By Brooke K. Mayer, PhD, PE

Throughout the course of our daily activities, humans are continuously exposed to a vast array of microorganisms, the majority of which are harmless or even beneficial. However, a small fraction of microorganisms, known as pathogens, cause disease or infection.

Waterborne diseases are transmitted through ingestion of contaminated water, i.e., a fecal-oral route. These microorganisms are referred to as enteric pathogens, or those that infect the gastrointestinal tract. The major groups of enteric pathogens include bacteria, viruses, and protozoa. Helminths, or parasitic worms, are another class of enteric pathogens, commonly found in the tropics or subtropics.[1]Examples of waterborne pathogens are shown in Table 1. Box 1 lists useful terminology for microbiological water-treatment process evaluations.

Publicly or privately owned public water systems serve more than 90 percent of people living in the United States.[2] These public water systems are subject to the U.S. Environmental Protection Agency’s (EPA’s) National Primary Drinking Water Regulations, which specify maximum contaminant level goals of zero for Cryptosporidium, Giardia, Legionella, total coliforms, and viruses. The enforceable maximum contaminant levels are predominantly determined by treatment technique rather than measuring effluent concentrations of the pathogens.

Many households in the U.S. and around the world utilize point-of-use (POU) or point-of-entry (POE) treatment devices in lieu of or in addition to public water systems. Unlike public water systems, there are no federal regulations for POU/POE performance.However, the need for accurate, objective POU/POE testing protocols and methods led to the establishment of recommendations, guidance, specifications, protocols, and procedures to set minimum requirements for drinking water microbiological safety and POU/POE performance.[3]

Among these efforts are the 1987 EPA Guide Standard and Protocol for Testing Microbiological Water Purifiers4, the World Health Organization (WHO) International Scheme to Evaluate Household Water Treatment Technologies, and NSF/ANSI standards and protocols. These agencies provide certification or other formal performance evaluations of POU/POE, and while not comprehensive for all practices around the world, they offer recognized international sources of information.[3] Table 2 includes brief descriptions of microbial-related POU/POE standards or protocols from these organizations, and Figure 1 shows select quantitative log reduction targets.

The EPA’s Guide Standard and Protocol for Testing Microbiological Purifiers focused on the performance of ceramic filtration candles, chemical disinfectants (halogenated resins), and UV disinfection, although applicability to other unit processes was explicitly indicated. The guide specifies microbial-reduction performance requirements; test microbes; sources and preparation methods for test microbes; microbial assays; compositions of test waters, including microbe concentrations; and specifications for setup design and use.[4]

The performance of different water-treatment technologies is impacted by water-quality parameters such as turbidity, total dissolved solids, dissolved organic matter, pH, and temperature. Accordingly, devices are tested with representative water compositions, as well as worst-case scenarios when evaluating performance. Bailey et al. (2021)[3] summarized the specific test-water qualities for several POU/POE testing protocols.

WHO categorizes processes using a tiered approach based on different levels of tolerable risk evaluated through quantitative microbial risk assessment (QMRA).[5] Risks are expressed as disability adjusted life years (DALYs) informed by local data to the extent possible or by default targets in the absence of local data. The QMRA-derived health-based targets were determined using reference pathogens selected for their global presence in contaminated drinking water, ability to cause disease, resistance to disinfection, and the availability of information on their occurrence and dose-response relationships. The QMRA reference pathogens are Campylobacter jejuni bacteria, rotavirus, and Cryptosporidium. The recommended test microbes include several surrogates in place of the pathogens, as shown in Table 2. The tiers of performance include the following:

  • Highly protective tier (***): 4-log reduction of bacteria, 5-log reduction of viruses, and 4-log reduction of protozoa (equivalent to a 10-6 DALY per person per year risk level).
  • Protective tier (**): 2-log reduction of bacteria, 3-log reduction of viruses, and 2-log reduction of protozoa (equivalent to a 10-4 DALY per person per year risk level).Targeted protection tier (*): Technologies that fulfill two of the three levels of microbial-reduction performance as specified in the protective tier.
  • Targeted protection tier (*): Technologies that fulfill two of the three levels of microbial-reduction performance as
    specified in the protective tier.

Figure 1. Log reduction targets for microorganisms in select standards and protocols. Note that targets for all three classes of microbial pathogens are included in some guidelines, whereas single classes are specified in others. Here, WHO is illustrated for “highly protective” performance in accordance with the International Scheme to Evaluate Household Water Treatment Technologies. For NSF/ANSI 55, the target for Class A systems is shown. Specific test organisms are listed in Table 2.

