fluoroquinolones in kentucky’s - university of kentucky wrri... · fluoroquinolones in...
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The Presence of 17-β Estradiol and
Fluoroquinolones in Kentucky’s
Surface Water
E.G. Beck1, R. DeJaco2, S. Webb1, A. Fogle3, D. Williams1, M. Silitonga4, and B. Lee5
1 University of Kentucky, Kentucky Geological Survey 2 University of Kentucky, Department of Chemical and Materials Engineering
3 University of Kentucky, Department of Biosystems and Agricultural Engineering 4 Kentucky State University, College of Agriculture, Food Science and Sustainable Systems
5 University of Kentucky, Department of Plant and Soil Sciences
Kentucky Water Resources Research Institute Symposium
March 18, 2013
Lexington, Kentucky
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17-β Estradiol
Potential Sources to the Environment
• Residential straight piping
• Runoff from fields where animals graze
• Wastewater treatment plant (WWTP) outfalls
• Runoff from fields where wastes (animal/human) are applied
• One of 3 main estrogens (steroids) produced by the human body
• Predominant and most potent sex hormone in females
• Produced by animals (dairy cows, etc)
• Synthetic estrogens (17 alpha EE2)
• Also called E2 or estradiol
18 carbons
2 hydroxyl
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Fluoroquinolones
• A subset of the synthetic broad-spectrum antibacterial drugs
known as quinolones. Fluoroquinolones have a fluorine atom
attached to the central ring system.
• Fluoroquinolones are used to treat humans
and animals.
Human Veterinary
Ciprofloxacin Ofloxacin Enrofloxacin
Fleroxacin Pefloxacin Danofloxacin
Enoxacin Norfloxacin Marboloxacin
Levofloxacin
Potential Sources to the Environment
WWTP outfalls, runoff, and straight piping
Ciprofloxacin
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Environmental Concerns
17-β Estradiol (most potent)
• Is released to the environment (Kolpin and others, 2002, etc)
• Half-life in river water of 0.2 to 9 days (Jurgens and others, 2002)
• Endocrine disruptor (Purdom and others, 1994; Jopling and others, 1998)
1 ppt shown to affect reproduction in trout (Hansen and others, 1998)
Fluoroquinolones
• Is released to environment (Andreozzi and others, 2003, etc)
• Bacterial resistance from use in poultry production
U.S. FDA banned use in poultry production (2005)
• Affects on wildlife are unknown
• Fate in environment is unknown
• Continuous input
• Not regulated
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Two Pilot Studies
(Screenings) Sampling Round 1 (June-July 2012)
USDA-NIFA Southern Regional Water Program
• 10 states (Kentucky, Tennessee, Mississippi, Oklahoma,
Arkansas, Texas, North Carolina, Georgia, Florida, and
Alabama)
• 19 sites sampled in the Floyds Fork watershed
KyWRRI Student Enhancement Grant
• 32 sites sampled in 5 watersheds (Bayou de Chien, Clarks River,
South Elkhorn Creek, Banklick Creek, and Licking River)
Sampling Rounds 2-5 (November-December 2012)
USDA-NIFA Southern Regional Water Program
• 20 sites in 4 watersheds (80 samples)
(Bayou de Chien, Clarks River, Floyds Fork, and South
Elkhorn Creek)
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Watersheds (HUC 11)
Round 1: All
Rounds 2-5: Bayou de Chien, Clarks River, Floyds Fork, and
South Elkhorn Creek
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Watershed Sampling Sites
