Fertilizer and Acid Rain
Effects on Algal GrowthJoshua Egan
9th Grade, Pittsburgh Central Catholic High School, 1st
Year in P.J.A.S.
Algal Importance
Base of the aquatic food chain
Bio-indicators of most stimuli present in marine environments
Studies upon this model may apply to other forms of life and provide vital information for protecting life
Chlamydomonas reinhardtii
Species of green algae
Tenfold increase in cells per day
Used as bio-indicator
Found in stagnant water, damp
soil, freshwater, seawater, and
even in snow as “snow algae”
Contains two flagella and large,
crescent shaped chloroplasts
Euglena gracilis
Genus of single-celled flagellate
protists, members of the class
Euglenoidea
Performs mitosis/reproduces
quicker in warm surroundings
Contain flagella and chloroplasts
Found in nutrient-rich freshwater
and significantly affected by
acidity
Runoff
Surface water movement and how
it goes through the water cycle
over land
Water neither evaporates nor
penetrates the surface
Creates floods and “watersheds”
with excess precipitation
Fertilizers and other chemicals
may be transported through the
cycle and in turn pollute water
sources
EutrophicationCaused by an overabundance of nutrients
in an ecosystem
No limiting factor on algal populations
Uncontrollable growth takes up resources
necessary for other organisms’ oxygen
Limits biodiversity
Can occur naturally
Occurs today by fertilizer runoff
Miracle-Gro Active Ingredients include:
Urea, Ammonium Phosphate, Urea Phosphate,
Potassium Chloride, Boric Acid, Copper Sulfate, Iron
EDTA, Manganese EDTA, Sodium Molybdate, Zinc
Sulfate, and Nuisance Dust
Phosphates: important for cellular processes including
the synthesis of ATP and ADP
Nitrogen: found in ammonia contained in fertilizers,
which may be a part of surface runoff; causes
eutrophication
Only aids plant growth at intended
concentrations
Acid Precipitation
Worldwide environmental
problem
Results from pollution by sulfur
dioxide and nitrogen oxide
Originates from smokestacks,
vehicle exhaust, and burning
fossil fuels
General pH is 5.6 (acidic)
Reduces biodiversity
Sulfuric Acid as a Pollutant
Common form of acid rain
Popular product in chemical industry
Factories produce and release the
substance
Emitted into the atmosphere; contaminates
precipitation and harms life as a result
Sulfur oxides can combine with water in the
atmosphere to create acid rain
Affects organisms such as algae in marine
environments
Spectrophotometry
Quantitative study of electromagnetic spectra
Visible light, near-ultraviolet, and near-infrared
and the use of a spectrophotometer: can measure
intensity as a function of color or light absorption
Important features include the spectral
bandwidth and linear range
Commonly used in scientific fields such as
chemistry, biochemistry, and molecular biology
Purpose
To determine if miracle-gro
fertilizer and acid rain (sulfuric
acid) have a significant effect on
algal growth
Either individually or
synergistically
Hypotheses
Null Hypothesis- Neither miracle-gro
fertilizer nor the sulfuric acid (acid rain)
will have significant effects on the algal
survivorship alone or synergistically
Alternate Hypothesis- Both the miracle-
gro fertilizer and the sulfuric acid (acid
rain) will have a significant synergistic
effect on the algal survivorship
