Fracking Chemical Influence on Algal Populations By: Sam Little Pittsburgh Central Catholic PJAS 2018 Grade 9

Problem Does a simulation formula of a fracking solution have an effect on algal populations?

Fracking Runoff into local watershed? A process of retrieving natural gas from deep below the Earth’s surface After the main bore hole is drilled, a mixture is pumped down it Water – 90% Sand – 9.5% Additives - 0.5% Companies use a “proprietary blend” of chemicals in the additives The primary concern is the impact of the additives on the environment Runoff into local watershed?

Variable A formula that has been used in other studies regarding the additives in fracking It is a simulation because fracking companies’ actual formula is not known Chemicals Mg/L Calcium Chloride 16.650 Magnesium Chloride 0.4750 Barium Chloride 0.1851

Algae Good model for showing over-all aquatic heath Used in this experiment Euglena Chlamydomonas These two species are common and excellent lab models Primary Producers

Euglena gracilis Unicellular, algal-like, Protist Very common in nature Live in fresh and brackish water rich in organic matter Autotroph and heterotroph

Chlamydomonas reinhardtii Unicellular, algal-like protists Biflagellate Cup-shaped Chloroplast Commonly found in soil  and fresh water ponds

Purpose The purpose of this experiment is to determine if a fracking simulation formula will have an effect on algal populations.

Hypothesis Null Hypothesis- The fracking simulation formula will not have a significant effect on algal populations. Alternative Hypothesis- The fracking simulation formula will significantly reduce algal growth.

Materials Euglena gracilis (Carolina Science) Chlamydomonas reinhardtii (Carolina Science) Test Tubes – 100mm x 13mm Borosilicate culture tubes Pipettes Micro Macro Spectrophotometer Test Tube Rack Fracking Simulation Formula Soil Water Spring Water Wax Paper

Procedure The test tube rack was filled with 24 tubes in a single row. The temperature of the testing room was set to 20 degrees Celsius and the rack was placed next to a window The variable, model, and waters were mixed in the following manner to create the desired concentrations.

Test Tube Mixtures Ingredients 0% 0.1% 0.5% 1% Algae 1 ml 1ml Soil Water Spring Water 3 ml 2.71 ml 1.53 ml 0.06 ml Formula 0 ml 0.29 ml 1.47 ml 2.94 ml

Procedure Each tube was mixed by inversion before it was placed into the spectrophotometer. The spectrophotometer was set to 430nm The absorbance was recorded for each test tube once a day for 10 days.

P-Value 0.09244 P-Value 0.15692 P-Value 0.06636

P-Value 0.80019 P-Value 0.09249 P-Value 0.40421

Conclusion ANOVA failed to reject the null hypothesis There was not significant variation among the groups The fracking simulation formula did not have a significant effect on the growth of algae

Limitations and Future Research Health of Culture- the model was not subjected to optimal growing conditions The variable – actual fracking additives Temperature – window? Optimal Lighting The Spectrophotometer and tubes- slight drift in readings Future Research Optimal lighting Test different species of algae Try other ingredients in the fracking simulation formula Vary simulation fracking formula exposures Interaction effect with other variables in watershed

Works Cited “Algae” Algae Facts, 2017, https://kids.britannica.com  The Editors of Encyclopædia Britannica. ?Chlamydomonas.? Encyclopædia Britannica, Encyclopædia Britannica, Inc., 2 Mar. 2017, www.britannica.com/science/Chlamydomonas. The Editors of Encyclopædia Britannica. ?Chlamydomonas.? Encyclopædia Britannica, Encyclopædia Britannica, Inc., 2 Mar. 2017, www.britannica.com/science/Chlamydomonas. The Editors of Encyclopædia Britannica. ?Euglena.? Encyclopædia Britannica, Encyclopædia Britannica, Inc., 27 Dec. 2017, www.britannica.com/science/Euglena. “What Is Fracking?” What Is Fracking?, 2017, www.what-is-fracking.com/.    “What Is Groundwater?” What Is Groundwater?, 2017, http://www.groundwater.org/.  

Spectrophotometry Machine used to measure the amount of light passing through a medium Useful in chemistry, physics, biochemistry, material and chemical engineering

Averages Euglena Chlamy 0% 0.10% 0.50% 1% 0.122 0.111 0.118 0.105 0.122 0.111 0.118 0.105 0.086 0.095 0.097 0.089 1 0.108 0.081 0.096 0.075 0.068 0.09 0.084 0.067 2 0.102 0.077 0.088 0.072 0.091 0.092 0.083 3 0.1 0.076 0.078 0.082 0.099 4 0.07 0.085 0.062 0.044 5 0.028 0.032 0.034 0.074 0.093 0.101 6 0.063 0.061 0.046 0.117 7 0.053 0.039 0.052 0.129 0.106 8 0.065 0.047 0.049 0.103 0.124 0.15 0.12 9 0.079 0.045 0.054 0.05 0.126 0.157 0.176 0.141 10 0.041 0.131 0.162 0.144

ANOVA Euglena Day 2 Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 3 0.309 0.103 0.000637 Column 2 0.23 0.076667 3.73E-05 Column 3 0.265 0.088333 5.43E-05 Column 4 0.216 0.072 0.000025 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.001721 0.000574 3.044081 0.092444 4.066181 Within Groups 0.001507 8 0.000188 Total 0.003228 11  

ANOVA Euglena Day 6 Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 3 0.188 0.062667 0.00017 Column 2 0.183 0.061 9.1E-05 Column 3 0.139 0.046333 9.23E-05 Column 4 0.137 0.045667 0.000105 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.000757 0.000252 2.198741 0.165915 4.066181 Within Groups 0.000918 8 0.000115 Total 0.001675 11  

ANOVA Euglena Day 10 Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 3 0.204 0.068 0.000048 Column 2 0.162 0.054 0.000523 Column 3 0.225 0.075 0.000175 Column 4 0.124 0.041333 6.33E-06 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.002018 0.000673 3.576872 0.066357 4.066181 Within Groups 0.001505 8 0.000188 Total 0.003523 11  

ANOVA Chlamydomonas Day 2 Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 3 0.269 0.089667 9.33E-06 Column 2 0.274 0.091333 0.000177 Column 3 0.276 0.092 0.000363 Column 4 0.248 0.082667 0.000105 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.000165 5.5E-05 0.335708 0.800188 4.066181 Within Groups 0.00131 8 0.000164 Total 0.001475 11  

ANOVA Chlamydomonas Day 6 Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 3 0.274 0.091333 2.13E-05 Column 2 0.354 0.118 0.000112 Column 3 0.346 0.115333 0.000464 Column 4 0.3 0.1 0.000036 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.001446 0.000482 3.043311 0.09249 4.066181 Within Groups 0.001267 8 0.000158 Total 0.002714 11  

ANOVA Chlamydomonas Day 10 Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 3 0.392 0.130667 0.000144 Column 2 0.468 0.156 5.7E-05 Column 3 0.47 0.156667 0.000849 Column 4 0.433 0.144333 0.000581 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.001345 0.000448 1.098788 0.404208 4.066181 Within Groups 0.003264 8 0.000408 Total 0.004609 11