Schmitz Orlando 1

Introduction
Algae normally shows that an ecosystem is healthy because it shows an abundance of
sunlight and nutrients, but sometimes algae can be invasive and destructive. Bryopsis is a marine
alga. It is normally seen as a nuisance in environments, whether it is a pond or a fish tank.
Bryopsis competes for resources such as space and sunlight. Using environmental factors, the
growth of Bryopsis may be able to be reduced, increased, or prevented in a controlled
environment. For example, If the salinity in the water is too high, then number of cells in the
algae can be reduced (Latala). This factor will be explained below.
This research was conducted to determine how to reduce the growth of Bryopsis using a
two factor DOE. These factors were the salinity and pH of the water that the Bryopsis grew in.
These factors were chosen because they are evident in all marine ecosystems and have a major
impact on the organisms that live in them. The experiment was completed in a three-week period
in controlled conditions. Though nothing was recognized as significant, the experiment was still
a success, and that will be explained in the conclusion section of the paper.
This experiment was important because it helps keep small ecosystems healthy and
controlled. For people who own fish, unwanted algae could be problem. The variables used in
the experiment can be changed to reduce or prevent the growth of unwanted algae.
The first factor that was taken into consideration in the experiment was the salinity of the
water that the algae was grown in. Salinity is the level of salt in a given amount of water,
normally measured in parts per thousand. The salt in the water can dehydrate the plant, because it
is hydrophilic. It absorbs water out of the cells. This is important to consider when setting up a
fish tank or any underwater ecosystem.

Schmitz Orlando 2
The second factor that was taken into consideration in the experiment was the pH of the
water that the algae was grown in. The pH of anything is a measure of the hydrogen ion content.
Lower levels of pH are more acidic, and higher levels of pH are more basic. Most algae live in a
pH of 0 to 7 (Hargreaves, Whitton). Setting the pH too low could kill the algae, because the
environment could be too acidic for certain types. Certain nutrients that are basic are lost when
the water becomes too acidic, such as calcium, potassium, and magnesium. These nutrients are
important to some plants.
The experiment was a two factor design of experiment. Three trials were completed over
a three-week period. Each week, five samples of Bryopsis were placed in cups filled with water.
Each cup represented a different condition, with low salinity and low pH, low salinity and high
pH, standard, high salinity and low pH, and high salinity and high pH. This will be changed and
the change in mass of the Bryopsis will be measured. The change in mass of the Bryopsis will be
used to determine whether either of the factors in experiment were statistically significant. The
entire process will be described in the experimental design section of this paper.

Schmitz Orlando 3
Problem Statement
Problem:
To determine the effect of salinity and pH on the growth of Bryopsis in a marine
ecosystem.

Hypothesis:
If the salinity is set to 25 parts per thousand and pH is set to 7 in a controlled
environment, then the growth of Bryopsis will be reduced.

Data Measured:
The levels of salinity and pH in a controlled environment will be the two factors used in
the experiment. The change in mass of samples of Bryopsis will be the data collected and
measured. The data will be analyzed by calculating the effect values of both factors, and the
interaction effect between the two factors. A prediction equation will also be calculated. The
three levels of salinity will be 25 ppt, 35 ppt, and 45 ppt. 35 ppt was chosen because it is the
normal ocean salinity level. The values above and below the 35 ppt were chosen because they
represent varying environments with less stable conditions. The three levels of pH were chosen
are 7, 8 and 9. 8 was chosen because it is the standard pH of ocean environments. The levels
above and below 8 were chosen because they represent varying levels in unstable environments.

Schmitz Orlando 4
Experimental Design
Materials:
1000 mL glass beaker
15 samples of Bryopsis
250 mL glass beaker
15-250 mL clear plastic cups
3-2 L bottles
ti nspire calculator
smartphone timer
citric acid
baking soda
Sartorius analytic scale (g)
distilled water
sea salt
tweezers
paper towel
API pH test strips
stirring magnet
Procedure:
1. To determine amount of salt needed, tare weight of 150 mL glass beaker. Add salt to beaker and
examine weight. If above the needed amount, remove salt until level. If below, add until level.
Repeat this until a hundredth of a gram.
2. To stir sample, place stirring magnet in 1000 mL glass beaker, add 1000 mL of water to
beaker, then add salt value for 25 ppt. Stir for 5 minutes.
3. To stir sample, place stirring magnet in 1000 mL glass beaker, add 1000 mL of water to
beaker, then add salt value for 35 ppt. Stir for 5 minutes.
4. To stir sample, place stirring magnet in 1000 mL glass beaker, add 1000 mL of water to
beaker, then add salt value for 35 ppt. Stir for 5 minutes.
5. Following the proper ratio of salt to water (3.8 grams per 150 mL), take one of three 2 L
bottle, fill to 1000 mL with distilled water stirred from beaker with 25 ppt salinity. Test
water with hydrometer to ensure 25 ppt salinity. If water is not exactly 25 ppt salinity,
start over until 25 ppt salinity is reached. Label 25 ppt.
6. Following the proper ratio of salt to water (5.8 grams per 150 mL), take two of three 2 L
bottle, fill to top of bottle with water stirred from beaker with 35 ppt salinity. Test water
with hydrometer to ensure 35 ppt salinity. If water is not exactly 35 ppt salinity, start over
until 35 ppt salinity is reached. Label 35 ppt.