Materials Sulfuric acid (concentration 10-1 molar)
Miracle-Gro fertilizer (concentration 10x)
X= recommended concentration for soil application
Chlamydomonas reinhardtii and Euglena gracilis algae
73 tubes (one with water for blanking the
spectrophotometer)
Two test tube racks
Micro-pipettes and tips
Soil water
Spring water
Spectrophotometer
Wax paper
Two lamps for elevated light source
Procedure 1. Two racks with a total of 72 tubes were set up
on a table under two lamps providing equal light
for all of the algae
2. The following ingredients were inserted into
the tubes to create different experimental
variable concentrations of miracle-gro fertilizer
and sulfuric acid
Each combination was made up four times each for
both types of algae, creating 72 tubes
Tube Concentration Table
0 0.1x (L) 1x(H)
0 00 0L 0H
pH=7
10-5 (L) L0
pH=6.5
LL LH
10-3 (H) H0
pH=5
HL HH
pH= 7.5
Miracle-gro Fertilizer
Sulf
uri
c A
cid
Tube Concentration Table
Control:
0x/0
0x/10-5 0x/10-3 0.1x/0 0.1x/10-5 0.1x/10-3 1x/0 1x/10-5 1x/10-3
Algae 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL
Soil
water
1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL
Spring
water
3 mL 2.995
mL
2.95 mL 2.995
mL
2.945
mL
2.945
mL
2.95 mL 2.9 mL 2.9 mL
Fertilizer
(10x)0 mL 0 mL 0 mL 5 µL 5 µL 5 µL 50 µL 50 µL 50 µL
Sulfuric
acid (10-1
molar)
0 mL 5 µL 50 µL 0 mL 0.05 mL 50 µL 0 mL 0.05 mL 50 µL
Total
volume
5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL
Procedure (continued) 3. The tubes were kept in the two racks under
constant, equal elevated lamp lighting for 14 days
4. The top of each tube was covered with wax
paper and mixed by inversion before each reading
5. Every two days, the tubes were measured in
the spectrophotometer at 430 nm (wavelength)
for 14 days
6. After the experiment, the results were
analyzed with stats and graphs
0x/10-5
0x/10-3
0x/0
0.1x/0
0.1x/10-5
0.1x/10-3
1x/0
1x/10-5
1x/10-3
Fert-
0.571
SA-
0.001
Synergy-
0.002
Fert-
0.107
SA-
0.000002
Synergy-
0.043
Fert-
0.013
SA-
0.00005
Synergy-
0.001
0x/0
0x/10-5
0x/10-3
0.1x/0
0.1x/10-5
0.1x/10-3
1x/0
1x/10-5
1x/10-3
Fert-
0.082
SA-
0.008
Synergy-
0.973
Fert-
0.142
SA-
0.003
Synergy-
0.853
Fert-
0.804
SA-
0.000006
Synergy-
0.201
P-Value Table
Chlamydomonas reinhardtii Euglena gracilis
Day 2: Fertilizer- 0.571
Sulfuric acid- 0.001
Synergy- 0.002
Fertilizer- 0.082
Sulfuric acid- 0.008
Synergy- 0.973
Day 4: Fertilizer- 0.107
Sulfuric acid- 0.000002
Synergy- 0.043
Fertilizer- 0.142
Sulfuric acid- 0.003
Synergy- 0.853
Day 14: Fertilizer- 0.013
Sulfuric acid- 0.00005
Synergy- 0.001
Fertilizer- 0.804
Sulfuric acid- 0.000006
Synergy- 0.201
Statistical Analysis (ANOVAS) for
Chlamydomonas reinhardtii
Varying fertilizer concentration effects:
Not significant: p-values >0.05 accept null
hypothesis
Varying sulfuric acid effects:
Not significant: p-values >0.05 accept null
hypothesis
Synergistic/interactive effects:
Significant: p-values <0.05 reject null
hypothesis
Statistical Analysis (ANOVAS) for Euglena
gracilis
Varying fertilizer effects:
Not significant: p-values >0.05 accept null
hypothesis
Varying sulfuric acid effects:
Significant: p-values <0.05 reject null
hypothesis
Synergistic/interactive effects:
Not significant: p-values >0.05 accept null
hypothesis
Limitations
Lighting inefficiencies
Algal health?
Living/thriving cells during study?
