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

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

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

4. 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;

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

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

15. 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

16. 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

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

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

19. Works Cited
12/documents/hfdwa_executive_summary.pdf
Article.Robbins.pdf

20. ANOVA Anova: Single Factor SUMMARY Groups Count Sum Average Variance
Column 1 10
1816
181.6
Column 2
1848
184.8
Column 3
3094
309.4
Column 4
1452
145.2
Column 5
1682
168.2
Column 6
2104
210.4
25.6
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 5
3.39E-30
Within Groups
12204 54
226
Total 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 FL
3094
1848
4942
309.4
184.8
247.1
4902
3300
245.1
165
ANOVA
Source of Variation SS df MS F
P-value
F crit
Sample 1
1.06E-17
Columns
4.88E-17
Interaction
6.65E-10
Within 36 39