Evaluation of Fecal Contamination in Strawberry River

Part of the Young Naturalist Awards Curriculum Collection.

by Joshua, Grade 11, Arkansas, 2011 YNA Winner


Western Lawrence County Waste Water Treatment Facility

Arkansas is a majestic state, with mountain ranges, delta regions, countless lakes and endless miles of rivers and streams. As a young child I spent many hours enjoying the cool refreshing water of Strawberry River. My favorite swimming hole was below an old abandoned bridge. A rope was tied to a tall oak tree, and I would use it to swing over the deepest spot and plunge into the river. Fish abounded in the waters, and everyone in the community picnicked in the shade by the sandy beach. It was the perfect spot to while away the summer. That is, until the Western Lawrence County Waste Water Treatment Facility (WLCWWTF) was constructed half a mile upstream. The wonderland where I spent so many hours as a child is deserted now, and nobody swims or fishes in that section of the river. I decided to find out for myself if the WLCWWTF had indeed contaminated the water, or if the community had overreacted.

Taking the temperature at site 1
A small kit with equipment used for field testing.
Equipment used for field testing

In November and December 2009, I tested the water quality at three sites and used the National Sanitation Foundation's Water Quality Index (Mitchell and Stapp, 1997) to determine if the water quality of Strawberry River in Lawrence County has been affected by the WLCWWTF. I tested the pH, temperature, turbidity and total solids of the water. I also tested for nitrates, phosphates, fecal coliforms, dissolved oxygen, and the biochemical oxygen demand of the river. I performed a visual evaluation of the vegetation, bank erosion and stability, and physical characteristics of each test site. Primary uses and impairments of the water, its odors and appearance, were also assessed. Land use and habitat assessment were also considered in determining the overall quality of the Strawberry River (Mitchell and Stapp 1997).

2011 Young Naturalist Award winner Joshua, 17, of Arkansas
Checking for phosphate at site 2

One test site was located five miles above the facility, the second a half-mile below the facility, and the third five miles below. I determined that all three sites had "good" water quality, and therefore the facility did not adversely affect the overall quality of the water. However, all three sites had high fecal coliform colony counts, which indicates the presence of pathogenic organisms. The high fecal coliform count signified that although the water quality was "good," it might not necessarily be safe for primary (full-body contact or swimming) and secondary (contact below the knees or wading) recreational contact by humans (Table 1 and Table 2). Excessive flooding during the spring, summer and fall of 2009 may have adversely affected the results. I decided to conduct further research on current water quality standards and perform additional tests to determine the risk to individuals during primary and secondary contact with the river.

A medium-narrow river with still looking water. Both side of bank have brown leafless trees.
Site 1
Young man standing near river writing in a notebook.
Performing visual evaluation at site 3

Water quality standards define the use of a body of water and describe the specific water quality criteria to achieve that use. These standards also contain antidegradation policies to protect existing water quality ("Pathogen TMDLs for Planning Segments 4G Reaches" 2007). Water-quality standards are established on the basis of an acceptable risk of illness that most people are willing to take to participate in water-related activities. The Environmental Protection Agency (EPA) administers the water quality standards program and is responsible for providing water quality criteria recommendations and enforcement of standards compliance by each state. In response to widespread public concern about the condition of our nation's waters, the United States Congress enacted the Federal Water Pollution Control Act Amendments of 1972 (Clean Water Act of 1972). The Clean Water Act of 1977 and the Water Quality Act of 1987 strengthened the law to better protect our nation's water ("Bacterial Water Quality Standards for Recreational Waters Status Report" 2003). In 1997, the EPA established the Beaches Environmental Assessment and Coastal Health (BEACH) program. The goals of BEACH were to strengthen beach standards and testing, provide faster laboratory test methods, predict pollution, invest in health and methods research, and inform the public ("Beach Monitoring and Notification" 2010). The BEACH Act of 2000 authorized the EPA to award grants for the development and implementation of programs to notify the public of potential exposure to disease-causing microorganisms ("Beaches Environmental Assessment and Coastal Health Act of 2000").

