Tricia Coakley 1 , Gail Brion 2 , and Alan Fryar 1 University of Kentucky

1. Relationships between indicators of fecal load, source, and age:Developing a multi-indicator approach for risk characterization Tricia Coakley1, Gail Brion2, and Alan Fryar1 University of Kentucky 1 Department of Earth and Environmental Sciences, 101 Slone Building Lexington, KY 40506-0053 USA 2 Department of Civil Engineering, 161 Raymond Building, Lexington, Kentucky 40506-0281 USA

2. Lexington, Kentucky Population: 280,000

3. 2007 study of Wolf Run watershed • Fields primers: • Bac 32F • CF 128F • HF 183F • HF 134F • General fecal marker detected in every sample • Human fecal markers detected frequently throughout the watershed • Unable to define most urgent locations for remediation

4. 3 Indicators rather than 1

5. 2008 Study Area

6. Methods • E.coli by Idexx Quantitray 2000 • AC/TC by mEndo broth and membrane filtration • Bacteroides host specific fecal markers by qPCR with primers and probes developed by Alice Layton (University of Tennessee) • Hubac = human specific • Allbac = general

7. Results • E.coli concentrations from <10 to >17,000 MPN/100mL • AC/TC ratios from 1 to 97 • Allbac found in all samples and ranging across 4 orders of magnitude • Hubac marker found in all but one sample and ranging across 5 orders of magnitude but with a lesser concentration than Allbac in each case • Hubac/Allbac apportionment allowed greater ablility to define hotspots of human sewage than Hubac concentration alone. • Screening samples with E.coli and AC/TC before analysis by qPCR would have reduced our molecular methods costs in half while providing necessary information to accurately locate the hotspots for immediate remediation.

8. Hubac and AC/TC relationship All samples with Hubac >20% also have AC/TC <20 All samples with AC/TC >20 also have Hubac <20% Some samples with Hubac <20% also have AC/TC <20 and this group typically had low E.coli loads

9. Wolf Run watershed (Hubac/Allbac)*100 <1% 1-10% 11-20% 21-30% 31-50% >50%

10. Wolf Run watershed • 15 samples with E.coli >500 MPN/100mL • 16 samples with AC/TC <20 • 13 samples meet both screening criteria • Of the 13 potential hotspots, 6 have >20% Hubac/Allbac • Only 1 hotspot identified by Hubac/Allbac of >20% failed the screening criteria

11. Glenns Creek Watershed (Hubac/Allbac)*100 <1% 1-10% Copies of DNA/mL 11-20% 21-30% 31-50%

12. Glenns Creek watershed • 3 samples with E.coli >500 MPN/100mL • 4 samples with AC/TC <20 • 2 samples meet both screening criteria for potential hotspots • Of those 2 samples, 1 was determined to be the only hotspot in this watershed with >20% Hubac/Allbac

13. Screening with E.coli and AC/TC across 3 watersheds Conclusion: E.coli and AC/TC are effective screening tools prior to molecular analysis for fecal source tracking. • Sensitivity = 87.5% • Specificity = 62.5% • Positive predictive value = 43.8% • Negative predictive value = 93.8%

14. Recommended Fecal Source Tracking Plan for reducing molecular methods expenses while maintaining necessary spatial sampling range • Sample a large number of locations canvassing the entire watershed. • Filter and archive DNA and analyze all samples for E.coli and AC/TC • Extract and analyze samples with greatest E.coli and lowest AC/TC values by qPCR for Allbac and Hubac markers. • Samples with greatest Hubac/Allbac percentages should be considered for immediate remediation.

15. Acknowledgements • Alice Layton and Dan Williams at the Center for Environmental Biotechnology, University of Tennessee, Knoxville • Kentucky Water Resources Research Institute • United States Geological Survey • Ken Cooke, Friends of Wolf Run, Kentucky River Watershed Watch, and Jean Watts,Tracy Knowles and students of Bluegrass Community Technical College