Comparison of 17a-Ethinylestradiol Levels in Denton Waste

Water
Katelyn Williamson, Enna Diaz, Omar Correa, Henry Lopez
TECM 1700 004
11/10/2014

Abstract
In this experiment, 17a-Ethinylestradiol (also known as EE2) was hypothesized to have no
noticeable difference in levels when college students are and are not attending school. To test
this we quantified EE2 levels through liquid chromatography mass spectrometry. Levels were
then found to be inconclusive during the month of July. However, during April and October, EE2
levels were detectable, indirectly concluding that months college students are attending school
directly affect the presence of EE2 in Denton waste water.
1

Introduction and Hypothesis

Introduction
17a-Ethinylestradiol (EE2) is a synthetic version of estrogen that is used in birth control
medication. Studies in fish have observed that EE2 causes male fish to show female
characteristics as well as dysfunction in the reproductive system. We consume this every time we
drink water that comes from the Wastewater Treatment. The following experiment was done to
find out if there is a connection between EE2 levels and the presence of college students. It will
focus on when students are in session (late August to the beginning of May) and when they are
not in school (mid-May to mid-August).

Hypothesis
There will be no difference in levels of EE2 present in waste water when students are attending
college and when they are not attending college.

Materials and Methods

The following section covers the materials used in the experiment as well as the methods used to
test for 17a-Ethyinylestradiol.

Materials List (Ragin, K. R, 2013 and Nelson et al, 2004)

1000 ml of April effluent sample

1000 ml of July effluent sample
1000 ml of October effluent sample
1800 ml of ethyl acetate
Twelve 1.5 ml Fisher Scientific amber glass vials
8 small glass beakers
Four 16 X 100 mm glass screw cap tubes
6 ml of methanol
21 ml of methylene chloride
1 canister of nitrogen gas
Four 13 X 100 mm glass tubes
An equal volume of 1 g/L dansyl chloride in acetone
45* C water bath
1 tube rack
60* C heating block
Vortexer
200 ul of sodium bicarbonate buffer (100 mmol/L. pH 10.5)
Mass Lynx software version 4.1
Micromass Quattro Ultima Mass detector
Waters 2695 separations module
Waters 2998 UV/vis detector
Waters Sunfire C18 Column (2.1 X 50 mm; 3.5 um particle size)
P200 micropipettor (20 ul -200 ul)
P1000 micropipettor (100 ul -1000 ul)
2

P200 micropipettor tip box

P1000 micropipettor tip box

Methods
This section describes the steps taken to gather the data discussed in the Results section. If
followed, these procedures should replicate the experiment exactly.
Sample Collection and Preparation (Ragin, K. R 2013)
1. Collect 1000 ml of wastewater treatment effluent from a Denton water treatment plant using
a beaker in April.
2. Record the date, time and collection location for future reference
3. Use the internal standard d3-17B-estradiol to spike the sample to determine the amount of
EE2
4. Extract the liquefied sample using 500 ml of ethyl acetate. Repeat one more time
5. Combine the extracted liquid and dry it using a gentle stream of nitrogen. There should be
solid residue present afterwards.
6. Transfer the solid residue to a smaller beaker
7. Set the P1000 micropipettor to 1000 ul and pipette 1000 ul of ethyl acetate into the smaller
beaker containing the residue.
8. Mix the residue is evenly distributed throughout the liquid, creating a thoroughly Solution
9. Using the P1000 micropipettor transfer 1.5 ml (1500 ul) of the resulting solution into each of
the 3 amber glass vials
10. Repeat Steps 5 and 6. Then continue with the remaining procedure
11. Using the P200 micropipettor pipette 100 ul of methanol into the small beaker
12. Mix the residue and methanol until they create a thoroughly mixed solution
Dansyl Chloride method (Nelson, R. E. 2004)
1. Use the P200 micropipettor to gather 50 ul of the solution previously created in step 12 of
Sample Collection and Preparation and transfer it into a 16 X 100 mm glass screw cap
2. Add 25 ul of the working stock mix of d4-E1 and d5-E2 as an internal standard using the
P200 micropipetter
3. Using the P1000 micropipetter transfer 6 ml (6000 ul) of methylene chloride into the tube
4. Remove the upper aqueous layer using the P100 micropipettor
5. Transfer the remaining solvent to a sterile 13 X 100 mm glass tube
6. Place the tube in a 45C water bath
7. While the tube is still in the water bath run a gentle stream of nitrogen in the tube to
evaporate the solvent. There should be a dried residue in the tube.
8. Remove the tube from the water bath and place it into a tube rack
9. Using the P200 micropipettor pipette 50 ul of sodium bicarbonate buffer (100 mmol/L. pH
10.5) into the tube to redisolve the residue
10. Then an an equal volume of 1g/L dansyl choloride in acetone to the tube and vortex for 1
minute
11. Place the tube in the 60C heating block for 3 minutes to incubate
12. Immediately place the entire sample to an autosampler glass vial, seal it then assay it
Column Installation (Waters (December 2010)
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1. Purge the system of any spare buffer and connect the inlet end of the column
2. Flush the column by running 100% methanol through the system by setting the flow rate to
0.1 mL/min and gradually increasing the flow rate to 1 mL/min over a period of 5 minutes
3. Stop the methanol flow it flows freely from the column outlet
4. Attach the column outlet to the Waters 2695 Separations Module
5. Repeat step 2 once the column is securely attached
6. Flush the column 5 times with a mixture of 60% methonal and water once the baseline
measurement had been reached
Setting up the MassLynx Software Waters (2002)
1. Select the system filed in the configuration screen, press enter to display a list of operating
modes
2. Select Control by MassLynx then press enter. The IEE 488 address field should be
highlighted.
3. Press enter to display a list of addresses
4. Select the address that corresponds to the Waters 2695 Separation module then press enter
5. Press exit and the machine is now controlled by the MassLynx software
6. Repeat steps 1-5 for the following machines: Waters 2998 UV/ vis detector and Micromass
Quattro Ultima mass detector