In stream
(bridge accesses and WWTP property)
From bridges
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Field Methods Recorded field measurements using
a Horiba U-22 water-quality meter* Measured discharge using a
Marsh-McBirney flow meter*
USGS stream gage data when
possible
Grab samples collected in 40-ml amber vials
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17-β Estradiol Analytical Method
• Enzyme-Linked Immunosorbent Assay (ELISA) kit from Abraxis LLC*
• Cross-reactivity
(n) 17-β Estradiol 100% (s) 17 alpha EE2 0.36%
(n) Estrone (E1) 75% E2-3 Glucoronide < 1%
(m) 16-Keto E2 27% E2-3 Sulfate < 1%
(n) Estriol (E3) 4.1% Cholesterol < 1%
• ChroMate® 4300 Microplate Reader by Awareness Technology, Inc.*
• Samples kept at 4°C and out of direct sunlight
• Samples analyzed within 7 days of collection
• 3 reps per sample bottle
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Fluoroquinolones Analytical Method
• Enzyme-Linked Immunosorbent Assay (ELISA) kit from Abraxis LLC*
• Cross-reactivity
(a) Enrofloxacin 100% (h) Ofloxacin 11%
(a) Danofloxacin 100% (h) Norfloxacin 7%
(h) Ciprofloxacin 66% (a) Marbofloxacin 6%
(h) Fleroxacin 25% (h) Levofloxacin 5%
(h) Enoxacin 17% (a) Sarafloxacin < 1%
(h) Pefloxacin 12% Oxolinic Acid < 1%
• ChroMate® 4300 Microplate Reader by Awareness Technology, Inc.*
• Samples kept at 4°C and out of direct sunlight
• Samples preserved with 10% methanol within 24 hours of collection
• Samples analyzed within 14 days of collection
• 3 reps per sample bottle
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17-β Estradiol
• Test sensitivity of 2.7 parts per trillion (ppt)
(Abraxis LLC, 2011*)
• Calculated MDL is 3.0 ppt
Kentucky Geological Survey Laboratory Services
(40 CFR Part 136, Appendix B, Revision 1.11, 2011)
Fluoroquinolones
• Test sensitivity of 0.016 parts per billion (ppb)
(Abraxis LLC, 2011*)
• Calculated MDL is 0.025 ppb
Kentucky Geological Survey Laboratory Services
(40 CFR Part 136, Appendix B, Revision 1.11, 2011)
Method Detection Limit (MDL)
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Confidence of Analytical Method for 17-β Estradiol
Step 2.
Step 1.
• Results at or near the MDL of 3.0 ppt are
suspect
• ELISA is an adequate screening method
only
Standard Concentrations
Rep 3.0 ppt 6.0 ppt 15.0 ppt
1 3.5 4.8 14.9
2 4.0 6.3 15.5
3 4.1 8.9 16.3
4 5.4 6.2 16.0
5 2.8 7.8 15.8
6 3.7 6.5 14.6
7 3.2 7.1 17.5
8 2.9 6.7 14.5
9 3.4 5.5 14.7
10 4.1 5.4 15.5
11 4.3 5.8 15.3
12 2.9 5.7 15.8
13 1.8 7.3 14.1
14 3.2 6.5 14.2
15 4.1 7.0 14.7
16 4.0 6.7 15.0
17 3.5 5.3 14.9
Std. Dev. 0.8 1.0 0.9
Mean 3.6 6.4 15.3
Bias 17% 6% 2%
3.0 ppt 6.0 ppt 15.0 ppt
Standard Error 0.19 0.25 0.21
1.96*Standard Error (CI) 0.4 0.5 0.4
Lower Confidence Limit 3.2 ppt 5.9 ppt 14.9 ppt
Upper Confidence Limit 4.0 ppt 6.9 ppt 15.7 ppt
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Statistical Analysis of Data
Step 1: Averaged analytical reps with all detects
Step 2: Used R (free statistical software) and maximum
likelihood estimation (MLE) method to average analytical
reps with nondects (Helsel, 2005)
Step 3: Used R and Kaplan-Meier method to create boxplots of
data (included detects and nondects) (Helsel, 2005).
Step 4: Used MLE tests within R to determine if there are
significant differences between sampling sites and
watersheds (p < 0.05 indicates 95% confidence level).
