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.
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
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