Effects of Road Salt Deicers on Yeast Survivorship

1. Effects of Road Salt Deicers on Yeast Survivorship James Brunner Pittsburgh Central Catholic High School Grade 9

2. Introduction • In this experiment, a yeast culture was tested under the effects of various chloride salts • Model- yeast, Variable- chloride salts, Interaction- yeast survivorship • Idea arose from wondering what effects salting roads has on local ecosystems

3. In PA, PENNDOT uses 96,367 tons of salt on roads in winter Several salts used as deicers These salts have harmful effects on everything from plants and animals to concrete and metals Road Salt Runoff

4. Standard salt used to de-ice roads Known damages- harmful to water, plants, metals, and even the roads it’s used on One of the cheapest salts to melt ice Chemical Symbol: NaCl Sodium Chloride

5. Also used as road salt de-icer Less harmful then Sodium Chloride Known to damage metal and concrete, but still known as best for metal and concrete Contains 17-56% more chloride ion than other salts Melts ice at slower rate than other chloride salts Chemical Symbol: MgCl2 Magnesium Chloride

6. Another salt used as de-icer Thought to be one of the “safer” salts to melt ice with Used as sodium-free substance for table salt Known as best for use with vegetation Chemical Symbol: KCl Potassium Chloride

7. Eukaryotic organism used in foods and drinks Most commonly studied organism in labs Easy to use- grows quickly, easy to count colonies, and nonpathogenic (safe to use) Less tolerable to extreme conditions- achieves better data Yeast

8. Past Studies • Several studies of the nature of this experiment have been conducted in the past, such as: • Effects of NaCl on yeast • Effects of NaCl on macro-invertebrates • In both studies, it was found that NaCl had a negative effect on the organisms’ survivorship

9. Purpose & Hypotheses • Purpose- to determine the effects of various chloride salts on a yeast culture • Null Hypothesis- The survivorship of the yeast cells will not be significantly affected by any of the concentrations of the three chloride salts • Alternative Hypothesis- all three chloride salts will reduce survivorship compared to the control

10. YEPD agar plates (1% yeast extract, 2% peptone, 2% glucose (dextrose), 1.5% agar) YEPD Media (1% yeast extract, 2% peptone, 2% glucose (dextrose)) Sidearm flask Spreading platform, spreader bar, ethanol 20 mL Sterile capped test tubes with Sterile Dilution Fluid (SDF) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) Saccharomyces cerevisiae (Yeast) 0.22 micron syringe filters + 10 mL syringe Weigh boat Scale Incubator Vortex Genie Stir Plate NaCl (10%) KCl (10%) MgCl2 (10%) Pipettes Sterile test tubes Water Ethanol (95%) Spreader Bar Micro Burner Materials

11. Procedure • Yeast was grown overnight in sterile YEPD media. • A sample of the overnight culture was added to fresh media in a sterile sidearm flask. • The culture was placed in an incubator(30 Degrees) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells/ml. • The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml. • The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations for a total of 9.9 ml. For Example:1 ml of 10% variable ‘z’ solution + 8.9 ml of SDF= final concentration of almost 1% ‘z’ (the addition of 0.1 ml of cell culture will result in a total of 10 ml and a 1% concentration). • 103 100 ml of cell culture was then added to the test tubes, yielding a final volume of 10 ml and a cell density of approximately 103 cells/ml • The solution was mixed by vortexing and allowed to sit at room temperature. • After vortexing to evenly suspend cells, 0/1 ml aliquots were removed from the tubes and spread on YEPD+LB plates. • The plates were incubated at 30 degrees for 48 hours. • The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

12. Test Tube Contents

13. Results • All salts affected the yeast; yeast survivorship declined as salt concentration rose • NaCl was the least harmful, while MgCl2 was the most harmful • All plates grew yeast well; except for the plates containing .5% KCl, which was contaminated and did not grow any yeast • The results in this experiment pertained to those of past studies of this nature

14. Effects of Salt on Yeast Survivorship # o f colonies 0.02 Salt Concentration 0.04 0.03 = P values lower then 0.05 Type of Salt

15. Pictures NaCl 1% KCl .1% MgCl2 .5% No Salt Added

16. ANOVA Chart

17. Conclusions • After the data was collected, the following concentrations of salt rejected the null hypothesis: 1%KCl, and 1% MgCl2 • All colonies affected negatively by the salts • Alternative Hypothesis correct- all salts reduced yeast survivorship • According to data, road salt de-icers inhibit the growth of yeast colonies

18. Conclusions (continued) • After ANOVA and Dunnett tests, null hypothesis rejected • Most ANOVA tests showed null hypothesis accepted, with no significant difference from control (most p values over .05), but Dunnett test showed that it was rejected, with significant difference from control (most t values less than t critical) • Dunnett test more powerful than ANOVA- compares better over two groups of data • ANOVA tests may have shown that null was accepted as decreases in yeast survival not very large, but clearly exist

19. Limitations and Extensions • All .5% KCl contaminated-data could not be obtained • Difficult to ensure exact exposure times to salt variables due to time variation in spread plating. This could be more easily overcome by using a team of people • More concentrations and various salts could be tested • Other cellular models could be tested • Another model, such as bacteria, added to achieve better data

20. References • “Rock Salt (NaCl) Sodium Chloride.” Peterschemical.com Dec. 21, 2007. <http://www.peterschemical.com/sodium-chloride/ • “Potassium Chloride and Urea.” Peterschemical.com Dec. 21, 2007. <http://www.peterschemical.com/potassium-chloride/ • “Magnesium Chloride.” Peterschemical.com Dec 21, 2007. <http://www.peterschemical.com/magnesium-chloride/ • “Yeast.” Wikipedia.org Dec. 21, 2007. <http://en.wikipedia.org/wiki/Yeast • “Sodium Chloride.” Wikipedia.org Dec. 21, 2007. <http://en.wikipedia.org/wiki/Sodium_chloride • “Magnesium Chloride.” Wikipedia.org Dec. 21, 2007 <http://en.wikipedia.org/wiki/Magnesium_chloride

21. References (continued) • “Potassium Chloride.” Wikipedia.org Dec. 21, 2007 <http://en.wikipedia.org/wiki/Potassium_chloride • “Road Salt and Water Quality.” Des.state.nh.us Dec. 21, 2007 <http://www.des.state.nh.us/factsheets/wmb/wmb-4.htm • “Field and laboratory investigations on the effects of road salt (NaCl) on stream macroinvertebrate communities.” Sciencedirect.com Dec. 21, 2007 <http://www.sciencedirect.com/science?_ob=ArticleURL&_u di=B6VB5- 455VM763&_user=10&_rdoc=1&_fmt=&_orig=search&_sort =d&view=c&_acct=C000050221&_version=1&_urlVersion= 0&_userid=10&md5=c9d9b919f172d211bbad1839fd5d0afe • “Effect of Sodium Chloride on Bakers' Yeast Growing in Gelatin.” Aem.asm.org Dec. 21, 2007 <http://aem.asm.org/cgi/content/abstract/43/4/757 • “Winter Maintenance- Notes and Other Interesting Information” Dot.state.pa.us Jan. 28, 2008. <http://www.dot.state.pa.us/Penndot/Districts/district6.nsf/winter_maint_notes?ReadForm