![[logo] US EPA](https://webarchive.library.unt.edu/eot2008/20081107084507im_/http://www.epa.gov/epafiles/images/logo_epaseal.gif){kind=logo}
Final Report: Understanding the Sources and Fate of Conventional and Alternative Indicator Organisms in Tropical Waters
EPA Grant Number: R828829Title: Understanding the Sources and Fate of Conventional and Alternative Indicator Organisms in Tropical Waters
Investigators: Harwood, Valerie J. , Rose, Joan B.
Institution: University of South Florida
EPA Project Officer: Nolt-Helms, Cynthia
Project Period: September 1, 2001 through August 31, 2003
Project Amount: $388,335
RFA: Recreational Water Quality: Indicators and Interstitial Zones (2000)
Research Category: Aquatic Ecosystems , Ecological Indicators/Assessment/Restoration
Description:
Objective:water quality indicator organisms in subtropical waters and sediments. The influence of the source of fecal pollution on indicator organism survival also was investigated. Inoculum sources included dog feces, untreated wastewater, and naturally contaminated soil. The indicator organisms included fecal coliforms (FC), Enterococcus spp. (ENT), Clostridium perfringens, and the F-specific RNA coliphage MS2. In addition, differential survival of certain Escherichia coli strains was investigated by genotypic (ribotyping) subtyping, which provided insight about the ecology of these organisms in aquatic environments, as well as information that will aid in refining microbial source tracking methodologies. Decay rates of vegetative and sporulating cultures of C. perfringens also were compared after observing very limited persistence of vegetative cells in initial experiments. We hypothesized that the persistence of C. perfringens may be affected by the physiological state of the organism, which would in turn affect its usefulness as an indicator of water quality. Summary/Accomplishments (Outputs/Outcomes):
The term persistence is used in this report to describe the extent to which a culturable indicator organism population was maintained over time. To make direct comparisons of the persistence of indicator organisms inoculated into mesocosms at different concentrations, average decay rates over a 4-week period (or to the time point at which organisms became undetectable) were calculated. Statistical analysis of decay rates showed that: (1) FC decay rates in freshwater mesocosms were significantly lower than ENT rates, indicating greater persistence of culturable FC; (2) decay rates were not significantly different between FC and ENT in saltwater mesocosms; (3) saltwater significantly increased the decay rates of both FC and ENT compared to freshwater; (4) FC decay rates were significantly lower in freshwater sediments than in the freshwater column; (5) in saltwater, FC decay rates were lower in sediments than in the water column, but the difference was not significant at = 0.05 (P = 0.083); and (6) decay rates of ENT were not significantly different in sediments compared to the water column in either water type, although the tendency was for greater persistence in sediments.
Inoculum type significantly affected FC and ENT decay rates under some conditions; however, these differences should be interpreted cautiously because not only were inoculum sources different, but the amount of inoculum material, and therefore organic carbon, differed between treatments. The trend was that lowest decay rates (greatest persistence) of FC and ENT were seen in mesocosms inoculated with contaminated soil, followed by wastewater, and finally dog feces. Differences in decay rates between mesocosms inoculated with the various materials were significant in the case of FC in the water column of freshwater and saltwater mesocosms and ENT in saltwater mesocosms.
Ribotyping analysis of E. coli isolates from the mesocosms demonstrated that certain phylotypes tended to persist longer than others in both the water column and sediments, regardless of the inoculum source. This phenomenon, termed differential survival, resulted in a shift in specific E. coli phylotypes within 24 hours of mesocosm inoculation, such that the E. coli population sampled from mesocosm waters and sediments was quite dissimilar from that initially sampled from fecal material.
Microcosms made from dialysis tubing were incubated in a freshwater lake on the University of South Florida campus over a 5-day period. Five randomly selected E. coli isolates and five ENT isolates from each of the three different sources (dog feces, wastewater, and contaminated soil) were cultured in the laboratory for inoculation into the microcosms. Microcosms containing nonsterile pond water plus inoculum were placed in a metal cage and suspended at one of two depths: 0.15 m below the surface or at the bottom of the pond (~1.2 m). Each microcosm contained E. coli or ENT from one source. Source did not significantly affect decay rates of organisms in the microcosms, nor did the indicator organism (E. coli vs. ENT). Although the trend was for longer persistence of culturable organisms at the deeper depth, differences in decay rates were not significant. The failure of the inoculum source and the indicator to influence persistence in this experiment may have been because of stochastic processes (i.e., only a small subset of isolates was cultured from the inoculum, and the more persistent phylotypes were not among them).
Dialysis tube microcosms made with sterile pond water also were employed to assess differential persistence of E. coli phylotypes. Each microcosm was inoculated with a mixed culture of three pure cultures derived from the initial isolates from one source. Sources for pure cultures were dog feces, Hillsborough River water, Hillsborough River sediment, contaminated soil, and wastewater. During the 5-day incubation period, some of the initial phylotypes became undetectable, demonstrating differential die-off, but no new or altered ribotypes were observed, demonstrating the stability of the patterns over time.
