH >7.2, at typical cold water temperatures (e.g. 0-20°C; 32-68°F) up to temperatures at which scalding becomes a risk (43.3°C/110°F). Simultaneous treatment with a complexing agent such as sodium silicate can further improve the compatibility of the treatment solution with plumbing system materials, especially metals such as copper and brass.

The release of harmful chemical vapors from the faucet into the environment can be avoided by using a gas containment device, such as a hose containing chlorine and chlorine dioxide vapors. The hose attaches to a faucet and ends near or attached to a drain or chemical. It provides containment of chemical vapors and directs the flow of treatment chemicals from the faucet to a drain or chemical scrubber. The gas containment device can be configured in many ways and includes beneficial features such as check valves, remote valves, and sensors for temperature, pH, and disinfectant concentration, as well as means for data acquisition, storage, and transmission. The gas containment device can be used in conjunction with flushing the treatment solution or with other volatile treatment chemicals such as hypochlorite (bleach) or chlorine dioxide which can be used to flush plumbing systems.

In certain plumbing systems with surfaces covered by a mixture of lime, iron sediment and biofilm, such as those receiving water with a high mineral content (hard water), the treatment can be carried out by first applying a low pH treatment to dissolve the lime deposits and iron, preferably in conjunction with a complexing agent such as sodium silicate. The first step is followed by using the treatment solution described here at a higher pH (eg >7.2) to remove the biofilm. This process can be repeated if necessary until lime, iron sediment and biofilm are removed by sampling or visual inspection.

EXAMPLES

Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative and not limiting. Those skilled in the art will readily appreciate that the specific examples are only intended to be illustrative of the invention as set forth in more detail in the following claims. Each embodiment and feature described in the application shall be understood to be interchangeable and combinable with each embodiment contained therein.

Example 1: Extinction ofPseudomonasBiofilm using solutions of chlorine, chlorine dioxide and a mixture of both

This example illustrates the use of a mixture of chlorine and chlorine dioxide to remove biofilmPseudomonas aeruginosa. These results show the effectiveness of this treatment solution even at pH 6.5 and the surprising advantage of such mixtures at pH 7.5 compared to treatment solutions containing either chlorine or chlorine dioxide alone in treating biofilms.

Overview of the test method

Pseudomonas aeruginosaBiofilm is tested with the reagents chlorine dioxide ("CD"), chlorine (sodium hypochlorite; "bleach") or a mixture of both reagents in equivalent volume fractions ("mixture") at approx. pH 6.5 and pH 7.5 to determine their ability to assess for biofilm inactivation as measured by a Minimal Biofilm Eradication Concentration (MBEC) assay. This MBEC assay measures the ability of a treatment solution to remove bacteria from established biofilms by killing the microbial cells and/or removing the biofilm.

The MBEC analysis protocol used is an adaptation of the ASTM standardized MBEC method. Briefly, biofilms are grown in a Calgary biofilm apparatus, which is a 96-well plate with a lid that, when in place, has 96 individual spikes protruding into the wells of the plate (see, e.g., (B. Ceri et al. , 1999). The bacterial cells in the wells form biofilms on the submerged pins. After a 20-hour growth period, these biofilms formed on the pins are challenged by placing the attached lid on a 96-well plate with serial dilutions of the disinfectants to be tested ( e.g. CD, Bleach or Mix) together with the voluntary controls. All challenges were performed in a biosafety level 2 cabinet with a challenge time of 30 minutes at a temperature of 22-25 °C. In the case of CD, the products are freshly prepared from a highly concentrated stock solution a few minutes before exposure. The pH and concentration stability of the reagents are tested for the CD working stock solutions with either water or a borate-boric acid buffer as solvent. Concentration ranges for disinfecting reagents are from approx. 3 ppm to approx. 50 ppm.

