By Ratul Saha, PhD, Richard Martin and Robert Donofrio, PhD

Water treatment systems have employed a number of mechanical and chemical approaches for microbial pathogen control. Traditionally, chemical disinfectants such as chlorine, chloramines or UV irradiation have been used for water treatment (Shannon et al., 2008). Recently, alternative means of disinfection, such as ozone, have gained importance (Gottschalk et al., 2009). Ozone was first used in 1893 in the Netherlands for the treatment of drinking water.

Ozone is mainly used to treat water for disinfection and chemical contaminant oxidation (US EPA, 1999). There are several advantages of using ozone. Ozone is more effective than chlorine in killing bacteria and viruses within a shorter contact time (CT). It is also effective against chlorine-resistant Cryptosporidium and Giardia. The CT value for 99.9-percent inactivation of Cryptosporidium is reported to be less than one mg min/L for ozone, whereas it is greater than 30 for chlorine (Donofrio et al., 2013; Langlais et al., 1991). Furthermore, ozone disinfection efficiency is not affected by pH. Ozone does have a short half-life, however, and decomposes to less reactive and effective molecules. Additionally, since ozone is consumed rapidly, there is no residual mode of disinfection and microbial re-growth cannot be prevented. The mode of action of ozone in aqueous solution is by direct oxidation of compounds by molecular ozone or oxidation of compounds by hydroxyl free radicals produced during the decomposition of ozone. The high oxidation potential of ozone oxidizes cell membrane materials, which allows ozone to enter the organism and damage enzymes, DNA and RNA, thus causing cell destruction (Khadre et al., 2001). In viruses, the first site of action is the inactivation of the capsid (mainly its proteins), leading to the release of the nucleic acid and disruption of adsorption of the virus particle to the host pili. In general, vegetative bacteria (such as gram-negative enteric pathogens) are most susceptible to ozone, while spore-forming bacteria and viruses are more resistant (Khadre et al., 2001).

One of the disadvantages of ozone, compared to traditional technologies, is that ozone is a more complex technology than UV and chemical disinfection (i.e. chlorine) and requires capital investment and maintenance (US EPA, 1999). Some chemical treatment systems require minimal or fairly low-technology equipment to create the disinfecting or oxidative effect (i.e., addition of liquid sodium hypochlorite). Note: In applications other than municipal drinking or wastewater, ozone systems are comparable to UV systems in terms of capital cost and more affordable in terms of maintenance cost.

Ozone use in water systems
Ozone is mainly used in water treatment systems because of its ability to disinfect without adding other chemicals. It can be produced in several ways:

  1. Electrical discharge. This is the most effective and economical way of producing ozone. In this process, ozone is produced by discharging high-voltage electricity across a gap through which filtered dry air or pure oxygen is flowing.
  2. Electrolytically. This method is more expensive and only produces small quantities of ozone relative to the electrical discharge process.
  3. Radiochemically. High-energy irradiation of oxygen will produce ozone. This method is still under development and not used to produce ozone commercially (Gottschalk, 2009).

Ozone can be dissolved in water with spargers or Venturi eduction. Ozone can be used to treat the following components present in water: bacteria; minerals (such as iron and manganese) that are easily filtered post-oxidation; dissolved metals; organics; protozoans; viruses and BOD and COD (Guzel-Seydim et al., 2004; Lenes et al., 2010). One of the major advantages of using ozone as a means of disinfection is that it can be used in conjunction with other disinfectants. For example, ozone used in combination with chlorine will reduce the concentration and consumption of chlorine necessary for routine treatment of water, thus reducing the formation of harmful chlorine byproducts. Not all impurities can be treated with ozone, such as bromine ions and complex pesticides. Caution should be taken with the use of ozone, as with any strong oxidizer, and water used for the treatment should be well characterized before the application of ozone, as pesticides, organics and inorganics may become more toxic or undesirable (US EPA, 1999).

Requirements of custom methodology for US EPA reference test methods
Incorporation of ozone as a control agent in the consumer product industry has been a recent phenomenon. The search for green disinfectants has given rise to wide varieties of ozone generators for various applications, such as treatment of recreational water (Donofrio et al., 2013) and food products (Khadre et al., 2001). Ozone is being used not only to decontaminate but also to increase shelf life. Though devices employing ozone for microbial control have become more commonplace, guidelines and methods for effective evaluation of these devices against their antimicrobial claims are lacking. The absence of a validated, peer-reviewed methodology could lead to mislabeling and manufacturing of ineffective products. In the US, any product or device that claims to have antimicrobial properties (mainly against pathogens) must be registered by US EPA under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). There are guidelines and methods set by the agency, according to which products must be tested to obtain US EPA registration. The antimicrobial test methods and procedures, however, are designed to determine the efficacy of disinfectants against commonly occurring pathogens for liquid, spray and towelette formulations (

