CICEET Progress Report for the period 9/01/01 through 3/01/02

Project Title: Microbial Source Tracking in Two Southern Maine Watersheds
Principal Investigator(s): Kristen Whiting, Michele Dionne, Stephen Jones

Accomplishments
Scheduled Tasks:

• Sept. - Oct. 2001: Identify and confirm project steering committee members.
• Sept. - Nov. 2001: Confirm sampling design; identify sampling sites in the Webhannet watershed; lay out sampling schedule; secure land owner permission for site access where necessary.
• Sept. - Nov. 2001: Confirm lab methods.
• Dec. - May 2001: Collect upstream water samples and conduct lab procedures to enumerate and isolate colonies of fecal coliform bacteria and E.coli.
• Oct. - April 2002: Collect fecal material from Webhannet watershed to build a local library of known samples for use in microbial source determination at Jackson Estuarine Lab (JEL).
• Feb. 2002: At JEL, conduct microbial source determination on isolates collected between Dec. 2001 and Feb. 2002.
• Aug. - Dec. 2002: Design and implement outreach strategies.

Progress on Tasks
Identify and confirm project steering committee members.
A nine-member steering committee (including the principle investigators) has been created to ensure that local, regional and state-level stakeholders are active participants in the design and implementation of the project. Their advisory role will result in greater awareness of project objectives and activities during the project as well as promoting community support of project results at its conclusion. Their input has thus far primarily consisted of recommendations for sample site locations in the estuarine portions of the Webhannet watershed and providing input regarding the selection of bacterial test methods. To date, communication with steering committee members has been managed via phone and email. To ensure timely and complete information sharing with members, a practice of forwarding bi-monthly project updates has been implemented. The focus of these updates is to summarize the findings from field sampling, describe tasks accomplished and tasks upcoming, and to pose strategic and technical questions of committee members.

Because this project employs innovative ribotyping methods that have not been widely used, our work has attracted the attention of many practitioners in the field of water quality analysis. To keep informed of our progress, these practitioners have asked to receive bi-monthly project updates also.

Confirm sampling design; identify sampling sites in the Webhannet watershed; lay out sampling schedule; secure landowner permission for site access where necessary.
By referring to baseline data established in Webhannet Estuary Watershed Non-Point Source Pollution Investigation (Mullan, C., Dionne, M., Whiting-Grant, K., 2001) and with guidance from the Principle Investigators and steering committee members, 21 sampling sites have been identified in the Webhannet watershed. (See Figure 1) Sample site locations were selected to provide comprehensive watershed coverage, ease of access and close proximity to suspected sources of fecal contamination. The sampling schedule was designed to provide for sampling before, during and after the local clam harvesting season from December to May and includes one pre-scheduled (random) and one post-precipitation event per month. The precipitation-related sampling takes place within two - 24 hours after the start of the event. Due to lab processing times, sampling must occur between Monday and Thursday, inclusive. All of these sites except four are located above the head-of-tide in freshwater/upstream sections of the watershed. The four sites located in estuarine sections of the watershed were sited at or near sampling sites of the Maine Department of Marine Resources (MDMR) in order to provide a common reference point with state-level fecal coliform monitoring.

After potential sampling sites were identified, project staff worked with GIS resources available at the Wells Reserve to view the watershed map with a tax map layer to identify those sampling sites on private property. Letters describing the project and requesting permission for access were forwarded to all affected property owners. The letter was worded to require a response only if access was prohibited; one landowner responded to deny access.

Confirm lab methods
Project staff are using the membrane filtration method with mTEC agar to allow for the enumeration of both fecal coliforms and Escherichia coli (E. coli). Other methods were initially considered, but none of them simultaneously tested for fecal coliform and E. coli. The Department of Environmental Protection uses E. coli as a fresh water standard while the Department of Marine Resources uses fecal coliform for marine waters. Because the sample sites for this project are located in both fresh and marine waters, the mTEC method accommodates all relevant water quality standards.