Only two concentrations of sulfuric acid and fertilizer
Miracle-gro had buffering effect on sulfuric acid
Extensions
Test other chemical effects
More varying variable
concentrations
Use other living species
Do the study at a larger scale
Longer experiment length
Bibliography http://www.ruf.rice.edu/~bioslabs/studies/in
vertebrates/chlamydomonas.html
http://toxics.usgs.gov/definitions/eutrophication.html
https://www.google.com/search?q=encyclopedia&gws_rd=ssl
http://www.all-science-fair-projects.com/print_project_1235_122
http://homeguides.sfgate.com/fertilizers-pollutants-78452.html
Mark Krotec, PTEI
ANOVA Statistical test that allows for the comparison of
means of different groups, to determine significant variation
Two factor ANOVA will determine where the significant effect is: either through one of the variables or both in synergy with each other
Utilize p-values as measure of significance
Confidence interval of the likelihood of seeing the same affect produced by the same variable with replication
p>0.05: not significant
p<0.05: significant
Day 2 ANOVAs
Anova: Two-Factor With Replication
SUMMARY 0 10(-5) 10(-3) Total
0x
Count 4 4 4 12
Sum 0.481 0.566 0.554 1.601
Average 0.12025 0.1415 0.1385 0.133417
Variance 2.96E-05 7E-06 0.000239 0.000171
0.1x
Count 4 4 4 12
Sum 0.597 0.389 0.524 1.51
Average 0.14925 0.09725 0.131 0.125833
Variance 0.00039 4.23E-05 0.000441 0.000745
1x
Count 4 4 4 12
Sum 0.567 0.396 0.579 1.542
Average 0.14175 0.099 0.14475 0.1285
Variance 0.000183 4.73E-05 0.001417 0.000926
Total
Count 12 12 12
Sum 1.645 1.351 1.657
Average 0.137083 0.112583 0.138083
Variance 0.000329 0.000483 0.000606
ANOVA
Source of VariationSS df MS F P-value F crit
Sample 0.000355 2 0.000178 0.571501 0.57136 3.354131
Columns 0.005006 2 0.002503 8.055186 0.001805 3.354131
Interaction 0.00686 4 0.001715 5.519101 0.002219 2.727765
Within 0.00839 27 0.000311
Total 0.020611 35
Anova: Two-Factor With Replication
SUMMARY 0 10(-5) 10(-3) Total
0x
Count 4 4 4 12
Sum 0.676 0.81 0.72 2.206
Average 0.169 0.2025 0.18 0.183833
Variance 0.000417 4.83E-05 0.000931 0.000593
0.1x
Count 4 4 4 12
Sum 0.775 0.924 0.791 2.49
Average 0.19375 0.231 0.19775 0.2075
Variance 0.000243 0.001794 0.001448 0.001254
1x
Count 4 4 4 12
Sum 0.737 0.839 0.724 2.3
Average 0.18425 0.20975 0.181 0.191667
Variance 0.000198 0.000362 0.000278 0.000409
Total
Count 12 12 12
Sum 2.188 2.573 2.235
Average 0.182333 0.214417 0.18625
Variance 0.000348 0.000761 0.000797
ANOVA
Source of VariationSS df MS F P-value F crit
Sample 0.003489 2 0.001744 2.7449 0.082191 3.354131
Columns 0.007352 2 0.003676 5.784721 0.008112 3.354131
Interaction 0.000309 4 7.73E-05 0.121627 0.973531 2.727765
Within 0.017158 27 0.000635
Total 0.028308 35
Chlamydomonas
reinhardtiiEuglena gracilis
Day 4 ANOVAs
Anova: Two-Factor With Replication
SUMMARY 0 10(-5) 10(-3) Total
0x
Count 4 4 4 12
Sum 0.633 0.618 0.808 2.059
Average 0.15825 0.1545 0.202 0.171583
Variance 0.000146 0.000184 0.000233 0.000661
0.1x
Count 4 4 4 12
Sum 0.72 0.439 0.811 1.97
Average 0.18 0.10975 0.20275 0.164167
Variance 4.47E-05 0.000993 0.000595 0.002155