Photo of the third of four test sites in the Strawberry River in Arkansas
Site 3
Site 2

Approximately 13% of surface waters in the United States do not meet designated use criteria because of high densities of fecal indicator bacteria ("Microbial Source Tracking Guide Document" 2005). It was recognized in 1930 that human excreta discharged to surface waters pose a health hazard to those who used the water for recreation ("EMPACT Beaches Project: Results from a Study on Microbiological Monitoring" 2006). Despite the uncertainty of the effects of animal fecal contamination of ambient waters to human health, microbiological contamination of recreational waters from human feces is regarded as a greater risk to human health as they are more likely to contain human-specific pathogens. Microbial source tracking (MST) is a method of examining the bacteria found in water samples for clues to determine the host species. These methods are used to trace the origin of fecal pollution using microbiological, genotypic, phenotypic and chemical processes. Fecal coliforms have been used for many years as indicators for determining the sanitary quality of surface and recreational water. Escherichia coli (E. coli) and Enterococci are considered to have a higher correlation with outbreaks of swimming-associated gastroenteritis than total and fecal coliforms, and were recommended as the basis for bacterial water quality standards in the 1986 report "Ambient Water Quality Criteria for Bacteria."

Measures of total coliforms are currently used for testing drinking water. Some coliforms are of fecal origin and others are found in the environment. Fecal coliforms are more fecal-specific than total coliforms and are used to determine shellfish-bed closures. Fecal coliforms by themselves are not pathogenic, but they are commonly found in the presence of viruses, bacteria, parasites and pathogens that can be harmful. They provide indirect evidence of the possible presence of pathogens in the water.

E. coli is a single species within the fecal coliform group. It is more fecal-specific than fecal coliforms and is less likely to give a false positive result. E. coli's presence in water is often associated with waterborne illness outbreaks caused by other pathogens. It does not live long in water, so its presence indicates a fairly recent contamination event (up to 48 hours). Normal testing for E. coli provides a sensitive measure of fecal pollution, but it cannot determine the source of the contamination (animal versus human) since it is present in all warm-blooded mammals (Wheeler et al. 2002).

Enterococci are 17 related species commonly found in the intestines of warm-blooded animals. They are not related to coliforms but are more fecal-specific than fecal coliforms. Each species of Enterococci can be traced to one or more hosts. Enterococcus faecalis is a good candidate bacterium for the identification of human fecal contamination because it is primarily found in humans and chickens (Wheeler et al. 2002).

There is a better reason for identifying Enterococci in recreational waters than simply reducing the occurrence of water-related illnesses. For many years, Enterococcus species were believed to be harmless to humans and considered medically unimportant. Because they produce bacteriocins, Enterococcus species have been used widely over the last decade in the food industry as probiotics or as starter cultures. Recently, Enterococci have become one of the most common nosocomial (hospital) pathogens, with infected patients having a mortality rate of up to 61 percent. The ability of Enterococcus species to survive a range of adverse environments allows for multiple routes of cross-contamination by Enterococci to cause human disease, including food, environmental and hospital sources (Fisher and Philips, 2009).

In 1986, the original test method for Enterococci was introduced by the EPA. It required the use of two media: m-Enterococcus (mE) agar as the primary isolation medium, and Esculin Iron Agar (EIA) for confirmation of the colonies. This method used a membrane filter (MF) procedure for direct counts of Enterococci. The filter containing the bacteria is placed on the mE agar and incubated for 48 hours, then transferred to the EIA and incubated for an additional 20 to 30 minutes for confirmation of the colonies ("Improved Enumeration Methods for the Recreational Water Quality Indicators Enterococciand Escherichia coli" 2000).

