THE EFFECT OF FERTILIZATION
CHEMICALS ON EUGLENA GRACILIS
Michael Halahurich
Pittsburgh Central Catholic
PJAS 2015
Grade 9
AMMONIUM NITRATE
Chemical
Formula: NH4NO3
Considered the best form of
nitrogen fertilizer
Contains 35% elemental
nitrogen
DIAMMONIUM PHOSPHATE
 Chemical
Formula: (NH4)2HPO4
 World’s most commonly used phosphorous
fertilizer
 Very popular because of it’s high nutrient
content
 Created in a controlled reaction of
phosphoric acid with ammonia
 Contains 18% nitrogen and 46%
phosphate
EUGLENA GRACILIS
 Single-celled
flagellate protists
 Commonly found in freshwater
environments such as lakes or ponds
 Harmless to humans, but often serve
as an indicator of water pollution
EUTROPHICATION
•
•
Ecosystem’s response to the
addition of natural or artificial
substances to aquatic systems
Cultural eutrophication- process
that speeds up natural
eutrophication through human
activity
ALGAL BLOOMS
A
rapid increase in the population of algae
in an aquatic system
 Mainly caused by fertilizer runoff
 Algae grow rapidly due to excess nutrients
 The algae eventually die and are
decomposed
 Causes hypoxia
 Dead zones are created
SPECTROPHOTOMETRY
Quantifies
amount of light that
passes through a test tube at a
predetermined wavelength
Commonly used in scientific
fields such as chemistry,
biochemistry, and molecular
biology
PURPOSE
The
purpose of this experiment
is to determine whether
ammonium nitrate and
diammonium phosphate have a
synergistic effect on Euglena
gracilis.
HYPOTHESIS
 Null
hypothesis: Diammonium
Phosphate and Ammonium Nitrate
will not have synergistic effects or
individual effects on the algae.
 Alternative hypothesis:
Diammonium Phosphate and
Ammonium Nitrate will have
synergistic and individual effects on
the algae.
MATERIALS
 Micropipette
 46
test tubes
 Tube racks
 Euglena Gracilis
 Spring water
 Ammonium nitrate
 Diammonium phosphate
 Artificial Light Source
 Carolina Spectrophotometer
 Notebook to record results
PROCEDURE
1.
2.
3.
Tubes with respective
concentrations of chemicals and
algae were placed 1.25 meters
under a light.
The room was kept at room
temperature (20-21C)
The following tube
concentrations were made:
TUBE MAKEUP
Control
0.1% 0.1%
DAP AN
1%
1%
DAP AN
0.1%
DAP
0.1%
AN
0.1%
DAP
1%
AN
1%
DAP
1%
AN
1%
DAP
0.1%
AN
4
ml
3.95
ml
3.95
ml
3.5
ml
3.5
ml
3.9
ml
3.45
ml
3
ml
3.45
ml
10%
0
DAP
ml
solution
0.05
ml
0
ml
0.5
ml
0
ml
0.05
ml
0.05
ml
0.5
ml
0.5
ml
10% AN 0
solution ml
0
ml
0.05
ml
0
ml
0.5
ml
0.05
ml
0.5
ml
0.5
ml
0.05
ml
Euglena 1
ml
1
ml
1
Ml
1
ml
1
ml
1
ml
1
ml
1
ml
1
ml
Total
Volume
5
ml
5
ml
5
ml
5
ml
5
ml
5
ml
5
ml
5
ml
Spring
Water
5
ml
PROCEDURE (CONT.)
4.
5.
6.
7.
The top of each tube was covered with a piece of
wax paper and gently inverted to mix
5ml of spring water was used to blank the
spectrophotometer
Absorbance was measured at 430 nm with a
spectrophotometer. Algae growth was indirectly
measured by quantifying the absorbance of the
tube.
Absorbance readings were taken every other
day for 15 days
EUGLENA GROWTH
0.25
Absorbance at 430nm
0.2
Day 7
P-value=0.038
Day 15 P-value=0.173
Control
0.15
lo AN
0.1
hi AN
0.05
0
Day 1 Day 3 Day 5 Day 7 Day 9 Day
11
Days Tested
Day
13
Day
15
EUGLENA GROWTH
0.45
Day 7
P-value=0.00003
Day 15
P-value=3.45E-05
Absorbance at 430nm
0.4
0.35
0.3
0.25
Control
0.2
lo DAP
hi DAP
0.15
0.1
0.05
0
Day 1 Day 3 Day 5 Day 7 Day 9 Day
Days Tested 11
Day
13
Day
15
EUGLENA GROWTH
0.4
Day 7
P-value=1.88E-06
Day 15 P-value=0.189
Absorbance at 430nm
0.35
0.3
0.25
Control
0.2
hi DAP lo AN
hi DAP hi AN
0.15
lo DAP hi AN
lo DAP lo AN
0.1
0.05
0
Day 1 Day 3 Day 5 Day 7 Day 9 Day Day Day
11
13
15
Days Tested
ANALYSIS OF DATA
The 2-Factor ANOVA suggested
that there were synergistic effects on
the algae for days 3, 5, 7, and 11, but
not on days 9, 13, and 15.
 Single factor ANOVAS were run to
determine if either of the chemicals
were significantly affecting the
algae.

