| 
|
When a body of water is found to contain high levels of fecal coliform, accusations may start to fly about who is responsible. Is it the local wastewater treatment plant? Or maybe the farmers are allowing their cattle to graze near the streams? Or could it be the deer in the wildlife park? Well, before you haul Bambi away in the paddy wagon, there is now a better way to identify the source of the contamination. Just as DNA technology is used to solve crimes, settle paternity disputes, and identify bodies, this same concept of DNA “fingerprinting” is being used in many parts of the US to identify the culprits of fecal contamination in watersheds.
The Need for BST
Bacterial Source Tracking (BST) is a relatively new methodology that has been developed and deployed in many parts of the US to identify sources of fecal contamination in watersheds. Fecal bacteria are isolated from water samples, and BST methodology is employed to determine the sources of those bacteria, whether it be human, livestock, or wildlife in origin. The implementation of the Total Maximum Daily Load (TMDL) by the US Environmental Protection Agency (EPA) has been the driving force behind BST development.
A TMDL is a calculation of the maximum amount of a pollutant that a watershed can receive and still meet water quality standards. Under section 303(d) of the 1972 Clean Water Act, states, territories, and tribes are required to develop lists of impaired waters. More than 40% of assessed watersheds do not meet the water quality standards these states, territories, and tribes have set for them. These impaired waters include approximately 300,000 miles of rivers and shorelines and approximately 5 million acres of lakes.
In an effort to achieve water quality standards and improve the TMDL program, in 1996 the EPA began a comprehensive evaluation of EPA’s and the states’ implementation of their Clean Water Act section 303(d) responsibilities. After this evaluation, new TMDL rules were established requiring states, territories, and tribes to come up with a plan for implementing load allocations for waters impaired solely or primarily by nonpoint sources.
Although it was relatively easy to find out if a watershed contained fecal bacteria, in many cases proving who or what were the sources of contamination was much harder, making the implementation of a plan for best management practices (BMPs) difficult. But now, by using BST to pinpoint the sources of contamination, implementation is no longer a crapshoot, so to speak.
Potato Creek
When the Potato Creek watershed in Georgia TMDL came back containing high levels of fecal bacteria, the source was not immediately known. The watershed covers approximately 237 square miles and flows from the city of Griffin to a confluence with the Flint River in Upson County. An Environmental Advisory Committee (EAC) was established by the McIntosh Trail Regional Development Center (RDC) in early 2001 to address the fecal coliform issue. Even though the source of the fecal coliform was not known, a preliminary TMDL implementation plan was submitted to the Georgia Environmental Protection Division of the Department of Natural Resources. The McIntosh Trail RDC subsequently committed to sponsoring a project to identify the sources of fecal bacteria in the watershed, to identify the BMPs to achieve the desired pollutant load reduction, and to draft a revised TMDL implementation plan.
Integrated Science & Engineering Inc. was contracted by the McIntosh Trail RDC to perform E. coli and fecal coliform sampling to identify the specific areas of concern with the Potato Creek watershed. “We suspected that the problem was several large cattle farms that are right along the creek, but at the head of the creek is the city of Griffin’s wastewater treatment facility,” says Dan Davis, president of the Fayetteville, GA-based Integrated Science & Engineering. “So the farmers were pointing their fingers upstream at the treatment plant as the source.”
Integrated Science & Engineering conducted water sampling along Potato Creek watershed to pinpoint stretches that contained high levels of fecal coliform. Three areas were identified as containing high levels of fecal contamination. Then Peter Hartel, professor of crop and soil sciences at the University of Georgia in Athens, employed bacterial source tracking methods to determine the sources. “Targeted sampling is nothing more than a children’s game of hot and cold to find the areas of contamination,” Hartel explains.
Results of the targeted sampling and BST indicated that in the Griffin reach, the most likely fecal sources were humans (a broken household sewer line), pets, and urban wildlife. In the Meansville reach, the most likely fecal sources were grazing cattle, and in the Thomaston reach the most likely fecal sources were dogs from a kennel and wildlife.
