Grantee Research Project Results
Final Report: Development and Evaluation of Three Simple, Low-Cost, Low-Tech Tests for Microbial Fecal Indicators in Drinking Water
EPA Grant Number: SU833548Title: Development and Evaluation of Three Simple, Low-Cost, Low-Tech Tests for Microbial Fecal Indicators in Drinking Water
Investigators: Sobsey, Mark D. , Stauber, Christine , Love, David , Tajuba, Julianne , McMahan, Lanakila , Casanova, Lisa , Chung, Sandra , Lusk, Tina
Institution: University of North Carolina at Chapel Hill
EPA Project Officer: Page, Angela
Phase: I
Project Period: September 30, 2007 through September 30, 2008
Project Amount: $10,000
RFA: P3 Awards: A National Student Design Competition for Sustainability Focusing on People, Prosperity and the Planet (2007) RFA Text | Recipients Lists
Research Category: Pollution Prevention/Sustainable Development , P3 Challenge Area - Safe and Sustainable Water Resources , P3 Awards , Sustainable and Healthy Communities
Objective:
No substance on Earth is as integral to human life and health as water. We use it for everything: drinking, cooking, feeding infants, personal hygiene, keeping our homes clean, and recreation. When water is contaminated with disease causing microorganisms (pathogens) the effects reach into every aspect of life. Better information about the quality of drinking and recreational water can reduce risks of waterborne and water-contact diseases and improve the health and lives of many. To address these needs, we propose to develop and evaluate three simple, inexpensive, user-friendly, portable tests for fecal bacterial and viral contamination of water that can be performed by the consumer at the point of use. Tests will detect and enumerate Escherichia coli (E. coli), coliphage, and hydrogen sulfide producing bacteria. Both E. coli and coliphage have been evaluated in peer-refereed journals and were shown to be reliable indicators of fecal contamination in water. Hydrogen sulfide producing bacteria have also been extensively evaluated as fecal indicators, but data are lacking on the extent to which their presence and concentrations in water predict risks of waterborne illness. If successful, our tests can reduce exposure to pathogens causing gastrointestinal and other waterborne diseases. Additional benefits include the assessment of water in disaster and other emergency situations where the equipment and personnel to perform standard drinking water analysis are not available.
Summary/Accomplishments (Outputs/Outcomes):
E. coli tests
A series of 19 experiments were performed to compare detection of E. coli on a variety of media across a range of temperatures. During each experiment, five methods were compared at six incubation temperatures. The experiments were repeated between two to four times for water spiked with pure culture strains of E. coli and natural water amended with sewage. All test media demonstrated growth after 24 hours at all temperatures tested. The exception was 20°C; after 24 hours there was no detectable growth (on average) for any method or strain at this temperature. At the five other temperatures, concentrations varied between methods when averaged across strains. The highest concentration of bacteria was detected using Colilert® at 35°C. Average concentrations detected for the five methods and six strains were compared using a non-parametric repeated measures analysis of variance for all repetitions of the experiments paired by temperature, repetition and strain. Based on this analysis, statistically significant differences (P<0.05) were found between the traditional methods (the two membrane filter methods and the broth method) compared to the two gel methods; the three traditional methods resulted in significantly higher concentrations of E. coli. Differences were not identified between the three traditional methods when compared to one another. There was also no significant difference between the two gel methods when compared to one another.
In addition to evaluation of five existing methods, research was performed to develop an absorbent pad format for E. coli detection. The new care-free utilization test for E. coli (CUTE) eliminates the need for reusable equipment and relies less on disposable equipment than traditional methods. CUTE works similarly to Petrifilm™ but can accommodate larger sample volumes. A series of 10 experiments have been performed to evaluate different media compositions for the absorbent pad format.
