The Effects of Fracking Chemicals and Nitrogen on Bacteria By Steele Mercer Central Catholic High School Grade 11

THE PROBLEM Hydraulic Fracturing and high concentrations of fertilizer Pollutes large areas around sites Are living organisms at risk from these two issues?

Objective To test the effects of Fracking Chemicals and Nitrogen Fertilizer on bacterial survivorship

Past Studies Surface water contamination from spills, leaks, and the disposal of inadequately treated wastewater or hydraulic fracturing fluids Accumulation of toxic and radioactive elements in soil and the sediments of rivers and lakes exposed to wastewater or fluids used in hydraulic fracturing;

Fracking Chemicals Substances used to pump natural gas out of the ground. Regulations prevented the obtaining of genuine fracking chemicals. Simulation (researcher approved) chemical mix substituted

Fracking Chemicals Simulation Composed of Sodium Chloride, Calcium Chloride, Magnesium Chloride, Potassium Chloride, Barium Chloride, Strontium Chloride Chemical Sodium Chloride Calcium Chloride Magnesium Chloride Potassium Chloride Barium Strontium Amount (mg/l) 75.65 16.65 0.475 0.3846 0.1857 0.20

Ammonium Nitrate NH₄NO₃ predominantly used in agriculture as a high-nitrogen fertilizer Ammonium nitrate's advantage over urea is that it is more stable and does not rapidly lose nitrogen to the atmosphere. Major cause of eutrophication

Escherichia Coli (DH5 Alpha) Gram(-) Model organism Rapid Growth Rate Ability to grow in the absence of oxygen Found in many environments intestinal tract of mammals Can synthesize all its own amino acids, purines and pyrimidines using the nitrogen donor molecules

Hypothesis Null Hypothesis- Fracking and Nitrogen exposure will have no significant effect on E. coli colonies Alternative Hypothesis- Fracking and Nitrogen Fertilizer will significantly reduce survivorship

Materials Escherichia coli (DH5 Alpha) Ammonium Nitrate Solution (50% stock solution) Fracking Chemical Simulation(100%(1x) stock solution) 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) YEPD Media and agar plates (1%yeast extract, 2% peptone, 2% glucose (dextrose) , 1.5% agar) Pipettes Incubator Sterile Pipette Tips Sterile Filter(0.2 Microns) Vortex

Procedure Grew E. coli overnight Diluted concentration to 10^5 cells per mL Sterile filtered fracking chemical and fertilizer mix Used stock solutions to create desired exposures Vortexed then incubated at room temp for 10 minutes

Exposures High- 5% Low- 1% Medium- 2.5% Fracking Chemical- 100%(1x) Nitrogen Fertilizer- 50% High High Low High Low High Medium Control Sterile Water 8.4ml 9.7ml 8.8ml 9.15ml 9.9ml Fracking Fluid 0.5ml 0.1ml 0.25ml 0ml Ammonium Nitrate 1ml 0.2ml E. Coli Total Vol 10ml

Procedure 6. Pipetted 0.1ml of each solution onto YEPD plates 7. Plates placed into incubator at 37C for 24 hours 8. Cell colonies counted 9. Statistical Analysis performed

Fracking Chemical and Nitrogen Fertilizer Effects on Survivorship Interaction P value-1.06E-17

Anova and Dunnett’s Results Single Factor P Value- 3.39E-30 Two Factor P Value- 1.06E-17 Dunetts Test: T Crit- 2.99 Solution T Value Significant Value? High High 4.3 Yes Low Low 3.8 nHifLo 14.73 nLofHi 9.7 Med Med 6.28

Conclusion The null hypothesis was rejected All tests significantly varied from control There appeared to be an interaction but it is not clear what this interaction was

Limitations One exposure time Only 2 concentrations were selected Fracking chemical was a simulation

Extensions Expand range of concentrations Use varying exposure time Different Microbe Models Other health effects

Works Cited https://www.epa.gov/sites/production/files/2016- 12/documents/hfdwa_executive_summary.pdf https://en.m.wikipedia.org/wiki/Ammonium_nitrate https://www.epa.gov/nutrientpollution/problem https://en.m.wikipedia.org/wiki/Hydraulic_fracturing https://www.cdc.gov/ecoli/index.html http://law.case.edu/journals/LawReview/Documents/63CaseWResLRev4.6. Article.Robbins.pdf

ANOVA Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 10 1816 181.6 24.71111 Column 2 1848 184.8 311.2889 Column 3 3094 309.4 173.3778 Column 4 1452 145.2 633.9556 Column 5 1682 168.2 187.0667 Column 6 2104 210.4 25.6 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 166603.7 5 33320.75 147.4369 3.39E-30 2.38607 Within Groups 12204 54 226 Total 178807.7 59   Anova: Two-Factor With Replication SUMMARY NH NL Total FH   Count 10 20 Sum 1808 1452 3260 Average 180.8 145.2 163 Variance 25.95556 633.9556 646.1053 FL 3094 1848 4942 309.4 184.8 247.1 173.3778 311.2889 4315.147 4902 3300 245.1 165 4446.516 860.4211 ANOVA Source of Variation SS df MS F P-value F crit Sample 70728.1 1 247.1762 1.06E-17 4.113165 Columns 64160.1 224.2228 4.88E-17 Interaction 19802.5 69.20456 6.65E-10 Within 10301.2 36 286.1444 164991.9 39