The m-E agar was later enhanced with triphenyltetrazolium chloride (TTC). This modified medium proved to be superior medium for the enumeration of Enterococci. It eliminated the need for a second medium for confirmation, and permitted a direct count of Enterococci in 48 hours ("M-Enterococcus Agar"). In 1997, a modified test method for Enterococci was recommended by the EPA. It required that a membrane filter be placed on the m-E agar and incubated for 24 hours ("Improved Enumeration Methods for the Recreational Water Quality Indicators Enterococci and Escherichia coli"). The modified method improved analytical quality and reduced analysis time from 48 hours to 24 hours. Use of this method can result in earlier notification to the public about health hazards at beaches.

The Question: This study attempted to evaluate the risk of water-related illnesses from fecal contamination to individuals coming into primary or secondary contact with the Strawberry River. It also compared the number of colonies attained with Enterococcus faecalisEscherichia coli, and fecal coliform microbial indicators.

The Hypothesis: I hypothesized that Strawberry River was not safe for primary or secondary contact, and that individuals using Strawberry River as a means for recreation were at risk of water-related illness from fecal contamination. I also hypothesized that of the two microbial indicators recommended by the EPA, Enterococcus faecalis would be a better microbial indicator of human fecal contamination than Escherichia coli.

Methods and Materials

Site 1

Sampling Sites and Protocols: In November 2010, I collected water samples from four sites located on Strawberry River in Lawrence County, Arkansas. Sampling sites were located a half-mile and five miles above the WLCWWT facility, as well as a half-mile and five miles below the facility. Water samples were collected in sterilized water bottles at all four test sites in the morning before school began. Each bottle was placed in the claw of a three-foot extension reach-and-grab; it was plunged below the surface of the water facing into the current to collect the sample, the lid was replaced, and the bottle was marked with the corresponding site allocation. Samples were kept cool and were processed within six hours of collection.

Site 2

Petri Plate Preparation: Fifty 50 mm-by-9 mm depth petri plates were prepared at Arkansas State University. Dehydrated difco m-Enterococcus agar (21 g) and deionized water (0.5 L) were heated and boiled for one minute on a hot plate in a 1.0 L flask. The agar was cooled to 42 degrees, then approximately 5.0 mL of the agar was distributed into each sterilized petri dish with a sterilized 6.0 mL pipette (m-Enterococcus agar, Screen 1). The m-Enterococcus agar petri plates were stored in a tightly sealed bag and refrigerated until needed.

Site 4
Site 3

Microbiological Analyses: A sterile pipette was used to transfer water samples to a sterile membrane filtration system, and each sample was filtered onto its respective 4.5-mm gridded membrane filter. Sterile forceps were used to place each filter in a petri dish of m-Enterococcus agar or in a petri dish of 2.0 mL Coliscan MF. Distilled water was used to rinse the membrane filtration system and forceps before processing samples from additional sites. All samples were incubated in a Quincy Lab Incubator at 35 degrees (0.5 deviation).

Plates containing Coliscan MF were removed after 24 hours. Each plate was placed under a microscope for enumeration of fecal coliforms and E. coli. Colony counts were recorded for analysis and verified using a fluorescent lamp and a magnifying lens. Plates containing m-Enterococcus agar were removed after a total of 48 hours of incubation. A microscope, a fluorescent lamp and a magnifying lens were also used to determine the E. faecalis colony count for each sample.

Heating and mixing de-ionized water and dehydrated m-Enterococcus agar
Collecting water samples
Conclusion and Discussion

The hypothesis was supported. Strawberry River was determined to be unsafe for primary and secondary recreational contact. Individuals using Strawberry River as a means for recreation are at risk of water-related illness from fecal contamination.