Results
Chart 1, showed below, measures EE2 levels in Denton waste water from April to October using
data obtained from samples (Table 1) prepared under Methods. EE2 levels are consistently
higher in samples taken in April and October, as opposed to ones taken in July. In the months
leading to April, college students have returned to school for spring semester. The same could be
said for the months leading to October, as college students are entering school for fall semester.
In July, most college students are on summer break, correlating with low levels of EE2. The
influx of EE2 levels is supported by the common use of oral contraceptive pills (birth control
pills) among female college students (Macdonald, N. et al.).

EE2 Levels in nanograms per liter (ng/L)

Chart 1: EE2 Levels in Denton Waste Water over Months

6
5
5
4
3
3
2
2
1

1
0
0
April

July

October

Months tested
Sample 1

Sample 2

Sample 3

Table 1: Measured EE2 Levels per Month

April
July
October
Sample 1
1 ng/L
*0 ng/L
1 ng/L
Sample 2
3 ng/L
*0 ng/L
2 ng/L
Sample 3
5 ng/L
*0 ng/L
2 ng/L
*Samples taken are inconclusive, meaning they are below analytical detection (.5 ng/L)

Discussion
From the samples that were collected, it can be concluded that the presence of college students
does affect the amount of EE2 in the Denton water. The hypothesis is rejected. In Table 1,
samples were taken during the months of April, July, and October. Samples of water were taken
during these months to see if the amount of EE2 would be higher or lower than others depending
on the month. The EE2 concentration present in water samples obtained in April ranged from 1-5
ng/l. In October, samples ranged from 1-2 ng/l of EE2. Lastly, in the month of July, the
concentration of EE2 levels in samples were 0 ng/l. Any sample with concentration below .5 ng/l
are too low to detect its EE2 level. In July, all three samples that were taken returned
inconclusive, meaning there was not enough EE2 in the water present to detect. In July, since
there are no classes the amount of EE2 is going to be significantly lower due to the fact that there
are not as many college students in Denton. With not as many students the levels of EE2, the
samples taken in July reflected that. In April and in October all the samples taken in those
months were beyond the minimum amount of 0 ng/l. October and April are months that students
are in class this proved that college students do affect the EE2 levels in water.

Works Cited
Brooks, B.W., Foran, C. M., Peterson, B. N., Wetson, J. La Point, T. W., and Huggett, D. B.
2003. Linkages between population demographics and effluent estrogenicity. Bulletin of
Enviornmental 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
Macdonald, N., Wells, G., Fisher, W., Warren, W., King, M., Doherty, J., & Bowie, W. (n.d.).

High-Risk STD/HIV Behavior Among College Students. JAMA: The Journal of the
American Medical Association, 3155-3159.
Nelson, R.E., Grebe S. K. et al. (2004). Liquid Chromatography-Tandem Mass Spectrometry
Assay for Simultaneous Measurement of Estradiol and Estrone in Human Plasma. In
Materials and Methods (Sample Preparation). Retrieved from
http://www.clinchem.org/content/50/2/373.full.pdf
Ragin, K. R., and Hugget H. (2013). Writing for Results: An Introduction to Writing in the Real
World of Science and Technology (2nd ed, pg 174). Dubuque, IL: Kendall Hunt Publishing
Company.
Waters (2002). Controlled by MassLynx Mode. Waters 2695 Separations Module Operators
Guide, Revision B. Retrived from http://www.vtpup.cz/common/manual/Extern_QUINTAANALYTICA_Waters_2695D_manual1_EN.pdf
Waters (December 2010). Reversed-phase Columns (Sunfire C18 and Sunfire C8). Sunfire
Columns Care and Use Manual. Retrieved from
http://www.waters.com/webassets/cms/support/docs/715000891.pdf