College Students Influence on the Concentration of 17a-Ethinylestradiol (EE2) in Wastewater. Lucas Hodde, Alex Elizalde, Yuval Parker, Garrett Reeves

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1 College Students Influence on the Concentration of 17a-Ethinylestradiol (EE2) in Wastewater Lucas Hodde, Alex Elizalde, Yuval Parker, Garrett Reeves Tecm November 3, 2014

2 2 Abstract We tested the concentration of EE2 (a synthetic version of estrogen that is used in many birth control medications) in the Denton wastewater in April, July, and October. Our hypothesis stated that there will not be a difference in the concentration of EE2 in the Denton wastewater. This was done by using liquid chromatography-mass spectrometry (LC-MS). Our results show that there is a difference in concentration of EE2 from April, July, and October. This means that the presence of college students does affect the concentration level of EE2 in the Denton wastewater. Introduction 17a-Ethinylestradiol (EE2) is a synthetic version of estrogen that is commonly used in many birth control medications. The main route by which EE2 is entering waterways is excrement (Brooks et al., 2003; Huggett et al., 2003). Since the presence of EE2 in Denton wastewater is likely correlated to the number of women using birth control, a better understanding of how college students influence the concentration of EE2 is needed. This experiment was performed to test if the presence of college students has an influence on the concentration of EE2 in the wastewater from the Denton wastewater treatment plant (WWTP). The concentration of EE2 in the WWTP wastewater will be measured when they are in session (late August to beginning of May) and when they are on summer break (mid- May to mid-august). Our hypothesis is that college students presence will have no effect on the concentration of EE2 in the Denton WWTP wastewater. Procedure For the procedure you will spike the samples with d3-17b-estradiol. Then for this experiment you will need the requested materials. First you need to combine layers then make sure they are dry. After that reconstitute the residues and transfer ethyl into the other container when the residues are reconstituted, when you are drying the residues make sure it is suspended and dried with nitrogen. Once everything is dried take a very small amount using the prescribed directions.

3 3 Chemicals These are the chemicals needed to replicate the experiment ml wastewater samples d3-17ß-estradiol 501 ml of ethyl acetate Compressed nitrogen 100 micro liters of methanol. 50 microliters of aliquot Dansyl chloride Equipment These are the machines and objects needed to replicate the experiment. Waters 2695 separations module Waters 2998 UV/vis detector Waters Sunfire C18 column (2.1 X 50mm; 3.5 micrometer particle size) Micromass Quattro Ultima mass detector MassLynx software, version ml amber glass vials (Fisher Scientific) Directions Below is the list of steps you will need to help you perform the experiment using liquid chromatography-mass spectrometry (LC-MS). 1. Spike all the wastewater water samples with d3-17b-estradiol. 2. Liquid-liquid extract samples twice using 500 ml of ethyl acetate. 3. Combine ethyl acetate layers and dry them under a gentle stream of nitrogen. 4. Reconstitute the resultant residues in 1 ml of ethyl acetate. 5. Transfer that same ml of ethyl acetate into the 1.5 ml amber glass vials (Fisher Scientific). 6. Dry the solvent under nitrogen with residue suspended in 100 microliters of methanol. 7. Take from this volume a 50 microliter aliquot using Nelson, Grebe, O Kane, and Singh s dansyl chloride method (2004).

4 4 Results This is a table of the data compiled from the experiment Replicate Sample # April July October 1 3ng/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 Table 1 * Below levels of analytical detection, which is 0.5 ng/l This a graph of the raw data. A corresponds with 1 from the replicate sample column in table 1 which is above. B corresponds with 2 and C corresponds with 3. The units used for the y-axis are in ng/l. Graph 1: Levels of EE2 in Denton Wasterwater The EE2 levels in fact were different and thus renders the hypothesis false. The data proved in three different samples that during the school year, the EE2 levels are actually higher. In all three samples in the month of july the levels were less than.5 nanograms per liter. This is constant for all three samples though when you look the months of october and april the levels are varied and higher than.5 nanograms per liter. Even though the levels differ and range from 1-5 nanograms in all of the samples, it still proves that the hypothesis is wrong.

5 Discussion The fact that the levels differ isn't necessarily a bad thing. To take the results in perspective, during the summer months women are using less birth control. Even though the hypothesis is wrong, this doesn't mean the experiment was a waste. There could have been multiple sources of error, depending on the type of tools that was used, there could have been a misreading. There could have been an unusual spike in the numbers that was not mentioned in the experiment. Also they did not take into account the possible other chemicals that could have reacted with the original chemicals causing a spike in the numbers. We gathered a lot of important information that can be used in future experiments based on finding chemical build up in the water. Next time we could modify the hypothesis with future experiments knowing what to expect causing a less surprising result. Also we can do a lot more research on the topic so that we know what we are actually doing. Similarities in the compound of chemicals can be used to make generalizations about possible problems with disposal of other disposable chemicals. This means that people are using more birth control during school time to be safe verses during the summer people are being less safe because the amount was smaller, it was so low that the numbers were undetectable. 5

6 6 References Brooks,B.W.,Foran,C.M,Peterson,B.N.,Weston,J. La Point,T.W., and Huggett, D.B Linkages between population demographics and effluent estrogenicity. Bulletin of Environmental Contamination and Toxicology 71: Huggett,D.B.,Foran,C.M. Brooks, B.W., Weston, J.,Peterson, B., Marsh, E.,and Schlenk,D 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 Liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma. Clinical Chemistry 50:373-84