Collect upstream water samples and conduct lab procedures to enumerate and isolate colonies of fecal coliform bacteria and E.coli
At this point, six sampling events have been conducted, two precipitation dates and four random dates.

Twenty-four volunteers have been recruited and trained to collect water samples using the WhirlPac bag method. Four volunteers with previous lab experience have also been intensively trained to assist project staff (graduate student Fred Dillon and AmeriCorps member Cayce Dalton) in conducting membrane filtrations of the samples and then to count the resulting fecal coliform and E.coli colonies. To ensure the highest possible sample quality, volunteers are subject to strict quality assurance protocols.

The protocols for isolating and enumerating target fecal bacterial contaminants from water samples have been discussed in detail and procedures have been finalized. Project staff select samples for E. coli isolation based on the DEP's seasonal (May-September) water quality standard of 64 colonies for Class B surface waters (all surface waters in the Webhannet watershed are Class B). Any samples with colony counts of 64 or greater are selected for isolation, which consists of removing individual E. coli colonies from the mTEC petri dishes and placing them on T-soy petri dishes. The T-soy is a non-selective growth medium that allows each isolated E. coli colony to grow more cell mass for eventual genetic analysis. Ten separate colonies are selected from each mTEC plate for isolation to provide a diversity of potential contamination sources. In theory, each isolated colony can originate from a different host species.

The logistics of transporting putative E. coli isolates from Wells, ME to the UNH ribotyping lab have been tested and optimized. All putative E. coli isolates provided have been tested by a battery of biochemical tests, and those confirmed as E. coli have been checked for purity and are being maintained on agar media or are preserved at -80°C. As of March 13, 2002, there had been 331 isolates that have been confirmed as E. coli. These were collected on five different dates from December 19, 2001 to February 21, 2002, and from as many as fourteen different sites.

Collect fecal material from Webhannet watershed to build a local library of known samples for use in microbial source determination at Jackson Estuarine Lab (JEL).
In addition to water sampling, the project also requires some collection of fecal material from potential source species in the watershed. With the help of a professional trapper from Wells, six scat samples were collected on October 25, 2001. Positively identified scat samples include that of coyote, deer, gray fox, red fox, raccoon and squirrel. Still needed are samples from humans, house pets such as dog and cat, and domesticated animals such as horse, cow, etc. Additionally, a large beaver dam on a tributary of the Webhannet and a duck pond on a tributary of Depot Brook indicate that samples from these species are also needed for the library of known samples. JEL has successfully isolated and preserved four E. coli isolates each from the feces of the six source species identified above. These feces samples were processed on October 26, 2001. This is a small number of isolates, and hopefully more samples can be collected. The range of source species also may not be representative of potentially significant source species, especially if humans are suspected sources. It is important that human fecal samples be collected, best from watershed septic systems.

At JEL, conduct microbial source determination on isolates collected to date. The nature of the ribotyping process is such that it is difficult to produce results early in the project. This is because of the need to build up a source library as a reference for identifying the source species of isolates from water samples. This project will greatly benefit from being in close enough proximity to other ongoing projects that have developed source libraries. These source libraries from other projects will soon be completely integrated into the JEL database. After that, isolates from water samples can be processed and the resulting DNA profiles compared to source library profiles for preliminary identification of source species. At the end of the project, a re-evaluation of source species with the full library that includes new source species profiles collected as part of the present project will be required.

Design and implement outreach strategies. To provide a forum for information sharing, a project web site is being designed (www.umseagrant-mst.org). In this early phase of the project, the target audiences for the site are members of the project steering committee, current volunteers, potential volunteers, interested community members and the project funders. Here, steering committee members can review archived project updates and view the latest data; current volunteers can check the schedule of sampling dates and view digital images of themselves in action; potential volunteers can email questions to the volunteer coordinator; interested citizens can learn about MST; and you as the funders, can review the digital versions of all project materials. In later phases of the project, the web site will also become an outreach tool to share project findings with local, regional and state-level officials who will use the findings to guide action planning for contaminant reduction. Additionally, the site will communicate project results to a national audience regarding the potential for applying microbial source tracking technology elsewhere in the county.

