Grantee Research Project Results

2003 Progress Report: Understanding the Sources and Fate of Conventional and Alternative Indicator Organisms in Tropical Waters

EPA Grant Number: R828829
Title: 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 , Michigan State University
Current Institution: University of South Florida
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 2001 through August 31, 2003
Project Period Covered by this Report: September 1, 2002 through August 31, 2003
Project Amount: $388,335
RFA: Recreational Water Quality: Indicators and Interstitial Zones (2000) RFA Text |  Recipients Lists
Research Category: Aquatic Ecosystems , Ecological Indicators/Assessment/Restoration , Water

Objective:

The objective of this research project is to investigate the fate (persistence of culturable organisms and potential for regrowth) of various water quality indicator organisms in subtropical waters and sediments. The influence of the source of fecal pollution on indicator organism survival also is under investigation. Inoculum sources include dog feces, untreated wastewater, and naturally contaminated soil. The indicator organisms include fecal coliforms (FCs), Enterococcus spp., Clostridium perfringens, coliphage MS2 (F-specific RNA), and bacteriophages of Bacteroides fragilis. In addition, the differential survival of certain Escherichia coli strains is being investigated by genotypic (ribotyping) and phenotypic (antibiotic resistance) subtyping, which will provide insight about the ecology of these organisms in aquatic environments as well as information that will aid in refining bacterial source tracking methodologies.

Progress Summary:

The term “persistence” is used to describe the extent to which a culturable indicator organism population is maintained over time. The mesocosm studies accomplished during Year 1 of the project suggested that FC and Enterococcus populations persisted better in freshwater than in saltwater over a 4-week incubation period under natural conditions. It also was evident that FCs persisted longer in freshwater mesocosms than enterococci (ENT) and that the reverse was true in saltwater mesocosms, in which ENT persisted longer than FCs. To make a direct comparison of the persistence of indicator organisms inoculated into mesocosms at different concentrations, average decay rates over 4 weeks were calculated. Statistical analysis of decay rates showed that: (1) FC decay rates in freshwater mesocosms were significantly lower than ENT rates, indicating a greater persistence of culturable FCs; (2) the relationship between the indicator organism persistence was reversed in saltwater; and (3) FC decay rates were significantly lower in freshwater sediments than in the freshwater column, but this difference was not statistically significant for FCs in saltwater, nor for ENT in either water type.

Inoculum source significantly affected decay rates of FCs in the water column of freshwater mesocosms; decay rates of FCs in mesocosms inoculated with contaminated soil were significantly lower than corresponding values in mesocosms inoculated with dog feces. The influence of inoculum source on decay rates was more pronounced in saltwater mesocosms; FCs and ENT from contaminated soil had the lowest decay rates (and highest persistence), whereas indicator organisms from dog feces had the highest decay rates. FCs from the soil inoculum also behaved distinctively in saltwater mesocosms, as culturable counts in both sediments and the water column were extremely low immediately after inoculation (day 0) and increased by two or more orders of magnitude by day 1. We plan to type these strains genetically by ribotyping to determine if the population that was sampled on day 1 is representative of the inoculum or represents a limited number of strains. The mesocosm studies demonstrated that the persistence of culturable FC and ENT are affected differently by both location (sediment versus water column) and water type. Furthermore, they demonstrated that the source of fecal organisms affects their persistence in environmental waters.

Microcosms made from dialysis tubing were incubated in a freshwater lake on the University of South Florida campus over a 5-day period. Five E. coli isolates and five Enterococcus spp. isolates from each of the three different sources (dog feces, wastewater, and contaminated soil) were cultured in the laboratory for inoculation into the microcosms. The strains were chosen randomly from pure cultures obtained from inoculum sources prior to inoculation of the mesocosms. In the first dialysis tube experiment, microcosms made with nonsterile pond water 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). Isolate source did not significantly affect decay rates in the microcosms, nor did the indicator organism (E. coli versus 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 indicator to influence persistence in this experiment may have been a result of stochastic process; that is, only a small subset of isolates was cultured from the inoculum, and the more persistent strains were not among them.

Dialysis tube microcosms made with sterile pond water also were employed to assess the stability of E. coli ribotypes over time. Each microcosm was inoculated with a mixed culture that was made 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. Over the 5-day incubation period, concentrations of some of the initial subtypes became undetectable, demonstrating differential die-off, but no new or altered ribotypes were observed, demonstrating the stability of the patterns over time.

A constructed pond containing nonsterile sediment and freshwater was inoculated with a nalidixic acid-resistant E. coli strain, an E. gallinarum strain, a mixed culture of five C. perfringens strains isolated from sewage, and coliphage MS2 (F-specific RNA). Culturable organisms were enumerated over a 4-week period in which ambient temperatures ranged from approximately 23-29ºC. C. perfringens became unculturable within 48 hours. Decay rates and persistence of FCs, ENT, and MS2 were much more similar to each other and to those observed in the mesocosm study. Decay rates for all indicators except C. perfringens were significantly lower in sediments than in the water column, indicating a longer persistence in sediments.

Future Activities:

We will repeat the pond study in saltwater and the results will be compared with those of the freshwater study. The E. coli strains isolated from the saltwater mesocosms inoculated with contaminated soil will be ribotyped to determine whether they are representative of the initial inoculum or are composed of one or more strains that were able to recover culturability or grow under the mesocosm conditions.

Journal Articles:

No journal articles submitted with this report: View all 12 publications for this project

Supplemental Keywords:

fecal coliform, FC, Escherichia coli, Enterococcus, bacteriophage, coliphage, bacterial source tracking, water quality, indicator bacteria, tropical, subtropical, source tracking, ribotype, microbial survival,, RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Geographic Area, Water, Waste, Ecological Risk Assessment, Health Risk Assessment, Fate & Transport, State, Recreational Water, Biology, Hydrology, Environmental Monitoring, E. coli, fecal coliform, fate and sources, recreational water monitoring, FLA, enterococci, tropical climate, indicator organisms, Florida, water quality, risk assessment, swimming-associated gastroenteritis, water quality criteria, fishing, nutrient rich conditions, fate and transport, microbes, human health risk, bacterial source typing, indicators, bacteria

Progress and Final Reports:

Original Abstract

2002 Progress Report

Final Report