After the challenge, the clamped lid is transferred to a receiving plate with a sodium bisulfite solution. Sonication of the recovery plate with the lid attached loosens the biofilm from the pegs into the wells of the recovery. The number of viable bacterial cells from each stick is determined by culturing the recovery plate. The extended MBEC assay protocol and the methods for preparing the disinfection reagent stock solutions used in the challenge are included in the Materials and Methods section below.

Results

The results for the CD, Bleach, and Mix challenges can be seen in Tables 1, 2, and 3, respectively. Bleach exposure shows a pH-dependent effect on biofilm reduction consistent with the literature, except for the anomalous results at -3 ppm. Using Mix (bleach and CD) (Table 3), the efficacy against biofilm at both pH 6.5 and pH 7.5 was superior to the efficacy of either of these disinfecting reagents alone. This is most clearly demonstrated by comparing the mixture dose of 6.25 ppm of each component (12.5 ppm total) with 12.5 ppm of each individual component.

Detailed materials and methods

Preparation of a concentrated CD solution: Pour, if available, approx. 50 ml of a CD solution in two 1 liter airtight glass containers. Otherwise, use commercial bleach to treat this glass. Moisten the entire inner surface of the flasks with the liquid and leave them in the dark at room temperature overnight. Add 2L of sterile distilled water to a bag containing the CD producing reagents. Close the bag and mix by turning it several times. Leave at room temperature for 2 hours. Rinse the jars from step 2 with sterile distilled water and cover with aluminum foil. Transfer the concentrated CD solution to the glass bottles so that the air space above the solution is as small as possible. Store the solution at 4°C until use. Prepare 30 ml of a 1:500 to 1:1000 dilution. Transfer 10 mL aliquots to 20 mL bottles with quantification system (CD colorimeter). Cover the bottles with aluminum foil. Add 3 drops of glycine to each bottle, cap and mix. Add the contents of one sachet of DPD to each bottle of CD solution. Mix by gently inverting the tubes. Use a bottle of water to empty the colorimeter (make sure you are using the CD colorimeter and not the chlorine (bleach) colorimeter), then measure the concentration. If the reading is >2.5, prepare a higher secondary dilution (1:1500) and repeat the process. Average the readings and multiply by the dilution factor to determine the strength (in ppm) of the concentrated solution. Label the bottles with the date and concentration.

Preparation of working CD stock solution: Fill a 50mL polypropylene tube with the concentrated solution and cover with aluminum foil. Allow the solution to reach room temperature protected from light. In a fresh 50 ml polypropylene tube, mix the concentrated solution with sterile distilled water to dilute to the working concentration (eg 100 ppm) in a final volume of 50 ml. Close the tube tightly and mix gently by inverting. Add 0.1 M NaOH in 10 μL increments, mix and measure pH. Repeat this step until the desired pH is reached. Also estimate pH using pH indicator paper strips. Prepare 1:200 dilutions (30 ml) and check the working concentration with the colorimeter (as described in the preparation of concentrated CD solutions). Use concentration calculation strips for double checking. Dip the amount of the strip into the solution for 2 seconds. Hold the colorimetric coupon, wait 10 seconds, then compare its color with the reference palette. Label the tube and store at 4 °C.

Preparation of 1x borate-boric acid buffer working CD stock solutions: In a fresh 50 ml polypropylene tube, add concentrated CD solution so that the final concentration after dilution to a volume of 50 mL is the working concentration (e.g. 100 ppm). Add 1x borate buffer to make 40-45 ml. Close the tube tightly and mix gently by inverting. Measure the pH and add 0.5 M boric acid in 1 ml increments (or less if necessary) until the desired pH is reached. Fill up to 50 ml with sterile, distilled water. Determine the final concentration as described above for the CD stock solution.

Stability test of working CD stock solutions: The pH and concentration stability test method consisted of diluting CD from a concentrated stock solution (about 590 ppm) with water to the concentration of the working stock solution (100 ppm) and adding 0.1 M NaOH in Add small amounts , until the desired pH is reached. Alternatively, the reagent was diluted to its working concentration in a combination of 1X borate buffer (measured pH 8.8) and 0.5M boric acid (measured pH 4.14). The desired pH values ​​were obtained by adjusting the proportion of boric acid in the solution.

