The Effects of Pesticide on Microbial Life Jose Esquivel 11th Grade Central Catholic High School

Pesticide Primary factors affecting the effectiveness of pesticides include: Type of pesticide Degradation Low temperatures

Non Target Effects Cause cancer in humans Negative interaction with vertebrate immune systems Passed on through food chains Removal of food sources

Pesticide Use DEET is the most common active ingredient in insect repellents across the market toxic for species of freshwater zooplankton

Pesticide (OFF Deep Woods) 25% DEET, 75% other ingredients (not revealed) Stored at room temperature Provides 8 Hours of protection

Yeast Single celled fungi Most commonly studied organism- similar biochemistry and genes as other Eukaryotes Saccharomyces cerevisiae (Yeast)

Purpose To examine the effects of OFF DEEP WOODS on Yeast cell survivorship

Hypothesis Null Hypothesis: The pesticide will not have a significant effect on Yeast cell survivorship Alternative Hypothesis: The pesticide will significantly reduce Yeast cell survivorship

Materials YEPD agar plates (YEPD media + 1.5% agar) YEPD media (1% yeast extract, 2% peptone, 2% glucose) Sterile Dilution Fluid [SDF] (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCI) Sterile pipette tips Micropippettes Vortex Incubator Sidearm flask Spreading turntable Spreader bar Ethyl alcohol Sterile capped test tubes with Sterile distilled water Saccharomyces cerevisiae (Yeast) Pesticide

Procedure 1. Pesticide was obtained from local Home Depot 2. The pesticide was sprayed into a 50 mL to achieve a fluid form of the pesticide 3. The pesticide was sterile filtered 4. Yeast cells were grown overnight in sterile YEPD media 5. The cultures were placed in an incubator until a density of 50 Klett spectrophotometer was reached. This is a density of approximately 10^7 cells/mL 6. The cultures were then diluted in Sterile Dilution Fluid to a concentration of 10^5 cells/mL

Procedure (Continued) 7. The different concentrations were made: 0% 0.1% 1% 10% Yeast 0.1 mL Dilution Fluid 8.9 mL Pesticide Solution 0 mL 0.01 mL 1 mL Sterile Water .99 mL 0.9 mL Final Volume 10mL 10 mL

Procedure (Continued) 8. The tubes were allowed to incubate at room temperature for 15 minutes 9. After vortexing to evenly suspend cells, 0.1 mL aliquots were removed from the tubes and spread on the YEPD agar plates 10. The Yeast cells incubated for 48 hours at 30C 11. The resulting colonies were counted, each colony is assumed to have arisen from one cell

Pesticide Effect on Yeast Cells 848 P Value- less than 10^-6 Colonies 521.14 334.29 131.43 1% 10% 0% 0.1%

Dunnett’s Test Table T crit.=3.01 Alpha=0.05 Yeast T Value Interpretation 0.1%. 11.693 Significant 1%. 18.378 Significant 10%. 25.635 Significant

Yeast Analysis Anova revealed significant variation between the means The Dunnett's test revealed significant negative effects on yeast survivorship at all concentrations

Limitations and Extensions Synchronization of spreading plates more precisely Limited exposure times Only tested liquid pulse Only survivorship was measured- not growth Extensions Increase trial size to 20 or 30 for more accurate results Vary exposure times to the water Conduct test on different micro organisms Agar infusion for prolonged exposure Synergistic effects?

References http://www.gmasw.com/chemlist.pdf http://www.ncbi.nlm.nih.gov/ http://www.biotecharticles.com/ http://www.off.com/en-US/Products/Pages/off-deep- woods.aspx#off-deep-woods-insect-repellent-v https://edis.ifas.ufl.edu/pdffiles/PI/PI12200.pdf

Yeast ANOVA