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Summary
of
Averaged
Data
Watershed Min Max
Number
of
Samples
Number of
Nondects
(percent)
Bayou de Chien < 3.0 3.6 12 9 (75)
Clarks River < 3.0 3.6 22 16 (73)
Floyds Fork < 3.0 8.2 43 26 (60)
South Elkhorn Creek < 3.0 15.3 43 23 (53)
Banklick Creek < 3.0 11.1 6 4 (67)
Licking River < 3.0 4.8 10 8 (80)
Estradiol
Watershed Min Max
Number
of
Samples
Number of
Nondects
(percent)
Bayou de Chien < 0.025 < 0.025 12 12 (100)
Clarks River < 0.025 0.169 22 15 (68)
Floyds Fork < 0.025 0.306 43 29 (67)
South Elkhorn Creek < 0.025 0.424 43 31 (72)
Banklick Creek < 0.025 < 0.025 6 6 (100)
Licking River < 0.025 0.052 10 9 (90)
Fluoroquinolones
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Southern Region Comparison
Estradiol (ppt) Fluoroquinolones (ppb)
State Min Max Min Max
Kentucky < 3.0 15.3 < 0.025 0.424
Alabama < 3.0 5.3 < 0.025 < 0.025
Arkansas < 3.0 11.9 < 0.025 < 0.025
Florida < 3.0 5.9 < 0.025 < 0.025
Georgia < 3.0 10.1 < 0.025 0.068
Mississippi < 3.0 7.1 < 0.025 0.033
North Carolina < 3.0 6.2 < 0.025 0.285
Oklahoma < 3.0 24.4 < 0.025 0.553
Tennessee < 3.0 < 3.0 < 0.025 < 0.025
Texas < 3.0 14.9 < 0.025 0.167
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Banklick
Creek
To few data points
to statistically
compare sites
within watershed
Sample
Site
Estradiol
(ppt)
Fluoroquinolones
(ppb)
1 11.1 < 0.025
2 < 3.0 < 0.025
3 < 3.0 < 0.025
4 3.9 < 0.025
5 < 3.0 < 0.025
6 < 3.0 < 0.025
Land Cover (percent)
Forest Developed Pasture Cultivated Other
36 36 24 1 3 6
5
4 3
2
1
2 sites 0 site
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Sample
Site
Estradiol
(ppt)
Fluoroquinolones
(ppb)
1 < 3.0 < 0.025
2 < 3.0 < 0.025
3 < 3.0 < 0.025
4 < 3.0 < 0.025
5 < 3.0 < 0.025
6 < 3.0 < 0.025
7 4.8 < 0.025
8 < 3.0 < 0.025
9 3.3 0.052
10 < 3.0 < 0.025
Licking River
1 2
3 4
5 6
7 8 9
10
Salyersville
outfall
Land Cover (percent)
Forest Pasture Developed Cultivated Other
82 12 4 1 1
To few data points to
statistically compare
sites within
watershed
2 sites 1 site
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Graves
County
≈ 125 poultry
houses
To few detects to make statistical
comparisons between sampling sites.
Bayou de Chien Land Cover (percent)
Cultivated Forest Pasture Developed Other
HUC 11 58 14 9 5 14
Site 2 41 27 20 5 7
Sample
Site
Estradiol
(ppt)
Fluoroquinolones
(ppb)
1 < 3.0 < 0.025
2 < 3.0 to 3.6 < 0.025
3 < 3.0 < 0.025
4 < 3.0 to 3.5 < 0.025
1 2
3 4
2 sites 0 site
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Sample
Site
Estradiol
(ppt)
Fluoroquinolones
(ppb)
1 < 3.0 to 3.6 < 0.025
2 < 3.0 to 2.8 < 0.025
3 < 3.0 to 3 < 0.025 to 0.169
4 < 3.0 to 3.2 < 0.025
5 < 3.0 < 0.025
6 < 3.0 < 0.025
Clarks River Land Cover (percent)
Cultivated Forest Pasture Developed Other
42 32 13 9 4
To few detects to make
statistical comparisons
between sampling sites.