BST Methods
While BST methods are constantly advancing and changing, the three basic types are molecular, biochemical, and chemical. Molecular (genotype) are all referred to as “DNA fingerprinting” and are based on the unique genetic makeup of different strains, or subspecies, or fecal bacteria. The fecal bacteria in any two animals are very much genetically similar. There are unique differences, but the differences are only in a small percentage of an organism’s total DNA. The key to molecular BST is finding these differences against a large background of similarity. Some types of molecular BST include ribotyping, pulsed-field gel electrophoresis (PFGE), and randomly amplified polymorphic DNA (RAPD).
 |
| Photos depict: Dog kennels (A, on Clay Street; B, between Forrest Avenue and Crescent Road; and C above Tilney Avenue), and cat box filler (D, below Crescent Road) located on the Griffin reach. |
One commonly used biochemical (phenotype) BST method is antibiotic resistance analysis (ARA). This method uses fecal streptococcus (enterococcus) and/or E. coli and patterns of antibiotic resistance for separation of sources. The premise is that human fecal bacteria will have the greatest resistance to antibiotics and that domestic and wildlife animal fecal bacteria will have significantly less resistance to the battery of antibiotics and concentrations used. Other biochemical methods include f-specific coliphage and sterols or fatty acid analysis, and fecal bacteria ratios.
Chemical BST methods do not detect fecal bacteria; instead these methods are designed to detect chemical compounds that are associated with humans. These chemicals are often found in wastewater effluent. Some of these methods detect optical brighteners that are in all laundry detergents, and there is a method to detect caffeine, which passes through the human digestive system.
With many BST methods it is necessary to first build a library or database of isolates taken from known sources such as humans, cattle, deer, dogs, etc. The size of the library is partially determined by the number of potential major sources of pollution in the targeted area.
One molecular method that does not require library of sources is the bacteroides fecal source tracking method. This method was created by Katherine Field, associate professor of microbiology, and her lab at Oregon State University. “The key thing is that we are using a different group of bacteria (bacteroides) than public health bacteria such as E. coli and the reason we are is that the bacteria we are using are more common and have more genetic diversity,” says Field. “The second key point is that it is PCR (polymerase chain reaction)-based, which means we are not growing bacteria and then administering some kind of test to tell them apart; we are simply taking water samples directly and testing them.”
Many researchers in the BST field do not solely use one particular method but instead use a combination of techniques. For example, in the Potato Creek watershed, Hartel used fluorometry, enterococcus specification, and detection of the enterococcal surface protein (esp) gene. Fluorometry identifies human fecal contamination by detecting the presence of fluorescing compounds, like optical brighteners in water from laundry and dishwashing detergents. Enterococcus speciation identifies sources of fecal contamination by determining the percentage of one of the fecal enterococci present in the water sample. A high percentage indicates human and bird contamination. And detection of the esp gene in enterococcus faecium isolates indicates human fecal contamination. “ I wanted to try several different inexpensive methods,” Hartel says. “We were not interested in any method that was expensive, and we wanted a method that anyone could use.”
Cost and Effectiveness of BST
There is some controversy over the effectiveness of BST methods, and this has prompted the EPA and US Geological Survey (USGS) to begin comparison studies of various BST methods to find out which is most effective. The general consensus is that biochemical BST methods are simpler, faster, less expensive, and allow large numbers of samples to be analyzed in a short period of time. Molecular BST methods may offer the most precise identification of specific types of sources, but are limited by high per-isolate cost and detailed, time-consuming procedures. However with the Bacteroides Fecal Source Tracking method Field reports that it is “cheaper and quicker than other methods” because you don’t have to grow the bacteria or create a library of sources.
According to a press release from the USGS concerning the comparison study of BST methods, when researchers sent E. coli challenge isolates (the sources of which were unknown to those conducting the tests) for testing, many isolates either remained unclassified or were classified to incorrect sources. In all, fewer than 30% of challenge isolates were classified to the correct source-animal species by any method.
Prior source tracking research reports cite accuracy ranges from 60% to 90% for various source tracking methods. The authors of the USGS study attribute the discrepancy between the 60%–90% accuracy rates and the 20%–30% accuracy rates they reported to a number of other factors:
- Different bacteria may be present in animal guts in different seasons. In the USGS study, challenge isolates were collected 9 months after the reference feces were collected;
- There may be too many strains of E. coli bacteria in each animal species for effective application with small reference libraries, such as the 900 reference strains in the USGS study. At a cost of $10 to $100 to analyze one reference strain, however, building large source libraries gets expensive rather quickly.