NSF/ANSI Standard 50
Currently for recreational water, there is one national standard for evaluating the efficacy of alternative disinfectants such as ozone when incorporated into a treatment device. NSF/ANSI Standard 50: Equipment for Swimming Pools, Spas, Hot Tubs and other Recreational Water Facilities contains evaluation, testing and certification criteria for ozone systems used in water treatment, specifically recreational water, pools and spa applications. The requirements include a number of design criteria, material health safety and corrosion resistance evaluation, as well as quantity of ozone output tests, durability and life testing, and of course, disinfection efficacy testing comprised of three-log kill of at least two target microorganisms, Enterococcus faecium and Pseudomonas aeruginosa (and other organisms if additional claims are made). In 2013, NSF will ballot new requirements to NSF/ANSI Standard 50 for the testing of ozone system disinfection efficacy performance related to Cryptosporidium parvum oocysts. The testing program is challenging and rigorous and utilizes the target organism, not a surrogate. The standard is a US EPA-recommended voluntary additional test in accordance with OCSPP 810.2600: Disinfectants and Sanitizers for Use in Water– Efficacy Data Recommendations.

Experimental design for assessing antimicrobial efficacy
There are certain issues that must be addressed when designing a test system to validate the disinfection efficacy of ozone systems. One of the major challenges with testing is that ozone automatically decomposes (Yousef et al., 2011). Therefore, appropriate guidelines are required for generation and handling of the ozonated solution intended for efficacy testing. Testing methods for closed-loop ozone treatment systems that incorporate critical variables can be reliably used to assess ozone system performance provided:

  1. They incorporate a disinfection efficacy-based quantification of ozone mixing and contact time performance.
  2. They incorporate a quantitative measure of ozone system output and qualitative impact on system water chemistry, such as ORP monitoring.
  3. There is accurate monitoring of system flowrates.

The methods also must be designed appropriately to fit the intended use of the disinfectants, as one method will not fulfill the requirements of all applications. For example, in order to evaluate an ozone spray treatment against current recommended US EPA methodology for germicidal spray disinfectants, the investigator must customize the test approach to account for the delivery method of the ozonated spray (and ensure appropriate operation of the ozone generating device), concentration of ozone that should be used for testing, appropriate contact time and choice of an appropriate neutralizer.

Irrespective of the active antimicrobial compound, the scope of existing efficacy protocols must also be considered when evaluating novel treatment devices. Many existing US EPA disinfection efficacy protocols involve assessing organism kill or inactivation while the organism is in suspension, or following a brief drying period following application of the microbial challenge suspension to a carrier. These protocols are effective for assessing antimicrobial efficacy of products designed for surface disinfection and in aqueous solutions. They are not designed, however, for the evaluation of the devices against microbial biofilms. A microbial biofilm consists of complex communities of microorganisms growing on surfaces within a myriad of environments, both biotic (i.e., at the teeth and gum interface within the human mouth) and abtioic (i.e., established on PVC piping within water distribution systems) (Saha and Donofrio, 2012). Biofilm formation is a naturally occurring process in the environment. Microorganisms form biofilms primarily for protection and to survive under extreme nutritional conditions. Biofilms allow for close interaction between multiple species of microorganisms and this close relationship promotes nutrient acquisition and gene transfer for increased antibiotic resistance. In nature, a majority of bacteria are found to be associated within biofilms and it is estimated that as little as 10 percent of the bacterial cells are found in the planktonic (i.e., free-swimming) form (Lucchesi et al., 2012).

Biofilm can be composed of multiple species. Microorganisms constituting biofilm communities are less susceptible to antimicrobial agents compared to their planktonic counterparts (Xu et al., 2000; Stewart and Costerton, 2001). Therefore, most of the existing standard methods for evaluation of disinfectants are not suitable for use with products claiming biofilm prevention or detachments. A number of ASTM standard methods have recently been published for growing of biofilm in a CDC biofilm reactor under laboratory conditions (ASTM E2562-12) and a single-tube disinfection assay (ASTM E2871-12). The methods, however, are designed around a single bacterium: Pseudomonas aeruginosa. Both the biofilm development method and the single- tube efficacy test must be modified in order to evaluate other bacteria. Also, as biofilms exist in multiple-species communities in nature, any efficacy testing performed on a single-species biofilm might not be adequate to make a product claim of preventing or removing biofilms. A key challenge is optimizing conditions for the formation of biofilms under laboratory conditions, as experimental parameters could influence biofilm development. In addition, current methodology is prone to large experimental variations; expensive imaging technologies are required for determining efficacy, and detachment claims are difficult to assess (biofilm formation is a dynamic process and goes through maturation and detachment processes naturally).

Rapid test methods versus traditional culture- based methods
Other limitations exist for current efficacy protocols, such as accuracy and prolonged length of time to final result. Monitoring and assessment of treatment technology efficacy have historically relied on culturing techniques. The traditional methods have also been used to study microbial diversity. Culturing techniques use different bacteriological media for recovery and enumeration of microorganisms. Studies have shown that culture-based techniques can recover less than five percent of a microbial population from any environment (Saha and Donofrio, 2012). This is primarily because microbial cells are in a different physiological state in the environment and there are injured cells. There are also cell populations that are viable but unculturable. Therefore, culture-based techniques underestimate the actual microbial load in any samples (Aman et al., 1995). Similar situations are encountered while conducting disinfection efficacy studies. The selection of appropriate media, incubation conditions and time plays a crucial role in the test methodology. If laboratories conducting such tests do not have a strict quality control regime, variation in test data is possible among laboratories performing the same test, thus compromising the overall performance of the disinfectants’ efficacy evaluation.