Coliphage test
The coliphage latex agglutination assay (CLAT) was performed on samples from Avalon Beach, CA as part of a larger project. Marine water samples were analyzed following 5-hr and overnight enrichment, with 86/324 and 179/324 samples positive for coliphage, respectively. When performing CLAT for F+ RNA coliphages, 0/324 and 27/324 positives were found for rapid enrichment and overnight enrichment, respectively. F+ DNA detection by CLAT on 5-hr enrichments and overnight enrichments were 0/324 and 52/324 positive, respectively. In an attempt to determine whether coliphage titer contributed to low CLAT results for 5-hr enrichments, an additional enrichment was performed on all samples and improved F+ RNA positivity to 26/324 and 58/324, respectively, for 5-hr and overnight enrichment. The detection rates for F+ DNA increased as well to 40/324 and 102/324, respectively, for 5-hr and overnight enrichment.
H2S Tests
A series of 15 experiments were performed to compare detection of H2S producing bacteria from sewage amended waters by two methods at two incubation temperatures and times. The experiments were repeated 2-4 times for replicate sets of conditions. Three variations on H2S media were also assessed in a most probable number format.
After 24 or 48 hours, there was no significant difference in the concentration of H2S bacteria when compared by media type (Hach vs. in house). In addition, the commercially prepared medium was compared at two temperatures over 24 and 48 hours. After 24 or 48 hours, there was no significant difference in concentration of H2S producing bacteria when compared by incubation temperature (25 vs. 35 °C). Statistically significant differences (P <0.05) were found between the Hach product at 24 hours when compared to the in-house autoclaved product after 48 hours at 35 °C. For the Hach broth significant differences were also found between incubation at 25 °C for 24 hours and 25 °C for 48 hours.
Conclusions:
Results from the laboratory experiments demonstrate robustness of the existing E. coli and H2S producing bacterial detection methods at a range of temperatures. This suggests that these tests could be applied at ambient temperatures without the need for incubators, especially in tropical settings. Traditional tests such as membrane filtration resulted in the detection of higher concentrations of E. coli compared to non-traditional test formats such as gels. Development of an easy to use, low-cost MPN format for the H2S test, such as plastic bags, can enhance the application of this method in the field. Additional field research would enhance the understanding of the conditions under which less traditional methods can and should be employed, especially in low-resource settings. These results also support the continued development of the less-resource intensive CUTE assay that employs absorbent pad detection of E. coli.
Proposed Phase II objectives and strategies:
Evaluation of these selected candidate tests for their ability to achieve health benefits via increased application of detection of fecal contamination of drinking water in actual field use will be the overarching goal of Phase II. Using laboratory results comparing the candidate fecal indicator tests proposed in Phase I, we can choose water quality tests that have the potential for successful application in field settings. Based on the laboratory data and a matrix used to characterize and prioritize the field tests in terms of their ability to enhance people, prosperity and the planet the following will be the specific aims for Phase II:
Specific Aim 1: Field evaluations of promising methods for bacterial indicators:
- Compare five methods for detection of fecal contamination in the field: one traditional E. coli method (either Colilert® or a membrane filter method) with Petrifilm™, Easygel®, CUTE (absorbent pad assay) and the bag format MPN H2S test
- Compare these methods at 35°C and ambient temperature incubation
- Compare these methods for contaminated drinking waters collected in three developing countries: Cambodia, Ghana and Honduras
Specific Aim 2: Further evaluations of CLAT assay for detection of coliphage in marine sample and contaminated drinking water:
- Compare and contrast CLAT method to spot-plate followed by reverse line blot hybridization for characterization of coliphage in marine samples during summer 2008 BEACHES project
- Compare the CLAT assay with a traditional membrane filtration method for E. coli and spot plate method for detection of coliphage for contaminated drinking water samples collected in Cambodia
- Assess application of CLAT at 35°C and ambient temperature incubation in Cambodia
Specific Aim 3: Determine the ability of the presence of fecal indicators in drinking water to correlate with increased health risk as a result of consumption of contaminated drinking water:
- Concurrently collect data on diarrheal disease from household that are providing drinking water samples for analysis in Aims 1 and 2 (data are to be collected in Cambodia, Ghana and Honduras)
- Collect additional data on other factors that may be associated to increased risk of diarrheal disease from households
- Attempt to understand and develop a logistic regression model that relates the presence and concentration of the fecal indicators of drinking water contamination to health risk
Supplemental Keywords:
drinking water, treatment, health effects, point of use technology fecal indicators, developing countries,The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.