TAML-Induced Degradation of Triclosan

1. TAML-Induced Degradation of Triclosan Luke Diorio-Toth 11th Grade Pittsburgh Central Catholic HS

2. TAML • GREEN catalyst engineered by Dr. Terry Collins at CMU • Accelerates H2O2 oxidation reaction • Highly selective • Made of common elements (C, H, O, N, Fe) • GREEN • Economical • Easy to synthesize

3. TAML (cont.) • Low working concentrations (nM-low μM) • 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

4. Possible Applications • Textiles • Pulp and Paper • Water Cleaning • Petroleum Refining • Biological Warfare • Unpublished studies suggest that TAML could be effective in degrading triclosan. Paper Mill releasing bleaches and other pollutants into water supplies

5. Triclosan • Potent antibacterial and antifungal agent • Increasing use in consumer products (soaps, textiles, toys, kitchen utensils) • Binds to ENR enzyme in cell, which increases affinity for NAD+, a stable conformation of the 3 chemicals which inhibits fatty acid synthesis (including synthesis of cAMP)

6. 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

7. Escherichia coli • Escherichia coli (E. coli) – very common, found in intestinal tract of most mammals • There are many strains of E. coli, most are non-pathogenic • Pathogenic strains can cause illness and death in humans • Frequently studied in biology – ubiquitous, simple structure, easily manipulated in the laboratory

8. Purpose • The purpose of this experiment was to determine the ability of TAML-activated H2O2 to degrade Triclosan into non-toxic bi-products

9. Hypotheses • Null - TAML itself will not have a significant effect on E. coli survivorship. • Null - H2O2 and Triclosan will have no significant effect on E. coli survivorship. • Null - TAML-activated H2O2 + Triclosan will have no significant effect on E. coli survivorship (compared to the positive control). • Alternative - TAML-activated H2O2 + Triclosan will have significant effect on E. coli survivorship (compared to the negative control).

10. Triclosan (99% pure, powdered) Fe-TAML B catalyst H2O2 (.88 M) Lab-Grade Catalase (Ward’s) E Coli DH5α Sterile 250mL sidearm flask NaHCO3 (Store bought baking soda) Sterile Dilution Fluid (SDF) Sterile Water De-ionized Water Sterile Polystyrene conical Sterile test tubes Micropipette and sterile tips Pipette pump and sterile tips Sterile filters 48 sterile Petri dishes, poured with LB media Tube racks Fine tip permanent marker Klett Spectrophotometer Metler Scale Weigh Boats Spoon Ethanol Vortex Materials LB Media (per 1 liter) 10g Tryptone (1%) 5g Yeast Extract (0.5%) 10g NaCl (1%) 2 mL 1N NaOH SDF (per 1 liter) 100mM KH2PO4 100mM K2HPO4 10mM MgSO4 1mM NaCl

11. Procedure • A culture of E Coli DH5α 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 108 cells/mL. • The culture was serially diluted in SDF to a concentration of approximately 103 cells/mL. • 48 Petri dishes, poured with LB agar were labeled and left to warm on the lab desk. • 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 ethanol 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.

12. Procedure (cont.) • Amounts of the various stock solutions and SDF were added to 12 tubes (2 tubes per variable):

13. Procedure (cont.) • Tubes were let sit for 15 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 40 min. • 0.1mL of the E Coli DH5α(at 103 cells/mL) was then added to each tube. • Tubes were vortexed and then 0.1mL from each tube was plated 4 times, yielding a total of 48 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.

14. Colony Counts

15. P value = 6.29E-28

16. Dunnett’s Test If t > t-crit, then data significant, reject null

17. P value = 1.50E-14

18. Dunnett’s Test If t > t-crit, then data significant, reject null

19. Conclusions • Null - TAML itself will not have a significant effect on E. coli survivorship. • ACCEPTED • Null - H2O2 and Triclosan will have no significant effect on E. coli survivorship. • REJECTED • Null- TAML-activated H2O2 + Triclosan will have no significant effect on E. coli survivorship (compared to positive control). • REJECTED • Alternative- TAML-activated H2O2 + Triclosan will have significant effect on E. coli survivorship (compared to negative control). • ACCEPTED, NULL REJECTED

20. Extensions/Limitations • Work with groups of students to speed up plating time would increase consistency of results. • E. Coli DH5α cell density was a little low when cultured, increased density may improve accuracy of tests. • Testing higher concentrations of Fe-TAML and lower concentrations of triclosan could improve results.

21. References • Dr. John Wilson, Assistant Professor, Biostatistics at the University of Pittsburgh • Dr. Terry Collins, Thomas Lord Professor of Chemistry at Carnegie Mellon University • http://en.wikipedia.org/wiki/Triclosan • www.beyondpesticides.org/pesticides/factsheets/Triclosan%20cited.pdf • 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