To overcome these problems associated with traditional culturing techniques, industry, regulatory bodies and laboratories are moving toward rapid microbiological methods, such as polymerase chain reaction (PCR). In PCR, mainly real-time PCR (qPCR)-based methodology has gained wide acceptance and importance due to its quantitative nature (Varma et al., 2009; Josefsen et al., 2010). One of the disadvantages of using qPCR is that it is not capable of differentiating between viable and non-viable cells as it measures the concentration of total recovered DNA from any samples. Researchers have overcome this problem, however, by taking advantage of DNA intercalating dyes, such as propidium monoazide and ethidium bromide (Rawsthrone et al., 2009). Due to the advent of molecular technologies, laboratories now have the opportunity to develop and implement rapid microbiological methods, which have not only shortened the turnaround time but also improved the quality of overall testing procedures.

Ozone has been demonstrated to be a viable chemical disinfectant for a number of applications, including drinking water, recreational water and surface sanitation. The evaluation of the efficacy of ozone treatment systems has relied on methodology initially designed for other target chemical compounds. Thus, in respect to ozone and other novel disinfection treatment systems, existing standards for assessing disinfectant efficacy have limitations. Furthermore, these protocols often need to be modified to accommodate these treatment technologies, to incorporate new detection or assessment approaches or to validate additional claims, such as biofilm prevention or destruction. As with any modification or creation of a new protocol, the investigator must subject the method to an appropriate validation procedure (US EPA 2009). It is only through the employment of properly vetted methodologies that accurate and meaningful interpretations can be made regarding the antimicrobial efficacy of a treatment system or device.


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About the authors
Dr. Ratul Saha, Research Scientist, Microbiology and Molecular Biology Division, Applied Research Center at NSF International, has 10 years of experi- ence conducting research and development focusing on all aspects of microbiology and biotechnology. He is also intensively involved with research in the area of microbial control and the development of biofilm test methods for evaluating the efficacy of wide varieties of antimicrobials simulating real-  life applications. Saha holds an MSc Degree in biotechnology from the Bangalore University as well as a second Master Degree and a PhD in microbiology from Michigan Technological University. He has published several peer-reviewed manuscripts and abstracts in the field of microbiology and molecular biology, serves on the Editorial Board of the Journal of Industrial Microbiology and Biotechnology and is an invited peer reviewer for the Journal of Environmental Health, Folia Microbiologica and World Journal of Microbiology and Biotechnology. Saha is a member of American Society for Microbiology, Society of Industrial Microbiology and Biotechnology and Bergey’s International Society for Microbial Systematics.

Richard A. Martin B.Sc., AFO, CPO is the Senior Business Development Manager at NSF International. He joined NSF in 1994 and has worked in and managed various segments of the NSF water business, including the plastics plumbing, mechanical plumbing, municipal drinking water systems and recreational water programs. For further information about NSF-certified UV systems, contact Martin via email, [email protected] or phone, (734) 769-5346.

Dr. Robert S. Donofrio is the Director of the Applied Research Center at NSF International. Previously, he served as Director for the NSF Microbiology and Molecular Biology Laboratories from 2004-2012 and has nearly 20 years of expertise in microbiology (environmental, water, food and clinical), lab design, technical operations management and method development and validation. During his tenure at NSF, Donofrio spearheaded initiatives to incorporate molecular techniques for pathogen detection in food and water testing, establish retail food testing, establish biofilm testing capabilities and the formation of virology and cell toxicity labs. He holds a PhD in biological sciences from Michigan Technological University and an MS Degree in environmental microbiology from Duquesne University. Donofrio has authored numerous publications, serves on several advisory boards (Healthy House Institute, NoroCore, US TAG for Nanomaterials) and is on the Board of Directors for the Society for Industrial Microbiology and Biotechnology.

About the company
NSF International is an accredited, third-party certification body that tests and certifies products to verify they meet public health and safety standards. Products that meet these standards bear the NSF Mark, which is respected by consumers, manufacturers, retailers and regulatory agencies at local, state, federal and international levels. Widely recognized for its scientific and technical expertise in the health and environmental sciences, NSF is a World Health Organization Collaborating Centre for Food and Water Safety and Indoor Environment. The company operates more than 165,000-square-feet of state-of-the-art laboratory space and serves companies in more than 150 countries worldwide. Its 1,200-plus staff includes microbiologists, toxicologists, chemists, engineers, environmental and public health professionals. NSF-certified UV systems that comply with NSF/ANSI Standard 50 can be found on NSF’s listing web page:


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