1. Fertilizer Effects on Acid Rain Stressed Algae
Andrew Haky
9th Grade, Pittsburgh Central Catholic HS
1st Year in P.J.A.S.

2. Runoff
Part of the water cycle and describes the water that flows over a land surface.
Surface runoff occurs on land, typically creating a ‘watershed.’
Materials that are transported on surface runoff are fertilizers, petroleum, pesticides, herbicides, and salt.
Pollution can come from this runoff

3. Algal Importance Base of aquatic food chain
Used as a bio-indicator for
aquatic environments

4. Eutrophication Caused by an overabundance of nutrients in an ecosystem
No limiting factor on algae populations
Uncontrollable growth takes up resources necessary for other organisms-oxygen.
Limits biodiversity
Can occur naturally
Occurs today by fertilizer run off
Serious Pollution Problem
Eutrophication is when a body of water has too many nutrients. The growth of algae no longer has a limiting factor and can grow uncontrollably, taking up resources necessary for other organisms, most often oxygen.
Eutrophication limits biodiversity. It does occur naturally, but with the quantity of fertilizers we use today, and their runoff, it has become a serious pollution problem.

5. Acid Precipitation
Worldwide problem
Caused by pollution of sulfur dioxide and nitrogen oxide
Often originates from smokestacks, vehicle exhaust, and burning fossil fuels
Average pH of 5.6
An increase of acid rain in many aquatic systems reduces biodiversity.

6. Acid Rain in Pennsylvania
Average pH of precipitation in PA: 4.5
Major problem due to industry
The many abandoned coal mines in PA create a large problem for the water bodies in the area

7. The main ingredients in Miracle-Gro are Nitrogen, Phosphates and Potash.
Nitrogen- found in ammonia used in fertilizers, causes algae populations to grow rapidly(eutrophication)
Phosphates are vital for cellular processes such as the synthesis of ATP and ADP.
Both phosphates and nitrogen are normally limiting factors of algal populations.

8. Potash and H2SO4 Potash Potassium oxide in fertilizers
93% used to make fertilizers
Important for food crops, gives them favorable qualities
Canada is the world’s leading producer
Sulfuric Acid
Top product in chemical industry
Can be combined with ammonia to create agricultural products
Corrosive, can bind with water droplets to create acid rain
The form of potash used in fertilizers is potassium oxide. Ninety-three percent of the world’s potash usage is in fertilizers. Potash is important for food crops because it has the properties of being able to retain water, yield a good harvest, create a good taste and color, help protect from disease, and can improve nutritional value. Canada is the world’s leading producer of potash. Sulfuric acid is the top product in the chemical industry. Its chemical formula is H2SO4. Reacting ammonia with sulfuric acid produces a product that can be sold to the agriculture industry. Sulfuric acid is very corrosive and can bind with water droplets creating acid rain.
Potash and H2SO4

9. Chlamydomonas Used as a bio-indicator Genus of green algae
Can create starch
Has an Eyespot to orient itself to light
Two flagella, swims with a breaststroke-like motion
10 μm in diameter
Large,crescent-shaped chloroplasts
Found in freshwater, soil, oceans, and snow

10. Euglena Protist Euglenophyte because of chloroplasts
μm in length
Endocytosis
Flagellum propels with whip-like motion
Can also make an inchworm-like movement
Has an Eyespot to orient itself towards light
Found in nutrient-rich freshwater
Effected by acidity
Asexual, prokaryotic fission
If photosynthesis is not possible, can absorb nutrients from decaying material in endocytosis

11. Purpose
Determine if fertilizers (Miracle-Gro) and acid rain (H2SO4 ) have an effect on algae populations (Euglena and Chlamydomonas) individually or in synergy.

12. Hypotheses
NULL- neither Miracle-Gro nor acidity will significantly affect algal population growth and they not act synergistically to affect growth
The hypothesis for this experiment is that Miracle-Gro fertilizer, due to the ingredients of nitrogen, phosphorus, and potash, will have a growth effect on the populations of algae as measured by the spectrometer.
It is hypothesized that acidity, resembling acid rain and in the form of sulfuric acid, will have a negative effect on the growth of algae.
The alternative hypothesis is that the Miracle-Gro will have an a growth effect on the Euglena, but not Chlamydomonas.
The null hypothesis is that neither Miracle-Gro nor acidity will have an effect on the algal types.

13. PROCEDURE 60 tubes were set up to receive equal light.
Chlamydomonas was added to half the tubes and Euglena to the other half. Tips were changed for each new content added. This was done throughout the experiment. Every tube for each group had equal concentrations of algae, although the Euglena concentration was half of the Chlamydomonas.
Miracle-Gro was added using a micropipet in 0,0.5, & 0.05 mL volumes to 10 tubes each to both Chlamydomonas and Euglena samples.
0.01x Sulfuric Acid was added using a micropipet to 5 out of every 10 tubes in 0.02 mL, but not the other 5.
Spring water was then added using a micropipet to make each tube 5 mL full.

