Fluoroquinolones in Kentucky’s - University of Kentucky WRRI... · Fluoroquinolones in...

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

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

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

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

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)


• 20 sites in 4 watersheds (80 samples)

(Bayou de Chien, Clarks River, Floyds Fork, and South

Elkhorn Creek)

Watersheds (HUC 11)

Round 1: All

Rounds 2-5: Bayou de Chien, Clarks River, Floyds Fork, and

South Elkhorn Creek

Watershed Sampling Sites

In stream

(bridge accesses and WWTP property)

From bridges

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

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

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

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


(40 CFR Part 136, Appendix B, Revision 1.11, 2011)

Method Detection Limit (MDL)

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

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).

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

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

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

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

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

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

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

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.


Can state there are differences

at the 90% confidence level.

MDL

MDL

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

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


Can state there are statistical

differences between sites at 95%

confidence level.



differences between sites at 95%

confidence level.

MDL

MDL

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



differences between watersheds

at 95% confidence level.


No statistical differences between

watersheds.

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.

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.

Acknowledgements

Funding


KyWRRI Student Enhancement Grant (USGS 104B Program)


Jason Backus and Andrea Mitchell

ArcMap Land Cover Data

Dan Carey, KGS retired; Bethany Overfield, KGS

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