It was also affirmed that of the two microbial indicators currently recommended by the EPA, Enterococcus faecalis provided the most consistent test results for fecal contamination. Each site on each test date exceeded bacterial water-quality standards for Enterococcus faecalis. E. coli only indicated unsafe levels of fecal contamination on the November 2 test date for Site 2 and Site 3. It also failed to show any levels of fecal contamination on November 9. The data obtained during the 2010 project indicated that each test site exceeded acceptable levels of fecal coliforms for each test date.

Preparing petri plates by transferring 5 mL of prepared m-Enterococcus agar to each dish

Sites were scheduled for testing during the first three weeks of November. Testing was rescheduled for the fourth week of November because 18 mm of rain fell immediately prior to the third test date. Water sample volumes of 5.0 ml, 2.0 ml, 1.0 ml, and 0.5 ml were used on 2 November 2010. These were the sample sizes the EP. recommends for testing rivers. Test results from last year indicated that these water volumes would produce sufficient colony counts (between 20 and 60) per plate. Each plate of E. faecalis produced less than 14 colonies, so the sample sizes for the 8 November 2010 were raised to 20 ml, 10 ml, 5 ml, and 2 ml. The E. coli colony count was less than nine colonies per plate, but the fecal coliform colony count was above 116 per plate; therefore the water sample volume for the Coliscan MF remained at 5 ml for the second sample date.

The sample sizes used during the second test date proved to be too small to provide adequate colony counts for the majority of the m-Enterococcus agar plates, so the sample sizes for the third test date were raised to 30 ml, 20 ml, and 10 ml. The Coliscan MF plate samples were also raised to 20 ml on the third test date due to the smaller fecal coliform colony counts and the absence of any E. coli colonies during the second test date.

Arkansas is in Region 4 of the EPA's Bacterial Water Quality Standards for recreational waters. In 1986 the microbial indicator assigned to Arkansas by the EPA was fecal coliforms. Arkansas, at its discretion, uses E. coli, as its primary indicator of fecal contamination ("Arkansas Swim Beach Program" 2010).

Using the membrane filtration method to transfer water samples to a sterile membrane filter

The Arkansas Department of Environmental Quality (ADEQ) and the Arkansas Pollution Control and Ecology Commission develop pollution limits that reflect the historic use of the state's waters. The designation and protection of specific uses is required by the Federal Clean Water Act and the Arkansas Legislature. The locations of the four test sites on Strawberry River in Lawrence County selected for the project had been assigned the following designations by the ADEQ: Extraordinary Resource Waters; Ecologically Sensitive Water Bodies (Strawberry River darter); Primary Contact Recreation (full-body contact); Secondary Contact Recreation (wading, boating, fishing); Fisheries; and Domestic, Industrial and Agricultural Water Supply ("Regulation No. 2" 2010).

Using the membrane filtter

Soils and Topography: Strawberry River is located in the Ozark Highlands, with a 4G designation. Its soils range from deep stony soils to shallow clay and loamy soils. The topography is characterized by rolling hills, steep valleys and ridges ("Pathogen TMDLs for Planning Segments 4G Reaches Segments" 2007).

Land Use: The predominate land use is forest (80.4%). Pastureland is the second most prevalent land use (15.1%). The remaining use is cropland (2%), transitional (2%) and wetland (0.01%) ("Pathogen TMDLs for Planning Segments 4G Reaches Segments" 2007).

Using tbrane filter

Climate: The average rainfall in Strawberry is 47.6 inches per year. The total amount of precipitation for Strawberry during 2009 was approximately 72 inches of rain, which resulted in numerous episodes of severe flooding throughout the year. Approximately 30 inches of rain fell during 2010, with drought conditions during September, October and November prior to water sampling.

Point Source Discharge: The Western Lawrence County Waste Water facility located within the testing area provides service to the towns of Strawberry and Lynn, with a combined population of 598. There are three facilities located several miles upstream from the test sites ("Pathogen TMDLs for Planning Segments 4G Reaches Segments" 2007).


