Running head: INFLUENCE OF POPULATION DEMOGRAPHICS ON ORAL

CONTRACEPTIVE IN WASTEWATER

Influence of Population Demographics on Oral Contraceptive in Wastewater

Lauren Guidry, Mark Mehany, Lauren Smith, Alex Wolski
TECM1700:004
November 12, 2014

Abstract
17-Ethinylestradiol (EE2) is a human contraceptive which can interfere with aquatic life. This
experiment analyzes and discusses the concentration levels of EE2 in the Denton Wastewater
Treatment Plant effluent in correlation to the presence of college students in Denton. Originally,
we hypothesized that there would be no change in the concentration of EE2 during the school
season and during the summer break. The experiment tests three different samples during
October, April, and July. The results falsified the hypothesis. The concentrations of EE2 in
Denton Wastewater Treatment Plant effluent were significantly lower in July than in October or
April. These data infer that there is a direct correlation between the levels of EE2 in the
wastewater effluent and the presence of college students.
1

Influence of Population Demographics on Oral Contraceptive in

Wastewater
Introduction
17-Ethinylestradiol (EE2) is a synthetic human contraceptive structurally similar to the
hormone estrogen which can be used in birth control medications. Studies have shown that when
male fish are exposed to EE2 they display female sexual characteristics and reproductive
dysfunction. EE2 is introduced to the water system through human excretion after consumption
of EE2 into the wastewater (Brooks et al., 2003; Huggett et al., 2003). Since many women use
birth control medications currently, research on how demographics influences EE2 water
concentrations is crucial. The purpose of this experiment is to determine if the concentration of
EE2 in the Denton Wastewater Treatment Plant (WWTP) sewage, or effluent, varies due to the
presence of college students. The experiment will be conducted when students are attending
college in October and April and when they are on summer break, away from college, in July.
We predict that there will be no difference in the concentration of EE2 in the Denton Wastewater
Treatment Plant effluent whether students are attending college or away from college.

Materials and Methods

The materials used in this experiment, some of which are specialized, include the following:

A Liquid chromatography-mass spectrometry (LC-MS)

1000 mL effluent samples
D3-17-estradiol
1 ml ethyl acetate
1.5 ml amber glass vials (Fisher Scientific)
100 l of methanol
Waters 2695 separations module
Waters 2998 UV/vis detector
Waters Sunfire C18 column
Micromass Quattro Ultima mass detector
MassLynx software, version 4.1
17a-Ethinylestradiol (EE2).

To test for the difference in the concentration of EE2 in the Denton Wastewater Treatment Plant
while college students are in school or not, we measured the concentrations of EE2 during the
school season and during the summer break using the following procedures:
1.
2.
3.
4.

Add a fixed amount of d3-17-estradiol to a 1000 mL of wastewater sample.

Once added, liquid-liquid extract the samples twice using 500 mL of ethyl acetate.
Combine and dry the ethyl acetate layers under a gentle stream of nitrogen.
Dissolve the resultant residues in a 1 mL ethyl acetate and transfer them to 1.5 mL amber
glass vials (Fisher Scientific).
5. Dry the new solution under nitrogen.
6. Suspend resultant residue in 100 L of methanol.
7. Take a 50 L portion of the solvent and test it using the Dansyl Chloride Derivatization
method detailed by Nelson, Grebe, OKane, and Singh (2004).
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8. Use MassLynx software version 4.1 and Micromass Quattro Ultima mass detector to
record the data.

Results
The experiment yielded successful results, which are recorded below in chart 1. In the month of
July, the concentration levels of EE2 were the lowest of the three months tested. During the
months of April and October, the concentrations were similar, yet far higher than the month of
July. These findings suggest that there is a correlation between the concentration of EE2 and
college students in Denton. The drop in concentration during the month of July allows us to infer
that when college students are on summer break, the concentration of EE2 significantly drops.
As seen below, all three samples have different concentrations each month that they were tested.
A visual representation is provided below in table 1 to show the correlation between the level of
EE2 and the months of testing.

Replicate
Sample #

April

July

October

1
2
3

3 ng/L
1 ng/L
5 ng/L

< 0.5 ng/L1

< 0.5 ng/L
< 0.5 ng/L

2 ng/L
1 ng/L
2 ng/L

This is below the levels of analytical detection, which is 0.5 ng/L.

Chart 1. Concentration of EE2 Based on Month

Table 1. Concentration (ng/L) of EE2 in the Denton WWTP Effluent in April, July, and October

Concentra<on"of"EE2"in"the"Denton"WWTP"(ng/L)"

Concentra<on"of"EE2"

6"
5"

5"
4"
3"

3"
2"

2"
1"

1"

1"

1"

<0.5" <0.5" <0.5"

0"
April"

July"

October"

Month"
Sample"1"

Sample"2"

Sample"3"

Discussion
Although we originally hypothesized that there would be no difference in the concentration of
17-Ethinylestradiol (EE2) during the April, July, and October, through the methods detailed
above, the results proved that the concentrations fluctuated over the three months of testing.
During the month of July, the concentrations of EE2 were below the levels of the analytical
detection, which is 0.5 ng/L. These findings not only support that there is a difference in the
concentrations of EE2 during April, July, and October, but there is also a direct correlation
between the population demographic, specifically college students, and the levels of EE2 in
wastewater. The results of this research can be further applied and used for future experiments to
reduce the effects of EE2 on the local aquatic life, such as altering the fishs reproductive system,
as well as other various harmful effects on the environment, such contaminating the fresh water.

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.c, and Schlenk,
D. 2003. In vitro and in vivo comparison of municipal effluent estrogenicity.
Toxicological Sciences 72:77-83.
Nelson, R., Grebe, S., OKane, D., and Singh, R. 2004. Liquid chromatography-tandem mass
spectrometry assay from simultaneous measurement of estradiol and estrone in human
plasma. Clinical Chemistry 50:373-84.