Investigation of 17a Ethinylestradiol in Surface Waters

1
Investigation of 17-a Ethinylestradiol in Surface
Waters

• Zachary Barnes
• Tony Flatness
• Heather Patterson
• Advisor Dr. Johanna Foster

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Oral Contraceptive Use

• Over 100 million women use oral contraceptives
• Largest contributor
• Key ingredient- 17-a Ethinylestradiol (EE)
• In the past 150 ug/dose
• Currently 20-35 ug

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About Ethinylestradiol

• 17 a-Ethinylestradiol (EE) is the most common
  hormone in oral contraceptives
• EE is a synthetic derivative of 17 ß-Estradiol
  (E2)
• E2 is the most abundant estrogen in the human
  body
• EE is an antagonist to E2
• Compete for estrogen receptors

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Chemical Structure
17 ß-Estradiol
17 a-Ethinylestradiol
http//www.all-science-fair-projects.com/science_f
air_projects_encyclopedia/ImageEstradiol.png
http//jeq.scijournals.org/cgi/content/full/30/6/2
077/FIG1
5

• Pathway
• Hypothalamus
• GnRH
• Pituitary
• LH and FSH
• Theca cells
• Cholesterol to DHEAto testosterone
• Granulosa cells
• Testosterone to E2
• Inhibitory effects
• Estrogen to Hypothalamus
• Progesterone to Hypothalamus
• Continues if fertilization occurs
• EEs effect
• Competes with E2

http//www.gravitywaves.com/chemistry/CHE452/23_Se
x20Steroid20Horm.htm
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Estradiol in Ovaries
http//www.gravitywaves.com/chemistry/CHE452/23_Se
x20Steroid20Horm.htm
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Excretion of EE

• EE discharged in urine
• 19 of dose
• Ends up in sewage treatment facilities
• Nitrifying bacteria responsible for microbial
  degradation
• 85 removed during wastewater treatment
• 1 to 7 ng/L in Europe, Japan, and the U.S.
• Combination of EE and other estrogenic compounds

8
Effects of EE ConcentrationsNash et al. 2004

• 0.1 to 15 ng/L
• Sexual development and differentiation
• 2 to 10 ng/L
• Fecundity
• 10 ng/L
• Reproductive behavior
• 1 to 10 ng/L
• Reduced viable embryos

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Effects of EE ConcentrationsNash et al. 2004

• 50 ng/L
• Fourteen hours- egg production and viability
• Ten days- complete reproductive failure
• 5 ng/L throughout entire life
• Complete reproductive failure
• 0.5 ng/L
• No effect on egg numbers

10
Vitellogenin (VTG) Production

• VTG- egg yolk precursor protein
• Female fish naturally carry VTG
• Males contain VTG gene

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EE and VTG concentrations

• Increased levels of VTG
• Males
• Decreased testicular growth
• Disruption of sexual differentiation
• Testicular atrophy
• Females
• Decreased egg production
• Kidneys and calcium in scales

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EE and Intersex fishJobling et al. 2006

• Males
• Reduced sperm density
• Reduced fertility and repopulation
• Developing oocytes and ovarian cavity
• Abnormal concentrations of sex steroid hormones
• Presence of two sperm ducts

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Effects on Fathead MinnowsBrian et al. 2005 and
Nash et al. 2004

• Increased VTG levels in concentration-dependent
  scale
• 0.2 to 1 ng/L
• 20 to 35 reductions in hatching success in
  embryos
• 5 ng/L throughout life
• No phenotypic males
• No normal testes in genetic males

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Effects on Fathead MinnowsBrian et al. 2005 and
Nash et al. 2004

• Spawning
• All fish phenotypically female
• Spawning and egg-laying occurred
• No change in male sexual behavior
• No viable embryos
• Histological analysis
• Malformed sperm ducts
• Ovarian cavities in testes
• Cilia in sperm ducts

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Why do we Care?

• Detrimental effects of fish populations
• The Bio-magnification affect of estrogen
• Increased concentrations of estrogens in Food
  Chain levels
• Possible reduction in number of lower level
  consumers

http//seattlepi.nwsource.com/dayart/20061010/Food
-Chain.gif
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Null Hypothesis

• The exposure of various levels of 17
  a-Ethinylestradiol will cause no physical change
  amongst the minnows and will not change offspring
  numbers.

