Tricia Coakley1, Gail Brion2, and Alan Fryar1

University of Kentucky

1 Department of Earth and Environmental Sciences, 101 Slone Building Lexington, KY 40506-0053 USA2 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

Lexington, Kentucky

Population:

280,000

2007 study of Wolf Run watershed2007 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

3 Indicators rather than 13 Indicators rather than 1

Load E.coliE.coli Allbac

Source HubacHubac CaffeineFecal

SterolsOther

Age AC/TCAC/TC

2008 Study Area2008 Study Area

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

ResultsResults

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.

Hubac and AC/TC relationship

0

10

20

30

40

50

60

70

80

90

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00

(Hubac/Allbac)*100

AC

/TC

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

Wolf Run watershed

(Hubac/Allbac)*100

<1%

1-10%

11-20%

21-30%

31-50%

>50%

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)*100 AC/TC E.coli

K054 /D03 19.65 8.93 10

K307/D19 9.3 6.76 1054

K461 /D01 7.87 1.13 359

K465/D18 11.33 8.99 2142

K466/D14 60.29 12.57 1376

K467/D20 1.75 12 1664

K468/D15 24.24 12.33 2035

K470 /D13 44.9 4.38 6488

K471/D10 7.91 3.49 6131

K472/D07 1 65.64 1071

D17 6.56 13.1 2247

K184/D16 12.6 8.68 1850

D11 31.03 3.87 5794

D12 14.25 2.69 5172

D02 5.78 N/A 17329

D06 23.97 18.24 4106

K305/D08 5.01 66.52 231

D04 21.42 4.62 148

D05 24.81 9.94 5172

Copies of DNA/mL

Glenns Creek Watershed

(Hubac/Allbac)*100

<1%

1-10%

11-20%

21-30%

31-50%

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)*100 AC/TC E.coli

K619 40.03 7.16 749

K615 0.72 40 331

K617 4.58 20 520

K616 0.44 12.86 465

K085 2.67 28.5 504

K096 0 5 0

K126 0.79 31.67 341

Screening with Screening with E.coliE.coli and AC/TC across 3 watersheds 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 >2015 1

E.Coli >500 &

AC/TC <209 7

Recommended Fecal Source Tracking Plan for reducing molecular Recommended Fecal Source Tracking Plan for reducing molecular methods expenses while maintaining necessary spatial sampling rangemethods 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.

AcknowledgementsAcknowledgements 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