1. The Synergistic Effects of Fertilizer and Pesticide on Algae Dominic Augello Central Catholic HS Grade 9 First Year in PJAS

2. Pesticides Include any chemical, antibacterial, or biological agent used to kill or repel unwanted insects Potentially harmful to non-target species Long-term effects unclear Bonide

3. Past Studies with Pesticides Studies found that 25 million agricultural workers are affected by pesticide poisoning yearly. Studies found that about 98% of insecticides and about 95% of herbicides reach a non-target species.

4. Fertilizer Nutrients that aid plant growth Primary nutrients: Nitrogen, Phosphorus, and Potassium Potential to harm non-target species Miracle-Gro

5. Past Studies with Fertilizers Studies found that fertilizers account for about 40% of corn yields, 40% of wheat yields, and 57% of grain yields across America. Studies found that using fertilizer regularly on plants affect the rhizobia of a plant.

6. Chlamydomonas Species of algae Usually circular in shape with few flagella Can live in saltwater, freshwater, damp soil, and snowy environments Used as a model for many studies on molecular biology.

7. Rationale To determine the synergistic effects of fertilizer and pesticide on the growth of Chlamydomonas.

8. Hypotheses Null Hypothesis: The fertilizer and pesticide will not have a synergistic effect on Chlamy. growth. Alternate Hypothesis: The fertilizer and pesticide will have a synergistic effect on Chlamy. growth

9. Materials Chlamydomonas Miracle-Gro Fertilizer Bonide Pesticide Soil Water Spring Water Distilled Water Spectrophotometer Micropipettes Test tubes Tube rack Vortex Lamp Wax Paper

10. Procedure 1. Soil water was placed in a 50ml conical tube. 2. Filled 45 tubes with 2ml of Chlamydomonas and 1ml of soil water. 3. The tubes were then filled with the following concentrations. Each concentration had 5 replicates.

11. Solution Table 0 0% Pesticide L.01 % Pesticide H 1% Pesticide 0 0x Fertilizer 2 ml Spring Water 0 ml Pesticide 0 ml Fertilizer 1.995 ml SW.005 ml Pesticide 0 ml Fertilizer 1.5 ml SW.5 ml Pesticide 0 ml Fertilizer L.01x Fertilizer 1.995 ml SW 0 ml Pesticide.005 ml Fertilizer 1.99 ml SW.005 ml Pesticide.005 ml Fertilizer 1.495 ml SW.5 ml Pesticide.005 ml Fertilizer H 1x Fertilizer 1.5 ml SW 0 ml Pesticide.5 ml Fertilizer 1.495 ml SW.005 ml Pesticide.5 ml Fertilizer 1 ml SW.5 ml Pesticide.5 ml Fertilizer

12. Procedure (continued) 4. Tubes were inverted and the absorbances, at 430 nanometers, were recorded with a Carolina spectrophotometer. 5. Placed tubes 21 cm below a fluorescent light at 20 degrees Celsius. 6. Recorded absorbance of each tube every other day for 14 days.

13. Effects of Fertilizer on Chlamy. Day 6 P-value = 8.75597E-05 Day 14 P-value = 6.73641E-07

14. Effects of Pesticide on Chlamy. Day 6 P-value = 1.32691E-05 Day 14 P-value = 0.909830175

15. Effects of Fertilizer and Pesticide on Chlamy. Day 6 P-value = 8.84154E-10 Day 14 P-value = 8.99708E-07

16. Pesticide Dunnett's Test ConcentrationsT-critT-valueSignificance Day 60.01%3.032.46Not Significant 1%3.035.47Significant Day 14.01%3.03.74Not Significant 1%3.035.31Significant

17. Fertilizer Dunnett's Test ConcentrationsT-critT-valueSignificance Day 6.01x3.031.07Not Significant 1x3.036.26Significant Day 14.01x3.03.31Not Significant 1x3.039.54Significant

18. Conclusion The alternate hypothesis was accepted for the synergistic effects of fertilizer and pesticide on algae. The null hypothesis was accepted for fertilizer and pesticide at low concentrations, but the null hypothesis was rejected for fertilizer and pesticide at high concentrations. Both fertilizer and pesticide appeared to have an effect on the algae when used in high concentrations.

