1. by Thomas O’Connor Central Catholic High School Grade 11 2011

2. Run Off and Pollution Risk Pesticides include any chemical, antibacterial, biological agent, or other similar substance used to kill or repel unwanted species. Pesticides can easily get into water supplies Toxic substances in water supplies kill life and disrupt ecosystems What technologies can we develop to treat this problem?

3. Tetra Amido Macrocyclic ligand (TAML) GREEN catalyst engineered through research led by Dr. Terry Collins of Carnegie Mellon Universtiy Accelerates H 2 O 2 oxidation reaction Highly selective Broad Range Made of common elements found in nature (C, H, O, N, Fe) GREEN Economical Easy to synthesize

4. TAML (continued) Structure Central iron atom, specific location of reactivity. Site to which hydrogen peroxide binds while transforming to a highly reactive form Attached to ringed carbon structure(“macrocycles”) by 4 nitrogen atoms (“tetra-amido”)

5. Possible Applications Textiles Pulp and Paper Water Cleaning Petroleum Refining Biological Warfare Paper Mill releasing bleaches and other pollutants into water supplies

6. Carbaryl Pesticide used was 1- naphthyl N-methylcarbamate, colloquially known as Carbaryl Used as insecticide Works by inhibiting acetylcholinesterase (AchE) Acetylcholinesterase degrades acetylcholine from the synaptic cleft between an affecter nerve and a muscle. Acetylcholine is the neurotransmitter that propagates the contraction signal from the central nervous system to muscle It binds to receptors on muscle cell, stimulating the cascade of reactions resulting in contraction Inhibition of AchE results in excessive acetylcholine in synaptic cleft, causing muscle to continually contract.

7. E. Coli Escherichia coli (E. coli) – very common, found in intestinal tract of most mammals One of the most Frequently studied prokaryotic organisms in biology. Can be easily manipulated, makes good subject for experimentation There are many strains of E. coli, most are non- pathogenic, although Pathogenic strains are quite dangerous and can cause illnesses. Frequently studied in biology – ubiquitous, simple structure, easily manipulated in the laboratory

8. Yeast Saccharomyces cerevisiae Single most studied eukaryotic cell Eukaryotic model, has similar biochemistry to other eukaryotes Easily manipulated Frequently studied

9. Purpose Do the ingredients of the TAML reaction significantly alter microbial survivorship? Does Carbaryl hurt microbial cell survivorship? Do products of 2 min reaction between activated TAML and pesticide affect microbial survivorship? Do products of 30 min reaction between activated TAML and pesticide affect microbial survivorship?

10. Hypotheses Null – Carbaryl will have no significant effect on Yeast or E. Coli survivorship Null - Activated TAML mix will not have a significant effect on E. coli or Yeast survivorship. Null- Activated TAML can not safely break down carbaryl

11. Materials Garden-Tech Sevin-5 pesticide 2.5 mM stock TAML solution H 2 O 2 (.88 M) Lab-Grade Catalase (Ward’s) E Coli DH5 α Saccharomyces cerevisiae Sterile 250mL sidearm flask Sterile Dilution Fluid (SDF) Sterile Water Sterile Polystyrene conical Sterile test tubes Micropipette and sterile tips Pipette pump and sterile tips Sterile filters 60 sterile Petri dishes, poured with LB media 60 sterile Petri dishes, poured with YEPD Media Tube racks Fine tip permanent marker Klett Spectrophotometer Metler Scale Weigh Boats Ethanol Vortex SDF (per 1 liter) 100mM KH 2 PO 4 100mM K 2 HPO 4 10mM MgSO 4 1mM NaCl

12. Procedure Preliminary Study 1. Cultures of E Coli DH5 α and Saccharomyces cerevisiae were grown overnight in a sterile 250mL sidearm flask in LB media or YEPD media at 37°C. 2. The cultures were left to grow to 50 Kletts units, which represents a cell density of about 10 8 cells/mL or 10 7 cells/mL. 3. The cultures were serially diluted in SDF to a concentration of approximately 10 3 cells/mL. 4. LB agar and YEPD agar plates were labeled and left to warm in incubator 5. 4g of Garden-Tech Sevin-5 pesticide was added to 20 mL of sterile water to create a stock solution 100 times as strong as recommended for home use.

