1
Effects of Calcium Compounds on
Algal Growth
PJAS 2015
Joe Ebbert
Pittsburgh Central Catholic
Grade 9
Surface Runoff
 Part
of the water
cycle
 Can
effect aquatic
life and soil life
 Common
form of
lake, stream, and
river pollution
2
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
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
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
Algal Models
Chlamydomonas
reinhardtii
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Euglena gracilis

Two flagella

One large flagellum

Contain just one chloroplast

Many chloroplasts

Commonly found in calm
waters and damp soil

Found mostly in rich organic
waste

Normally 10-30um wide

Over 100 species

Normally 35-50um long
Purpose
To determine the effects
of different
concentrations of calcium
chloride and calcium
nitrate on the growth of
Chlamydomonas and
Euglena.
7
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
Materials
9

3 test tube racks

150mL of spring water

48 test tubes

60mL of soil water (Carolina)

200µl micro-pipette

Calcium Chloride

1000µl micro-pipette

Calcium Nitrate

Carolina Spectrophotometer

Pipette tips

Euglena gracilis (from Carolina)

Window sill lighting

Chlamydomonas reinhardtii
(from Carolina)
Procedure
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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)
11
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
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 mL
Repeat 3 times for each variable for both Chlamydomonas and Euglena.
Chlamydomonas Growth Chart
p-value day 6: 3.48E-06
p-value day 14: 4.2E-15
12
Dunnett’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
13
Dunnett’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
14
Euglena Growth Chart
p-value day 6: 0.47
p-value day 14: 3.04E-08
15
Dunnett’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
16
Chlamydomonas Results
17
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 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 (pvalue>0.05) for day 6 CaCl2 0.01%, 0.1%
and Ca(NO3)2 0.01%, 0.1%.
Euglena Results
18
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%.
Conclusion
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The alternative hypothesis is
accepted because both
calcium chloride and calcium
nitrate had concentrations
that varied significantly in
algal growth from the control.
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
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/ChemicalStructure.23336.html
http://www.nist.gov/pml/div685/grp03/spectrophoto
metry.cfm
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Chlamydomonas ANOVA day 6
22
Anova: 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
MS
F
ANOVA
Source of
Variation
SS
df
Between
Groups
0.019984
6
0.003331
Within Groups
0.003494
17
0.000206
Total
0.023478
23
16.20789
P-value
3.48E-06
F crit
2.69866
Euglena ANOVA day 6
23
Anova: 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
MS
F
0.976888
ANOVA
Source of
Variation
Between
Groups
SS
df
0.00219
6
0.000365
Within Groups
0.006351
17
0.000374
Total
0.008541
23
P-value
0.470291
F crit
2.69866
Chlamydomonas ANOVA day 14
24
Anova: 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
Between
Groups
0.061663
6
0.010277
Within Groups
0.000805
17
4.73E-05
Total
0.062468
23
F
217.0775
P-value
4.2E-15
F crit
2.69866
Euglena ANOVA day 14
25
Anova: 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
MS
F
30.93271
ANOVA
Source of
Variation
SS
df
Between
Groups
0.021689
6
0.003615
Within Groups
0.001987
17
0.000117
Total
0.023676
23
P-value
3.04E-08
F crit
2.69866