1. Effects of Calcium Compounds on Algal Growth PJAS 2015 1 Joe Ebbert Pittsburgh Central Catholic Grade 9

2. Surface Runoff  Part of the water cycle  Can effect aquatic life and soil life  Common form of lake, stream, and river pollution 2

3. Introduction  Calcium Chloride and Calcium Nitrate used as variables.  Calcium chloride: used in road salts  Calcium nitrate: used in fertilizers  Both major runoffs in the region  Chlamydomonas and Euglena alga used as experimental subjects.  Two types of algae are common algal models 3

4. Effects of Calcium Chloride on Aquatic life  Elevated chloride levels pose a risk to species survival, growth, and/or reproduction.  Chloride toxicity increases when associated with other cations, such as calcium  Increases release of toxic metals from sediment  Can inhibit nutrients and DO in aquatic systems 4

5. Effects of Calcium Nitrate on Aquatic life  Some fertilizers contain nitrogen, which plants need in order to grow.  Nitrate is a eutrophic agent  Can create algal blooms leading to hypoxic zones 5

6. Algal Models Chlamydomonas reinhardtii  Two flagella  Contain just one chloroplast  Commonly found in calm waters and damp soil  Normally 10-30um wide Euglena gracilis  One large flagellum  Many chloroplasts  Found mostly in rich organic waste  Over 100 species  Normally 35-50um long 6

7. Purpose To determine the effects of different concentrations of calcium chloride and calcium nitrate on the growth of Chlamydomonas and Euglena. 7

8. Hypotheses  Null: The calcium compounds tested WILL NOT have significant effects on the growth rates of chlamydomonas and euglena.  Alternative: The calcium compounds tested WILL have significant effects on the growth rates of chlamydomonas and euglena. 8

9. Materials  3 test tube racks  48 test tubes  200µl micro-pipette  1000µl micro-pipette  Carolina Spectrophotometer  Euglena gracilis (from Carolina)  Chlamydomonas reinhardtii (from Carolina)  150mL of spring water  60mL of soil water (Carolina)  Calcium Chloride  Calcium Nitrate  Pipette tips  Window sill lighting 9

10. Procedure 1. Arranged 48 test tubes across 3 racks (each tube received equal sunlight) and pipetted 2 mL of chlamydomonas into 24 tubes and pipetted 1 mL of euglena into 24 tubes (euglena is denser). 2. Pipetted spring water and calcium chloride or calcium nitrate into each test tube, following the chart on the next slide. 3. Recorded absorbance on days 0, 2, 4, 6, 8, 10,12, and 14 using spectrophotometer set at 430 nm wavelength. 10

11. Procedure (continued) Concentration 0%0.01%0.1%1% Chlamy. 2 mL Soil water 1 mL Spring water 2 mL1.995 mL1.95 mL1.5 mL Chlor./nitrate 0 mL0.005 mL0.05 mL0.5 mL Total 5 mL 11 Concentration 0%0.01%0.1%1% Euglena 1 mL Soil water 1 mL Spring water 3 mL2.995 mL2.95 mL2.5 mL Chlor./nitrate 0 mL0.005 mL0.05 mL0.5 mL Total 5 mL Repeat 3 times for each variable for both Chlamydomonas and Euglena.

12. Chlamydomonas Growth Chart 12 p-value day 6: 3.48E-06 p-value day 14: 4.2E-15

13. Dunnett’s Test (Chlamy day 6) Concentrationt-value Significance Calcium Chloride 0.01%0.21Not Significant Calcium Chloride 0.1%1.15Not Significant Calcium Chloride 1%5.85Significant Calcium Nitrate 0.01%1.46Not Significant Calcium Nitrate 0.1%3.55Not Significant Calcium Nitrate 1%5.33Significant 13 t-crit=4

14. Dunnett’s Test (Chlamy day 14) Concentration t-valueSignificance Calcium Chloride 0.01%3.05Not Significant Calcium Chloride 0.1%4.79Significant Calcium Chloride 1%12.86Significant Calcium Nitrate 0.01%22.7Significant Calcium Nitrate 0.1%11.12Significant Calcium Nitrate 1%12.21Significant 14 t-crit=4

15. Euglena Growth Chart 15 p-value day 6: 0.47 p-value day 14: 3.04E-08

16. Dunnett’s Test (Euglena day 14) Concentrationt-valueSignificance Calcium Chloride 0.01%1.25Not Significant Calcium Chloride 0.1%1.8Not Significant Calcium Chloride 1%3.46Not Significant Calcium Nitrate 0.01%4.85Significant Calcium Nitrate 0.1%3.32Not Significant Calcium Nitrate 1%12.62Significant 16 t-crit=4

17. Chlamydomonas Results The null hypothesis that the Chlamydomonas experimental groups would not significantly vary from the control is rejected (p-value 0.05) for day 6 CaCl2 0.01%, 0.1% and Ca(NO3)2 0.01%, 0.1%. 17

18. Euglena Results The null hypothesis that the Euglena experimental groups would not significantly vary from the control is accepted (p-value>0.05) for ALL day 6 concentrations and day 14 CaCl2 ALL concentrations and Ca(NO3)2 0.1% and is rejected (p-value<0.05) for day 14 Ca(NO3)2 0.01%, 1%. 18

19. Conclusion The alternative hypothesis is accepted because both calcium chloride and calcium nitrate had concentrations that varied significantly in algal growth from the control. 19

20. Limitations and Extensions Limitations  Only two types of algae were used  Algal health?  Longer experimentation time, more readings Extensions  Test different types of algae  Test for synergistic effects of variables  Health of the cells 20

21. References http://www.biodiversitylibrary.org/item/44537#page /79/mode/1up http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1126 904/ http://www.drugs.com/pro/calcium-chloride.html http://www.chemspider.com/Chemical- Structure.23336.html http://www.nist.gov/pml/div685/grp03/spectrophoto metry.cfm 21

22. Chlamydomonas ANOVA day 6 Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column 160.9010.1501670.000421 Column 230.4580.1526679.43E-05 Column 330.4160.1386670.000192 Column 430.2810.0936672.23E-05 Column 530.4920.1646.4E-05 Column 630.5520.1840.000292 Column 730.2980.0993333.03E-05 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups0.01998460.00333116.207893.48E-062.69866 Within Groups0.003494170.000206 Total0.02347823 22

23. Euglena ANOVA day 6 Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column 160.8790.14650.000136 Column 230.4410.1470.000217 Column 330.4490.1496670.00019 Column 430.4580.1526670.001412 Column 530.5030.1676670.000422 Column 630.4850.1616670.00043 Column 730.4030.1343330.000162 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups0.0021960.0003650.9768880.4702912.69866 Within Groups0.006351170.000374 Total0.00854123

24. Chlamydomonas ANOVA day 14 Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column 160.9410.1568331.46E-05 Column 230.510.179.1E-05 Column 330.4020.1340.000013 Column 430.2920.0973330.00014 Column 530.780.269E-06 Column 630.6230.2076671.23E-05 Column 730.3010.1003330.0001 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups0.06166360.010277217.07754.2E-152.69866 Within Groups0.000805174.73E-05 Total0.06246823 24

25. Euglena ANOVA day 14 Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column 161.3780.2296670.000282 Column 230.6590.2196670.00016 Column 330.7250.2416672.03E-05 Column 430.6110.2036672.03E-05 Column 530.5830.1943334.13E-05 Column 630.6150.2052.8E-05 Column 730.4160.1386671.73E-05 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups0.02168960.00361530.932713.04E-082.69866 Within Groups0.001987170.000117 Total0.02367623 25