INTRODUCTION

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Identification of sources of Escherichia coli in water bodies of urban systems is confounded by the presence of multiple sources as well as by the many factors that influence the ultimate fate of the microbe once it is released into the environment. Some important factors that have been described in prior studies include temperature (2, 34), light (25), salinity (19, 33), rainfall (21, 32), predation (3, 4, 7, 22), available nutrients (20), and environmental pollutants (24). Given the complexities of environmental systems, the influence of each factor in regulating the survival and growth of E. coli is difficult to predict for different field settings, although empirical models have been developed for some situations (5, 26). When one is faced with a river that is experiencing elevated E. coli levels, identifying the cause(s) of the microbial contamination is thus a formidable challenge, since the concentration of E. coli observed in the river is a function of the sources active at that time, chemical and biological factors, and the changing hydro-climatologic conditions that influence the fate of the organisms once they are released. This paper summarizes the approach utilized to identify the source(s) of E. coli for one urban river system. The approach is easily applicable to other river systems, and the results obtained from this study are potentially applicable to other tidally influenced rivers located in tropical or subtropical environments. The primary objective of the present investigation was to identify the source(s) of E. coli in a river located in Ft. Lauderdale, Fla., such that appropriate measures could be taken to improve water quality to meet recreational standards (14, 35). Previous analysis had shown that recreational standards were exceeded in over 90% of the samples collected from the river (fecal coliform levels were >200 CFU/100 ml). Suspected sources of contamination at the outset of the present investigation included septic tank systems, sanitary sewers, wastewater from live-aboard boats, inflows to the river from upstream and downstream water bodies, and animal sources. The basic approach chosen for the present investigation was to intensely monitor E. coli concentrations in the field under various hydro-climatologic conditions. From the data gathered during spatially and temporally intense sampling, more-focused sampling and laboratory efforts were developed, which targeted and defined the ultimate source(s) of contamination.

	MATERIALS AND METHODS

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Site description. This investigation focused on the North Fork of the New River, which is located in a highly urbanized area of Ft. Lauderdale, Fla. Ft. Lauderdale, Fla., is characterized by a subtropical climate with an average yearly rainfall of 147 cm and an average temperature of 29.4°C. The North Fork is a 6-km tidally influenced tributary of the New River, which on its east end is defined by the split of the New River into its North and South Forks. A control structure, S33, defines its western boundary (Fig. 1). Conductivity measurements within the North Fork indicate that the river is brackish, with a maximum proportion of 10% seawater. A large portion of the North Fork is characterized by a natural meandering shoreline which is lined with dense vegetation. Seawalls are present in some areas and are especially prevalent on the river's east end. Live-aboard boats are found near the central portion of the river. Bird populations, especially ducks, are found throughout the length of the North Fork. Other animal populations, which include domesticated and wild animals, have been observed along the channel banks. The sanitary infrastructure is characterized by two distinct regions. The northern portion of the site (north of Broward Boulevard) is served primarily by sanitary sewers. This is in contrast to the areas to the south, which are served primarily by septic tanks.

View larger version (44K): [in this window] [in a new window]   FIG. 1.   Location of the North Fork of the New River.

Sampling strategy. Sources of E. coli in the North Fork of the New River were investigated through five focused efforts. These efforts included (i) a series of quality control experiments aimed at establishing confidence limits of the analytical procedures and for evaluating the impact of an autosampler on sample quality, (ii) sampling the length of the North Fork to identify the river reach contributing high E. coli levels, (iii) autosampler experiments at two locations to determine temporal variations, (iv) spatially intense sampling efforts at hot spots, and (v) a laboratory effort aimed at further isolating suspected sources. Field sampling efforts included simultaneous measurements of relevant hydrologic parameters, including groundwater and canal elevations to determine the direction of lateral flows from the river, rainfall to determine storm water impacts, and tidal cycles to determine the effects of diurnal fluctuations in water levels. Furthermore, basic physicochemical parameters, including temperature, pH, conductivity (model 600R instrument; YSI, Yellow Springs, Ohio), and turbidity (model 40 instrument; Turner Designs, Sunnyvale, Calif.) were measured to establish correlations with E. coli concentrations.

