College Students Influence on Contraceptive-Concentration Found in Denton Waste Water

Callum Muir, Calvin Rowe, Donovan Street, Raven Walker

TECM 1700:006
November 4, 2014

Abstract
This study involves the effects of college students on the concentration of 17a-Ethinylestradiol
(EE2), a drug mainly found in birth control medication, in the waste water. This drug has been
observed to cause reproductive dysfunction in male fish and cause them to develop female
characteristics. The study is being performed to evaluate if college students have an effect on the
level of (EE2) in the waste water system. The hypothesis was that there would be no effect on
the concentration of (EE2) due to college students. Data is taken from values found at the Denton
Wastewater Treatment Effluent. The method of receiving the data used taking several samples
and breaking it down to find the concentration of (EE2) in the samples. The data from the
samples show that there is a small increase in the amount of (EE2) in the waste water when
college students are present in the area.

Introduction
17-a Ethinylestradiol or (EE2) is a synthesized version of estrogen, commonly used in birth
control, that is taken orally and absorbed through the small intestine, then metabolized in the
liver since estrogen by itself is quickly inactivated by the liver if taken by regular means. Since
(EE2) is excreted through urine it can make its way to bodies of water through the sewer
systems and other irrigational systems. This drug has been observed to cause reproductive
dysfunction in male fish populations, as well as cause male fish to display female characteristics.
Since humans are the main perpetrator in the drug entering the water system, and college
students are more likely to be using these birth control medications, this experiment was
conducted to find if more (EE2) was prevalent in the wastewater system in the presence of
college students. The experiment will take data when students are in session (mid-August to midMay) and when they are on break (mid-May to mid-August).

Hypothesis
The presence of college students will have no effect on the amount of EE2 in the wastewater
system.

Methods
This experiment was performed to test the concentration of the synthetic version of estrogen,
17-Ethinylestradiol (EE2), commonly used in birth control, when college students are attending
college.
The experiment used a Waters 2695 separations module connected to a Waters Sunfire C18
column to produce all chromatographic separations. A quadrupole-hexapole-quadrupole
instrument (Micromass Quattro Ultima mass detector) assisted in preforming the electrospray
ionization and mass-spectrometric analysis. The MassLynx Software, version 4.1, determined
criteria to set limits of detection.

Materials
Use the following materials to perform the experiment.

1000 ml of 17-Ethinylestradiol (EE2)

500 ml of ethyl acetate

2 1.5 ml amber glass vials

100 l of methanol

50 l of aliquot

A Waters 2695 separations module

Waters 2998 UV/vis detector

Waters Sunfire C18 column (2.1 x 50 mm; 3.5 m particle size)

Micromass Quattro Ultima mass detector

MassLynx software (Version 4.1)

Procedure
Follow these instructions to perform the experiment
1.

Quantify the EE2 using liquid chromatography-mass spectrometry (LM-MS).

2.

Spike 1000 ml of effluent with the internal standard, d3-17-estradiol.

3.

Once the liquid is spiked, extract the samples twice using 500 ml of ethyl acetate.

4.

Dry the combined ethyl acetate layers under a gentle stream of nitrogen.

5.

Reconstruct the resultant residues to 1 ml of ethyl acetate.

6.

Transfer the 1 ml of ethyl acetate to 1.5 ml amber glass vials.

7.

Dry the reconstituted vials under nitrogen with residue re-suspended in 100 l of
methanol.

8.

Then take 50 l of aliquot from the previous step for estrogen quantifications using a
dansyl chloride derivatization detailed by Nelson, Grebe, OKane, and Singh.

Results
We have found that in the month of
April, the level of EE2-concentration
in the tested waste water was much
higher comparatively to the months
of July and October (see Fig. 1). We
have also found that, overall, the
month of July shows the lowest
EE2-concentration out of the tested
months. For all three samples, the
month of April varied the greatest,
ranging between 1 ng/l to 5 ng/l. The
month of July showed no significant
change in any of the three trials,
measuring at a concentration level of
0.5 ng/l. Finally, Octobers
variability in concentrations were at
2 ng/l for two out of three trials and
1 ng/l for the other trial.

Fig. 1 Chart of EE2-Concentration levels found in tested waste water

Discussion
The hypothesis states that the presence of college students will have no effect on 17aEthinylestradiol (EE2) concentration in the Denton wastewater system. However, it was found
that over the summer vacation months when students are seldom present, the concentration of
EE2 dropped to less than 0.5 ng/l, while the concentration of EE2 ranged between 1 and 5 ng/l
during the spring and fall semesters. The data was taken over a time period of one school year,
but it would be more conclusive with more research. Conducting the experiment for a number of
years would provide more knowledge of EE2 and show if the variation of EE2 concentration in
water is relatively similar each year. Based on the data, the hypothesis was incorrect in the sense
that EE2 concentration levels reduced when college students were absent from campus. These
results give a better understanding of how population demographics effect water conditions, and
how those conditions change once the demographic is changed. The resulting data give reason to
reduce the concentration of EE2 in the Denton water system in order to lower the risks of
reproductive dysfunction, especially in males. This process now has the backing of significant
data, giving it reason to be enacted in many more cities. With more knowledge of EE2 comes
better resources on how to reduce its presence in not just the wastewater of Denton, but
throughout the state of Texas or even all of the United States.

References
Raign, K., & Huggett, H. (2013). Writing Lab Reports. In Writing For Results: An Introduction
to Writing in the Real World of Science and Technology (Second ed., pp. 173-175). Dubuque,
IA: Kendall Hunt.