Effects of Population on Estrogen Concentration in Denton Wastewater

Brigido Montemayor, Yousif Nahash, Triston Williams, Jameson Reno

TECM 1700: 502
11-10-14

Abstract
This experiment was to test for the concentration of 17a-Ethinylestradiol in Denton
wastewater due to the population in college students. 17a-Ethinylestradiol is a type
of estrogen, female hormone, generally found in birth control medicine. To test for
this, filter out the wastewater by taking three samples in each month of in April, July,
and October. The population of Denton fluctuates due to the college students and
increase of the number of women taking birth control medication. However, the
hypothesis was rejected. The concentration of 17a-Ethinylestradiol increased in the
months of April and October. July had no trace of the hormone. This is probably the
cause of the absence of college students in the summer.

EFFECTS OF POPULATION ON ESTROGEN CONCENTRATION

Introduction

17a-Ethinylestradiol, otherwise known as EE2, is an artificial form of estrogen that

is used in many birth control medications. Studies in fish have shown that EE2 can
give male fish female features and can cause a decrease in fertility and cause
humans to have cardiovascular and endocrine problems (Drugs 1). EE2 is most
commonly released into the water through human bodily waste. Given that the rise
of EE2 in the Denton water supply is most likely caused by an increase in the
number of women using birth control medication, a better understanding of how a
larger population in one area, as opposed to a smaller one, effects the amount of EE2
in the water supply is needed. The experiment was done to find the relation
between the concentration of EE2 in the Denton wastewater treatment plant
(WWTP) emissions and an increased presence of college students. If the presence of
college students is causing the high concentration of EE2, then the months of April
and October will have the highest concentration of EE2. Specifically, the
concentration of EE2 will be measured in the WWTP emissions during the months
of late August to early May.
Materials And Methods
Materials:
These are the necessary materials needed for this experiment, to test for the
concentration of EE2.

Waters 2695 separations module

Waters 2998 UV/vis detector

EFFECTS OF POPULATION ON ESTROGEN CONCENTRATION

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

Quadrupole-hexapole-quadrupole-instrument

Procedure:
1) To test for the concentration, take samples in the months of April, July, and
October. In each of these months take three samples of the water. EE2 was
quantified using liquid chromatography-mass spectrometry. A total of 1000mL
effluent samples were spiked with internal standard d3-17B-estradiol, which
was used to quantify EE2.
2) Once spiked, samples were liquid extracted twice using 500mL of ethyl acetate
layers were combined and dried under a gentle stream of nitrogen. The
resultant residues were reconstituted in 1mL ethyl acetate and transferred to
1.5mL amber glass vials (Fisher Scientific).
3) Once the solvent dried under nitrogen with residue suspended in 100l
methanol. From this volume, 50l aliquot was taken for estrogen quantifications
using a dansyl chloride derivatization method detailed by Nelson, Grebe,
OKane, and Singh(2004). All chromatographic separations were conducted
using a Waters Electrospray ionization and mass-spectrometric analysis were
conducted using a quadupole-hexapole-quadrupole- instrument (Micromass
Quattro Ultima mass detector, Manchester, UK). For all EE2, instrument
determined (MassLynx Software, Version 4.1) criteria were used to set the
limits of detection.

EFFECTS OF POPULATION ON ESTROGEN CONCENTRATION

Results
Three samples were taken during each of the three months of April, July, and
October. The three samples of April in order from first to third were 3 ng/l, 1 ng/l,
and 5 ng/l. For July, all the results were less than 0.5 ng/l, with the first sample
actually being so low that it was not detected. For October, the samples in order
were 2 ng/l, 1 ng/l, and 2 ng/l. The green bar above each month represents the first
sample, the purple bar represents the second, and the orange represents the third.
According to the table, the samples were extremely concentrated in the months of
April and October when compared to the samples taken in July, which all registered
lower than 0.5 nanograms per liter. This shows that there is a significantly less
amount of EE2 in the water during the months in which students are not attending
college, contrary to the original hypothesis of the experiment.

EFFECTS OF POPULATION ON ESTROGEN CONCENTRATION

Discussion:
The hypothesis stated that there will be a higher concentration of EE2 in Denton
wastewater treatment plant when college students are attending college and not
attending college, likewise the data supports the hypothesis. The graph above shows
that there is an increase in EE2 in the months students are attending college. On the
other hand, the months that students are not attending college there is a decrease in
EE2.The data that were collected is based on a short term and more data is required

EFFECTS OF POPULATION ON ESTROGEN CONCENTRATION

to be accurate. Further studies to determine if the increase is directly linked to the
college students and more data need to be collected to confirm the hypothesis.

EFFECTS OF POPULATION ON ESTROGEN CONCENTRATION

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 Enviromental Contamination and Toxicology 71:504-51
Ethinyl estradiol Side Effects in Detail - Drugs.com. (n.d.). Retrieved November 5, 2014.

Huggett, D. B., Foran, C. M. Brooks, B. W., Weston, J., Peterson, B.N., Marsh, E.,
Schlenk, D. 2003. In vitro and in vivo comparison of municipal effluent
estrogenicity. Toxical Sciences 72:77-83
Nelson, R., Grebe, S., OKane, 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