Pesticide Runoff Effects on Microbial Life Tyler Barkich

Pesticide Runoff Effects on Microbial Life Tyler Barkich Grade 9 Pittsburgh Central Catholic High School

Pesticides � Any chemical, antibacterial, biological agent, or other similar substance used to kill or repel unwanted species. � Used commonly in agriculture and around homes � 877 million pounds of pesticides were used agriculturally in 2007. � Bayer Pesticide Active Ingredients: β-cyfluthrin and imidacloprid.

Previous Studies � Research suggests over 100 active pesticide ingredients can cause cancer or birth defects. � Pesticides have been linked to endocrine disruption. � Pyrethroids, a type of ingredient found in the pesticide used in this experiment, are toxic to most forms of life. � An estimated 98% of sprayed insecticide and 95% of herbicide are not used on target species.

Escherichia coli (E. coli) � Rod shaped cells, usually 2 nanometers in length � Prokaryotic � Gram-negative � Found in the intestines of many mammals � Commonly used model � Reproduces rapidly, usually within thirty minutes � Many strains, most non-pathogenic

Staphylococcus epidermidis � Bacteria that is mostly harmless and lives normally on skin and mucous membranes of humans � Gram-positive � Many forms are considered non-pathogenic � Pathogenic forms can be lethal

Gram+ vs. Gram- Bacteria Gram+ • Most pathogenic • Simple cell wall. • Antibiotics work against the formation of the cell wall. • Staphylococcus epidermidis Gram- • The cell wall is a thin layer of lipopolysaccharide, which adds extra protection. • This layer protects the cell from certain antibiotics. • Escherichia coli

Question � Do pesticides have an effect on the microbial populations in natural ecosystems?

Purpose �To determine if the Bayer Advanced Pesticide in different concentrations will affect the survivorship or Escherichia coli or Staphylococcus epidermidis.

Hypotheses �Null Hypothesis: The pesticide will not significantly reduce the survivorship of Escherichia coli or Staphylococcus epidermidis. �Alternative Hypothesis: The pesticide will significantly reduce the survivorship of Escherichia coli or Staphylococcus epidermidis.

Materials Sterile test tubes � Micropipettes � Bayer Pesticide (0. 015% β � -cyfluthrin, 0. 012 % imidacloprid) Vortex � Ethanol � Spreader � LB Agar Plates � LB Media (0. 5% yeast extract, 1% tryptone, 1% sodium chloride) � Incubator � Labeling Tape � Marker � � Escherichia coli (DH 5 Alpha) � Staphylococcus epidermidis Test tube rack � Sidearm flask � Sterile filters � Klett Spectrophotometer � Pipette tips � Sterile Dilution Fluid (100 m. M KH 2 PO 4, 100 m. M K 2 HPO 4, 10 m. M Mg. SO 4, 1 m. M Na. Cl) � Bunsen Burners � Matches � Latex gloves �

Procedure 1. Escherichia coli and Staphylococcus epidermidis were 2. A sample of those cultures were added to media in a sidearm flask. 3. The cultures were incubated until a density of 50 Klett spectrophotometer units was reached. This represents a density of 108 -109 cells/ml. 4. This cultures were diluted in sterile dilution fluid to a density of 105 cells/ml. 5. The pesticide was sterile filtered and was diluted to concentrations of 0%, 0. 01%, 0. 1%, and 1% in sterile dilution fluid. grown overnight in sterile LB media.

Chart of Liquid Concentrations 0% Pesticide 0. 01% Pesticide 0. 1% Pesticide SDF 9. 9 m. L 9. 89 m. L 9. 8 m. L Pesticide 0 m. L 0. 001 m. L 0. 1 m. L E. Coli/Staph 0. 1 m. L Total 10 m. L

Procedure (cont. ) 6. 0. 1 m. L of the E. coli culture was added to 4 tubes of each concentration of pesticide and 0. 1 m. L of the Staphylococcus was added to 4 tubes of each concentration of pesticide. This produced a final volume of 10 m. L, and a cell density of 103 cells/ml. 7. After vortexing to evenly suspend cells, 0. 1 m. L aliquots from each tube was spread evenly on LB agar plates. 8. The plates were incubated at 37 °C overnight. 9. The resulting colonies were counted. Each colony is assumed to have risen from one cell.

Agar Infusion Procedure 1. Sterilized pesticide was spread evenly over LB agar plates in two concentrations, a stock concentration (200 µL of pesticide) and a 10% concentration (20 µL of pesticide and 180 µL of sterile dilution fluid. ) 2. The plates were incubated at 37 °C for two hours. 3. Escherichia coli and Staphylococcus epidermidis 4. 5. were grown overnight in sterile LB media. 100 u. L aliquots of Control cell suspension were spread onto the infused plates. The plates were incubated at 37 °C overnight. Resulting colonies counted.