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Significance?

• Possible changes in current wastewater treatment
  requirements
• The impact on fish occurring now in the Cedar
  River
• Future effects of reproductive disorders
• Lower population of Fathead minnows means less
  food for other fish species
• If this effects fish what else could it effect?

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Goals of our Study

• To aid in research efforts on the effects of
  wastewater effluents
• To represent effects and conditions on fish
  locally
• To observe the effects of 17 alpha-ethinylestradio
  l on Fathead Minnows
• Anatomically
• In Vivo
• In offspring successes

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Procedure

• After acquiring Fathead Minnows 15 will be
  distributed to each 20 gallon tank
• In each tank will be an aerator, and a constant
  identical atmosphere
• There will be a 1 week acclamation period for the
  minnows to become accustomed to their new
  environment
• After the acclamation week, contamination of
  tanks water with predetermined levels of 17-alpha
  Ethinylestradiol will begin

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Contamination

• There will be 8 tanks total
• Two tanks of each contamination level will be
  present
• Contamination levels 5ng/L, 39ng/L, 73ng/L and a
  dechlorinated and conditioned tap water control
  group.
• Chosen contamination levels represent the minimum
  found to cause mutation, the mean levels found in
  urban rivers in the U.S. and a moderate level
  between the two (Fent et. al., 2006)
• Three time frames
• Two 21 day contamination periods
• Single 42 day contamination

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Project Timeline

• September
• 8th Begin by acquiring fish and allowing 1 week
  acclimation period
• 16th Begin investigation by exposing fish to
  various contaminations
• 23rd 75 Water change in all tanks with
  contamination level checks
• 30th 75 Water change in all tanks with
  contamination level checks
• October
• 7th End of first 3 week study in half of all
  tanks Induction of spawning begins. 75 water
  change for other half of tanks
• 14th Upon adequate time to spawn, adult fish will
  be euthanized for testing, fish eggs will be
  collected and counted and permitted to mature
  into fry and the second three week study will
  begin, 75 water change for other half of tanks
• 21st Second group will be exposed, 75 water
  change for the other half of tanks
• 28th Conclusion of 6 week exposure to the second
  half of tanks, spawning to be coerced, 75 water
  change for the other half of tanks
• November
• 4th Upon adequate time to spawn adult fish will
  be euthanized for testing, fish eggs will be
  collected and counted and permitted to mature
  into fry for further fecundity testing, 75 water
  change for remaining tanks
• 11th Conclusion of contamination for remaining
  tanks. All remaining fish will be coerced into
  spawning
• 18th Upon successful spawning the remaining adult
  fish will be euthanized for testing and the eggs
  will be permitted to grow into fry for final
  tests of fish fecundity
• 25th Data Collection is complete, statistical
  analysis of tests will begin

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Fathead Minnow Lifestyles

• Feeding will occur twice per day with a
  consistent diet of flake food and brine shrimp
• 10 hour light period and 14 hour dark period per
  day
• Daily visual checks to ensure that ick or other
  diseases are not affecting the minnows
• 75 water changes will occur on a weekly basis
• Contamination levels checked biweekly

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Following Contamination

• Spawning will be induced at the end of each
  groups contamination period.
• Light and temperature changes will occur to match
  natural breeding months
• 15 hours of light and 9 hours dark in correlation
  with a temperature change from 65 degrees F to a
  minimum of 73 degrees F
• Following spawning the minnows will be euthanized
  with a lethal dose of benzocaine.
• 500mg/L applied for 10 minutes

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What are we looking for?