Estimation of the initial concentration of the initial bleach solution: Bleach may be present at a concentration of 1 to 8% (ie, 10,000 to 80,000 ppm). The following initial concentration calculation method is used to determine this initial concentration. In a new polypropylene tube, prepare 15 mL of a 1:500 dilution in ultrapure water (UPW). Add 10 mL of UPW to the 20 mL bottles of the quantification system (Cl₂colorimeter). Set one of the bottles aside to use as a subject. Replace either 100 or 50 μL of UPW with the 1:500 dilution from step (1) (ie, make additional 1:100 or 1:200 dilutions, giving final dilutions of 1:50,000 or 1:100,000, respectively). Add the contents of one sachet of DPD reagent to each bottle of CD solution. Mix by gently inverting the tubes. Empty the colorimeter (make sure you are using chlorine and not the CD colorimeter) and then measure the concentration. If the reading is >2.5, prepare a higher secondary dilution (eg 1:1500) and repeat the process. Average the readings and multiply by the dilution factor to determine the strength (in ppm) of the concentrated solution. Label the original bottle with the date and estimated concentration.

Preparation of bleach working stocks: Fill a 50 ml polypropylene tube with the concentrated bleach solution. Allow the solution to reach room temperature. In a fresh 50 ml polypropylene tube, mix the concentrated solution with sterile distilled water to dilute to the working concentration (eg 100 ppm) in a final volume of 50 ml. Close the tube tightly and mix gently by inverting. Add 1 N HCl in 10 μL increments, mix as in (9.2), and measure pH. Repeat this step until the desired pH is reached. Also estimate pH using pH indicator paper strips. Prepare 1:200 or 1:100 dilutions and check the working concentration as before (steps 2-6). Label the tube and store at 4 °C until use.

MBEC-Assay-Protocol

The following equipment, reagents, and methods are used to perform the MBEC test protocol to test the disinfection effectiveness of chlorine dioxide (CD), chlorine (Bleach), and a mixture of chlorine dioxide and bleach (Mix).Pseudomonas aeruginosaBiofilm.

A. Test Method: All steps must be performed using aseptic techniques and in an aseptic environment.

1. Bacterial culture (2 days before MBEC analysis).

• • 1.1. Thaw an aliquot of a working listPseudomonas aeruginosa(ATCC 27835) and used to streak a plate on Tryptic Soy Agar ("TSA") prepared according to the manufacturer's instructions.
  • 1.2. Incubate for 16-18 hours at 35°C.
  • 1.3. Select an isolated colony and inoculate 200 ml of sterile tryptic soy broth ("TSB") prepared according to the manufacturer's instructions.
  • 1.4. Incubate at 35°C and 150 rpm for 16 to 18 hours. The density of viable bacteria should be 10⁸CFU/ml or higher and can be verified by serial dilution and plating.
  • 1.5. Prepare a 25 mL 1:1000 dilution in TSB to adjust the cell density to approximately 10⁵CFU/ml. Vortex the diluted sample for approx. 10 p.
  • 1.6. Perform seven 10-fold serial dilutions from step (1.5) in triplicate.
  • 1.7. Spot plate 20 μL serial dilutions from 10° to 10⁻⁷on a number of TSA signs. Label the plates and incubate at 35°C for 16-18 hours.

2. Biofilm growth

• • 2.1. Open a package containing a new MBEC device.
  • 2.2. Transfer 25 mL of the inoculum prepared in (1.5) to a sterile reagent container.
  • 2.3. Add 150 μL to each well of the 96-well plate packed with the MBEC device, except columns 9 to 11 and A12, B12, and C12.
  • 2.4. Place the pin cover on the microplate, ensuring that the orientation of the wells matches the orientation of the pin cover (ie, pin A1 should be placed in well A1).
  • 2.5. Maintain the device at 33-37°C using the orbital shaker and humidified incubator.
  • 2.6. For optimal biofilm quantification, the replicate MBEC units should be prepared according to Section 7.

