Running Head:17a-Ethinylestradiol in Denton
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Population influence on the concentration of 17a-Ethinylestradiol in the Denton Effluent
Evan Devora, Marco Duarte, Gadiel Espada
University of North Texas
TECM 1700
November 12, 2014
Abstract
In this experiment our purpose was to test the amount of 17a-Ethinylestradiol in the water
supply at the Denton Wastewater Treatment Effluent. Ethinyl estradiol also known as 17aEthinylestradiol (EE2) is a synthetic version of estrogen that is commonly used in birth control
medication. We believed that the water would show no change. To conduct this experiment we
used mass spectrometry. We also spiked the sample with the internal standard d, 17β-estradiol.
The results from this experiment concluded that the water is getting contaminated when college
students are more actively present in Denton.
17a-Ethinylestradiol in Denton
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Introduction
The purpose of this experiment was to measure the content of 17a-Ethinylestradiol (EE2)
or Ethyl Estradiol in the Denton Wastewater Treatment Effluent (WWTP). (EE2) is found in
most birth control medication that is used to today. Because Denton is home to the University of
North Texas we wanted to test to find if a larger concentration of (EE2) could be found around
the time school was open for the fall and spring semesters. We also tested during the summer
semester as well to find if there was any connection between them.
The reason we are testing at the (WWTP) is because something like this gets exposed to
the water system is through post-human consumption (i.e. consumed and excreted) (Brooks et
al., 2003; Huggett et al., 2003). Since women are more present during the fall and spring
semesters we are trying to see if the amount of women on campus affects the amount of (EE2) in
the water supply. This leads for the experiment to take place during the fall and spring semester
to try and find a connection. The summer semester is used as the control for the experiment..
It has been observed that this compound has been linked with causing the male fish to
display female characteristics and it also causes the reproductive system dysfunction. This could
lead to problems for the local ecosystem if large amounts of (EE2) are found in the water. Our
hypothesis is that there will be no correlation between when the students are in school and when
they are not.
Materials and Methods
Materials
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1 Waters 2695 separations module
1 Waters 2998 UV/vis detector
1 Waters Sunfire C column
1 Micromass Quattro Ultima mass detector
3 1000 ml water samples
3 d3-17ß-Estradiol samples
1 Source of nitrogen gas
3 1.5ml amber glass vials
100 µl methanol
Methods
The following list is describing the steps used to test the amount of (EE2) in the water supply:
1. Take the 3 1000 ml water samples retrieved from the WWTP and spike each sample with
d3-17ß-Estradiol
17a-Ethinylestradiol in Denton
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2. Make sure the Waters 2695 separations module is paired with a Waters 2998 UV/vis
detector and a Waters Sunfire C column. Once done, take the spiked samples and place
them in the separator and extract them with 500 ml of ethyl acetate.
3. Take the ethyl acetate layers and dry them in a gentle stream of nitrogen.
4. The layers should be reconstructed into 1 ml ethyl acetate samples. Take the 3 different
samples and place them into 3 different 1.5 ml amber glass vials
5. Take the 3 vials and dry them under nitrogen while being suspended in 100 µl of
methanol.
6. From this, take 50 µl of the ethyl acetate from each sample and place them into the
Micromass Quattro Ultima machine.
7. Use the Micromass Quattro Ultima machine to determine the concentration of EE2 in
each separate sample
8. Repeat steps 1-7 for the month desired.
Results
Below is a table explaining our findings for each month tested:
Table 1. Concentration (ng/l) of EE2 in the WWTP in April, July, and October
Sample #
April
July
October
1
3 ng/l
<0.5 ng/l*
2 ng/l
2
1 ng/l
<0.5 ng/l
1 ng/l
3
5 ng/l
<0.5 ng/l
2 ng/l
According to the separate samples taken in the months of April, July and October, the
readings vary in each month. In July, the concentration is noticeably lower than in October or
April. The concentration is highest in April, near the end of the academic school year. The star
in sample 1 of July notifies that the detection of EE2 is below the criteria of detection, which is
0.5 ng/l.
Discussion
The results of the experiment do not go with the hypothesis stated earlier. The hypothesis stated
that the concentration would be the same when college is in session and when it is not.
According to the data, the concentration is noticeably higher when school is in session compared
to when school is not. Thus, college students while attending college affect the EE2
concentration in the waste water. This could be due to the use of popular use of birth control in
college women. Future experiments will narrow where the EE2 is coming from, and how it is
getting into the waste water.
17a-Ethinylestradiol in Denton
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References
Brooks, B.W., Foran, C.M, Peterson, B.N., Weston, J.La Point, T.W., and Huggett, D.B. 2003.
Linkages between population demographics and effluent estrogenicity. Bulletin of
Environmental Contamination and Toxicology 71:504-51
Huggett, D.B., Foran, C.M. Brooks, B.W., Weston, J., Peterson, B.N., Marsh, E., and Schlenk,
D. 2003. In vitro and in vivo comparison of municipal effluent estrogenicity.
Toxicological Sciences 72:77-83.
Nelson, R., Grebe, S., O’Kane, D., and Singh, R. 2004. Liquid chromatography-tandem mass
spectrometry assay for simultaneous measurement of estradiol and estrone in human
plasma. Clinical Chemistry 50: 373-84.