1

Effects of Calcium Compounds on Algal GrowthPJAS 2015

Joe Ebbert

Pittsburgh Central Catholic

Grade 9

2Surface RunoffPart of the water

cycle

Can effect aquatic life and soil life

Common form of lake, stream, and river pollution

3Introduction

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

4Effects 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

5Effects 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

6Algal ModelsChlamydomonas 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

7Purpose

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

8Hypotheses

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.

9Materials 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

10Procedure

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.

Procedure (continued)Concentration

0% 0.01% 0.1% 1%

Chlamy. 2 mL 2 mL 2 mL 2 mL

Soil water 1 mL 1 mL 1 mL 1 mL

Spring water

2 mL 1.995 mL 1.95 mL 1.5 mL

Chlor./nitrate

0 mL 0.005 mL 0.05 mL 0.5 mL

Total 5 mL 5 mL 5 mL 5 mL

11

Concentration

0% 0.01% 0.1% 1%

Euglena 1 mL 1 mL 1 mL 1 mL

Soil water 1 mL 1 mL 1 mL 1 mL

Spring water

3 mL 2.995 mL 2.95 mL 2.5 mL

Chlor./nitrate

0 mL 0.005 mL 0.05 mL 0.5 mL

Total 5 mL 5 mL 5 mL 5 mLRepeat 3 times for each variable for both Chlamydomonas and Euglena.

Chlamydomonas Growth Chart 12

p-value day 6: 3.48E-06

p-value day 14: 4.2E-15

13Dunnett’s Test (Chlamy day 6)Concentration t-value Significance

Calcium Chloride 0.01%

0.21 Not Significant

Calcium Chloride 0.1% 1.15 Not Significant

Calcium Chloride 1% 5.85 Significant

Calcium Nitrate 0.01% 1.46 Not Significant

Calcium Nitrate 0.1% 3.55 Not Significant

Calcium Nitrate 1% 5.33 Significant

t-crit=4

14Dunnett’s Test (Chlamy day 14)

Concentration t-value Significance

Calcium Chloride 0.01%

3.05 Not Significant

Calcium Chloride 0.1% 4.79 Significant

Calcium Chloride 1% 12.86 Significant

Calcium Nitrate 0.01% 22.7 Significant

Calcium Nitrate 0.1% 11.12 Significant

Calcium Nitrate 1% 12.21 Significant

t-crit=4

Euglena Growth Chart 15

p-value day 6: 0.47

p-value day 14: 3.04E-08

16Dunnett’s Test (Euglena day 14)Concentration t-value Significance

Calcium Chloride 0.01%

1.25 Not Significant

Calcium Chloride 0.1% 1.8 Not Significant

Calcium Chloride 1% 3.46 Not Significant

Calcium Nitrate 0.01% 4.85 Significant

Calcium Nitrate 0.1% 3.32 Not Significant

Calcium Nitrate 1% 12.62 Significant

t-crit=4

17Chlamydomonas ResultsThe null hypothesis that the Chlamydomonas experimental groups would not significantly vary from the control is rejected (p-value<0.05) for day 6 CaCl2 1% and Ca(NO3)2 1% and day 14 CaCl2 0.1%, 1% and Ca(NO3)2 0.01%, 0.1%, 1%, and is accepted (p-value>0.05) for day 6 CaCl2 0.01%, 0.1% and Ca(NO3)2 0.01%, 0.1%.

18Euglena 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%.

19Conclusion

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

20Limitations and ExtensionsLimitations

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

21References

http://www.biodiversitylibrary.org/item/44537#page/79/mode/1up

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1126904/

http://www.drugs.com/pro/calcium-chloride.html

http://www.chemspider.com/Chemical-Structure.23336.html

http://www.nist.gov/pml/div685/grp03/spectrophotometry.cfm

22Chlamydomonas ANOVA day 6Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 6 0.901 0.150167 0.000421

Column 2 3 0.458 0.152667 9.43E-05

Column 3 3 0.416 0.138667 0.000192

Column 4 3 0.281 0.093667 2.23E-05

Column 5 3 0.492 0.164 6.4E-05

Column 6 3 0.552 0.184 0.000292

Column 7 3 0.298 0.099333 3.03E-05

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 0.019984 6 0.003331 16.20789 3.48E-06 2.69866

Within Groups 0.003494 17 0.000206

Total 0.023478 23       

23Euglena ANOVA day 6Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 6 0.879 0.1465 0.000136

Column 2 3 0.441 0.147 0.000217

Column 3 3 0.449 0.149667 0.00019

Column 4 3 0.458 0.152667 0.001412

Column 5 3 0.503 0.167667 0.000422

Column 6 3 0.485 0.161667 0.00043

Column 7 3 0.403 0.134333 0.000162

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 0.00219 6 0.000365 0.976888 0.470291 2.69866

Within Groups 0.006351 17 0.000374

Total 0.008541 23       

24Chlamydomonas ANOVA day 14Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 6 0.941 0.156833 1.46E-05

Column 2 3 0.51 0.17 9.1E-05

Column 3 3 0.402 0.134 0.000013

Column 4 3 0.292 0.097333 0.00014

Column 5 3 0.78 0.26 9E-06

Column 6 3 0.623 0.207667 1.23E-05

Column 7 3 0.301 0.100333 0.0001

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 0.061663 6 0.010277 217.0775 4.2E-15 2.69866

Within Groups 0.000805 17 4.73E-05

Total 0.062468 23       

25Euglena ANOVA day 14Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 6 1.378 0.229667 0.000282

Column 2 3 0.659 0.219667 0.00016

Column 3 3 0.725 0.241667 2.03E-05

Column 4 3 0.611 0.203667 2.03E-05

Column 5 3 0.583 0.194333 4.13E-05

Column 6 3 0.615 0.205 2.8E-05

Column 7 3 0.416 0.138667 1.73E-05

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 0.021689 6 0.003615 30.93271 3.04E-08 2.69866

Within Groups 0.001987 17 0.000117

Total 0.023676 23