3. Biofilm growth control

• • 3.1. Using sterile (flaming) forceps, grasp pin D12 close to the lid to avoid contact with the biofilm. Break off the pin and place in a sterile microcentrifuge tube containing 1.0 mL of buffer water ("buffered water" = 0.0425 g of carbohydrates).₂AFTER₄/L distilled water, filter sterilized and 0.405 g MgCl.6H₂O/L distilled water; filter sterilized according to ASTM method 9050 C.1.a).
  • 3.2. Repeat step (3.1) with wells E12 to H12 in the corresponding microcentrifuge tubes.
  • 3.3. Place the stainless steel insert tray in the ultrasound unit. Place the tubes from 3.1 and 3.2 in the dish and sonicate on high power for 25-35 minutes.
  • 3.4. Make 1.0 mL serial 10-fold dilutions in buffered water and spot plate on TSA. Incubate for 16-18 hours at 35°C.

4. Prepare the challenge board

• • 4.1. Use a double-cornered sterile 96-well plate to prepare the assay plate according to the assay plate setup map shown below.

• • 4.2. Prepare 20 ml of the desired disinfection stock solution.
  • 4.3. Add 200 μL of sterile TSB to well A12 of the challenge plate. This will be Sterility Control (SC).
  • 4.4. Add 200 µL of sterile neutralizer to column 7 and well B12. These are neutralizer toxicity control (N) and sterility control.
  • 4.5. Add 100 µL of sterile neutralizer to column 6, followed by 100 µL of disinfectant. This is the check of the effectiveness of the neutralizer.
  • 4.6. Add 200 µL of buffered water to column 8 and well C12. These are an untreated control (UC) and a buffer water sterility control.
  • 4.7. Add 100 µL of buffered water to columns 1 through 5 (rows B through H).
  • 4.8. Add 200 μl of stock disinfectant to columns 1 to 5 (row A).
  • 4.9. Add 100 µL of the disinfection stock solution to columns 1 through 5 (rows B and C).
  • 4.10. Using a multichannel micropipette, mix the contents of columns 1 to 5 (row C) by pipetting up and down. Keep the tips in the micropipette for the next step.
  • 4.11. Transfer 100 μL from the wells in row C to the corresponding wells in row D. Discard the tips.
  • 4.12. Mix with fresh tips by pipetting the contents into row D, columns 1 through 5.
  • 4.13. Transfer 100 μL from row D to row E. Discard the tips after each transfer and mix with fresh.
  • 4.14. Repeat the process along the plate up to row H.
  • 4.15. Discard 100 μL from row H, columns 1 through 5.
  • 4.16. Add 100 μL of buffered water to rows C through H of columns 1 through 5.

5. Biofilm disinfection challenge

• • 5.1. Prepare a wash plate by adding 200 μL of buffer water to each well of a new 96-well double-corner plate.
  • 5.2. Prepare the recovery plate by adding 200 μL of neutralizer to each well of a new 96-well double-corner plate.
  • 5.3. Rinse the planktonic bacteria from the biofilm formed on the lid of the MBEC unit by placing the lid in the rinse plate for 10 s.
  • 5.4. Transfer the MBEC lid to the assay plate and incubate at room temperature for the manufacturer's recommended contact time.
  • 5.5. After the contact time, the MBEC lid is transferred to the recovery plate with the neutralizer.

