Measuring Estradiol/Ethinylestradiol in Water

[GraceLiu]

I'm new here, so if I commit any dramatic breaches of etiquette, please let me know.

For my Junior year research project, I'd like to work with pharmaceuticals--in particular, phytoremediation of freshwater contaminated with the estrogen from birth control pills. Unfortunately, I'm a little hazy on the specific compounds in a typical estrogen (not progesterone) birth control pill, though I do believe that either Estradiol or Ethinylestradiol is present. Vetiver grass, according to Michigan Tech research, has been shown to absorb almost all tetracycline and monensin from water when grown in a hydroponic system for 12 weeks. I would like to use it to remove hormones from water as well.

In particular, what I'd like to know is:
-How do I perform an estrogen test in water? Is it possible to find a testing kit of some sort that does not require university equipment? If not, what sort of university equipment would I need?
-What type of estrogen would I find in a typical estrogen birth control pill?
-What should I use as a comparison for the Vetiver grass water system? I'm looking for a relatively affordable, extremely effective way of removing estrogen compounds from water, like maybe an activated carbon filter or a reverse osmosis system?
-Does Vetiver grass have a good chance of removing hormonal compound from water in the first place? If not, should I revert to a variation of the original Michigan Tech antibiotic experiment? What equipment would I use in that case then?

Thank you for any help you can provide.

[deleted-71417]

Hi,

You sound like you have a great idea for a project. Vetiver grass has long been studied for use as a phytoremediation plant for heavy metal contaminants and agrochemicals, and has been shown to work pretty well.

Here are sites that discuss the specific active ingredients of birth control pills:

http://www.wdxcyber.com/ncontr13.htm

http://www.natazia.com/index.html?WT.mc ... &WT.srch=1

Here is a review of estrogen assay techniques:
http://toxsci.oxfordjournals.org/cgi/re ... /2/288.pdf

There is current interest in phytoremdiation of estrogens, though I am unsure whether vetiver grass has been studied. Here is an example paper link that explicitly looked at removing estradiol from wastewater:

http://www.sciencedirect.com/science?_o ... c9fde08b9e

The analysis technique they used was HPLC/MS (high performance liquid chromatography coupled to mass spectrometry).

Another potentially relevant article is:

http://eg.geoscienceworld.org/cgi/conte ... ct/16/2/61

This abstract might hint at another assay technique:

“CATHERINE HARTUNIAN (KATHLEEN ARCARO), DEPARTMENT OF ANIMAL SCIENCES, UMASS AMHERST, AMHERST, MA 01003
EFFECTS OF ESTROGENIC TOXINS ON VITELLOGENIN EXPRESSION IN MALE MEDAKA FISH
The goal of this project is to develop an efficient, inexpensive protocol for assessing the presence of estrogenic pollutants in waterways.
Exposure to estrogenic pollutants may be correlated with reproductive problems, including infertility, reduced sperm count, cryptorchidism and
testicular cancer. Estrogens are routinely added to the environment through the daily use of pharmaceuticals, oral contraceptives, pesticides,
and industrial and household chemicals. Water treatment plants do not always detect and filter out all these estrogenic compounds, causing
them to remain in the waterways where animals and plants are exposed. Male fish exposed to the estrogenic pollutants respond by
producing vitellogenin, a precursor egg-yolk protein, which is normally expressed only in female fish. In the present study male medaka
(Oryzias latipes) were used because they are a small, hardy freshwater fish that survive in a wide range of temperatures. Livers of adult male
medaka fish were excised and RNA was extracted using Qiagen’s RNeasy mini kit. Due to the small amount of starting tissue (<2ml/liver),
established protocols for RNA extraction were insufficient and modifications had to be resolved to obtain a better yield of RNA. Levels of
vitellogenin expressed in the RNA were measured using MLT’s Vitellogenin mRNA Assay kit. Fish will be exposed to solutions containing known
concentrations of β-estradiol in the laboratory. A correlation between levels of vitellogenin detected and estrogen concentration will be
examined and used as a standard against vitellogenin levels detected from fish exposed to samples from local waterways, leading to a way
for estimating estrogen levels in the field. “

Other related citations:

http://www.osti.gov/energycitations/pro ... _id=827358

http://www.sciencedirect.com/science?_o ... 19f30886f9

You may find this manual on using Vetiver grasses helpful, particularly Section 4 on
its used to treat contaminated water:

http://www.vetiver.org/TVN-Manual_Vf.pdf

This abstract/paper discusses analysis of the estrogens you mentioned:

“Environ Sci Technol, 2004 Dec 1, 38(23), 6377 - 84
Dairy wastewater, aquaculture, and spawning fish as sources of steroid hormones in the aquatic environment; Kolodziej EP et al.; A suite of androgens, estrogens, and progestins were measured in samples from dairy farms, aquaculture facilities, and surface waters with actively spawning fish using gas chromatography-tandem mass spectrometry (GC/MS/ MS) to assess the potential importance of these sources of steroid hormones to surface waters . In a dairywaste lagoon, the endogenous estrogens 17beta-estradiol and estrone and the androgens testosterone and androstenedione were detected at concentrations as high as 650 ng/L . Samples from nearby groundwater monitoring wells demonstrated removal of steroid hormones in the subsurface . Samples from nearby surface waters and tile drains likely impacted by animal wastes demonstrated the sporadic presence of the steroids 17beta-estradiol, estrone, testosterone, and medroxyprogesterone, usually at concentrations near or below 1 ng/L . The endogenous steroids estrone,testosterone, and androstenedione were detected in the raceways and effluents of three fish hatcheries at concentrations near 1 ng/L . Similar concentrations were detected in a river containing spawning adult Chinook salmon . These results indicate that dairy wastewater, aquaculture effluents, and even spawning fish can lead to detectable concentrations of steroid hormones in surface waters and that the concentrations of these compounds exhibit considerable temporal and spatial variation.”

I regret I cannot give you a comprehensive answer to all your questions. The time commitment to do so is too great. However the forgoing citations should give you a feel for the typical methods used to assay for these estrogens at the levels encountered in wastewaterand some background on the use of vetiver grasses in phytoremediation.

Your project sounds like it has a lot of potential to me, though I suspect you will need a good mentor from a university who can help arrange access to lab facilities. I suggest reading the following link:
https://www.sciencebuddies.org/science- ... tors.shtml

I wish you every success in pursuing this very interesting project!

Best regards,

Barrett L. Tomlinson

[GraceLiu]

Thank you so much for your very helpful response. A lot of these articles look like they're dealing with exactly what I'm looking for. I hope you don't mind if I show up later with follow-up questions once I finish reading all the articles thoroughly.

[MelissaB]

Just to add to what Barrett has said, I agree that you will need a mentor for this project since you will be dealing with prescription drugs (birth control). You need permits in order to use controlled drugs in experiments.

Luckily, though, there is a LOT of research going on on this topic, so you should be able to find someone who is interested in this topic and willing to mentor you.

Good luck!