Detection of E. coli. E. coli from all environmental samples was enumerated by using a defined substrate technology (11-13) which is commercially known as Colilert-18 (IDEXX, Atlanta, Ga.). This technique has been shown to correlate very well with the traditional membrane filter and multiple-tube fermentation methods when used to test both freshwater (8, 10) and marine water (23). The method is based upon sample fluorescence under long-wavelength UV light when MUG (5-methylumbelliferyl--D-glucuronide) is metabolized by E. coli. The analytical method involved adding indicator nutrients containing 5-methylumbelliferyl--D-glucuronide to a 100-ml volume of water which included 10 ml of sample and 90 ml of sterile phosphate-buffered water prepared as described in Standard Methods for the Examination of Water and Wastewater (1). The solution was then placed in a distribution tray which separated the sample into a series of test wells. The trays were incubated for 18 h at 35.5°C, and wells that fluoresced were counted at the end of the incubation period to provide a most probable number (MPN) per 100 ml of water. All sets of analyses for E. coli determinations included blanks and replicate samples for quality control purposes.

Water and soil sampling. To isolate the river reach contributing to E. coli contamination, the length of the North Fork was extensively sampled on six different occasions over a period of 1 year, which included different antecedent rain and tidal conditions. Samples were collected from a boat every 150 m along the 6-km length of the North Fork by submerging a presterilized pipette to a depth of 0.3 m below the surface and extracting a 10-ml sample. Each sample was immediately placed on ice inside a presterilized 100-ml bottle. Samples were analyzed within 4 h of collection. The results indicated that E. coli contamination is distributed throughout the length of the North Fork, with two notable hot spots: interstate highway 95 (I-95) and the Argyle Canal.

Laboratory evaluation of E. coli multiplication in soil. The results of these earlier efforts supported the hypothesis that the source of E. coli brought to the water column was the soils along channel banks. Therefore, during the final effort of the study, soil samples were collected from the I-95 area and subjected to a set of laboratory experiments aimed at establishing whether E. coli was capable of growing in riverbank soils. The experiments consisted of collecting riverbank soils by aseptically scraping off the upper layer of soil from the north bank at I-95. Two soil samples were allowed to air dry. The first sample was dried to a moisture content of 0.8%, and the second was dried to a moisture content of 14%. A third soil sample was maintained wet (35% moisture content) by covering the sample with plastic. These moisture contents were chosen to simulate the range that would be observed along the banks of the river. Each sample was placed in a tray and then subjected to periodic wetting and drying in an effort to simulate tidal conditions in the river. The first sample analyzed was the sample that was air dried to a 0.8% moisture content. The initially wet sample was analyzed next, and then the soil sample that was air dried to a moisture content of 14% was analyzed. Wetting and drying cycles were simulated by submerging and removing each soil sample from a 20-liter fish tank filled with unsterilized water collected from the North Fork. The fish tank was kept in the dark inside an incubator maintained at 25°C. The wetting and drying schedule was as follows: 6 h wet, 6 h dry, 12 h wet, 6 h dry, 6 h wet, 12 h dry, etc.. This sequence was continued over a 4-day period. Four soil samples were collected for E. coli determinations during each wetting or drying cycle. Two samples were collected half an hour after the beginning of the cycle, and two were collected half an hour before the end of the cycle. E. coli was also measured immediately before the first wetting cycle to obtain an initial concentration in the water and soil. E. coli cells were enumerated by using Colilert-18 reagents. A negative control with presterilized water and presterilized soil was subjected to the same wetting and drying sequence described above. No E. coli growth was observed for the negative control. Fecal coliforms in samples were also enumerated, by using the standard membrane filter method (1), for the experiment conducted with the soil sample initially dried to 14% moisture.