• Irregular size for the age of the minnow
• Weight, length
• Reduced fecundity in exposed minnows
• Tested through coerced spawning results
• Uneven sex distribution in fry
• Increased levels of estrogen in adult minnows in
  vivo
• Abnormal gonadal developments such as but not
  limited to
• Males developing female tissues
• Both sexes having underdeveloped sex organs
• Both sexes having deformed reproductive organs

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Analysis

• Upon completion of observations statistical
  analysis will be used to determine whether our
  results are truly significant or not.
• T-Test will be used to determine if any
  deviations from the sex ratio, fish with deformed
  reproductive organs, and weight/length
  distribution is significant.
• with a level of a.05

http//www.biologyforlife.m/images/website/t-test.
gif serc.carleton.edu
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Implications

• If our expected findings are true this could
  effect
• Tolerated contamination levels in surface waters
• Wastewater treatment plants methods and minimum
  treatment protocols
• The finding could aid in increasing the need for
  knowledge in human pharmaceutical waste research
• Could also assist in environmental biologists
  maintaining fish population levels in Iowa and
  beyond
• Leading to more research in the effects of
  contaminated surface waters on other vertebrates

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Works Cited

• Brian, J. V., Harris, C. A., Scholze, M.,
  Backhaus, T., Booy, P., Lamoree, M., Pojana, G.,
  Jonkers, N., Runnalls, T., Bonfa, A., Marcomini,
  A., and Sumpter, J. P. 2005. Accurate
  predictions of the response of freshwater fish to
  a mixture of estrogenic chemicals. Environmental
  Health Perspectives, 113.6 721-728.
• Casey, P. M., Cerhan, J. R., and Pruthi, S.
  2008. Oral contraceptive use and the risk of
  breast cancer. Mayo Clinic Proceedings, 83.1
  86-91.
• Fent, K., Weston, A. A., Caminada, D. 2005.
  Ecotoxicology of human pharmaceuticals. Aquatic
  Toxicology 76.2006 122-159
• Liney, K. E., Hagger, J. A., Tyler, C. R.,
  Depledge, M. H., Galloway, T. S., and Jobling, S.
  2006. Health effects in fish of long-term
  exposure to effluents from wastewater treatment
  works. Environmental Health Perspectives, 114.1
  81-88.
• Liney, K. E., Jobling, S., Shears, J. A.,
  Simpson, P., and Tyler, C. R. 2005. Assessing
  the sensitivity of different life stages for
  sexual disruption in roach (Rutilus rutilus)
  exposed to effluents from wastewater treatment
  works. Environmental Health Perspectives, 113.10
  1299-1306.
• Jobling, S., Williams, R., Johnson, A., Taylor,
  A., Gross-Sorokin, M., Nolan, M., Tyler, C. R.,
  Aerie, R., Santos, E., and Brighty, G. 2006.
  Predicted exposures to steroid estrogens in U.K.
  rivers correlate with widespread sexual
  disruption in wild fish populations.
  Environmental Health Perspectives, 114.1 32-40.
• J. R. 2000. Estrogen effects linger in male
  fish. Science News 158.6 94.
• Nash, J. P., Kime, D. E., Van der Ven, L. T. M.,
  Wester, P. W., Brion, F., Maack, G.,
  Stahlschmidt-Allner, P., and Tyler, C. R. 2004.
  Long-term exposure to environmental
  concentrations of the pharmaceutical
  ethynylestradiol causes reproductive failure in
  fish. Environmental Health Perspectives, 112.17
  1725-1733.
• Stancel, G. M., Boettger-Tong, H. L., Chiappetta,
  C., Hyder, S. M., Kirkland, J. L., Murthy, L.,
  and Loose-Mitchell, D. S. 1995. Toxicity of
  endogenous and environmental estrogens What is
  the role of elemental interactions.
  Environmental Health Perspectives, 103.7 29-33.
• Dott, W., Weber, S., Leuschner, P., Kampfer, P.,
  and Hollender, J. 2005. Degradation of
  estradiol and ethinyl estradiol by activated
  sludge and by a defined mixed culture. Applied
  Microbiology Biotechnology, 67 106-112.
• Wheeler, J. R., Gimeno, S., Crane, M.,
  Lopez-Juez, E., and Morritt, D. 2005.
  Vitellogenin A review of analytical methods to
  detect (anti) estrogenic activity in fish.
  Toxicology Mechanisms Methods, 15.4 293-306.
• Zhang, H., Cui, D., Wang, B., Han, Y., Balimane,
  P., Yang, Z., Sinz, M., and Rodrigues, A. D.
  2007. Pharmacokinetic drug interactions
  involving 17a-Ethinylestradiol A new look at an
  old drug. Clinical Pharmacokinetics, 46.2
  133-157.