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

Accomplishments
Scheduled Tasks:

• March - May 2002: Complete collection of upstream water samples from the Webhannet watershed 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.
• Throughout: Conduct outreach efforts related to project methods and preliminary results.

Progress on Tasks
Complete collection of upstream water samples from the Webhannet watershed and conduct lab procedures to enumerate and isolate colonies of fecal coliform bacteria and E.coli.
Upstream water sampling ended in May, 2002. Between March and May six sampling events occurred, with four of them experiencing 0.5" of precipitation or greater during the preceding 36 hours and the two remaining dates experiencing virtually no precipitation during the preceding 36 hours. A total of eleven sampling events in the upper Webhannet watershed were conducted from December to May. Our hope was that roughly one-half of all sampling events would follow a significant runoff or precipitation event, which can vary considerably depending on a variety of factors.

Membrane filtration, water bath and colony enumeration procedures have been conducted on each of the 21 samples collected on each of these dates. Samples exhibiting colony counts greater than 64 have been isolated and sent to Jackson Estuarine Lab (JEL) for E.coli confirmation. (See Fecal Results and Maps)

In order to provide the most complete data set possible, the decision was made to wait until all upstream water samples were collected before selecting specific isolates to put through the ribotyping process. The experimental design for this project calls for ribotyping to be conducted on 160 to 200 isolates. Based on the proportion of sampling dates conducted in upstream sections of the watershed (versus estuarine sections), this segment of the data was allocated with 60% of the total isolates for ribotyping, leaving the remaining 40% for estuarine-originating isolates. With the upstream sampling phase of the project complete, criteria for isolate selection were designed. The method involved identifying those sampling sites that consistently yielded the highest E.coli counts as indicated by geometric and arithmetic means. Weighted most heavily were sampling sites from tributaries with greater flow contributions while selecting isolates from across the entire sampling period.

A goal of our work is to help resource managers develop targeted mitigation strategies to reduce fecal loadings in the estuary and hopefully reopen closed shellfish harvesting areas. We hypothesize that those sites with the highest counts potentially represent the greatest fecal loading to the estuary. These isolates are currently awaiting ribotyping at JEL.

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).
Fecal material from eight species have been collected, ribotyped and confirmed. We plan to add human, goose and moose samples to this local library in the immediate future.

Design "hot spot" focused watershed survey.
To design an effective watershed survey, project staff consulted with resource managers at the Maine Departments of Marine Resources and Environmental Protection. During a spring, 2002 review of existing data (including data from a Webhannet watershed survey conducted in 2000), several areas were identified for field investigation based on recent evidence of wildlife activity (e.g. beaver dams) and historically high fecal results.

Confirm estuarine sampling design; identify sampling sites in the Webhannet estuary; lay out sampling schedule; secure land owner permission for site access where necessary.
In the experimental design for this project, freshwater and estuarine portions of the watershed were divided into separate sampling periods to focus on snow melt and freshet runoff before, during and after the clam harvesting season (January to April). All freshwater sampling occurred from December to May, and in estuarine problem sites from June to Sept.

Estuarine sampling has been established to identify unknown sources of contamination in a tidally influenced region near Deptula Drive in Wells. Water quality monitoring conducted by the Wells National Estuarine Research Reserve (WNERR) since 1991 consistently turns up extraordinarily high fecal coliform counts during summer months at two sites in the Webhannet Marsh. Nine sampling events will have occurred in the estuary by the end of September. Estuarine sampling generally occurred two to four hours after high tide to allow for easier access to sample sites and to account for any fecal contamination originating from adjacent marsh areas. Because colony counts in the summer months are usually significantly and predictably higher than those in winter, the higher cut off level of 427 colonies was considered for isolate samples. However, the lack of precipitation during this period yielded below average sample results so the decision was made to continue using the cut off level of 64 colonies per 100 mL.

Estuarine sample sites were selected over a broad geographical area to obtain a complete and comprehensive data set. They were also selected based on ease of access and historical problem areas (Deptula Drive).

Conduct Webhannet watershed survey.
On April 19th, May 1st, May 13th, May 14th and May 28th project staff conducted surveys of the watershed. After consulting with DMR and DEP staff they concluded that the greatest sources of observable fecal contamination would most likely originate from the undeveloped portions of the watershed. A map of the sewered areas in the watershed was developed and project staff visited a few of these representative developed areas but were unable to identify any specific sources of fecal contamination. They also documented the most significant findings with digital photographs as a standardized watershed survey form did not prove very useful in documenting potential sources of fecal contamination.