Schmitz Orlando 5

7. Following the proper ratio of salt to water (7.5 grams per 150 mL), take three of three 2 L
bottle, fill to 1000 mL with water stirred from beaker with 45 ppt salinity. Test water with
hydrometer to ensure 45 ppt salinity. If water is not exactly 45 ppt salinity, start over until
45 ppt salinity is reached. Label 45 ppt.
8. For trial 1, take 5 uniform, clear plastic cups that can hold 250 mL water from materials.
9. Take two of the plastic cups, unused, fill with 25 ppt salinity water from 2 L bottle to
exactly 250 mL water. Label one (-,+), label the other (-,-).
10. Take one of the plastic cups, unused, fill with 35 ppt salinity water from 2 L bottle to
exactly 250 mL water. Label it (S).
11. Take two of the plastic cups, unused, fill with 45 ppt salinity water from 2 L bottle to
exactly 250 mL water. Label one (+,+), label the other (+,-).
12. The second factor of the experiment is the pH balance of the water. So, (-) pH is 7. (S) ph
is 8. (+) pH is 9.
13. Take cup (-,-), test pH with testing strips labelled above. if pH reads above 7, then add
citric acid until pH reads 7. If pH reads below 7, then add baking soda until pH reads 7.
For each solution, stir for 30 seconds, then check again.
14. Take cup (+,-) test pH with testing strips labelled above. if pH reads above 7, then add
citric acid until pH reads 7. If pH reads below 7, then add baking soda until pH reads
7. For each solution, stir for 30 seconds, then check again.
15. Take cup (S), test pH with testing strips labelled above. if pH reads above 8, then add
citric acid until pH reads 8. If pH reads below 8, then add baking soda until pH reads 8.
For each solution, stir for 30 seconds, then check again.
16. Take cup (+,+) test pH with testing strips labelled above. if pH reads above 9, then add
citric acid until pH reads 7. If pH reads below 9, then add baking soda until pH reads 9.
For each solution, stir for 30 seconds, then check again.
17. Take cup (-,+) test pH with testing strips labelled above. if pH reads above 9, then add
citric acid until pH reads 7. If pH reads below 9, then add baking soda until pH reads 9.
For each solution, stir for 30 seconds, then check again.
18. Label 5 spots in the area you are growing A-E. These should be on a table. Make sure
they get the same sunlight per day, in a greenhouse environment. Keep temperatures and
humidity consistent to ensure accurate results.
19. Label cup (-,+) 1, cup (-,-) 2, cup (S) 3, cup (+,+) 4, and cup (+,-) 5. These numbers are
used for randomization.

Schmitz Orlando 6
20. Use ti nspire to generate a random number between 1-5 using the randint(1,5) function.
Generate until all 5 numbers are chosen. In the correct order, the first number chosen is
tested on spot 1, second on spot 2, third on spot 3, fourth on spot 4, and fifth on spot 5.
21. Measure the weight of each sample of Bryopsis, write down on paper to hundredth of a
gram. Label each sample weight as start weight to their designated cup, (1, 2, 3, 4, 5),
and carefully place in center of cup. The current weight will be subtracted from final
weight to determine growth in that environment.
22. Leave samples in greenhouse environment overnight.
23. Day two, inspect samples for any unwanted influences, such as objects in the water, etc.
With the proper environment, these shouldnt be an issue. These will be factored as noise,
which will decrease the accuracy of the experiment.
24. Day three, inspect samples again for any unwanted influences.
25. Test pH of cup 1 with testing strips labelled above.
26. If pH of cup 1 is above 9, use citric acid to lower until pH reads 9. For each solution, stir
for 30 seconds, then check again.
27. If pH of cup 1 is below 9, use baking soda to raise until pH reads 9. For each solution, stir
for 30 seconds, then check again.
28. Test pH of cup 2 with testing strips labelled above.
29. If pH of cup 2 is above 7, use citric acid to lower until pH reads 7. For each solution, stir
for 30 seconds, then check again.
30. If pH of cup 2 is below 7, use baking soda to raise until pH reads 7. For each solution, stir
for 30 seconds, then check again.
31. Test pH of cup 3 with testing strips labelled above.
32. If pH of cup 3 is above 8, use citric acid to lower until pH reads 8. For each solution, stir
for 30 seconds, then check again.
33. If pH of cup 2 is below 8, use baking soda to raise until pH reads 8. For each solution, stir
for 30 seconds, then check again.
34. Test pH of cup 4 with testing strips labelled above.
35. If pH of cup 4 is above 9, use citric acid to lower until pH reads 9. For each solution, stir
for 30 seconds, then check again.

