Correlation between Population Demographics and 17a-Ethinylestradiol in the Denton

Wastewater Treatment Plant

Ross Nelson, Sharbria Blanche, Opeoluwa Bamgbose, Alex Martinez
TECM 1700:007
11/12/14

Abstract
The objective of this experiment was to figure out whether college students using oral
contraceptives in the area had an effect on the concentration of EE2 in the Denton wastewater
treatment plant at the times where they were and not attending school. This experiment is
being performed because a previous study indicated that a high concentration of EE2 causes
male fish to display female characteristics and some reproductive dysfunctions. The EE2 enters
the water system through the excretion of post-human consumption. Therefore, if the EE2 is in
the birth control when the pill or shot is passed through the system it will then be released
along with the exiting waste. In order to figure out if the presence of college students in the
area affects the concentration of EE2: an experiment will be conducted to then test the stated
hypothesis, There will be no difference in the concentration of EE2 in the Denton wastewater
treatment plant effluent when college students are attending college and not attending
college.
1

Introduction
17a-Ethinylestradiol (EE2) is a synthetic version of estrogen that is commonly used in
many birth control medications. Laboratory studies of fish have shown that male fish exposed
to EE2 display female sexual characteristics and reproductive dysfunctions. According to the
article, Linkages between population demographics and effluent estrogenicity, EE2 enters
waterways via post-human consumption. Given that the presence of EE2 correlates to number
of women who use birth control; a better understanding of how population demographics
influences the concentration of EE2 in wastewater are needed. The experiment was conducted
to determine if the concentration of 17a-Ethinylestradiol (EE2) in the Denton wastewater
treatment plant (WWTP) effluent varies due to the presence of college students. Specifically,
the concentration of EE2 will be measured in the WWTP effluent when students are in session
(late August to the beginning of May), and during summer break (mid-May to mid-August). The
stated hypothesis is that, there will be no difference in concentration of EE2 in the Denton
wastewater treatment plant effluent whether college students are attending college or not.

Materials and Methods

Materials:
1000 mL effluent samples of EE2
500 mL ethyl acetate
1.5 mL amber glass vials
100 ul methanol
50 ul aliquot
d3-17B-estradiol
Compressed Nitrogen gas (at least 5 cubic feet)
Liquid chromatography-mass spectrometry machine
Quadrupole-hexapole-quadrupole instrument (Micromass Quattro Ultima mass
detector, Manchester, UK)
Methods:
We are conducting this experiment to find out the concentration of EE2 in the Denton
wastewater treatment plant effluent when there is a higher concentration of college students
in comparison to when there is not.
1. Spike the effluent samples with the internal standard d3-17B-estradiol and the samples
will become liquid
2. Extract the liquid twice using 500 mL of ethyl acetate
3. Combine the ethyl acetate layers and dry them under a gentle stream of nitrogen
4. Use the resultant residue and reconstitute in 1 mL ethyl acetate and then transfer it to a
1.5 mL amber glass vials
5. Take the reconstituted solvent residue and dry it under nitrogen with residue
resuspended in 100 ul of methanol
6. Take 50 ul aliquot using a dansyl chloride derivatization method for the estrogen
qualification
2

Results
According to our findings, there is an apparent difference in the levels of EE2 in the
Denton WWTP between April, July, and October. When students are not present on campus in
July, the concentration of EE2 are below levels of analytical detection (< 0.5 ng/l). However, the
levels of EE2 effluent in the Denton Wastewater Treatment Plant were between 2-5 ng/l when
students were attending college in April and October. The varied outcomes in the three trials
suggests that the previously stated hypothesis was not valid as a result of this experiment.
Table 1&2: Concentration (ng/l) of EE2 in the Denton WWTP Effluent in April, July, and October

Replicate Sample #

April

July

October

3 ng/l

< 0.5 ng/l*

2 ng/l

1 ng/l

< 0.5 ng/l*

1 ng/l

5 ng/l

< 0.5 ng/l*

2 ng/l

*Below levels of analytical detection, which is under 0.5 ng/l

Discussion
As observed in the experiment, it can be concluded that there actually was a difference
in the concentration of EE2 in the Denton wastewater treatment plant effluent when there is a
higher concentration of college students in comparison to when there is not. When using liquid
chromatography-mass spectrometry to quantify the EE2, it became apparent that the previously
stated hypothesis was not valid as a result of performing this experiment. The concentration
levels of EE2 varied from April to October according to the data taken from samples 1, 2, and 3
during those months. There were in fact higher levels of EE2 concentration present when
students were in school from late August to the beginning of May. After comparing the results
from each sample, it became clear that there shouldve been several more samples taken for
further observation. The samples shouldve also been recorded consecutively for every month of
the year and on a certain day. By doing so, the results of this experiment will therefore become
more reliable. The experimental design used does adequately address the hypothesis; however,
faulty assumptions can be made based on the lack of data which lessens the credibility of the
experiment as a whole. The only question prompted by the results of this experiment would be:
if there was another experiment, would the outcome be the same if more samples were taken?

Conclusion
Our hypothesis at the beginning of this experiment was that there will be no difference
in the concentration of EE2 in the Denton wastewater treatment plant effluent whether college
students are attending school or not. Using the liquid chromatography-mass spectrometry
method to quantify the EE2, three separate samples were created to see if there was a
difference in the EE2 during the months where students were and not attending school. After
testing these three samples, it became apparent that there was in fact a difference in the
concentration levels of EE2 when students were and not attending college. According to our
data, there were higher and detectable levels of EE2 from late August to the beginning of May
when school was in session. In contrast, the levels of EE2 measured during the month of July
where students were on summer break were undetectable (below 0.5 ng/l). Due to the
findings stated above, we are able to conclude that our hypothesis was incorrect and more
samples will be taken in the future to solidify the outcome of this experiment.

References
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