14. Steps 2-5 created 30 tubes of Chlamydomonas
Steps 2-5 created 30 tubes of Chlamydomonas. 15 tubes had a pH of 7 and the other 15 tubes had a pH of 5. For both of these groups, 5 tubes had a fertilizer concentration of 0x, 5 had a concentration of 0.001x, and five had a concentration of 0.1x. The same was also done using Euglena.
6.Each tube was marked with a black line so that it would enter the spectrophotometer the same direction every time.
7.Wax paper was used to cover each tube. The tubes were each inverted 3 times.
8. The spectrophotometer was allowed to warm up for 30 minutes at 430 nm.
9.Each tube was placed into the spectrophotometer and the absorbance was taken.
10. After 19 days, the results were analyzed using statistics and graphs.

18. Did the pH have an effect on the Chlamydomonas Samples at either 0x, 0
Did the pH have an effect on the Chlamydomonas Samples at either 0x, 0.001x, or 0.1x fertilizer concentrations?
- Insignificant, the groups had p-values of >0.05 (p-values were at 0x, at 0.001x, and at 0.1x ACCEPT NULL
Did the fertilizer concentrations have an effect on Chlamydomonas at a pH of 5 or 7?
- Significant, the p-value were: 7.714E-7 at pH7 & 3.37E-4 at pH5 REJECT NULL
Was there an interaction between pH and fertilizer concentration with the Chlamydomonas Samples?
- Insignificant, the p-value of is >0.05 ACCEPT NULL
Statistical Analysis
ANOVAS
Chlamydomonas

19. Did the pH have an effect on the Euglena Samples at either 0x, 0
Did the pH have an effect on the Euglena Samples at either 0x, 0.001x, or 0.1x fertilizer concentrations?
-Significant at 0x (p-value of 0.002). REJECT NULL Insignificant at 0.001x or 0.1x because p-values were > than (0.473 at 0.001x & at 0.1x) ACCEPT NULL
Did the fertilizer concentrations have an effect on Euglena at a pH of 5 or 7?
-Insignificant, the p-values were at pH7 & at pH5 ACCEPT NULL
Was there an interaction between pH and fertilizer concentration with the Euglena Sample?
- Insignificant, the p-value of is >0.05 ACCEPT NULL
Statistical Analysis
ANOVAS
Euglena

20. Dunnet’s Test
For the significant ANOVA’s, Dunnet’s tests were conducted to find which group(s) varied significantly from the control.
Chlamydomonas, pH7- The samples with a fertilizer concentration of 0.001x did not vary significantly from the control but the 0.1x samples did.
-T 2.53<3.03 T-crit [0.001x] T 10.22>4.63 T-crit [0.1x](very sure)
Chlamydomonas, pH5- The samples with a fertilizer concentration of 0.001x did not vary significantly from the control but the 0.1x samples did.
-T 1.616< 3.03 T-crit [0.001x] T 5.620>4.63 T-crit [0.1x] (very sure)
Euglena, 0x fertilizer- the pH difference was found to be significant.
-T 4.378> 4.03 T-crit (very sure)

21. Conclusions
The null hypothesis is rejected because fertilizer concentration had an effect on Chlamydomonas and because pH did have an effect on Euglena at 0x. The null is accepted in that pH did not effect Chlamydomonas and fertilizer concentration had no effect on Euglena.
Research indicated Euglena would be effected by pH level. This was supported at a 0x fertilizer concentration.
Eutrophication may have played a role in Euglena’s spike and drop. It is possible it could have effected Chlamydomonas at a later time.
The null hypothesis is rejected in that fertilizer concentration had an effect on Chlamydomonas. It can also be rejected in that pH did have an effect on Euglena at 0x. The null is accepted in that pH did not effect Chlamydomonas and fertilizer concentration had no effect on Euglena.

22. Notes/Variables/Limits
EXTENSIONS
Algae shock?
Euglena Tube <pH 5, 0.001x>
Spectrometer accuracy
Cloudy and overcast days
Sample size limit
Could test thermal water pollution
Could test salt concentration
Use other organisms

23. Bibliography
" The Columbia Electronic Encyclopedia. © 1994, , on Fact Monster. © 2000–2007 Pearson Education, publishing as Fact Monster. 01 Jan <
Cloern, James E. "Eutrophication." Encyclopedia of Earth. 18 Dec Web. 30 Dec <
Jones, Andrew Z. "Visible Light Spectrum." Physics. New York Times Company. Web. 01 Jan <
Mackean, D. G. "Biology: Protista, Amoeba, Malaria, Paramecium, Spirogyra, Chlamydomonas, Euglena, Educational notes & drawings by D G Mackean." Biology Teaching & Learning Resources. Educational articles, drawings, experiments & PowerPoint presentations by D G Mackean. D G
Mackean and Ian Mackean. Web. 30 Dec <
Ophardt, Charles E. "Acid Rain." Elmhurst College: Elmhurst, Illinois Web. 01 Jan <
"sulfuric acid." The Columbia Encyclopedia, Sixth Edition Encyclopedia.com. 1 Jan <
Kitajima, Yasuko. "Fertilizer." Formal Reasoning Group. 19 June Web. 01 Jan <