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EPA Guidance on Interpretation of the Nooksack River Watershed MST Project Data

October 20, 2017

The EPA Region 10’s Manchester Environmental Laboratory (MEL) has completed its analysis on
microbial source tracking (MST) samples collected in the Nooksack River watershed, in partnership with
the Lummi Nation and Whatcom County.
EPA and our partners are engaged in a broader effort to increase our understanding of fecal
contamination in the Nooksack watershed; this MST study is one part of that work, and additional work
is expected/planned.
MEL has provided the following guidance to assist with interpreting the laboratory results:
1. The study was designed around using end-point Polymerase Chain Reaction (PCR) (the MST PCR
method that has been in use for > 10 years at MEL), to analyze genetic markers for human and
ruminant sources (e.g. deer, elk, cattle, etc.). During the course of the project, a quantitative
PCR (qPCR) methodology was mobilized at MEL, allowing for the ability to detect additional
markers. The last half of the samples collected for this project were analyzed using qPCR not
only for human and ruminant, but also for avian, cattle, and dog to see if additional data could
be garnered. It is important to note that the original sampling plan did not take into account the
additional markers (i.e., avian, canine, and cattle). For example, since the ruminant marker has
been reported to occur in higher abundance than the cattle markers, the ruminant marker
should be more easily detected than the cattle markers. The limitations of the ruminant and
cattle markers related to animal age and diet also need to be considered prior to the study
design and in the interpretation of the data. Thus, cattle markers may have been present, even
though they were not detected.
2. Utilizing end-point PCR for the first half of the project, the ruminant marker was detected in
9.4% of the samples (Note: the cattle marker was not available at that time). Applying qPCR to
the last half of the samples, the ruminant marker was detected in 12.9% of the samples while
the cattle marker was not detected. The non-detect of the cattle marker could mean:
a) cattle fecal material was not present,
b) cattle genetic material was present, but not at a sufficient level to be detected by these
markers, or
c) cattle fecal material was present but did not contain the genetic sequence targeted by the
cattle markers.
3. MST projects with a watershed-wide approach have historically been conducted in phases at
MEL in an effort to hone in on problem areas; Phase I is an overall coverage of the watershed,
Phase II uses Phase I data to close in on problem areas, and Phase III (if necessary) uses all of the
data generated to more closely pin-point the sources of pollution. The Nooksack MST Project is
a Phase I project. Sections 3.1.1 and 4.3 of the Quality Assurance Project Plan (QAPP) discuss the
potential need for additional sampling sites and studies.
4. Samples were collected at eight locations once each month for twelve months. Sampling began
in January 2016, and the last sample was collected December 2016.
5. The November/December MST data show an increased frequency of positive results; however,
the fecal coliform data (from a concurrent sampling effort by Whatcom County) are moderate to
low during the same collection days. Given the difference in concentration and frequency at

which the ruminant and cattle markers occur in cattle fecal material, and the moderate to low
levels of fecal coliform in the samples, one should be careful interpreting the cattle marker as
absent when it could be present, but at concentrations too low to detect.
6. Additionally, when interpreting MST data, a number of factors must be carefully considered,
such as historical fecal data, sample site selection, land use, weather, river flows, tidal currents,
and an understanding of method limitations. These other factors have not yet been evaluated in
relation to these MST lab results.
7. MEL staff have clarified the definition section of the final Lab report to read: “U - Undetermined,
identifies the contaminant, if present, as being below the level of detection, lacking the target
marker, or coming from a source other than ruminant, cattle, human, dog, or avian feces.” The
final Lab report is attached.