Team KERMIT Research Proposal

Amy ChenChristina Gonzalez

David HuRicha Kalsi

Tanya KapoorHaeMin Park

Sam ParkAlex ProctorAlec Ridgway

Andrew TavernerNick Vujcic

Mentor: Steve TurleyLibrarian: Jim Miller

• Approximately 1.6 billion lbs of poultry litter (chicken manure mixed with bedding) is generated annually in the Delmarva area

• Large amounts are used as fertilizer for agricultural lands as a substitute for commercial fertilizers

• Excessive application to agricultural fields often results in runoff of poultry litter associated hormones (PLAH)

• Main component of PLAH: estradiol• Other components: estrone• Endocrine-disrupting compound (EDC)

•Amphibians in the Delmarva area are being found to display intersex characteristics including malformed gonads.

• Feminization of male fish and frogs • Mixed ovarian and testicular tissues in gonads

Intersex tissue: oocytes mixed with seminiferous tubules.

CONCENTRATION of Estradiol LOCATION

5-25 ng/L Pond that received runoff (general)

19 to 75 ng/L MD Eastern Shore streams/ rivers receiving agricultural runoff

20-2330 ng/L Runoff near litter pastures

38.7-196 ng/L Runoff from freshly manured field

245 ng /L Pond following a rain event after poultry litter application

730 ng/L Eluant from pile of poultry litter

It has been shown that at 200ng/L, feminization effects are visible.

• Focuses on effects of estradiol only

What has not done yet been done…What has not done yet been done…

• Use environmentally relevant concentrations and allow for natural biodegradation

• Observing synergistic effects among the various chemicals in poultry litter—Most studies look at the effects of estradiol alone

• Directly connect poultry litter to frog deformations

Will exposure to environmentally relevant concentrations of PLAH in poultry litter negatively affect the sexual development of Xenopus laevis?

• Model organism for EDC research• Skin is semi-permeable• Growth cycles sensitive to hormonal

control• Easy to induce reproduction

– Large number of offspring• Entire life cycle is aquatic so

exposure can be maximized

X. Laevis tadpole

X. Laevis adults; female on left and male on right

Initial Concentrations Setup:1.Send poultry litter subsamples to University of Buffalo for gas chromatography- mass spectroscopy analysis

• Estradiol• Estrone• Estriol• Testosterone

2.Dilute to desired concentrations based on estradiol concentration data

Days 1-30 Days 30-120

Send 2 homogenized, aqueous subsamples to setup initial concentrations

Send 2 subsamples for analysis every week

Send 2 subsamples for analysis biweekly

•Xenopus laevis (African clawed frogs) tadpoles at 5 DPF•Testing conditions:

• 23 ± 2 ⁰C• 16:8 hrs of light: dark• Vented to decrease ammonia concentrations• Grown in 5 gal. aquaria

0 ng/L estradiol 15 ng/L 60 ng/L 300 ng/L

25 tadpoles 25 tadpoles 25 tadpoles 25 tadpoles

X 5 replicates

5 DPF: expose to poultry litter

17 DPF: sex differentiation sensitive stage

55-75 DPF: Metamorphosis occurs.

120 DPF: Sacrifice juvenile frogs with tricaine methanesulfonite (MS-222)

• Feed floating tadpole food

• Monitor survival• Observe external malformations• Monitor behavior

• Feed floating frog food

• Monitor survival • Observe external malformations• Monitor behavior

• Monitor time to metamorphosis • Measure wet weight and snout-vent length

Sex Differentiation Determination

Histology phase: determining sex ratios.• Stain gonads with hematoxylin and eosin solution

– Ovarian and testicular tissues will stain selectively• Observe gonadal abnormalities (as below)

Statistical Analysis

Shapiro Wilk’s Test for Normality

Bartlett’s Test for NormalitySteel’s Many-One Rank Test

(non parametric test)

Dunnett’s Test (Parametric test)

Normal Non-normal

Homogenous variance

Heterogenous variance

• Threats to the ecosystem – destruction of frog populations

• Groundwater contamination – can lead to eventual human intake of

EDCs

• Increased EDCs in environment linked to – Higher breast cancer rates, – Higher prostate cancer rates– Higher testicular cancer rates, – Decreased sperm counts……in HUMANS.

• Lab space secured

• IACUC Proposal submitted and approved

• Grants applications submitted:•ACCIAC•Sea Grant

• Grant application in Progress:•HHMI Grant

• Pilot Study has been started

• Tadpole/frog care training has been scheduled

Fall 2011• Begin research

• Feed frogs daily• Periodic water changes• Sacrifice frogs

• Present at Colloquium

• Complete thesis• Prepare & practice for thesis defense

Fall 2012

Spring 2012• Begin analyses of gonadal tissues• Complete a poster of our work• Present at Undergraduate Research Day• Begin writing thesis

Spring 2013*

• Defend thesis• Revise thesis for publication

*Provided world does not end in 2012

Item Quantity Unit Cost Overall Cost

5-Gallon Aquaria 20 Donated $0.00

Histology Materials n/a n/a $500.00

X. Laevis Tadpole and Adult Powdered Food

n/a n/a $40.00

Tank Maintenance Items n/a n/a $210.00

Poultry Litter I pile n/a <$50.00

X. Laevis Specimens 400 Donated $0.00

pH/ DO2 1 $500 $500

Lighting 20 $10 $200

Lab Space 1 n/a $0.00

Poultry Litter Analyses 25 300 $7500.00

TOTALS ~$9000.00

References• Harrison, P. T. C., Holmes, P., & Humfrey, C. D. N. (1997). Reproductive health in

humans and wildlife: are adverse trends associated with environmental chemical exposure? Science of The Total Environment, 205(2-3), 97-106. doi:doi: DOI: 10.1016/S0048-9697(97)00212-X

• Houlahan, J. E., Findlay, C. S., Schmidt, B. R., Meyer, A. H., & Kuzmin, S. L. (2000). Quantitative evidence for global amphibian population declines. Nature, 404(6779), 752-755. doi:10.1038/35008052

• Lutz, I., Kloas, W., Springer, T., Holden, L., Wolf, J., Krueger, H., & Hosmer, A. (2008). Development, standardization and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of <i>Xenopus laevis</i>. Analytical and Bioanalytical Chemistry, 390(8), 2031-2048.

• Welshons, W. V., Thayer, K. A., Judy, B. M., Taylor, J. A., Curran, E. M., & Saal, F. S. V. (2003). Large Effects from Small Exposures. I. Mechanisms for Endocrine-Disrupting Chemicals with Estrogenic Activity. Environmental Health Perspectives, 111(8), 994-1006.

• Wolf, J. C., Lutz, I., Kloas, W., Springer, T. A., Holden, L. R., Krueger, H. O., & Hosmer, A. J. (2010). Effects of 17 β-estradiol exposure on Xenopus laevis gonadal histopathology. Environmental Toxicology and Chemistry, 29(5), 1091-1105.

• Seth (2009). Intersex fish and how we’re causing it. City Renewed, http://www.cityrenewed.com/2009/04/intersex-fish-and-how-were-causing-it/