National Exposure Research Laboratory & 

National Risk Management Research Laboratory 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

Legionella                        cells growing within an amoeba that may colonize piped water biofilms

RESEARCH PROJECT COLLABORATION

National Exposure Research Laboratory 

Microbiological & Chemical Exposure Assessment Research Division & 

National Risk Management Research Laboratory 

Water Supply and Water Resour ce Division 

INFLUENCE OF DISINFECTANT RESIDUAL ON BIOFILM DEVELOPMENT, MICROBIAL 

ECOLOGY, AND PATHOGEN FATE AND TRANSPORT IN DRINKING WATER INFRASTRUCTURE 

IMPACT STATEMENT

One of the U.S. Environmental Protection Agency’s (EPA) 

primary roles is that of an advocate for sustainable water 

infrastructure. Biofilms, which form on interior surfaces 

of the drinking water infrastructure, can sequester, 

accumulate and protect viruses, bacteria, fungi, and 

parasitic protozoa. Release of biofilms may therefore 

release a more infectious dose back into drinking water. 

The experimental studies performed through this project 

are designed to evaluate risks to human health posed by 

pathogens associated with these biofilms, initially 

focusing on bacterial pathogens that may grow in 

association with amoebae in biofilms. By providing the 

science and engineering information that we and our 

partners need, EPA research contributes measurable 

results that advance our efforts to ensure safe drinking 

water. 

BACKGROUND:

Biofilms form on interior surfaces of the drinking water infrastructure, including storage facilities and distribution 

systems. Pathogens may enter drinking water systems in the bulk water or through intrusions or physical breaches. 

Biofilms can sorb pathogens (viruses, bacteria and parasitic protozoa) and serve as a protective niche for their survival. 

In addition, opportunistic pathogens, principally respiratory pathogens such as nontuberculous mycobacteria [NTM] and 

Legionella-like bacterial pathogens, may grow within biofilms in storage facilities and distribution systems, including 

premise plumbing. Recent data suggests that disinfection practices may selectively control opportunistic pathogens. 

For example, chloramination is reputed to control Legionella, but may be less effective for NTM bacteria. However, 

there are limited data on how disinfection strategies impact the biofilm community structure that may support 

opportunistic pathogens and that may also provide a niche for sequestering fecal pathogens. Current models assessing 

risks from drinking water are limited by not accounting for pathogen biofilm effects. 

DESCRIPTION:

This project focuses on providing basic data to bound risk estimates resulting from pathogens associated with pipe 

biofilms. Researchers will compare biofilm pathogen effects under two different disinfection scenarios (free chlorine or 

chloramines) for a conventionally treated source water (Potomac River). Samples will be collected from storage 

facilities, dedicated sampling taps used for TCR compliance monitoring, and biofilm swabs from household cold water 

taps and shower heads (premise plumbing). Microbial community structures, NTM, and Legionella-like bacteria and their 

amoebae hosts will be characterized. Potential novel biomarkers of exposure will be collected and used to explore the 
 

 

National Exposure Research Laboratory & 

National Risk Management Research Laboratory 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

potential for salivary and urine antibody tests. Water quality data, operations information, and system condition 

information will also be collected to aid in interpretation of microbial data. In addition to disinfectant residual, key 

water quality variables including nitrogen species (ammonia, nitrite, nitrate, and organic-nitrogen), phosphorus, metals 

(lead, copper, arsenic), fluoride, organic carbon, pH, alkalinity, temperature, sulfate, chloride, and other inorganics will 

be assessed. All potential pathogens will be genotyped and stored for future reference. In addition, if a water main 

break or other ingress event occurs during the project-sampling phase, material will also be stored for molecular 

identification of fecal pathogens and indicators. 

EPA’s Office of Research and Development has funded this research project in support of its Aging Water Infrastructure 

Research Program. Phase I of this project will run from March 2010 through March 2011, and dependent on project 

success, Phase II will run for a second year. 

EPA GOAL: Goal #2 - Clean & Safe Water; Objective 2.1.1- Water Safe to Drink 

ORD MULTI YEAR PLAN: Drinking Water (DW), Long Term Goal (LTG) - DW-1 Characterize risks associated with DW sources, 

distribution, treatment, and use 

RESEARCH PARTNERS: 

• EPA’s National Exposure Research Laboratory: Stacy Pfaller (NTM); Shay Fout (salivary antibodies) 

• EPA’s National Risk Management Research Laboratory: Laura Boczek (biofilm disinfection); Randy Revetta (biofilm community 

structure) 

• EPA’s National Health and Environmental Effects Research Laboratory: Elizabeth Hilborn & Andrey Egorov (human health 

effects) 

• EPA’s Office of Water: Susan Shaw (coordination of EPA staff/home access) 

• Washington Aqueduct & WSSC contacts 

EXPECTED OUTCOMES AND IMPACTS:

It is expected that this study will have a direct impact on public health by providing utilities information on pathogen 

risks associated with biofilm growth in storage facilities and water distribution systems. This information will facilitate 

better management decisions, which will result in a decrease in the risk of waterborne illness. 

OUTPUTS:

Primary outputs from this project will be journal and technical papers describing key parameters that will enable 

quantitative microbial risk assessments of opportunistic and fecal pathogens associated with biofilms in drinking water 

systems. This information will enable updates to EPA’s hydraulic models for drinking water distribution systems, so that 

the role of pathogen-biofilm interactions can be included. 

RESOURCES:

Aging Water Infrastructure Research Program: http://www.epa.gov/awi/ 

National Exposure Research Laboratory: http://www.epa.gov/nerl/ 

National Risk Management Research Laboratory: http://www.epa.gov/nrmrl/ 

CONTACTS:

Nicholas Ashbolt, Principal Investigator - (513) 569-7318 or ashbolt.nick@epa.gov 

Steven Doub, Media Relations - (513) 569-7503 or doub.steven@epa.gov 

Michelle Latham, Communications - (513) 569-7601 or latham.michelle@epa.gov 

EPA/600/F-10/012 

February 2010