Project staff will be presenting an overview of the research program along with preliminary findings in May at the New England Interstate Water Pollution Control Commission's 13th Annual Nonpoint Source Meeting. They will also be discussing the various alternative microbial source tracking techniques used by researchers elsewhere.

Maine Public Radio is also planning to conduct a feature story on the project. Correspondent Naomi Schalit will join project staff in the field on May 16 and the coverage should air soon after.

Difficulties Encountered
Collection of known samples for the development of a local fecal library has been challenging. Project staff do not possess expertise in scat identification, therefore knowledgeable resource people must be recruited. Coordinating field time with these experts is difficult and as a result project staff have not yet successfully gathered all the samples needed.

The lack of an "interagency standard" for bacterial testing has been problematic. MDEP tests for E. coli in freshwater and MDMR tests for fecal coliform in estuarine and marine waters, each using a different and incompatible test method. This does not interfere with the central project goals, but it limits the potential for wider use of the bacterial monitoring done in the process. By testing for both E. coli and fecal coliform we hope our results will have the maximum utility possible.

Anticipated Success in Meeting Project Objectives in Scheduled Project Period
We do not anticipate problems in meeting project objectives (see below for upcoming schedule).

Preliminary Results
The bacterial levels in the freshwater portion of the watershed have shown a general decline as the winter has progressed, with a few exceptions. High bacterial counts resulted from the February 21, 2002 post precipitation sampling, and average counts resulted from the January 24, 2002 post precipitation sampling. Despite the general trend of lower counts, the data is quite variable, ranging from a maximum of approximately 550 CFU/100mL, to minimums of <15 CFU/100mL (See individual charts by date). Ribotyping analyses of Webhannet watershed and estuarine samples will be completed in the next reporting period.

Tasks and activities for next reporting period

Tasks for the next reporting period

• March - May 2002: Continue to collect upstream water samples and conduct lab procedures to enumerate and isolate colonies of fecal coliform bacteria and E.coli.
• March - May 2002: Continue to collect fecal material from Webhannet watershed to build a local library of known samples for use in microbial source determination at Jackson Estuarine Lab (JEL).
• April - May 2002: Design "hot spot" focused watershed survey.
• April - May 2002: Confirm estuarine sampling design; identify sampling sites in the Webhannet estuary; lay out sampling schedule; secure land owner permission for site access where necessary.
• June 2002: Conduct Webhannet watershed survey.
• June - Sept. 2002: Collect estuarine water samples and conduct lab procedures to enumerate and isolate colonies of fecal coliform bacteria and E.coli.
• June 2002: At JEL, conduct microbial source determination on isolates collected in the Webhannet watershed between March and May 2002.
• Sept. - Oct. 2002: Identify and confirm steering committee members for Little River watershed project.
• Sept. - Nov. 2002: Confirm sampling design; identify sampling sites in the Little River watershed; lay out sampling schedule; secure land owner permission for site access where necessary.
• Sept. - Nov. 2002: Confirm lab methods for Little River watershed project.
• Oct. 2002: At JEL, conduct microbial source determination on isolates collected from the Webhannet estuary between June and Sept. 2002.
• Nov. 2002: Prepare and disseminate final Webhannet watershed report and begin to provide presentations on findings at the local, regional and state-levels.
• Nov. - Dec. 2002: Design and begin to implement community outreach strategies regarding the watershed-specific results of the Webhannet project.
• Dec 2002: Begin to share results of the Webhannet project with regional and national professional organizations.

Work plan to accomplish tasks
Work plan is described in attached time line.