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

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

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

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.

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.

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

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.

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

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

Euglena Protist Euglenophyte because of chloroplasts 20-300 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

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

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.

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.

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.

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 0.720 at 0x, 0.670 at 0.001x, and 0.249 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 0.110516 is >0.05 ACCEPT NULL Statistical Analysis ANOVAS Chlamydomonas

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 0.05. (0.473 at 0.001x & 0.275 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 0.476 at pH7 & 0.333 at pH5 ACCEPT NULL Was there an interaction between pH and fertilizer concentration with the Euglena Sample? - Insignificant, the p-value of 0.429 is >0.05 ACCEPT NULL Statistical Analysis ANOVAS Euglena

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)

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.

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

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