Anthony Tirone
Pittsburgh Central Catholic
PJAS 2013
Grade 9



One of the largest and fastest growing
threats to global water resources.
Rainwater collects and carries chemicals
into lakes or streams, possibly causing
eutrophication or toxicity.
Eutrophication results in depletion of
oxygen in the water, killing off many
other species.



Used very often in production farming,
gardens, homes, and businesses.
May harm non-target species.
Pesticide runoff could potentially effect
algal growth due to toxins in it.

May possibly harm aquatic organisms.

Long-term effects are still unclear.

Spectracide®




Contains the active ingredients:
Gamma-Cyhalothrin & Lauryl sulfate.
Is advertised to kill more than 180 types of
insects.
Is used often for personal pest control.
Is advertised for use on plants which
suggests that it will not harm them.




Fertilizer runoff enters aquatic ecosystems
each day.
Contains high amounts of nitrogen which
may aid eutrophication.
From aiding in cell division to providing
essential nutrients, algae can use fertilizers
the same way many other plants do.
Miracle-Gro®




Contains the active ingredients:
Urea Phosphate, Ammonium Phosphate, &
Potassium Chloride.
Is advertised to aid all plant growth.
Only aids plant growth at intended
concentrations.
Is used often as a fertilizer in homes
and at businesses.

In a study entitled Effects of Nitrogen
Fertilizer and Pesticide Management on
Floodwater Ecology, it was observed that
transient green algal blooms were observed
after the broadcast application of Nitrogen
fertilizer 7 days after transplanting




Flagellated, unicellular, green, algae.
Commonly found in fresh water and
sometimes even in soil or in snow on
mountain tops.
Can grow on a simple medium of salts in
the light, using photosynthesis to provide
energy.
Common experimental cell model.





Extremely common algae.
Generally cylindrical in shape with many
flagella.
Can obtain food through endocytosis and
photosynthesis.
Live in nutrient-rich freshwater or in
sewage systems.
Frequently employed as an experimental
model.

To discern the effect(s) of pesticide
and fertilizer runoff on the
survivorship of Chlamydomonas
reinhardtii and Euglena gracilis.

Both individual and synergistic effects.
•
Null: The presence of fertilizer and pesticide will
NOT have a significant effect on algal growth.
• Alternative 1: The presence of the fertilizer or
pesticide WILL significantly alter algal growth.
• Alternative 2: There will be significant synergistic
effects of the variables resulting in a change in
algal growth.












Chlamydomonas reinhardtii
Euglena gracilis
Soil water (sterile)
Spring water
Miracle Grow® brand fertilizer
Spectracide® brand pesticide
Test tubes (13 x 100 mm borosilicate culture
tubes)
Pipettes (macro + micro)
Pipette tips
Spectrophotometer (Carolina Educator)
Test tube rack
Sunlight Source
1.
The following ingredients were added
to the culture tubes to create the
desired concentrations of variables:
Tube Tube
1
2
Tube
3
Tube
4
Tube
5
Tube
6
Tube
7
Tube
8
Tube
9
2.5
2
2
2
1.5
1.5
2
1.5
1.5
1
1
1
1
1
1
1
1
1
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
Fertilizer
(mL)
0
0.5
10%
0.5
100%
0
0.5
10%
0.5
100%
0
0.5
10%
0.5
100%
Pesticide
(mL)
0
0
0
0.5
10%
0.5
10%
0.5
10%
Total % [F]
0
.001
0.1
0
.001
0.1
0
.001
0.1
Total % [P]
0
0
0
.001
.001
.001
0.1
0.1
0.1
Spring
water
(mL)
Soil Water
(mL)
Algae
(mL)
0.5
0.5
0.5
100% 100% 100%
2.
The tubes were gently inverted and their
absorbance was measured in the
spectrophotometer (Wavelength 430 nm).
3.
Absorbance readings were taken every
other day for 11 days.
0.25
Absorbance
0.2
Control
0.15
0.001% P
Day 5
1.2E-14
0.1
0.1% P
Day 11
8.91E-10
0.001% F
0.05
0.1% F
0
Day 1
Day 3
Day 5
Day 7
Days Measured
Day 9
Day 11
0.25
Absorbance
0.2
Control
0.15
Low P-LowF
Day 5
8.09E-21
0.1
Day 11
2.69 E-06
Low F-Hi P
Low P-Hi F
Hi P-Hi F
0.05
0
Day 1
Day 3
Day 5
Day 7
Days Measured
Day 9
Day 11
0.24
Absorbance
0.2
0.16
Control
0.001% P
0.12
Day 5
3.77E-17
0.08
Day 11
0.0395
0.1% P
0.001% F
0.1% F
0.04
0
Day 1
Day 3
Day 5
Day 7
Days Measured
Day 9
Day 11
0.25
Absorbance
0.2
Control
0.15
Low P-Low F
Day 5
0.01995
0.1
Day 11
7.27E-05
Low F-Hi P
Low P-Hi F
Hi P-Hi-F
0.05
0
Day 1
Day 3
Day 5
Day 7
Days Measured
Day 9
Day 11
Chlamydomonas