1x
Count 4 4 4 12
Sum 0.677 0.468 0.658 1.803
Average 0.16925 0.117 0.1645 0.15025
Variance 0.000679 0.000227 0.002103 0.001428
Total
Count 12 12 12
Sum 2.03 1.525 2.277
Average 0.169167 0.127083 0.18975
Variance 0.000323 0.000802 0.001147
ANOVA
Source of VariationSS df MS F P-value F crit
Sample 0.002815 2 0.001408 2.434485 0.106645 3.354131
Columns 0.024487 2 0.012244 21.17589 2.95E-06 3.354131
Interaction 0.006573 4 0.001643 2.841938 0.043533 2.727765
Within 0.015611 27 0.000578
Total 0.049486 35
Anova: Two-Factor With Replication
SUMMARY 0 10(-5) 10(-3) Total
0x
Count 4 4 4 12
Sum 0.806 0.956 0.854 2.616
Average 0.2015 0.239 0.2135 0.218
Variance 0.000942 6.13E-05 0.001353 0.000909
0.1x
Count 4 4 4 12
Sum 0.84 1.077 0.946 2.863
Average 0.21 0.26925 0.2365 0.238583
Variance 0.002065 0.001412 0.001852 0.002094
1x
Count 4 4 4 12
Sum 0.798 0.982 0.782 2.562
Average 0.1995 0.2455 0.1955 0.2135
Variance 0.00098 0.000228 0.000327 0.00098
Total
Count 12 12 12
Sum 2.444 3.015 2.582
Average 0.203667 0.25125 0.215167
Variance 0.00111 0.000648 0.00127
ANOVA
Source of VariationSS df MS F P-value F crit
Sample 0.004292 2 0.002146 2.09523 0.142599 3.354131
Columns 0.014794 2 0.007397 7.221212 0.003075 3.354131
Interaction 0.001363 4 0.000341 0.332767 0.853461 2.727765
Within 0.027657 27 0.001024
Total 0.048106 35
Chlamydomonas
reinhardtiiEuglena gracilis
Day 14 ANOVAs
Anova: Two-Factor With Replication
SUMMARY 0 10(-5) 10(-3) Total
0x
Count 4 4 4 12
Sum 0.769 0.828 1.027 2.624
Average 0.19225 0.207 0.25675 0.218667
Variance 0.000158 3.27E-05 0.004402 0.002083
0.1x
Count 4 4 4 12
Sum 1.344 0.579 1.488 3.411
Average 0.336 0.14475 0.372 0.28425
Variance 0.004909 0.000442 0.002259 0.012926
1x
Count 4 4 4 12
Sum 1.386 0.563 0.613 2.562
Average 0.3465 0.14075 0.15325 0.2135
Variance 0.005872 0.000399 0.014749 0.01541
Total
Count 12 12 12
Sum 3.499 1.97 3.128
Average 0.291583 0.164167 0.260667
Variance 0.008385 0.001242 0.014548
ANOVA
Source of VariationSS df MS F P-value F crit
Sample 0.037334 2 0.018667 5.056905 0.013636 3.354131
Columns 0.106012 2 0.053006 14.35953 5.65E-05 3.354131
Interaction 0.128925 4 0.032231 8.73152 0.000117 2.727765
Within 0.099667 27 0.003691
Total 0.371938 35
Anova: Two-Factor With Replication
SUMMARY 0 10(-5) 10(-3) Total
0x
Count 4 4 4 12
Sum 0.959 1.24 0.979 3.178
Average 0.23975 0.31 0.24475 0.264833
Variance 0.001014 0.000149 0.001005 0.001708
0.1x
Count 4 4 4 12
Sum 0.947 1.338 0.977 3.262
Average 0.23675 0.3345 0.24425 0.271833
Variance 0.002425 0.001486 0.001218 0.003551
1x
Count 4 4 4 12
Sum 1.082 1.176 0.911 3.169
Average 0.2705 0.294 0.22775 0.264083
Variance 0.000838 0.000624 0.000254 0.001288
Total
Count 12 12 12
Sum 2.988 3.754 2.867
Average 0.249 0.312833 0.238917
Variance 0.00142 0.000919 0.000743
ANOVA
Source of VariationSS df MS F P-value F crit
Sample 0.000439 2 0.000219 0.218976 0.804753 3.354131
Columns 0.03856 2 0.01928 19.25601 6.35E-06 3.354131
Interaction 0.00643 4 0.001608 1.605576 0.201609 2.727765
Within 0.027034 27 0.001001
Total 0.072463 35
Chlamydomonas
reinhardtii Euglena gracilis