	RESULTS

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River length sampling. The results from full-length sampling of the North Fork (Fig. 2) indicate that E. coli baseline numbers were similar during five of the six sampling runs. During these runs, baseline E. coli numbers were below 500 MPN/100 ml on each end of the North Fork between 31st Avenue and Delevoe Park and between 11th Avenue and the junction with the South Fork. The primary exception to the low baseline levels corresponded to the December sampling trip, for which baseline values at each location almost doubled. The December samples were the only samples collected after a rain event and during high-tide conditions. These results suggest that E. coli entered the river during wet conditions and that during high tide the source is more pronounced. Stage measurements of the canal and groundwater during these tidal conditions indicated that groundwater contributions are strongest during low tide, thereby eliminating this water body, including contamination of groundwater from septic tanks and sanitary sewers, as a source of E. coli contamination. Although E. coli concentrations within the North Fork varied, the concentration observed between 11th Avenue and the junction with the South Fork were consistently low under all sampling conditions, indicating that the source of E. coli contamination to the North Fork does not come from the New River.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Results of river length sampling effort.

Autosampler experiments. The results of the autosampler experiments (Fig. 3) show two distinct relationships between E. coli levels and hydrologic conditions. The first and most obvious relationship is the extremely high E. coli concentrations observed during periods of rainfall. The second trend occurred from h 120 to 168, where E. coli concentrations correlated with tidal cycles after a storm. During high tide, concentrations of E. coli were elevated, whereas the opposite was observed during low tide. It is interesting that the cyclical pattern in E. coli concentrations occured 2 days after the storm had ceased. The peaks during high tide were significantly higher than the values observed during the 2 days immediately after the storm.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Results of 1-week autosampler experiment. Conc., concentration; H, high tide; L, low tide.

Source-specific sampling effort. The storm sewer sampling effort showed that water within the sewers contained lower E. coli concentrations than river water, indicating that storm sewers are not a likely source between storms. Results of the intensive grid sampling at both I-95 and the Argyle Canal indicated that the highest E. coli concentrations were along the channel banks. This observation was consistent for both shallow and deep samples. For example, a maximum value of 5,200 E. coli organisms per 100 ml was observed in a shallow-water sample on the south bank near I-95, whereas concentrations of fewer than 1,000 E. coli organisms per 100 ml were observed toward the center of the river (Fig. 4). The results of the field soil sampling effort indicated that the clean-area control at 27th Avenue had the lowest level of E. coli, with 14 E. coli organisms per g of dry soil. This low concentration in soil is in contrast to the case for the other sampling sites, which had higher E. coli levels. The soil collected from the banks of the Argyle Canal contained 87 E. coli organisms per g of dry soil. The highest E. coli value was observed on the north bank of I-95, which contained 200 E. coli organisms per g of dry soil. This value is in contrast to the lower results obtained on the south bank (37 E. coli organisms per g of dry soil).

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Results of intensive grid sampling effort at I-95 (contour interval, 200 E. coli organisms per 100 ml; shallow-water samples only).

Laboratory soil testing. A comparison of the air-dried sample (initial moisture of 14%) with the sample that was maintained wet (initial moisture of 34%) shows a marked difference in growth characteristics (Fig. 5). At the beginning of the experiment, the water and soil E. coli concentrations in the air-dried sample (initial moisture of 14%) were 520 per 100 ml and 440 per g (dry weight), respectively. Six hours after the soil sample was lowered into the water, the concentrations in the water and soil rose to 812 per 100 ml and 9,600 per g (dry weight), respectively. During the next drying cycle, concentrations in the water and soil rose to values of 4.4 × 10⁴ and 2.1 × 10⁴ per 100 ml, respectively. After the first 24 h, the E. coli concentrations in water appeared to stabilize at roughly 4 × 10⁵ E. coli organisms per 100 ml. The data for the water column are in contrast to the soil E. coli concentrations. After the rapid increase during the first 24 h, the soil E. coli concentrations would generally drop to lower values immediately after the soil was either lowered into or removed from the water. After lowering or removal, the E. coli concentrations generally increased until the soil setup was again changed. Apparently, once the E. coli concentrations reached values of greater than 10³ per 100 ml, a change in soil moisture resulted in the loss of E. coli.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Results from laboratory experiments designed to simulate tidal conditions.