Pesticide Effects on E. coli Survivorship 350 309. 5 290. 25 275 239 Number of Colonies 300 P value: 8. 19 E-06 250 200 150 Colonies 100 50 0 0% 0% [Pesticide] 0% 1%

Pesticide Effects on Staphylococcus epidermidis Survivorship 250 228. 25 191 200 Number of Colonies P value: 2. 23 E-06 154. 5 150 138. 5 Colonies 100 50 0 0% 0% [Pesticide] 0% 1%

Dunnett’s Tests T critical = 3. 5 Alpha =. 05 Pesticide concentration Escherichia coli T value Interpretation 0. 01% 2. 47 Not significant 0. 1% 4. 43 Significant 1% 9. 06 Significant Staphylococcus epidermidis Pesticide concentration T value Interpretation 0. 01% 4. 03 Significant 0. 1% 7. 98 Significant 1% 9. 72 Significant

Pesticide Effects on E. coli and Staphylococcus Survivorship 100 Percent Survivorship 90 80 70 60 50 E. coli 40 Staph 30 20 10 0 0% 0% [Pesticide] 0% 1%

Pesticide Effects on E. coli and Staphylococcus Survivorship: Infused Plates 400 P-value: 1. 07 E-08 333. 875 Number of Colonies 350 300 250 200 288. 625 P-value: 1. 8 E-09 198. 375 178. 5 150 248. 75 146. 125 100 50 0 E. Coli Control Staph Low Concentration High Concentration

Dunnett’s Tests: Infused T critical = 2. 67 Alpha =. 05 Escherichia coli Pesticide concentration T value Interpretation Low 4. 16 Significant High 10. 93 Significant Staphylococcus epidermidis Pesticide concentration T value Interpretation Low 5. 3 Significant High 9. 97 Significant

Conclusions A. B. Liquid Exposure • The null hypothesis that pesticide will not affect E. coli or Staphylococcus epidermidis survivorship was rejected for all of the concentrations of pesticide, except for the 0. 01% exposure to E. coli. • Evidence suggests that the pesticide significantly reduces the survivorship in both species. Agar Infusion • The null hypothesis was rejected. • Evidence suggests that the pesticide significantly reduces the survivorship in both species.

Limitations, Extensions, and Future Studies Limitations � � It was difficult to exactly synchronize the plating for the microbes, possibly contributing to the varying colony counts. Infusion of pesticide into LB agar was not exact. Extensions and Future Studies � � � � Higher and lower concentrations of pesticide Different types of pesticides More species of bacteria More replicates Liquid agar infusion Vary pesticide exposure time Perform a growth rate experiment

References � http: //www. epa. gov/opp 00001/pestsales/07 pestsales/market_estimates 2007. pdf � http: //www. epa. gov/pesticides/about/ � http: //www. greennetworld. com/facts/pollution. htm � http: //www. beyondpesticides. org/pesticides/ factsheets/Synthetic%20 Pyrethroids. pdf � http: //www. docstoc. com/docs/9271628/Effe ct-of-pesticides-on-environment

E. coli ANOVA SUMMARY Groups Count Sum Average Variance 0% 4 1238 309. 5 127 0. 01% 4 1161 290. 25 80. 25 0. 1% 4 1100 275 124 1% 4 956 239 153. 3333 ANOVA Source of Variation Between Groups Within Groups Total SS df MS 10686. 19 3 3562. 063 1453. 75 12 121. 1458 12139. 94 15 F P-value 29. 4031 F crit 8. 19 E-06 3. 490295

Staphylococcus ANOVA SUMMARY Groups 0% 0. 01% 0. 1% 1% ANOVA Source of Variation Between Groups Within Groups Total Count Sum 4 4 SS 913 764 618 554 df Average 228. 25 191 154. 5 138. 5 Variance 188. 25 270. 6667 203. 6667 19. 66667 MS 19226. 19 3 6408. 729 2046. 75 12 170. 5625 21272. 94 15 F P-value 37. 57408 F crit 2. 23 E-06 3. 490295

E. coli ANOVA: Infused SUMMARY Groups Count Sum Average Variance Control 8 1587 198. 375 114. 2679 Low 8 1428 178. 5 57. 14286 High 8 1169 146. 125 102. 4107 ANOVA Source of Variation Between Groups Within Groups Total SS df MS 11128. 58 2 5564. 292 1916. 75 21 91. 27381 13045. 33 23 F P-value 60. 96263 F crit 1. 8 E-09 3. 4668

Staphylococcus ANOVA: Infused Groups Count Sum Average Variance Control 8 2671 333. 875 166. 4107 Low 8 2309 288. 625 304. 8393 High 8 1990 248. 75 403. 3571 ANOVA Source of Variation Between Groups Within Groups Total SS df MS 29023. 58 2 14511. 79 6122. 25 21 291. 5357 35145. 83 23 F P-value 49. 77706 F crit 1. 07 E-08 3. 4668