13. Procedure (cont.) 8. Amounts (in mL) of the various stock solutions and SDF were added to 8 tubes (2 tubes per variable): 9. Tubes were vortexed and then 0.1mL from each tube was plated 5 times, yielding a total of 20 plates each for yeast and for E. Coli 10. Plates were then placed in an incubator set at 37°C overnight. 11. Colonies were then counted, each colony was assumed to have arisen from one cell. Control 10x Concen- tration1x.1x Yeast or E.Coli.1 Pesticide Stock01.1.01 SDF for E.Coli SW for Yeast 9.98.99.89.89 Total10.0

14. Procedure (cont.) 9. Tubes were vortexed and then 0.1mL from each tube was plated 5 times, yielding a total of 20 plates each for yeast and for E. Coli 10. Plates were then placed in an incubator set at 37°C overnight. 11. Colonies were then counted, each colony was assumed to have arisen from one cell.

15. Procedure (Focus Study) 1. Cultures of E Coli DH5 α and Saccharomyces cerevisiae were grown overnight in a sterile 250mL sidearm flask in LB media or YEPD media at 37°C. 2. The cultures were left to grow to 50 Kletts units, which represents a cell density of about 10 8 cells/mL or 10 7 cells/mL. 3. The cultures were serially diluted in SDF to a concentration of approximately 10 3 cells/mL. 4. LB agar and YEPD agar plates were labeled and left to warm in incubator 5. 4g of Garden-Tech Sevin-5 pesticide was added to 20 mL of sterile water to create a stock solution 100 times as strong as recommended for home use. 6. 100mg of solid lab-grade catalase was added to 10 mL of sterile water to create a working concentration of catalase.

16. Procedure (cont.) 8. Amounts (in mL) of the various stock solutions and SDF were added to 8 tubes (2 tubes per variable): Control Activated TAML onlyCarbaryl only2 minute30 minute H2O20.50 TAML0.50 Carbaryl00111 SDF for E.Coli SW for Yeast 9.98.88.97.8 Total9.99.89.99.8

17. Procedure (cont.) 9. The tube with TAML mix only and 2 minute experimental tube was left to sit for 2 min (and the 30 min tube for 30 min) while the TAML oxidation reaction occurred. 10. 0.1mL of catalase was added to each TAML containing tube to quench the reaction, and degrade any residual H 2 O 2. Tubes were then left to quench for 2 min. 11. 0.1mL of the E. Coli or Yeast were then added to each tube. 12. Tubes were vortexed and then 0.1mL from each tube was plated 8 times, yielding a total of 40 plates each for yeast and for E. Coli 13. Plates were then placed in an incubator set at 37°C overnight. 14. Colonies were then counted, each colony was assumed to have arisen from one cell.

18. Yeast ReplicateControl.1xx10x 113113410644 21271428986 3141129111102 413713812197 513513711592 Average134.2136108.484.2 E.Coli Control.1xx10x 210202152110 204206163124 18618815494 194 17098 197186148106 Average198.2195.2157.4106.4 Preliminary study data

19. Preliminary Study Yeast ANOVA Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Control5671134.229.2.1x568013623.5 x5542108.4147.8 10x542184.2540.2 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups8999.432999.816.199814.17E-053.238872 Within Groups2962.816185.175 Total11962.219 F value> F-crit Interpretation: at least two means vary significantly.

