Effects of TAML Treated Water on Yeast Survivorship Michael Gemmell 9th Grade Pittsburgh Central Catholic High School

Problem Modern technology such as factories and water refineries have posed potential environmental risks. Scientist have searched for means of degrading these risks into harmless substances.

TAML GREEN catalyst engineered by Dr. Terry Collins at CMU Accelerates H2O2 oxidation reaction Made of common elements found in nature. (C, H, O, N, Fe) GREEN Easy to synthesize

TAML Low working concentrations Breaks itself down after reaction Highly water soluble Produces non-toxic byproducts Breaks down stable chlorine compounds Step towards a sustainable society Reduce exotic elements Think in terms of nature’s chemistry

Possible Uses Textiles Water Cleaning Petroleum Refining Biological Warfare TAML could be effective in degrading triclosan.

Triclosan Antibacterial and anti-fungal agent Increasing use in consumer products (soaps, textiles, kitchen utensils) Binds to ENR enzyme in cell, which increases affinity for NAD+ (nicotineamide- adenine-dinucleotide), which is a stable conformation of the 3 chemicals which inhibits fatty acid synthesis.

Triclosan Health Concerns Can be degraded in water by UV radiation to form several highly-toxic dioxins that act as potent endocrine disruptors. Only partially degraded by current water treatment methods Significant amount is released into environment Increases the risk of infertility, early puberty, and other hormone related problems in humans.

Purpose Determine the ability of TAML + H2O2 to degrade Triclosan, and test that reaction's residue on the survivorship of yeast.

Yeast Eukaryotic, unicellular fungi. Used in genetics and cell biology, in light of the fact that the cell cycle of a yeast cell is similar to the cell cycle of humans. Saccharomyces cerevisiae- common yeast. Similar genetic makeup, biochemistry, and cell cycle to other eukaryotes, including human cells. Most studied cell in the world.

Hypothesis Null: The residue of TAML-Activated H2O2 and Triclosan reaction will not have a significant effect on the survivorship of yeast. Null: Triclosan by itself will not have a significant effect on the survivorship of yeast cells. Null: TAML-Activated H2O2 will not have a significant effect on the survivorship of yeast cells. Alternate: The residue of TAML-Activated H2O2 and Triclosan will have a significant effect on the survivorship of yeast cells.

Materials Tube Racks Permanent Marker Ethanol Matches Vortex Triclosan (99% pure, powdered) Fe-TAML B catalyst H2O2 (.88 M) Lab-Grade Catalase (Ward’s) Saccharomyces cerevisiae Sterile 250mL sidearm flask NaHCO3 Sterile Water Test tubes Micropipette and sterile tips Pipette pump and sterile tips 60 Sterile YEPD Agar Petri Dishes Tube Racks Permanent Marker Ethanol Matches Vortex YEPD Media Bacto peptone 20 g 2% Yeast extract 10 g 1% Dextrose 20 g 2% H2O to 1L

Procedure A culture of yeast was grown overnight in a sterile 250mL sidearm flask in LB media at 37°C. The culture was left to grow until it reached an absorbance reading of about 50 Kletts, which represents a cell density of about 107 cells/mL. The culture was serially diluted to a concentration of approximately 103 cells/mL. 0.0353g of solid Fe-TAML B was added to 10mL of sterile water to create a 5*10-3M stock solution. 0.0289g of solid triclosan (99%) was added to 1mL of alcohol and 9mL of sterile water to create a 1*10-2M stock solution. 0.89g of solid NaHCO3 was added to 10mL of sterile water to create a 1M stock solution. 0.025g of solid lab-grade catalase was added to 50mL of sterile water to create a working concentration of catalase.

Tube Contents Control 0.01% Triclosan 0.1% 1% 10% RXM 0.1% 0mL 0.01mL CRXM 0.1% 10% RXM 0.1% Variable 0mL 0.001mL 0.01mL 0.1mL 1mL Sterile Water 9.8mL 9.79mL 9.7mL 8.8mL NaHCO3 Yeast Total 10mL

Procedure TAML activators and H2O2 were mixed with Triclosan Tubes were let sit for 20 min while the TAML oxidation reaction occurred. 0.1mL of catalase was added to each tube to quench the reaction, and degrade any residual H2O2. Tubes were then left to quench for 20 min. 0.1mL of yeast (at 103 cells/mL) was then added to each tube. Tubes were vortexed and then 0.1mL from each tube was plated 5 times, creating a total of 60 plates. Plates were then placed in an incubator set at 37°C overnight. Colonies were then counted, each colony was assumed to have arisen from one cell.

Triclosan Effects on Yeast Survivorship P=2.33E-05 Colonies Concentration

TAML+ H2O2 Effects on Yeast Survivorship Colonies Concentration

TAML+ H2O2 +Triclosan (Reaction Mix) Effects on Yeast Survivorship Colonies Concentration

Summary of ANOVA Data Analysis Variable P-Value Significance TAML+H2O2+Triclosan P=0.477695 Insignificant Triclosan P=2.33E-05 Significant TAML+H2O2 P=0.392405

Dunnett’s Test for Triclosan Concentration t-value T-crit value Significance 0.01% 0.0136 2.78 Insignificant 0.1% 5.16 Significant 1% 5.17

Interpretations TAML+ H2O2 +Triclosan did not have a significant effect on the survivorship of yeast. The TAML accelerated the oxidation of the H2O2 and degraded the Triclosan. The Triclosan only significantly affected the survivorship of yeast when the concentration exceeded 0.1%. TAML+ H2O2 by itself did not have a significant effect on the survivorship of yeast. Next slide is limitations and further study

Conclusions Null: The residue of TAML-Activated H2O2 and Triclosan reaction will not have a significant effect on the survivorship of yeast. Accepted Null: Triclosan by itself will not have a significant effect on the survivorship of yeast cells. Rejected Null: TAML-Activated H2O2 will not have a significant effect on the survivorship of yeast cells. Alternate: The residue of TAML-Activated H2O2 and Triclosan will have a significant effect on the survivorship of yeast cells.

Extensions/Limitations Spread plating limited not only by human error, but by unequal staggering. Limited to only one model. Limited to one anti-bacterial substance. Extensions Testing higher concentrations of Fe-TAML and lower concentrations of Triclosan could improve results. Using different bacteria could improve results. Test TAML on other anti-bacterial substances. Vary the length of the reaction and compare results.

References www.beyondpesticides.org/pesticides/factsheets/Triclosan%20cited.pd f antoine.frostburg.edu/chem/senese/101/consumer/faq/triclosan.shtml www.sciencenetlinks.com/sci_update.cfm?DocID=178 linkinghub.elsevier.com/retrieve/pii/S0025326X08001707 www.ehponline.org/members/2006/114-11/innovations.html www.chem.cmu.edu/groups/Collins/ www.chem.cmu.edu/groups/collins/about/about.html http://scienceforkids.kidipede.com/biology/cells/yeast.htm

TAML+ H2O2 +Triclosan P=0.477695

Triclosan P=2.33E-05

TAML+ H2O2 P=0.392405

TAML+ H2O2 Colonies Concentration

Triclosan Colonies Center title Concentration

TAML+ H2O2 +Triclosan Reaction Mix Colonies LABEL AXES Concentration