A freshwater constructed pond containing nonsterile sediment and water from the Hillsborough River was inoculated with a nalidixic acid-resistant E. coli strain, an Enterococcus gallinarum strain, a mixed culture of five C. perfringens strains isolated from sewage, and MS2 (an F-specific RNA) coliphage. The inoculum densities were approximately 107-8 CFU/100 ml for all of the organisms. Culturable organisms were enumerated over a 4-week period in which ambient temperatures ranged from approximately 23 to 29°C. C. perfringens became undetectable (water: < 0.2 CFU/100 ml; sediment: < 4 CFU/100 g wet weight) within 48 hours and 72 hours in water and sediments, respectively, and had a significantly higher decay rate than any of the other organisms. MS2 coliphage decay rates were significantly lower in water and sediments (persistence was greater) than any of the bacteria, remaining detectable for 21 days. E. coli decay rates were lower than those of Ent gallinarum in sediments; however, decay rates of the organisms did not differ significantly in water column samples. Decay rates for all indicators except C. perfringens were significantly lower in sediments than in the water column, indicating longer persistence in sediments.
A saltwater constructed pond containing nonsterile sediment and water from the Gulf of Mexico was inoculated with the same suite of indicator organisms as the freshwater pond at approximately 106 CFU/100ml for each indicator. Ambient temperatures during this 4-week period ranged from approximately 19 to 25°C. As previously observed, C. perfringens became undetectable rapidly, within 24 hours in the water column, and within 48 hours in sediments. MS2 was culturable for 96 hours, demonstrating markedly less persistence than in the freshwater pond, and decay rates increased over time, producing a biphasic curve. Decay rates of E. coli and Ent gallinarum in the water column were very high and similar to that of C. perfringens, as these organisms were no longer detected after 24 hours; however, increased persistence was seen in sediments. In sediments, decay rates of Ent gallinarum were significantly lower than the other indicator organisms, as culturable cells were recovered for 14 days. Decay rates of all indicators were significantly lower in sediments than in the water column, indicating longer persistence in sediments.
To test the hypothesis that the physiological state of C. perfringens affects its survival in water, two constructed freshwater ponds were inoculated with C. perfringens cultures that were either fresh (containing vegetative cells) or aged (containing endospores). Both C. perfringens cultures were derived from the same mixed culture of five strains isolated from sewage used in the fresh and saltwater ponds. The fresh culture was incubated approximately 18 hours before inoculation of the pond to introduce a culture consisting of mainly vegetative cells. The aged culture was incubated at 37°C for 7 days before being added to the pond. Both ponds were filled with nonsterile sediment and freshwater, and 106 CFU/100 ml of their respective culture was added. Background (natural) levels of C. perfringens in the water column and sediment were below detection limits (< 1 CFU/100 ml and < 12 CFU/100 g wet weight, respectively). Culturable cells were no longer detected after 48 hours in either the water column or the sediment of the fresh and aged inoculum treatments. No differences were detected in decay rates between the vegetative and aged cultures in water column samples. In sediments, the decay rate of the aged culture increased between days 2 and 3, producing a biphasic curve, while the vegetative culture decreased linearly over the course of the experiment.
Conclusions:Results from this study indicate that many biological and environmental factors influence the fate of indicator organisms in aquatic environments. Decay rates were affected by indicator organism, inoculum, water type, sediment versus water column location, and E. coli strain. In general, indicator organism decay rates were greater in the water column than in sediments, and were greater in saltwater than in freshwater. Although the former is consistent with observations in the literature, unexpected findings include: (1) no evidence for positive decay rate (growth) for any indicator organism; (2) C. perfringens, which by virtue of endospore formation is generally considered a highly persistent organism, was less persistent than any of the other indicator organisms; (3) differential survival of E. coli phylotypes in freshwater and sediments suggests that microbial source tracking methods in which E. coli is used as the source identifier face significant challenges.
These results have implications for the accuracy of microbial source tracking methods, modeling of microbial population dynamics in water, and efficacy of regulatory standards for the protection of water quality. Recognition and exploration of the complex relationships between indicator organisms, pathogens, and their habitats are crucial to improving the indicator system for microbial water quality to better predict pathogen presence.
Journal Articles on this Report: 1 Displayed | Download in RIS Format
| Other project views: | All 13 publications | 1 publications in selected types | All 1 journal articles |
| Type | Citation | ||
|---|---|---|---|
|
|
Anderson ML, Whitlock JE, Harwood VJ. Persistence and differential survival of fecal indicator bacteria in subtropical waters and sediments. Applied and Environmental Microbiology 2005;71(6):3041-3048 |
R828829 (Final) |
not available |
fecal coliform, Escherichia coli, Enterococcus, Clostridium perfringens, bacteriophage, coliphage, water quality, indicator bacteria, tropical, subtropical, source tracking, ribotype, microbial survival, persistence, freshwater, E. coli strains, mesocosms, saltwater, inoculum material, water quality indicator organisms, contaminated soils, sediments, aquatic environments, wastewater.
,
Ecosystem Protection/Environmental Exposure & Risk, Water, Geographic Area, Scientific Discipline, Waste, RFA, Biology, Health Risk Assessment, Recreational Water, Ecological Risk Assessment, Fate & Transport, Hydrology, Environmental Monitoring, State, bacteria, recreational water monitoring, risk assessment, water quality, fecal coliform, fate and transport, swimming-associated gastroenteritis, indicator organisms, fate and sources, organisms, E. coli, enterococci, human health risk, FLA, water quality criteria, fishing, bacterial source typing, nutrient rich conditions, Florida, indicators, microbes
Relevant Websites:
None.
Progress and Final Reports:
2002 Progress Report
2003 Progress Report
Original Abstract