1. Tricia Coakley 1, Gail Brion 2, and Alan Fryar 1 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 Relationships between indicators of fecal load, source, and age: Developing a multi-indicator approach for risk characterization

2. Lexington, Kentucky Population: 280,000

3. 2007 study of Wolf Run watershed K54+K461 K54+K465 K54 K465+ K54+K465 K54+K465 K184+K465 K184 K470+ K184 K470+ K184+ K470 duplicate+ K184+K470 K461 K470+ K461 K470 duplicate+ K461 K470+ K461 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 LoadE.coliAllbac SourceHubacCaffeine Fecal Sterols Other AgeAC/TC

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 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 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 All samples with Hubac >20% also have AC/TC <20

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 site ID(Hubac/Allbac)*100AC/TCE.coli K054 /D0319.658.9310 K307/D199.36.761054 K461 /D017.871.13359 K465/D1811.338.992142 K466/D1460.2912.571376 K467/D201.75121664 K468/D1524.2412.332035 K470 /D1344.94.386488 K471/D107.913.496131 K472/D07165.641071 D176.5613.12247 K184/D1612.68.681850 D1131.033.875794 D1214.252.695172 D025.78N/A17329 D0623.9718.244106 K305/D085.0166.52231 D0421.424.62148 D0524.819.945172

11. Copies of DNA/mL Glenns Creek Watershed (Hubac/Allbac)*100 <1% 1-10% 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 site ID(Hubac/Allbac)*100AC/TCE.coli K61940.037.16749 K6150.7240331 K6174.5820520 K6160.4412.86465 K0852.6728.5504 K096050 K1260.7931.67341

13. Screening with E.coli and AC/TC across 3 watersheds Sensitivity = 87.5% Specificity = 62.5% Positive predictive value = 43.8% Negative predictive value = 93.8% Conclusion: E.coli and AC/TC are effective screening tools prior to molecular analysis for fecal source tracking. <20% Hubac >20% Hubac E.Coli <500 or AC/TC >20 151 E.Coli >500 & AC/TC <20 97

14. Recommended Fecal Source Tracking Plan for reducing molecular methods expenses while maintaining necessary spatial sampling range 1. Sample a large number of locations canvassing the entire watershed. 2. Filter and archive DNA and analyze all samples for E.coli and AC/TC 3. Extract and analyze samples with greatest E.coli and lowest AC/TC values by qPCR for Allbac and Hubac markers. 4. 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