EE2 and its effects on waste waters in Denton

Kevin Dodson, Gxara Munda, Matthew OBrien, Malena Reyes

TECM 1700.007
11/10/14

Through our experiment we expected to show that there will be no change in the level of EE2
that will be found in Dentons WWTPs effluent while students are attending college. We
hypothesised that the level of females that attend college during the academic year will not cause
an impact on the levels of EE2. Our experiment will shows how proceeded to examine the levels
of EE2 that we are able to extract from different effluent samples. Our results are as accurate as
we could get when attempting to trying to find the amounts of this substance present in varying
samples of effluent. Even though our hypothesis was not correct; our findings were coherent and
show that there is variation in the amounts of EE2 that are found in the samples that we tested
during varying portions of the year. So while our prediction about the lack of impact the female
population would have on the levels of EE2 in Dentons WWTPs effluent, we concluded that the
female population could be the cause of the changing levels of EE2 that we found.

Introduction
17a-Ethinylestradiol (EE2) is a synthetic version of estrogen that is commonly used in many
birth control medications. Laboratory studies in fish have observed that EE2 causes male fish to

Running head: EE2 AND ITS EFFECTS ON WASTE WATERS IN DENTON

display female sexual characteristics, as well as causes reproductive dysfunction. EE2 is entering
waterways primarily through post-human consumption (i.e. consumed and excreted) (Books et
al. 2003; Hugget et al., 2003). Because EE2 is found in birth control medication; it can be
assumed that there could be a relation between the amount of EE2 found in waste treatment
plants and the density of women in an area. This experiment was conducted to observe EE2
concentrations found in Dentons water waste treatment plant (WWTP) during periods of
heightened female population during the school year. We feel that the concentration of EE2 in
the Denton WWTP will not be affected by the influence of the higher female population during
the school year.

Methods
This section will go into detail about the materials and process used in our experiment to find the
amounts of EE2 in the tested samples.

Materials

1000 single ml waste samples

d3-17b-estradiol (synthetic estrogen)
500 ml of ethyl acetate
Nitrogen based drying system
1 ml of ethyl acetate
1.5 amber glass vial (Fisher Scientific)
100 l of methanol
dansyl chloride
Waters 2695 separations module
heating block
Waters 2998 UV/vis detector
Waters Sunfire C column (2.1 x 50mm; 3.5 um particle size)
quadrupole-hexapole-quadrupole instrument (Micromass Quattro Ultima mass detector
Manchester, UK)
MassLynx software, version 4.1

Procedure
1. Separate the hormone d3-17B-estradiol from the EE2 solution using liquid
chromatography-mass spectrometry (LC-MS).
a. Separate the EE2 solution into its individual chemical compounds using a Waters
2695 separations module coupled to a Waters 2998 UV/vis Detector and a Waters
Sunfire C column (2.1x50mm; 3.5 m particle size).
2

Running head: EE2 AND ITS EFFECTS ON WASTE WATERS IN DENTON

b. Measure the mass of each individual chemical compound using a quadrupolehexapole-quadrupole instrument.
2. Collect 3 waste samples from the local water waste treatment plant. Each sample should
contain 1000 ml of water waste.
3. Pour the internal standard, d3-17B-estradiol, into each of the 3 waste samples.
4. Extract the upper liquid layer from the samples using 500 ml of ethyl acetate. Repeat this
process twice.
5. Combine the ethyl acetate layers within each of the samples.
6. Dry the ethyl acetate layers under a gentle stream of nitrogen until they produce a
resultant residue.
7. Transfer the resultant residues to 1.5 ml amber glass vials (Fisher Scientific) after the
residues are reconstituted in 1 ml ethyl acetate.
8. Resuspend the residue in 100l of methanol.
9. Dry the reconstituted solvent under nitrogen along with the resuspended residue.
10. Take a 50l aliquot for estrogen quantifications using a dansyl chloride derivatization
method detailed by Nelson, Grebe, OKane, and Singh (2004).
a. Add 1 g/L dansyl chloride in acetone.
b. Mix equal measures of the dansyl chloride- acetone mixture into each of the
individual waste samples.
c. Mix each of the waste samples for 1 minute.
d. Incubate the samples in a heating block at 60 C for 3 minutes.
e. Transfer the waste samples into sealed glass vials.
f. Regularly examine the vials for changes in EE2 concentration.
g. Record how much EE2 is present in each vial.

Results
The following graph will show the concentration of EE2 that was found in three of the samples
of effluent liquid in the months of April, July, and October.

Running head: EE2 AND ITS EFFECTS ON WASTE WATERS IN DENTON

Graph 1: Concentration of EE2 in Waste Samples during the months of April, July, and October
The observations showed that there was a higher concentration of EE2 in the Denton WWTP
effluent in each replicate sample during the month of April compared to the months of July and
August. During the month of April, sample 1 showed a concentration of 3 ng/1, sample 2 showed
a concentration of 1 ng/1, and sample 3 showed a concentration of 5 ng/1. The month of July
exhibited the lowest concentration of EE2, 0.5ng/1 for each of the months: April, July and
October. The month of October exhibited a concentration of 2 ng/1 in sample 1, a concentration
of 1 ng/1, and a concentration of 2 ng/1. Based on the observations, the hypothesis can be
rejected because there was a change of concentration of EE2 in the Denton WWTP Effluent
between the months of April, July, and October.

Discussion
The results in all three replicated samples showed a relation between EE2 levels and population
demographics. During the spring and fall semesters, in which student populations had increased,
the experiment exemplified elevated EE2 concentration levels, negating the original hypothesis.
In regards to repeating another experiment to ensure that the hypothesis would be correct would
be completely unnecessary due to the fact that the data is accurately depicted through the current
experiment. The data shows that levels of EE2 concentrations would increase when student
population increases. The only thing, if any that should be changed in future experiments might
be the placement of where the data was drawn from. In order to assure more accuracy, a
designated location for each year the EE2 concentrations were measured would prove to be
4

Running head: EE2 AND ITS EFFECTS ON WASTE WATERS IN DENTON

effective. In relation to fish reproductive dysfunctions, the increasing concentrations of EE2
levels would greatly affect the male fishs ability to reproduce. This is because the presence of
EE2 cause male fish to gain feminized characteristics. This is a problem when attempting to
mate. When Male fish lose their vivid colors and size the female fish are less likely to become
attracted to the male fish causing for a low outcome of production in assorted fish. Future
experiments in regards to the fish and their outcomes with affected EE2 concentration levels
show to be a necessary experiment.

Running head: EE2 AND ITS EFFECTS ON WASTE WATERS IN DENTON

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., C. M. Brooks, B. W., J. Peterson, B. N., Marsh, E., and Schlenk, D. 2003. In
vitro and in vivo comparison of municipal effluent estrogenicity. Toxicology 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.