Collect estuarine water samples and conduct lab procedures to enumerate and isolate colonies of fecal coliform bacteria and E.coli.
By Aug 31, five estuarine sampling events have occurred, three random and two precipitation dates. Membrane filtration, water bath and colony enumeration procedures have been conducted on each of the eighteen samples collected on each of these dates. Samples exhibiting colony counts greater than 64 per 100 mL have been isolated and sent to JEL for E.coli confirmation. (See Fecal Results and Maps). Isolate selection for ribotyping will be postponed until all estuarine samples have been collected and a data comparison is possible.

At JEL, conduct microbial source determination on isolates collected in the Webhannet watershed between March and May 2002.
The Jackson Estuarine Lab (JEL) recently purchased a riboprinter that will automate their ribotyping process. This will allow them to analyze isolates at a reduced cost. This service will be made available to this project for ribotyping of estuarine samples, but not for upstream samples. This is due to the fact that the scat samples collected from the Webhannet watershed have been processed using the manual ribotyping process and method consistency requires that these same procedures be used for ribotyping isolates.

The ribotyping of high priority Escherichia coli isolates from the Webhannet watershed has been completed. The interpretation of the ribotyping results is currently underway.

Design and implement outreach strategies related to project methods, goals and preliminary results.

• The project web site www.umseagrant-mst.org continues to be updated.
• Project staff presented an overview of this project along with preliminary findings on May 23rd at the New England Interstate Water Pollution Control Commission's 13th Annual Nonpoint Source Meeting held in Booth Bay, ME.
• Project staff participated in a poster session during the second State-of-the Bay Conference held in Freeport, ME on June 19th.
• Staff will present on the background and methods of the project at the Maine Wastewater Control Association's Fall Conference in Phippsburg on September 20th.
• Abstracts will be submitted for presentations at the following conferences:
  • Emerging Technologies, Tools, and Techniques to Manage Our Coasts in the 21st Century - January 2003 in Coco Beach, FL
  • First National Coastal and Estuarine Habitat Restoration Conference - April 2003 in Baltimore, MD
  • Coastal Zone03: Coastal Zone Management Through Time - July 2003 in Baltimore, MD
• An article in the Portland Press Herald's Sunday Telegram on July 21st . featured the project. See the following link to read the story directly: www.pressherald.com/news/state/020721pollution.shtml).

Difficulties Encountered
There were some logistics to overcome related to decisions on which of the large collection of E.coli strains would be the highest priorities for ribotyping. The time spent on making this decision was extremely useful in helping to focus efforts and relating goals for watershed water quality assessments to the type of information that results from conducting ribotyping analysis.

Anticipated Success in Meeting Project Objectives in Scheduled Project Period
The few difficulties encountered were normal types of problems that need to be solved in this type of project, where the use of a new technology is being applied in a novel application. The frequent communication between the water sampling and study design participants with the ribotyping lab have been extremely useful in ensuring a successful project.

Preliminary Results

• There are currently 36 E.coli isolates from source species fecal samples collected from the Maine study site that have useful ribotypes. The database includes patterns for eight species, including seven wildlife species and one pet species (dog). This is a useful start for a source species database, but it still requires a better balance of source species, especially needed are isolates from humans in the watershed. There were fifteen isolates from sewage influent that were unsuccessfully ribotyped, for a variety of reasons. Overall, 65 isolates from source species were analyzed. Not including the influent isolates, ribotyping was successful for 72% (36/50) of the other source species isolates.
• This watershed-specific source species database was used to analyze the patterns of isolates from high priority water samples. Samples collected during the one 2001 date (12/4/01) included high priority isolates from three sites. Ribotyping resulted in seventeen different isolates for which useful patterns were found. During 2002, isolates collected on ten different dates from twleve sites were deemed high priority. Ribotyping resulted in 80 useful ribotype patterns form the water sample isolates.
• Interpretation of the results is currently underway using the Maine host species database. The results will then be re-interpreted using a database with isolates from Maine and a much larger database from New Hampshire.

Tasks and activities for next reporting period

Tasks for the next reporting period

• 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, initiate ribotyping of isolates from the Little River watershed, and finish the interpretation and reporting of results for the Webhannet watershed study.
• 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.
• Dec. 2002 - May 2003: Collect Little River watershed water samples and conduct lab procedures to enumerate and isolate colonies of fecal coliform bacteria and E.coli.

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

Concerns or difficulties
The significant amount of time spent to discuss all logistics and goals for the project have allowed us to overcome all concerns and difficulties encountered to date.

Expenditures
Expenditures were in the range anticipated for the work accomplished to date