ANOVA RESULTS
DAY
2 FACTOR ANOVA
(INTERACTION?)
SINGLE FACTOR
ANOVA
Day 3
P-value= 0.0002
Interaction? Yes
DAP P-value=0.0001
AN P-value= 0.712
Day 5
P-value= 0.048
Interaction? Yes
DAP P-value=0.0005
AN P-value=0.557
Day 7
P-value= 1.88E-06
Interaction? Yes
DAP P-value=0.00003
AN P-value=0.038
Day 9
P-value= 0.225
Interaction? No
DAP P-value= 6.77E-05
AN P-value=0.562
Day 11
P-value=0.047
Interaction? Yes
DAP P-value=0.023
AN P-value=0.0756
Day 13
P-value= 0.338
Interaction? No
DAP P-value=0.005
AN P-value=0.670
Day 15
P-value= 0.189
Interaction? No
DAP P-value=3.45E-05
AN P-value=0.173
DUNNETT’S TEST
 Further
statistical analysis
 Follows Single Factor ANOVA if Pvalue is significant
 Compares each experimental group
back to the control
DUNNETT’S TEST RESULTS T-CRIT=3.90
DAY
T-VALUE
SIGNIFICANT?
Day 3
0.1%=5.36
1%=6
Significant
Significant
Day 5
0.1%=3.89
1%=5.39
Not Significant
Significant
Day 7
0.1%=1.66
1%=8.16
0.1%=1.35
1%=2.35
Not Significant
Significant
Not Significant
Not Significant
Day 9
0.1%=2.76
1%=8.03
Not Significant
Significant
Day 11
0.1%=-1.1
1%=2.45
Not Significant
Not Significant
Day 13
0.1%=2.03
1%=4.48
Not Significant
Significant
Day 15
0.1%=1.97
1%=7.43
Not Significant
Significant
CONCLUSIONS
There appeared to be significant synergistic
effects on days 3, 5, 7, and 11.
 Single-factor ANOVA revealed that DAP
appeared to have significant effects on all days.




0.1% concentration not significant for all days except
for day 3
1% concentration significant for all days except day
11
No significant effects were present for
ammonium nitrate on any day.
LIMITATIONS & EXTENSIONS

Limitations
Unwanted microbes could have entered test tubes
 Spectrophotometer readings might not have been
exact
 Health of Algae?


Extensions





Test other types of algae (Chlamydomonas
Reinhardtii)
Test organic fertilizers vs. artificial fertilizers
Increase length of experiment
Test different concentrations
Use more replicates
REFERENCES
http://www.mosaicco.com/images/NSS__17_Diam
monium_Phosp.pdf
 http://www.ammoniumnitrate.org/
 http://www.fcps.edu/islandcreekes/ecology/euglen
a.htm
 http://toxics.usgs.gov/definitions/eutrophication.h
tml
 http://www.sciencedaily.com/articles/a/algal_bloo
m.htm

2-FACTOR ANOVA
ANOVA
ource of VariationSS
Sample
0.080417
Columns 0.137733
Interaction 0.055739
Within
0.068748
Total
0.342637
df
2
2
4
36
44
MS
F
P-value
F crit
0.040208 21.05535 8.8E-07 3.259446
0.068866 36.06221 2.53E-09 3.259446
0.013935 7.297058 0.000207 2.633532
0.00191
SINGLE FACTOR ANOVA
Anova: Single Factor
SUMMARY
Groups
Count
Column 1
5
Column 2
5
Column 3
5
ANOVA
Source of VariationSS
Between Groups
0.084445
Within Groups
0.024706
Total
0.109152
Sum
Average Variance
1.511
0.3022 0.001952
1.601
0.3202 0.004085
0.764
0.1528 0.00014
df
MS
F
P-value
F crit
2 0.042223 20.50769 0.000134 3.885294
12 0.002059
14