19. Limitations and Extensions Limitations: Only one model was used Few concentrations were tested Algal health may have varied Extensions: Use more models in future experiments Use more concentrations of the variables Use multiple brands of fertilizers and pesticides Have more replicates

20. References http://blog.chron.com/sciguy/2013/04/what-is-ammonium-nitrate-and-why-do-we-use-it/ http://people.oregonstate.edu/~muirp/pesthist.htm http://news.berkeley.edu/2012/04/02/fertilizer-use-responsible-for-increase-in-nitrous-oxide-in- atmosphere/ http://news.berkeley.edu/2012/04/02/fertilizer-use-responsible-for-increase-in-nitrous-oxide-in- atmosphere/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4025000/ http://www.cropnutrition.com/understanding-fertilizer-and-its-essential-role-in-high-yielding-crops https://news.illinois.edu/blog/view/6367/204407 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946087/ http://sciencenetlinks.com/science-news/science-updates/farmers-pesticides/ https://www.ocf.berkeley.edu/~lhom/organictext.html http://www.chlamy.org/info.html http://www.sciencedaily.com/releases/2015/02/150226084942.htm

21. Two-Factor ANOVA Day 6 Anova: Two-Factor With Replication SUMMARY00.0001 0.01Total 0x Count55 515 Sum0.9760.935 1.0672.978 Average0.19520.187 0.21340.198533333 Variance0.00002270.000011 4.93E-050.000154124.01x Count55 515 Sum1.0131.081 1.1773.271 Average0.20260.2162 0.23540.218066667 Variance2.23E-050.0002277 0.00020630.000324352 1x Count55 515 Sum1.1931.311 1.7214.225 Average0.23860.2622 0.34420.281666667 Variance0.00031487.22E-05 0.00020120.002362381 Total Count15 Sum3.1823.3273.965 Average 0.212133 333 0.2218 0.264333 333 Variance 0.000487 838 0.001115 457 0.003634 095 ANOVA Source of Variation SSdfMSFP-valueF crit Sample 0.056688 311 2 0.028344 156 226.2504 656 4.11025E -21 3.259446 306 Columns 0.023136 844 2 0.011568 422 92.34217 295 6.69202E -15 3.259446 306 Interacti on 0.012125 156 4 0.003031 289 24.19654 102 8.84154E -10 2.633532 094 Within0.0045136 0.000125 278 Total 0.096460 311 44

22. Two-Factor ANOVA Day 14 Anova: Two-Factor With Replication SUMMARY00.00010.01Total 0x Count55515 Sum1.0181.0051.1113.134 Average0.20360.2010.22220.208933333 Variance2.93E-050.00001754.52E-050.000121781.01x Count55515 Sum1.0271.1061.1843.317 Average0.20540.22120.23680.221133333 Variance0.00001430.00032920.00021570.000335838 1x Count55515 Sum1.291.341.7014.331 Average0.2580.2680.34020.288733333 Variance0.0002030.00030157.47E-050.001602352 Total Count15 Sum3.3353.4513.996 Average 0.22233333 3 0.23006666 70.2664 Variance 0.00075252 4 0.00102892 4 0.00305168 6 ANOVA Source of VariationSSdfMSFP-valueF crit Sample0.055433220.0277166202.7384592.54156E-20 3.25944630 6 Columns 0.01660893 32 0.00830446 7 60.7446358 92.90354E-12 3.25944630 6 Interaction 0.00730906 74 0.00182726 7 13.3658972 78.99708E-07 2.63353209 4 Within0.004921636 0.00013671 1 Total0.084272844

23. Single Factor ANOVA Anova: Single FactorPesticideDay 6 SUMMARY GroupsCountSumAverageVariance Column 150.9760.19520.0000227 Column 250.9350.1870.000011 Column 351.0670.21344.93E-05 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups 0.0018257 332 0.0009128 67 32.995180 72 1.32691E- 05 3.8852938 35 Within Groups0.00033212 2.76667E- 05 Total 0.0021577 3314 Anova: Single FactorFertilizerDay 6 SUMMARY GroupsCountSumAverageVariance Column 150.9760.19520.0000227 Column 251.0130.20262.23E-05 Column 351.1930.23860.0003148 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups 0.00539053 32 0.00269526 7 22.4730405 8 8.75597E- 05 3.88529383 5 Within Groups0.001439212 0.00011993 3 Total 0.00682973 314

24. Single Factor ANOVA Anova: Single FactorPesticideDay 14 SUMMARY GroupsCountSumAverageVariance Column 161.018 0.1696666 67 0.0069322 67 Column 261.0051 0.1675166 67 0.0067408 02 Column 361.121 0.1868333 33 0.0075409 67 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups 0.0013449 012 0.0006724 51 0.0950951 42 0.9098301 75 3.6823203 44 Within Groups 0.1060701 7515 0.0070713 45 Total 0.1074150 7617 Anova: Single FactorFertilizerDay 14 SUMMARY GroupsCountSumAverageVariance Column 151.0180.20362.93E-05 Column 251.0270.20540.0000143 Column 351.290.2580.000203 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups 0.00954893 32 0.00477446 7 58.0835360 9 6.73641E- 07 3.88529383 5 Within Groups0.0009864128.22E-05 Total 0.01053533 314

25. Day 6 Results Average Absorbance P-value = 8.84E-10 Concentrations of Fertilizer Concentrations of Pesticide

26. Day 14 Results Average Absorbance P-value = 9E-07 Concentrations of Fertilizer Concentrations of Pesticide