Running Head: The Effects of Birth Control in Wastewater

The Effects of Birth Control in Wastewater

Caitlin Schultz, Sierra Shepard, Sydney Ricks University of North Texas

Author Note This lab report was prepared for TECM 1700, section 006, taught by Professor Nogay

Running Head: The Effects of Birth Control in Wastewater

Abstract
Traces of EE2 were found in the Denton area to have impacted the sexual characteristics of male fish. The experiment was conducted to determine if the concentration of EE2 in the Denton wastewater treatment plant (WWTP) effluent varies due to the presence of college students. Specifically the concentration of EE2 was measured in the WWTP effluent when students were in session (late August to beginning of May) and when they are on summer break (mid-May to mid-August). The hypothesis states that there will be no difference in the concentration of EE2 in the Denton wastewater treatment plant effluent when college students are attending college and not attending college. Results showed that there were higher concentrations of EE2 in the wastewater during the times when students were attending college in the fall and spring. This showed that the population or demographics of an area can influence the environment.

Introduction
When humans take medications, traces of those medications go into waste water, which will go into lakes. Lakes are the natural habitat to many animals including fish, so we studied the effect of 17a-Ethinylestradiol (EE20, or known as oral contraceptives) in freshwater. EE2 causes male fish to display female characteristics, as well as causing reproductive dysfunction. Do the demographics of the area influence the concentration EE2 in the water? We think that there is no difference in the concentration of EE2 in the water based on when students are attending college, but this experiment will give us the true answer.

Running Head: The Effects of Birth Control in Wastewater

Materials
9 beakers 1000mL of d3-17-esradiol(EE2) from April, July, and October 1.5ml amber glass vials (Fisher Scientific) Ethyl acetate Nitrogen

Methods
The following procedure was done to find the concentrations of EE2 in the wastewater that was filtered out into the lakes near the Denton area.
1. Add a 1000mL effluent sample to the standard 2. Once spiked, samples were liquid-liquid extracted twice using 500mL of ethyl acetate 3. The ethyl acetate layers were combined and dried under a gentle stream of nitrogen. 4. The resultant residues were reconstituted in the 1mL ethyl acetate and transferred to 1.5ml amber glass vials (Fisher Scientific). 5. Reconstituted solvent was dried under nitrogen with residue re-suspended in 100l of methanol. 6. From this volume, a 50l aliquot was taken for estrogen quantifications using a dansyl choride derivatization method detailed be Nelson, Grebe, OKane, and Singh (2004). 7. All chromatographic separations were conducted using a Waters 2695 separations module coupled to a Waters 2998 UV/VIS detector and a WatersSunfire C18 column (2.1 x 50mm; 3.5 m particle size). 8. Electrospray ionization (positive ion mode) and mass-spectrometric analysis was conducted using a quadrupole-hexapole-quadrupole instrument (Micromass Quattro Ultima mass detector, Manchester, U.K.).

Running Head: The Effects of Birth Control in Wastewater

9. For all EE2, instrument determined (MassLynx software, version 4.1) criteria were used to set limits of detection.

Results
One experiment was conducted using three different samples of water. Using the methods above, they measured the concentrations found in the samples and recorded the results for each month that the same samples were tested.

Concentrations of EE2 in chosen water samples

6

Concentration of EE2 in ng/L

5 5 4 3 3 2 2 1 1 0.2 0 April July October 0.1 0.2 1 2

Water Sample # During Each Month

1 2 3

Table 1. Shows concentrations of EE2 during each month

Replicate Sample # 1 2 3

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

July <0.5 ng/L* <0.5 ng/L <0.5 ng/L

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

Table 2. Shows data in original table format

Running Head: The Effects of Birth Control in Wastewater This shows that the birth control during the times that students were in college was significantly higher than during the summer time. This shows that when school starts, many students take birth control since it is measured from the waste water. This has an effect on many of the fish in the lakes are that are exposed to the waste water, as they begin to have more female sexual characteristics.

Discussion
This study was used to show the influence of population demographics on the concentrations of the oral contraceptive, 17a-Ethinylestradiol, in Denton wastewater Treatment Effluent. 17aEthinylestradiol (EE2) is a synthetic version of estrogen that is commonly used in many birth control medications. Laboratory studies on fish have shown that EE2 causes male fish to display female sexual characteristics, as well as causes reproductive dysfunction. The main route by which EE2 is entering waterways is via post-human consumption (i.e. consumed and excreted). The experiment was conducted to determine if the concentration of EE2 will be higher or lower in the WWTP (Waste Water Treatment Plant) effluent when students are in session (late August to beginning of May) and when they are on summer break (mid-May to mid-August). The results showed a significant increase in the amount of EE2 found in the water during the months in which students were at school. Although this disproves the hypothesis, it does show that the demographics of an area can impact the environment in an odd way. The effects of male reproductive abilities could negatively or positively impact the environment as a whole.

Running Head: The Effects of Birth Control in Wastewater

References
Brooks, B. W., Foran, C. M., Peterson, B. N., Weston, J. La Point, T. W., & Huggett, D. B.. (2003). Linkages between population demographics and effluent estrogenicity. Bulletin of Environmental Contamination and Toxicology, 71, 504-551. 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. Toxicological Sciences, 72, 77-83. Nelson, R., Grebe, S., OKane, D., &Singh, R. (2004). Liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma. Clinical Chemistry, 50, 373-384.