Deployment of Microbial Source Tracking to Identify Sources of Fecal Pollution in WaterC. Hagedorn, A. Hassall, M. Saluta, J. Dickerson, and T. Wade

1Department of Crop and Soil Environmental Sciences, VPI & SU, Blacksburg, VA

ABSTRACTMicrobial source tracking (MST) is a new technology that is being developed, tested, and deployed to identify sources of fecal pollution in water. The latest National Water Quality Inventory from 2000 reported that approximately 40% of streams, 45% of lakes, and 51% of estuaries in the U.S. were not clean enough (impaired) to support recreational uses such as fishing and swimming. The leading cause of impairments is fecal microorganisms, and recreational exposure to fecal material in polluted water carries substantial health risks. Establishing the sources of fecal contamination is crucial for the evaluation of health risks, sustainable management of water, achieving acceptable water quality and sanitation, and directing clean-up efforts. Enforcement of the Total Maximum Daily Load (TMDL) process in the U.S. has provided the incentive for the development and deployment of MST. Costs associated with TMDLs over the next 15 years are estimated to be approximately $1.0 billion for development of TMDL plans, $255 million for additional monitoring to support TMDLs, and $13.5 to $64.5 billion for TMDL implementation (a total of $15 to $66 billion). Many states are developing TMDL plans in compliance with court-ordered implementation, including Virginia. It is now clear that MST data will be heavily used in the TMDL process nationwide, and there is great expectation that MST will provide definitive answers, regardless of the present status of MST methodology. Is current MST technology adequate for such a task? This poster describes the MST multi-investigator method comparison studies conducted to date, along with a tiered system for evaluating each method, and some examples of successful uses of MST in urban and rural watersheds.

The Virginia Tech Source Tracking Laboratory has performed over 35 Total Maximum Daily Load (TMDL) projects in Virginia plus projects in AR, CA, FL, MD, NC, NY, SC, TN, TX, Wash. D.C., and WVA. Our program employs a variety of phenotypic and genotypic source tracking methods, and has recently developed fluorometry (detection of optical brighteners in detergents) to locate sources of human-derived pollution. Projects sponsored by EPA, NOAA, USDA, and USGS have resulted in method development and evaluation, and sampling procedures for source tracking. Sampling procedures include determination of the number of samples required, the frequency samples are collected, the sampling period duration, the impact of seasonality, and the numbers of isolates per sample required to obtain statistical confidence (and legal defensibility) in source tracking and source load allocation results. Our lab has participated in the design and performance of all multi-state method comparison studies conducted to date, and pioneered the concept of using blinded challenge tests in all field-oriented projects. Our program has cooperated on projects, trained lab personnel, and introduced method improvements for MapTech, Inc., a private-sector environmental engineering company.

SOURCE TRACKING AT VIRGINIA TECH

Phenotypic Methods

Sereogr ouping of organisms based on different somatic O antigenic determ inants

E.coli, SalmonellaNoImmunologica l Methods (sereotyping)

Based on d ifferences in bacteria l metabolism of a wide range of carbon sourc es

E.coli orEnterococcus

YesCUP (BIOLOG)

Based on antiob ioticresistance patterns unique to different sources of pollution

E.coli orEnterococcus

YesARA

DescriptionTargetLibrary Dependent

Method

Genotypic Methods

Based on the detection of flourescentlylabeled 16S rDNA PCR products using an automated DNA sequencer

BacteroidesPrevotella

NoLH-PCR and T-RFLP

DNA fingerprinting using cutting restriction enzymes coupled with electrophoresis analysis

E.coli orEnterococcus

YesPFGE

Conserved sequences in bacterial repetitive elements are used as PCR primers to distinguish among different strains of the same bacterial species

E.coliYesRep-PCR

Genetic fingerprint of the genes that code for rRNA, and are highly conserved in microbes. DNA is extracted and fragments are separated by gel electrophoresis to form patterns of 4-12 bands

E.coli or Enterococcus

YesRibotyping

DescriptionTargetLibrary Dependent

Method

•Equipment and lab facilities required

•Training required

•Library size required

•Implementation time

•Cost of ensuring results are legally defensible

•Cost per sample

•Turnaround time

Tier 3: Cost and Logistics

•Relationship to actual source of contamination

•Relationship to public health outcomes

•Relationship to commonly used water quality indicators

•Ease of communication to public

•Ease of communication to management audiences

Tier 2: Management Relevance

•Reproducibility of results

•Accuracy of correct classification of isolates into correct group

•Confidence that identified indicator is from presumed source

•Level of resolution

•Matrix stability

•Geographic stability

•Temporal stability

Tier 1: Measurement Reliability

Specific Evaluation CriteriaCategory of Criteria

Status of Source Tracking• The VT program will participate in a large-scale multi-state

project to evaluate and improve water quality along the entire Gulf Coast. This will be the largest deployment of source tracking yet attempted.

• The best approach for source tracking is to use multiple methods to increase confidence, perform appropriate QA/QC on the methods used, and deploy an alternative approach, such as fluorometry to detect optical brighteners, whenever possible.

• The source tracking community is diverse and growing, consisting of a small core of senior investigators plus many junior scientists who are new to the field. One role of a new on-line journal (Environmental Detection News) will be to serve as a single “how to do source tracking” reference for new researchers and to keep the entire community updated as new developments occur.