6. Quantification of biofilm growth (from replicate biofilm plate)

• • 6.1. Prepare a staining plate by adding 200 µL of a 0.1% crystal violet solution to columns 1 to 8 and column 12 of a fresh 96-well 2-corner plate.
  • 6.2. Transfer the attached lid of the replica recovery plate to the staining plate.
  • 6.3. Incubate 30 minutes at room temperature.
  • 6.4. Prepare two wash plates by adding 200 μL of ultrapure water to columns 1 to 8 and column 12 of two 96-well 2-corner plates.
  • 6.5. Transfer the lid to the first rinse plate and let sit for 10 seconds to remove excess stains and plankton bacteria. Pour onto the second rinse plate and repeat.
  • 6.6. Allow the clamped lid to air dry upside down for 30 minutes.
  • 6.7. Add 150 µL of 95% ethanol to a new 96-well 2-corner plate in columns 1 to 8 and column 12.
  • 6.8. When dry, place the pin cap on the ethanol plate and incubate for 10 minutes.
  • 6.9. Remove the attached lid and discard it along with the staining and rinsing plates used in this section.
  • 6.10. Transfer 100 μL of each well from the plate in (6.8) to the corresponding well of a fresh 96-well plate (ONE CORNER).
  • 6.11. Use a plate reader to determine absorbance at 600 nm.

7. Quantification of MBEC

• • 7.1. Place the recovery plate with the lockable lid (from step 6.5) into the stainless steel bowl and the bowl into the ultrasonicator. Sonicate at high power for 25 to 35 minutes to remove and dissolve biofilm.
  • 7.2. Eight single-corner sterile 96-well plates are used for this step (columns 1 to 8 only).
    • 7.2.1. Add 180 µL of buffered water to rows B through H in all 8 plates.
    • 7.2.2. After sonication and using a multichannel micropipette, transfer 100 μL from each well in row A of the recovery plate to row A of a sterile plate prepared in 7.2.1.
    • 7.2.3. Transfer 100 μL from each well in row B of the recovery plate to row A of another sterile plate prepared in 7.2.1.
    • 7.2.4. Repeat for rows C to H on the recovery plate.
    • 7.2.5. Serial dilution with a multichannel pipette (10° to 10⁻⁷) by transferring 20 μL to each of the 8 rows for each plate.
  • 7.3. Spot plate the serial dilutions from each of the eight microtiter plates onto TSA to determine the number of viable cells. Use one square TSA plate per microplate. Use a multichannel pipette to withdraw 5 µL from each well and spread on the TSA plate.
  • 7.4. Incubate the TSA plates at 33-37°C for 18-20 hours and count the colonies.
  • 7.5. Discard the attached MBEC lid and the 96 plates used to make the serial dilutions.

8. Qualitative determination of MBEC

• • 8.1. Add 100 μL of sterile TSB to each well of the recovery plate.
  • 8.2. Cover the collection plate with a new sterile, non-glued lid and place in a humidified incubator at 33–37°C for 24 h.

9. Data analysis

• • 9.1. Quantitative MBEC results with log₁₀reduction:
    • 9.1.1. Count the 5 μL spots on each of the 8 spot plates where individual colonies are visibly different from each other within the plated spot. Note the column (1-8) and the dilution row (10).⁰to 10⁷), where each location is located.
    • 9.1.2. Calculate the log₁₀Density for each pin as follows:
      Protocol₁₀(KBE/mm²)=Protocol₁₀[(X/B)(V/A)(D)]
    • Wo:
      • X=CFU counted on site,
      • B=volume plated (0.01 ml),
      • V = Well volume (0.20 ml),
      • A=pin area (46.63 mm2) and
      • D = dilution
    • 9.1.3. Average the counts from columns 1 through 5 for row A to find the logarithm of the mean₁₀Density of the undiluted disinfectant.
    • 9.1.4. Average the counts from columns 1 through 5 for row B to find the logarithm of the mean₁₀Density for 50% disinfectant. Repeat the calculation for the remaining rows (C-H).
    • 9.1.5. Average the counts from column 6, rows A through H to find the logarithm of the mean₁₀Density to check the effectiveness of the neutralizer according to the procedure described in TSA Test Method E1054.
    • 9.1.6. Average the counts from column 7, rows A through H to find the logarithm of the mean₁₀Density to control neutralizing toxicity.
    • 9.1.7. Average the counts from column 8, rows A through H, and determine the mean log₁₀Density of the untreated control.
    • 9.1.8. Calculate the log₁₀Reduction for each disinfectant concentration as follows:
      reduction = mean log₁₀Untreated control pins - mean log₁₀Treated pins
  • 9.2. Qualitative MBEC results are determined by visual assessment (+/- growth) after 24 hours incubation of the recovery plates. To determine minimum biofilm eradication (MBEC) values, inspect the recovery plate wells (visually) for turbidity. Alternatively, use a microplate reader to obtain optical density readings at 650 nm (OD).₆₅₀). Klare Brunnen (OD₆₅₀=0.1) is evidence of biofilm elimination. MBEC is defined as the minimum concentration of disinfectant that will remove the biofilm. This is the lowest concentration where no growth was observed in most of the five boreholes.