Microbial selection for performance evaluations of POU/ POE technologies is critical, as it is not feasible nor desirable to test every species and strain of concern, and each microorganism has different physical, chemical, and biological properties that influence its susceptibility. For example, Cryptosporidium is very resistant to chlorine, but it is susceptible to UV disinfection. However, adenovirus is effectively treated using chlorine but has enhanced resistance to UV.

In general, resistance to chemical disinfection is greatest for protozoan (oo)cysts and bacterial spores, followed by viruses. Vegetative bacteria is the least resistant. For physical removal through filtration, protozoa, which are larger than the other microbe types, are typically more effectively removed than bacteria. Viruses exhibit the lowest removal due to their small size. Given the diversity in microbial responses to water-treatment processes, there is no single optimal selection for test microorganisms, leading to variations in recommended methodology among standards and protocols.

In many cases, surrogate or indicator microorganisms are tested as representatives of waterborne pathogens given the inherent difficulty and risk of regularly testing the actual pathogens. Pathogens are often present at low concentrations, which are difficult to detect. Yet low doses can still be infectious depending on the organism, and the methods used to detect and quantify them are often very complex, time- and cost-intensive assays.

Thus, more easily detected and quantified microorganisms are evaluated, and their presence, in turn, provides evidence of the potential presence of enteric pathogens or is suggestive of the level of microbial treatment provided by a given technology. The presence of indicator organisms (e.g., E. coli) is indicative of the potential presence of human fecal contamination, and their concentration is an indicator of the extent of contamination.

Ideal indicators are consistently and exclusively associated with fecal contamination because they do not exist or grow naturally in water, are present in large quantities, and are easily detectable. Surrogates are subtly different from indicators in that they are used as conservative test organisms to model the treatability of the target pathogens. Ideal surrogates are less well treated relative to pathogenic organisms (giving conservative estimates of performance) while also more easily assayed. In general, indicator and surrogate microorganisms are more convenient, simpler, and cost-effective options to evaluate POU/POE technology performance.[3]

Approaches to microbial detection and quantification can be broadly grouped as microscopic, cultural, or molecular techniques. Each method is associated with advantages and disadvantages with respect to the information it conveys, as well as ease and cost of the protocol.

Microscopy continues to play an integral role in the study of microorganisms but is not widely used for the direct counts needed for process performance testing; it is time intensive, and microbial viability is unknown in the absence of additional preparation steps, such as the use of fluorescent dyes such as propidium iodide. This question of infectivity is vital for accurate evaluations of many water-treatment processes.

Microbial mitigation can take the form of physical removal, such as extraction by sedimentation or filtration, or inactivation, when the microbes remain in the water but are rendered unable to replicate and are thus noninfectious. Molecular methods are limited in their ability to reliably detect and quantify infectious microorganisms, such that cultural methods remain central to POU/POE technology evaluation (as shown in Table 2). Further investigation of methods that overcome the challenges associated with physiological microbial damage yielding viable but not culturable organisms, as well as the occurrence of light and dark repair mechanisms, is warranted.


  1. Maier, R. M.; Pepper, I. L.; Gerba, C. P. Environmental Microbiology, 2nd ed.; Academic Press: Burlington, MA, 2009.
  2. S. Environmental Protection Agency (EPA). Drinking Water Requirements for States and Public Water Systems. 2020. dwreginfo/information-about-public-water-systems (accessed 2021-05-06).
  3. Bailey, E. S.; Beetsch, N.; Wait, D. A.; Oza, H. H.; Ronnie, N.; Sobsey, M. D. Methods, Protocols, Guidance and Standards for Performance Evaluation for Point‐of‐Use Water Treatment Technologies: History, Current Status, Future Needs and Directions. Water 2021, 13 (8), 1–85. w13081094.
  4. Guide Standard and Protocol for Testing Microbiological Water Purifiers; Washington, D.C., 1987.
  5. World Health Organization. Evaluating Household Water Treatment Options: Health-Based Targets and Microbiological Performance Specifications.

About the Author
Dr. Brooke K. Mayer is an Associate Professor in the Department of Civil, Construction and Environmental Engineering as part of the Opus College of Engineering at Marquette University. She holds Bachelors, Masters and Doctorate degrees in civil engineering with an emphasis in environmental engineering from Arizona State University. She is a registered Professional Engineer in the state of Arizona.


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