Nonpoint Source Contamination: Wildlife resources of fecal coliform contamination for Strawberry River include raccoons, beavers, muskrats, river otters and minks. White-tailed deer are also contributors, although most of their time is spent in terrestrial habitats, allowing time for decomposition between storm events. The greatest contributors of fecal coliform are waterfowl, most notably ducks and geese. Agricultural livestock often have direct access to the streams that pass through the pastures of grazing animals. Chicken litter that is applied to cropland can contribute fecal material during storm events. Site 4 is located between Reeds Creek and the WLCWW facility. There are 50 poultry houses on 19 farms in the Reeds Creek watershed. Site 4 was selected because Reeds Creek has the highest potential for negative impact on water quality because of the concentration of poultry farms in the small tributary watershed ("Pathogen TMDLs for Planning Segments 4G Reaches Segments" 2007).

Chart 1: Enterococus faecalis

In November 2009, I completed a water quality project on three test sites of Strawberry River using the National Sanitation Foundation's Water Quality Index. Fecal coliform was the bacterial indicator used to determine if the water quality of Strawberry River in Lawrence County Arkansas had been affected by the Western Lawrence County Waste Water facility.

Chart 2: Escherichia coli

In November 2010, I tested four sites on Strawberry River. The microbial indicator fecal coliform was used to obtain fecal colony test results for comparison with results documented in the 2009 water study. Both EPA-recommended microbiological indicators, E. coli and E. faecalis, were also used to determine the fecal contamination of the water.

Chart 3: Fecal coliform

During 2011, I plan to conduct my testing during the spring, summer and fall. This schedule of testing will allow me to determine the potential health risk before, during and after the season when individuals are most likely to participate in recreational activities involving primary contact with the Strawberry River. I will continue to test at the four sites on Strawberry River, and I will include two test sites on Reeds Creek. E. faecalis will be the microbial indicator I use because it provided the most consistent results in the 2010 study.

Insufficient colony counts of Enterococcus faecalis on November 2 test date.Sample sizes were: 0.5mL, 1mL, 2mL, 5mL
L: November 2 colonies - 5 mL Middle: November 8 colonies - 5 mLRight: November 22 colonies - 20 mL Fecal coliform indicated unsafe microbial levels on November 2 and November 22 test dates. E-coli failed to indicate any unsafe levels and no colonies were observed on November 8 test date.

First and foremost I thank Mr. Alan Smith, science instructor at Hillcrest High School, for challenging me to undertake this project, and for providing laboratory space, equipment and assistance during my research. I convey my gratitude to Dr. David Gilmore, professor of microbiology; Ms. Valerie Campbell, graduate student; and Arkansas State University in Jonesboro, Arkansas, for providing the dehydrated m-Enterococcus agar and the laboratory space, equipment and assistance needed for preparation of the m-Enterococcus agar petri plates. I thank Jeff Bristow for allowing me to cross his property to gain access to Site 4. In addition, I extend my appreciation to my mother, Yota Shaw, for providing financial support, transportation, verification of microbial colony counts and assistance with the research.

Acceptable quantity of colony growth of Enterococcus faecalis on November 22 test date.Sample sizes were 10mL, 20mL, and 30 mL

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Fisher, K., and C. Phillips. "The Ecology, Epidemiology and Virulence of Enterococcus." Microbiology155 (2009): 1749-1757. Retrieved on 10 August 2010 from 

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Mitchell, M.K., and W.B. Stapp. Field Manual for Water Quality Monitoring. 11th ed. Dubuque, IA: Kendall/Hunt Publishing, 1997.

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Wheeler, A.L., P.G. Hartel, D.G. Godfrey, J.L. Hill, and W.I. Segars. "Potential of Enterococcus faecalis as a Human Fecal Indicator for Microbial Source Tracking." Journal of Environmental Quality 31 (2001): 1286-1293. Retrieved on 5 May 2010 from http://www.water.rutgers.edu/Source_Tracking/Enterococcus