- E. coli strains may not be truly specific to one animal source. Some E. coli strains have been found in more than one animal source, such as when animals live in close proximity with one another, though no evidence to support this premise was found in the USGS study.
 |
| A section of broken sewer pipe sags near Springer Drive in the Griffin reach. |
“All too often, results of these analyses are offered without enough validation of accuracy,” explains Dan Stoeckel, USGS technical lead for the project. “Source tracking study results should always be supported by quality control data. Interpretation of the results can be supported by multiple lines of evidence, such as land-use data and presence of wastewater chemicals such as caffeine, when pointing to sources of contamination. Both the client and the analyzing laboratory have a part in making sure interpretations are accurate.”
Hartel believes that if targeted sampling were used during this study the results would have been much better. “Targeted sampling would have reduced all the environmental complexities,” he says.
Another factor that is often overlooked, according to Hartel, is sediment. “The federal government is not paying much attention to sediment, even though we have known for years that it is a serious problem. I can literally play patty cake with the sediment and cause virtually any stream to go out of compliance,” Hartel says.
Another way to help insure that BST is accurate is to use a “toolbox” approach, selecting the method that seems warranted to the particular situation. In addition, using more than one BST method also improves the chances that the source identifications are correct.
 |
| Cows wander through a small, unnamed tributary of Potato Creek. |
 |
| One of Potato Creek's tributaries lies directly behind this pit bull kennel. |
BST Helps With BMPs
Charles Hagedorn, professor of environmental sciences at Virginia Tech in Blacksburg, VA, feels that BST can be a valuable resource and should be mandatory. “I think that source tracking should be used in every impairment that’s on the nation’s impaired waters list where the impairment is due to fecal contamination,” Hagedorn says. In part due to Hagedorn’s urging, the state of Virginia is now requiring BST be performed on every impaired watershed.
Hagedorn conducted BST on Page Brook Creek in Clarke County, VA. The watershed is located in a rural area with an agricultural economy. The dominant source of the fecal contamination was cattle. Since that time, BMPs have been deployed. There are roughly 18 farms in the vicinity, and most have done some type of management to reduce livestock access to the stream. Hagedorn says he has been monitoring the creek regularly, and in areas where farmers have either fenced the stream or limited livestock access to the stream, the cattle fecal coliform signature has disappeared.
However, Hartel does not believe BST should be conducted on every impaired watershed. In many cases, just by observing the surroundings, the sources of fecal contamination are obvious. For instance, in Potato Creek a broken sanitary pipe, cattle access to the stream, and a dog kennel were easily spotted. “Most of the time it is obvious where the contamination is coming from. There are one or two cases that you might come up against where you don’t know if it is a leaking septic tank or a swine lagoon,” Hartel says.
Now that the sources of the fecal coliform have been uncovered in the Potato Creek watershed, the job of how to clean it up begins. The final step in the development of the TMDL implementation plan for Potato Creek is to develop BMPs that target the specific fecal bacteria sources that were identified through the BST. For the Griffin reach, Integrated Science & Engineering has recommended the replacement of sanitary pipe, as well as the implementation of illicit discharge detection and elimination program and a sanitary sewer inspection program. In the Meansville reach the company recommended a grazing management plan that includes exclusion fencing, removed watering systems, and riparian buffer restoration and protection. And finally, for the Thomaston reach, a pet waste education campaign was recommended, along with site inspections for dog kennels. Once these BMPs are implemented, the fecal coliform levels should be reduced.
As with any technology, BST will continue to evolve, change, and be refined. And when a community is faced with a contaminated water supply, residents and officials naturally want to clean it up quickly and painlessly. When used correctly, Bacterial Source Tracking can be a key tool in doing that. Hopefully, in the future, it can become a quick, easy, and inexpensive way for testing all watersheds.
NIKKI STILES is a freelance writer based in the city of Fairmont, WV.
OW - July/August 2006 |