Schmitz Orlando 7
36. If pH of cup 4 is below 9, use baking soda to raise until pH reads 9. For each solution, stir
for 30 seconds, then check again.
37. Test pH of cup 5 with testing strips labelled above.
38. If pH of cup 5 is above 7, use citric acid to lower until pH reads 7. For each solution, stir
for 30 seconds, then check again.
39. If pH of cup 5 is below 7, use baking soda to raise until pH reads 7. For each solution, stir
for 30 seconds, then check again.
40. Day four, inspect samples for any unwanted influences.
41. Repeat steps 24-40 on day 5, Friday.
42. Remove each Bryopsis sample carefully with tweezers, place on one strip of paper towel
next to each cup.
43. Tare weight of a cylindrical beaker on scale.
44. Measure the weight of each sample of Bryopsis by putting in beaker and on scale, write
down as changed weight to their designated cup, (1, 2, 3, 4, 5).
45. Subtract start weight from changed weight to get each difference in mass. These
numbers will be used in the math portion of this experiment.
46. Repeat steps 1-45 three times, to come up with three sets of five changes in mass of
Bryopsis. These are the pieces of data used to determine the effect of each variable.

Schmitz Orlando 8
Diagram:

Figure 1. Experimental Setup

Above is the experiment setup complete with the cups, random spots, and samples of
algae, and paper towel. It is located in the greenhouse.

Schmitz Orlando 9

Data and Observations

Data:
Table 1
Design of Experiment Values
Salinity (ppt)
25

Standard
35

+
45

PH
Standar
d
8

+
9

This table shows our standards for each independent variable. These numbers were
determined because research showed the numbers that would give us the best results. To prepare
these tests baking soda and citric acid were used to raise and lower the pH. Sea salt was used to
change the salinity.

Table 2
Response Variable Measures
Mass of Bryopsis (g)
(Salinity, PH)
DOE
(+,+)
(+,-)
(-,+)
(-,-)
1

0.363
5

0.223
2

0.2851

0.100
9

Standar
d
0.6070

Schmitz Orlando 10

2
3
Averag
e

0.954
6
0.634
9
0.651
0

1.031
0
0.578
8
0.611
0

0.9756
0.3022
0.5209

0.540
3
0.284
6
0.308
6

0.7663
0.3061
0.5598

Table 2 shows the data from each DOE in the sequence they were completed.

Observations:
Table 3
Observations
Date

Observation

3/9/16

Particles in high pH water, foggy

3/11/16

Plants are a yellowish tint, opposed to the dark green original tint

3/14/16

PH was difficult to set to 9, most likely off by as much as a half

3/16/16

Plants are light green, water is musty

3/18/16

Time running out, pH was set low for long weekend

3/21/16

Each set of pH was very high, in the nines

3/22/16

Data collection finished

Schmitz Orlando 11

Figure 2. Cup Samples

This figure shows an example of the five cups used in a test. Image taken on Wednesday,
March Sixteenth. The Bryopsis is a medium green tint. When the sample was placed, it was a
dark green tint. When the sample was scaled, it was a yellowish tint. This was most likely due to
the lack of nutrients for the plant. Weight of each Bryopsis was different before each test. This
may have affected the growth rate. Each cup has paper towel over it to ensure that there will be
nothing going into the cup.

Schmitz Orlando 12
Data Analysis and Interpretation
Table 4
Table of Factors
Salinity (ppt)
Standard
25
35

+
45

pH
Standard

+
9

Table 4 above shows the factors of the varying salinity and pH levels used throughout the
tests. For salinity, the low value was 25 parts per thousand, the standard value was 35 parts per
thousand, and the high value was 45 parts per thousand. For pH, the low value was a pH of 7, the
standard value was a pH of 8, and the high value was a pH of 9. It is estimated that the standard,
standard trails will yield the highest change in mass, as it is most like the natural conditions that
Bryopsis thrives in.

Table 5
Table of Averages
Runs
Salinity

pH

+ (45)

+ (9)

- (25)

- (7)

+ (45)

- (7)

- (25)
+ (9)
Grand Average

Firs
t
DOE
0.36
35
0.10
09
0.22
32
0.28
51

Seco
nd
DOE
0.954
6
0.540
3
1.031
0.975
6

Third
DOE

Aver
age

0.634
9
0.651
0.284 0.308
6
6
0.578
8
0.611
0.302 0.520
2
99
0.5228975

This table shows the data in

the three different runs that were
tested, the averages of all of the
trials, and the grand average. The
grand average is 0.5228975 grams.