Euglena
Yes/No
Does Pesticide Aid Algal Growth?
3.13E-06
7.3E-08
Yes
Does Fertilizer Aid Algal Growth?
2.16E-08
6.27E-09
Yes
Do Pesticide & Fertilizer Have a Synergistic
Effect on Algal Growth? (2-Factor ANOVA)
0.005716
7.63E-06
Yes



The ANOVA stat analysis suggests that
the null hypothesis should be REJECTED.
All Concentrations of Pesticide and
fertilizer SIGNIFICANTLY affected the
growth of both algae.
There was a significant effect of fertilizer
and pesticide in combination; the
synergy increased the growth of both
algae.


Limitations
◦ Chlamydomonas reinhardtii and Euglena
gracilis not fresh, 2 day travel time.
◦ Algal health and longevity might vary
◦ Optimal Light?
Extensions
◦ Use more diverse groups of algae
◦ Use other concentrations of variables
◦ Cell counts with hemacytometer
◦ Use different kinds fertilizer and pesticide






www.waterencyclopedia.com
www.scientificamerican.com
Modern Biology, Austin TX, Holt Rinehart and
Winston, 2000
http://ga.water.usgs.gov
http://www.ncbi.nlm.nih.gov/
Biology and Fertility of Soils February 1994,
Volume 17, Issue 2, pp 129-137
Ian C. Simpson, Pierre A. Roger, Roberto Oficial,
Ian F. Grant
ANOVA
Source of Variation
Sample
Columns
Interaction
Within
SS
0.00071
0.00109
0.00017
0.00014
Total
0.00220
ANOVA
Source of Variation
SS
df
2
2
4
36
MS
0.000396
0.000548
4.295566
3.988899
F
P-value
F crit
99.31476
2.22104E-15
3.2594463
137.3871
1.41069E-17
3.2594463
10.76880
7.63E-06
2.6335320
44
df
MS
F
P-value
F crit
Sample
5.15111E-05
2
2.57556E-05
2.148285449
0.0131407 3.259446
Columns
0.000557511
2
0.000278756
23.25115848
3.289E-07 3.259446
Interaction
Within
0.000208356
0.0004316
4
36
5.20889E-05
1.19889E-05
4.34476367
0.0057164 2.633532
Total
0.001248978
44

Single Variable Effects on Chlamydomonas
P-Value
1.2E-14
Significant

Synergistic Variable Effects on Chlamydomonas
P-Value
8.09E-21
Significant

Single Variable Effects on Euglena
P-Value
3.77E-17

Synergistic Variable Effects on Euglena
P-Value
0.019955
Significant
Significant




Single Variable Effects on Chlamydomonas
P-Value
8.91E-10
Significant
Synergistic Variable Effects on Chlamydomonas
P-Value
2.69 E-06
Significant
Single Variable Effects on Euglena
P-Value
0.039513
Significant
Synergistic Variable Effects on Euglena
P-Value
7.27E-05
Significant