1. Effects of Over-the-Counter Medications on E-coli Nathaniel Preston Houck Grade 12 Central Catholic High School

2. The Problem: Contaminates Entering The Water Everyday millions of people around the world take a wide variety of pharmaceuticals. But these might be harmful to other organisms. Question: Where do these drugs end up and what are their affects.

3. The Problem Starts With Your Toilet 50% to 90% of a medication is not absorbed by the body and is excreted as waste. This waste is then flushed away. People also dispose of unused medications by flushing them down the toilet. Sewage lines and septic tanks empty into aquatic environments.

4. Water Treatment Can’t Remove All Contaminates Wastewater is treated through settling, disinfection, and activated sludge. None of these processes are designed to remove dissolved pharmaceuticals in water. Treatment water is discharged into reservoirs, rivers or lakes.

5. Studies Show Drugs in the Water 100 pharmaceuticals found in rivers, lakes, and coastal waters throughout Europe and the United States. Includes antibiotics, sex hormones, and medications for epilepsy and depression. A recent study found 21 pharmaceuticals in a stream in India. Enough ciprofloxacin, a powerful antibiotic, was dumped into the stream each day to treat 90,000 people.

6. Nature Contaminated Pharmaceuticals have still unknown effects. Potential for toxicity endocrine disruption and other dangers to fish, plants and other aquatic organisms. Kidney failure and impaired reproduction found in some fish and other wildlife. Currently, there are no federal regulations requiring that water be tested for pharmaceutical residue. No laws have been set up to limit the amounts of drug residue in water supplies.

7. The Common Drugs Painkillers - Aspirin, Ibuprofen, and Tylenol Reducing the transmission of pain information to the brain, modulators hypothalamic thermostat, and inflammation suppressors.

8. E-Coli Escherichia coli Gram-negative bacterium found in the lower intestine of warm blooded animals. Easily grown and its genetics are comparatively simple and easily-manipulated, making it one of the best-studied prokaryotic model organisms. E-coli are an ideal organism to perform this test with because of its similarities to bacteria present in natural waterways and soils.

9. Purpose Test the effects Aspirin, Ibuprofen, and Tylenol on the survivorship of e-coli in vitro.

10. Hypotheses Alternative Hypothesis: The different drugs (Aspirin, Ibuprofen, Tylenol) at the two concentrations will have adverse effects on survivorship of the E-coli. Null Hypothesis: The different drugs (Aspirin, Ibuprofen, Tylenol) at the two different concentrations will not have adverse effects on the survivorship of the e-coli.

11. Drugs and Concentrations The E-coli in this experiment were subjected to the drugs diluted from the original concentration of 65g/mL. ▫High Concentration - 0.0001% - 0.0065mg/mL ▫Low Concentration - 0.00001% - 0.00065mg/mL

12. Materials LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride). 40 Luria Broth agar plates Liquid Tylenol Liquid Ibuprofen (Schein pharmaceuticals) Liquid Aspirin (Walgreens generic) Stock E. coli DH5 Alpha Micropipette and tips Vortex Klett Spectrophotometer Incubator 14 Sterile Capped test tubes Sterile Dilution Fluid (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl) Spreader bar Ethanol Bunsen Burner

13. Procedure 1. E-coli was grown overnight in sterile LB media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 5. The following chart was used to create the high and low concentrations of the drugs:

14. Chart of Concentration 0.0001% Concentration 0.00001% Concentration Painkiller0.1mL0.02 mL Sterile Dilution Fluid 9.8mL9.88mL E-coli0.1mL Total10mL

15. Procedure 6. Final volume in the tubes equals 10 mL and a cell density of approximately 10 3 cells/mL. 7. The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 8. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB plates. 9. The plates were incubated at 37 C for 24 hours. 10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

16. P-value = 2.27E-06

17. Results: Dunnett’s Test Tylenol Low4.545 Significant Ibuprofen High2.641 Not significant Tylenol High5.526 Significant Aspirin Low1.077 Not significant Ibuprofen Low2.641 Not significant Aspirin High4.448 Significant T Critical = 4.48

18. Conclusions In the null hypothesis was rejected for Tylenol (high and low) and Aspirin (high) These drugs at these concentrations seem to have significant negative effects on the survivorship of e-coli in veto. The Ibuprofen showed no significant effects on the e-coli at these concentrations.

19. Conclusions The release of antibiotics into the environment through human involvement can be harmful to all of nature.

20. Limitations/Extensions Plating time was not exactly timed and could be remedied by a teem of students performing the procedure. Different concentrations of the drugs. Other types of common drugs. Ground water levels of contaminates could be tested and compared. Studies to find out what organisms are most harshly effected by the contamination.

21. Lessons/Preventions Proper disposal of unused medications. New ways of treating water. Water should be tested frequently for a wide variety of contaminants to find what can be most harmful to the environment.

22. Sources Kolpin DW, Furlong ET, Meye MT, Thurman EM, et al. 2. “Pharmaceuticals, Hormones, and Other Organic Wastewater Contaminants in U.S. Streams, 1999–2000: A National Reconnaissance.” Environmental Science and Technology 2002;36 (6). Hemminger P. 3. “Damming the Flow of Drugs into Drinking Water.” Environmental Health Perspective 2005 October;113 (10);A678–81. Barber LB, Murphy SF, Verplanck PL, Sandsrom MW, et al. 4. “Chemical Loading into Surface Water Along a Hydrological, Biogeochemical, and Land Use Gradient: A Holistic Watershed Approach.” Environmental Science & Technology 2006 Jan 15;40 (2);475-86. Godfrey E, Woessner WW, Benotti MJ. 5. “Pharmaceuticals in On-Site Sewage Effluent and Ground Water.” Ground Water 2007 May–June;45 (3);263–71. Gros M, Petrovic M, Barcelo D. 6. “Wastewater Treatment Plants as a Pathway for Aquatic Contamination by Pharmaceuticals in the Ebro River Basin (Northeast Spain).” Environmental Science & Technology 2007 July 15;41 (14):5077–84. Mendoza M. 7. “Providers, Researchers Keeping Secrets.” Associated Press, March 10, 2008. Ternes TA, Meisenheimer M, McDowell D, et al. ➤➤ “Removal of Pharmaceuticals During Drinking Water Treatment.” Environmental Science and Technology 2002 September 1;36 (17);3855–63. Stackelberg PE, Gibs J, Furlong ET, et al. ➤➤ “Efficiency of Conventional Drinking- Water-Treatment Processes in Removal of Pharmaceuticals and Other Organic Compounds.” Science of the Total Environment 2007 May 15;377 (2–3);255–72.