20. Preliminary Study E.Coli ANOVA Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Control5991198.285.2.1x5976195.275.2 x5787157.479.8 10x5532106.4136.8 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups27520.239173.497.33051.73E-103.238872 Within Groups15081694.25 Total29028.219 F value> F-crit Interpretation: at least two means vary significantly.

21. Preliminary study ANOVA results Yeast ANOVA P=4.17*10 -5 E.Coli ANOVA yields P=1.73*10 -10 P value cutoff : alpha =.05 Interpretation: At least two means vary significantly in both the yeast and E. Coli data.

23. Dunnett’s Test (prelim study) If t > t-crit, then data significant, reject null ConcentrationT-ValueT-CritIntepretation.1x0.2091473.29 Not significant 1x2.9977723.29 Not significant 10x5.8096353.29 Significant ConcentrationT-ValueT-Crit.1x0.4885973.29 Not Sgnificant 1x6.6449163.29 Significant 10x14.951063.29 Signficant Yeast E. Coli

24. Replicate No Pesticide and No TAMLTAML + No Pesticide Pesticide +No TAML2 minute exposure 30 minute exposure 1160153131152157 2164151139156154 3148162126142161 4156158124147153 5152 135155151 6 146129149155 7146156118153148 8157149128144152 Average154.25153.375128.75149.75153.875 Yeast Data

26. Yeast ANOVA F value> F-crit Interpretation: at least two means vary significantly. Anova: Single Factor SUMMARY GroupsCountSumAverageVariance No Pesticide or TAML81234154.2537.35714 TAML + No Pesticide81227153.37526.26786 Pesticide +No Taml81030128.7542.21429 2 minute exposure81198149.7526.21429 30 minute exposure81231153.87515.55357 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups3808.754952.187532.254112.69E-112.641465 Within Groups1033.253529.52143 Total484239

27. E.Coli Data Replicate No Pesticide or TAMLTAML + No Pesticide Pesticide +No Taml 2 minute exposure 30 minute exposure 1250256225248253 2257259215262258 3249253218253251 4253273212260264 5255262218249247 6253257214257258 7250253208259252 8254250220249257 average252.625257.875216.25254.625255

29. Anova: Single Factor SUMMARY GroupsCountSumAverageVariance No Pesticide or TAML82021252.6257.696429 TAML + No Pesticide82063257.87551.55357 Pesticide +No Taml81730216.2527.07143 2 minute exposure82037254.62531.125 30 minute exposure8204025528 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups9737.8542434.46383.689322.04E-172.641465 Within Groups1018.1253529.08929 Total10755.9839 E.Coli ANOVA F value> F-crit Interpretation: at least two means vary significantly.

30. ANOVA results Yeast ANOVA yields P=2.69*10 -11 E.Coli ANOVA yields P=2.04*10 -17 P value cutoff : alpha =.05 Interpretation: At least two means vary significantly in both the yeast and E. Coli data.

31. Dunnett’s Test If t > t-crit, then data significant, reject null Variable T-ValueT-CritIntepretation Activated TAML Only 1.5390863.02 Not significant Pesticide Only 10.663663.02 Significant 2 minute exposure 0.5863183.02 Not significant 30 minute exposure 0.6962533.02 Not significant Yeast E. Coli Variable T-ValueT-CritIntepretation Activated TAML Only 0.254633.02 Not significant Pesticide Only 7.4206423.02 Significant 2 minute exposure 1.3095253.02 Not significant 30 minute exposure 0.1091273.02 Not significant

32. Conclusions Carbaryl is harmful to microbial life Activated TAML alone does not harm microbial life Break down products of Carbaryl are safe to microbes.

33. Limitations/ Extensions Difficulty in synchronous plating One exposure time Could be done with other species A field study could be conducted Different reaction mixes

34. References Dr. Terry Collins, Thomas Lord Professor of Chemistry at Carnegie Mellon University Dr. John Wilson, Assistant Professor, Biostatistics at the University of Pittsburgh Mr. Mark Krotec, PTEI 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