	DISCUSSION

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This study has shown that the soils along the riverbanks were the primary source of E. coli in the river between storm events. This finding is supported by the facts that intensive grid sampling showed the highest E. coli levels near channel banks, site-specific soil sampling showed that sites with elevated E. coli levels in the water were also characterized by high E. coli concentrations in the soil, and E. coli concentrations were observed to correlate with tidal cycles, with the highest concentrations observed during high tide. During high tide the river is at a higher stage, thereby causing the water column to come in contact with soils that were previously dried. Laboratory experiments confirmed that upon soil drying, E. coli is capable of multiplying by several orders of magnitude. Predation has been shown to play an important role in E. coli survival within natural systems (7, 20, 22) and soil moisture appears to be associated with predator survival. It is likely that E. coli can survive at a lower soil moisture than its predators. Therefore, upon soil drying, conditions are suitable for E. coli growth. It is hypothesized that the degree to which the soil is dried plays a critical role in regulating growth. It is likely that the outer fringes of the channel banks, which experience the most extreme drying conditions, dominate the contribution of E. coli to the water column.

Another curious aspect of the field data is the fact that the cyclical variations in E. coli concentrations in water were observed 2 days after the storm had ceased. If fecal deposition were the primary source of contamination, then why are there no peaks during these 2 days? The scenario where E. coli multiplies within the bank soils is more likely. One plausible explanation for this observation is that the storm flushes E. coli from the soil banks and that it takes E. coli roughly 2 days to increase to levels that affect the water column to a noticeable degree.

It is also likely that local conditions affect the degree to which E. coli is capable of multiplying to elevated levels. The banks along most of the North Fork of the New River are characterized by relatively steep channel embankments or retained by seawalls. The large shallow and shaded embankments along I-95 and the Argyle Canal distinguish these sites from the remaining portions of the North Fork. These conditions are conducive to E. coli growth, since light tends to inhibit growth (25) and the long, shallow embankments provide a large surface area where wetting and drying periodically occur. South Florida's climate, characterized by warm and humid conditions, also plays a likely role in E. coli growth in soil. Such growth has been documented in other studies conducted in tropical and subtropical regions of the world (6, 16, 28-30, 37, 38).

For the North Fork of the New River, the initial list of suspected E. coli sources has been narrowed as a result of this effort, and a new, unanticipated source, i.e., soils within the riverbanks, also was identified. Riverbank soils were found to be a significant source between storms, with the largest E. coli concentrations observed during high tide. High E. coli concentrations were also observed during storms. Such correlations with rainfall have been observed in other studies (21), some of which identified overland flow (9, 18) and sewerage overflows (27, 32) as potential contributors of E. coli. Likely sources of E. coli in the North Fork of the New River during storms therefore include direct runoff from the riverbanks, storm sewer inflows, and sanitary sewer overflows. The riverbank soils were found to contain E. coli, and these contaminated soils can be washed in by direct runoff. Another mechanism by which direct runoff acts as a source is by washing in fecal matter deposited by animals along the banks. It is also recognized that storm sewers contribute water to the river during storms, and this added flow can very possibly contain elevated E. coli concentrations. A sanitary sewer source is also a possibility, since storms can aggravate infiltration and inflows, thereby causing an indirect connection to the river through sewer overflows.

Additional study is recommended to further pinpoint the source of E. coli in the North Fork of the New River during storms. These additional studies should begin by capturing and analyzing storm sewer flows and direct runoff prior to their entering the river. A comprehensive study to investigate the growth patterns of E. coli within riverbank soils from the North Fork and other water bodies in climates similar to that of South Florida is further recommended. Efforts should focus on the impacts of periodic wetting and drying on the growth of E. coli and the impact of soil properties (e.g., clay versus sands [15, 17, 31]) on supporting regrowth of the microbe. If the results of such a comprehensive study confirm that E. coli is capable of multiplying in soils of tropical and subtropical regions to the point that it affects E. coli levels within the water column, perhaps the use of E. coli as an indicator organism for these areas is flawed and other, more novel indicators of human wastewater contamination should be considered for regulating water quality in such regions.