Example 2: Biofilm removal in a hot water circulation system using a treatment solution mixture of chlorine and chlorine dioxide

This example illustrates the method of applying a mixture of chlorine and chlorine dioxide to a hot water recirculation system to treat biofilm. The physical and chemical parameters in the example, such as treatment solution chemistry, treatment solution temperature, treatment solution pH, flow rates, treatment time and sequence, are for illustrative purposes and are not intended to limit the scope of the invention.

A dosing tap is installed on the outlet side of the building's central water heater. A chemical feed pump compatible with the treatment solution is connected to the dosing tap. A sampling tap is installed on the hot water return. Fixtures (faucets, shower heads) throughout the building are primed by removing aerators and final filters. Exhaust gas prevention devices are installed at each outlet – e.g. B. flexible tubing that acts as a conduit from which the treatment solution exits the device to the drain. Unheated domestic water circulates through the hot water distribution system at a rate of 2-8 feet per second (fps).

Chlorine and chlorine dioxide are added to the circulating water at the dosing tap, resulting in a treatment solution with a concentration of 50 mg/L (~25 mg/L each of chlorine and chlorine dioxide) at pH 7.5. Sodium silicate, a complexing agent, is added to the circulating water to achieve a concentration of 25 mg/L. The treatment solution circulates through the hot water system for one hour. The concentration of the treatment solution is measured every 5 minutes on the hot water return; If the concentration is >5% below the target value of 50 mg/L, additional chemicals are added at the dosing tap until the target concentration of the treatment solution, measured at the hot water return, is reached.

As it flows through the system, starting at the dosing point, the stopcocks are opened to full flow until the concentration of the treatment solution reaches the set point of 50 mg/L; The flow is then reduced to 0.25 gallons per minute (gpm) and the water is allowed to flow for another 5 minutes and then turned off. Due to the design of the EGR, the treatment solution remains in contact with all wettable surfaces of the faucet.

After all faucets have been flushed with the treatment solution and closed, the treatment solution is circulated through the system for an additional hour. The chemical feed pump is switched off and the hot water system is flushed with clean potable water for 30 minutes.

Beginning at the dosing point and continuing through the system, all faucets are opened to full flow and flushed with clean, unheated service water until chemical concentrations in the water are below the EPA Maximum Contaminant Level (MCL) and Maximum Disinfectant Residual Level (MRDL - Limits, i.e. the limits to which disinfectants or disinfectant by-products are regulated. Clean water is then allowed to run through the tap for an additional 5 minutes. The concentration of the treatment solution is measured again and documented as being below the MRDL for each regulated disinfectant/disinfectant by-product/MCL The water tap is closed and the gas tank is removed.

After acute treatment, the hot water system can be treated to ensure continuous microbial control.

All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually incorporated by reference for all purposes. Full citations for these references are at the end of the specification immediately before the requirements.

While various specific embodiments have been illustrated and described, it should be understood that various changes may be made without departing from the spirit and scope of the invention(s).

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