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