1. By: Kayleigh Makoid

2.  Nicotine  Epinephrine  Daphnia  Previous testing

3.  Are there inhibitory effects of epinephrine on nicotine toxicity?

4.  My hypothesis is if the nicotine concentration increases, then the daphnia will die for both molar amounts of epinephrine.

5.  Microscope  100–1000 μL micropipette  Pipettes  Distilled Water  Nicotine solution  Epinephrine  15 mL centrifuge tubes  1.5 mL centrifuge tubes  Stand for centrifuge tubes  Goggles  Gloves

6. Preparation of the Nicotine Solution Preparation of the Epinephrine 1. 2mL of Nicotine + 8mL of H20= 1M (1000mM) 2. 1mL of above solution + 19mL of H20= 10mM 3. 1mL of above solution + 19mL of H20=.1mM 4. 1mL of above solution + 9mL of H20=.01mM 5. 1mL of above solution + 9mL of H20=.001mM 6. 1mL of above solution + 9mL of H20=.0001mM 7. Insert each solution into five separate 1.5 mL centrifuge tubes 1. 4.5 mL of H20 in 3 flasks 2. Add.5mL of epinephrine to first flask 3. Add.5mL of the above solution to the second flask 4. Add.5mL of the above solution to the third flask

7.  Allow 12 daphnia to soak in each solution of epinephrine prepared for 10 minutes  Put daphnia under microscope  Add drop of each concentration of nicotine to different daphnia (two for every dilution of nicotine)  Count heartbeats for 1 min per Daphnia  Record results heart

8. .1 Epinephrine solution 12 Daphnia 2 in 1000mM2 in 10mM2 in.1mM2 in.01mM 2 in.001mM.001 Epinephrine solution 12 Daphnia 2 in 1000 mM2 in 10mM2 in.1mM2 in 1mM 2 in.001mM

9. Nicotine (mM) Heartbeats per min 1000mM78, 84 10mM101, 98.1mM107, 102.01mM106, 112.001mM104, 110.001M Epinephrine Nicotine (mM) Heartbeats per min 1000mM103, 106 10mM111, 109.1mM114, 112.01mM123, 131.001mM160, 143.1M Epinephrine Heartbeats with H20142, 151.1M Epinephrine.001M Epinephrine 158, 154157, 164 Nicotine (mM) Heartbeats per min 1000mM81, 87 10mM94, 97.1mM109, 98.01mM110, 107.001mM108, 104 Controls

10. IC50 for Nicotine=4.9990 Standard Deviation for Nicotine= 2.3604 IC50 for Epinephrine=10.9100 Standard Deviation for Epinephrine=1.6994 IC50 for Nicotine=4.9990 Standard Deviation for Nicotine= 2.3604 IC50 for Epinephrine=10.9100 Standard Deviation for Epinephrine=1.6994

11. IC50 for.001M epinephrine competition 27.9300 IC50 for.1 M epinephrine competition 6.3520 x 10 -3 Standard deviation for.001 M is 0.2635 Standard deviation for.1 M is 0.2187 IC50 for.001M epinephrine competition 27.9300 IC50 for.1 M epinephrine competition 6.3520 x 10 -3 Standard deviation for.001 M is 0.2635 Standard deviation for.1 M is 0.2187

12. Independent variable  Concentration of epinephrine Dependent variable  Heartbeats of the Daphnia Controls  Water, epinephrine, and nicotine Constants  Room temperature, fume hood, microscope, and daphnia species

13.  Epinephrine is effective at.1M concentration to treat low dosages of Nicotine poisoning  Hypothesis partially supported

14. Factors the Affected Accuracy Future Experimentation  Death of Daphnia  Aging solutions .1M Epinephrine’s potency on Daphnia  Inability to be precise with counting  Test other molar amounts of epinephrine on nicotine

15.  Clare, John, P.. D, “Daphnia.” Caudata. N.p., July 2002. Web. 25 Jan. 2011..  “Epinephrine Injection.” MedlinePlus. N.p., 1 Sept. 2008. Web. 25 Jan. 2011..  Meeker-O’Connell, Ann. “How Nicotine Works.” Discovery Health. N.p., 2011. Web. 25 Jan. 2011..  “Nicotine Information.” Home Drug Testing Kit. N.p., 10 Dec. 2002. Web. 25 Jan. 2011..  Scott, Elizabeth. “The Definition Of Epinephrine.” About. com. N.p., 22 Dec. 2007. Web. 25 Jan. 2011..