1
2 3
4
5
6
Murray outfall
4 sites 1 site
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Sample Site
Estradiol (ppt)
Fluoroq (ppb)
1 < 3.0 < 0.025
2 < 3.0 < 0.025
3 < 3.0 < 0.025
4 < 3.0 < 0.025
5 < 3.0 < 0.025
6 < 3.0 < 0.025
7 < 3.0 to 2.9 < 0.025
8 < 3.0 to 7.8 < 0.025
9 < 3.0 < 0.025
10 < 3.0 < 0.025
11 < 3.0 < 0.025
12 < 3.0 to 7.2
< 0.025 to 0.160
13 < 3.0 to 2.9 < 0.025
14 < 3.0 to 8.2
< 0.025 to 0.306
15 < 3.0 < 0.025
16 < 3.0 to 3.9
< 0.025 to 0.206
17 < 3.0 < 0.025
19 < 3.0 < 0.025
20 < 3.0 < 0.025
Floyds Fork
Land Cover Percent
Forest 43
Pasture 32
Developed 18
Cultivated 5
Other 2
Jeffersontown
outfall
1 2
3
4 5
6 7
8
9 10
11
12
13 14
15 16 17
19 20 6 sites 3 sites
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FF08 FF12 FF14 FF16
34
56
78
FF12 FF14 FF16
0.0
50
.10
0.2
0
Floyds Fork 17-β Estradiol Fluoroquinolones
Kaplen-Meier method used to
generate boxplots.
Based on MLE test p = 0.2
Therefore, cannot state that the
4 sites are statistically different.
Based on MLE test p = 0.08
Can state there are differences
at the 90% confidence level.
MDL
MDL
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Sample
Site Estradiol (ppt) Fluoroq (ppb)
1 < 3.0 to 3.8 <0.025
2 < 3.0 to 15.3 < 0.025 to 0.424
3 < 3.0 to 2.9 < 0.025
4 < 3.0 to 8.5 < 0.025 to 0.261
5 < 3.0 to 3.7 < 0.025
6 <3.0 < 0.025
7 < 3.0 to 5.2 < 0.025
8 < 3.0 to 4.4 < 0.025 to 0.092
9 < 3.0 <0.025
10 3.6 <0.025
11 4.1 <0.025
South Elkhorn Creek
Land Cover Percent
Pasture 63
Developed 24
Forest 8
Cultivated 4
Other 1 1
2 3
4
5 6
7
8 9
10
11
Lexington
outfall
9 sites 3 sites
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South Elkhorn Creek
SE02 SE04 SE05 SE08
46
81
01
4
SE02 SE04 SE08
0.0
10
.05
0.2
0
17-β Estradiol Fluoroquinolones
Kaplen-Meier method used to
generate boxplots
Based on MLE test p = 0.001
Can state there are statistical
differences between sites at 95%
confidence level.
Based on MLE test p = 0.005
Can state there are statistical
differences between sites at 95%
confidence level.
MDL
MDL
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BC CR FF SE
12
51
0
CR FF SE
1e
-04
1e
-03
1e
-02
1e
-01
Watershed Comparison
17-β Estradiol Fluoroquinolones
MDL
MDL
Based on MLE test p = 0.02
Can state there are statistical
differences between watersheds
at 95% confidence level.
Based on MLE test p = 0.13
No statistical differences between
watersheds.
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Conclusions
• ELISA kit for 17-β Estradiol and Fluoroquinolones
is an adequate screening method.
• Estradiol was detected in all 6 watersheds.
• Fluoroquinolones were detected in 4 of the 6 watersheds.
• Watersheds with the most developed land cover
South Elkhorn Creek (24%), Banklick Creek (36%),
and Floyds Fork (18%) had the highest estradiol
concentrations 14.3, 11.1, and 7.8 ppt, respectively.
• To few detects in 4 of the 6 watersheds makes it difficult
to analyze data statistically.
• Using the nondects, a handful of sites in the Floyds
Fork and South Elkhorn Creek watersheds could be
analyzed statistically.
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Future Research
• Screen additional watersheds using ELISA method.
• Collect additional surface water samples and analyze
for specific compounds.
• Collect samples from karst springs.
• Use passive samplers in streams and karst springs
to calculate time-weighted averages.
• Collect groundwater samples from water wells and
screen using ELISA method and analyze
for specific compounds.
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Acknowledgements
Funding
USDA-NIFA Southern Regional Water Program
KyWRRI Student Enhancement Grant (USGS 104B Program)
Kentucky Geological Survey Laboratory Services
Jason Backus and Andrea Mitchell
ArcMap Land Cover Data
Dan Carey, KGS retired; Bethany Overfield, KGS
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Karst