This is used to predict what would happen if values were interpolated or extrapolated.

Table 6

Schmitz Orlando 13

1
0.8
0.63
0.6

Mass (g) 0.41

0.4
0.2
0

-1

Salinity

Effect of Salinity
Salinity (ppt)
(-) 25
(+) 45
0.3086
0.651
0.52099
0.611
0.414795
0.631

Figure 3. Graph of Salinity

This figure shows the effect value of salinity on the mass of the Bryopsis. The data shows
that as salinity rises, the change in the mass of Bryopsis rises. The effect of salinity is 0.216205
grams. This is done by adding the averages where the two of the lesser variables are averaged
and the two of the greater variables are averaged, and the low variable average is subtracted from
the high variable average. On average, as salinity increases, the mass of Bryopsis increases by
0.216205 grams. According to the test of significance, this piece of data is not significant. This is
important because low-salinity waters could yield less Bryopsis growth, something to consider
when finding a way to control the algae growth.

Schmitz Orlando 14

Table 7
1
0.8
0.6
0.59

Mass (g) 0.46

0.4
0.2
0

-1

PH

Effect of pH
pH
(-) 7
0.308
6
0.611
0.459
8

(+) 9
0.651
0.52099
0.58599
5

Figure 4. Graph of pH
Figure 4 and Table 7 show the effect pH had on the mass of the Bryopsis. Similar to the
effect before, the data shows that as pH rises, the change in mass of Bryopsis rises. The effect of
pH is 0.126195 grams. This is calculated the same way as table 3. On average, as pH increases,
the mass of Bryopsis increases by 0.126195 grams. According to the test of significance, this

Schmitz Orlando 15
piece of data is not significant. This is important because lowering the pH in water could also
lower the change in mass of Bryopsis, which is also something to consider.

Table 8
1
0.8
0.65
0.61

0.52

0.6

Mass (g)
0.31

0.4
High pH
0.2

Low pH

-1

Salinity

Interaction Effect

p
H

Solid
Segment
Dotted
Segment

(+) 9
(-) 7

Salinity
(+)
(-) 25
45
0.520 0.6
99
51
0.308 0.6
6
11

Figure 5. Interaction Effect Graph

Figure 5 and Table 8 are a visual representation of the interaction effect that salinity and
pH had on the algae. The dotted segment represents the pH held low. The solid segment
represents the pH held high. The X axis represents salinity. The interaction effect of the two
effect values is -0.086195. This is calculated by dividing the change of Y over the change of X.
When both the salinity and pH was held low, the change in mass was 0.3086 grams. When the
pH was held low and the salinity was held high, the change in mass was 0.611 grams. When the
pH was held high and the salinity was held low, the change in mass was 0.52099 grams. When

Schmitz Orlando 16
the pH was held high and the salinity is held high, the change in mass was 0.651 grams.
According to the test of significance, this piece of data is not significant. The data suggests that
there was somewhat of an interaction between the two variables, because both slopes on the
graph are positive. When the salinity was held high, the change in mass was higher. This
suggests that salinity played the larger role of the two factors.

Schmitz Orlando 17

12
10
8
6
Mass (g) 4
2
0
0

3
2

5
4

7
6

9
8

11
10

Trials

Figure 6. Graph of Standards

This graph shows the change in weight of the standard tests in trial one, two, and three.
With only three sets of standards, it is more difficult to determine what was actually statistically
significant, and what does not matter. The experiment was restricted by the amount of time
available to conduct the experiment, the cost of materials for the experiment, and the availability
of samples of Bryopsis. With more standards, it may be determined that 0.3061 could be an
outlier, but without more standards to prove that something occurred in that sample, it will be
used as a standard. In the end out of each DOE, the standard was near to having the most growth
change.

I.E

pH

Salinity

-0.086195

0.126195

0.216205

-0.15

-0.1

-0.05

0.05

0.1

0.15

0.2

0.25

Figure 7. Dot Plot of Effects

This figure shows effect of salinity, 0.216205 grams, effect of pH, 0.126195 and the
interaction effect, -0.086195. This graph shows that salinity had the greatest impact on the

Schmitz Orlando 18
growth of Bryopsis, and the interaction effect was the least significant. This also suggests that
salinity is the leading factor in this experiment.

Fence1

I.E

pH

Salinity

Fence 2

-0.9204

-0.086195

0.126195

0.216205

0.9204

-1.5

-1

-0.5

0.5

1.5

Figure 8. Test of Significance

This figure shows if there are any pieces of data that lie outside of the range of standards
times two. The two areas that are equal to the range of standards times two are called the fences.
The range of standards was calculated by subtracting the lowest change in standards, 0.3061,
from the highest change in standards, 0.7663. This results in the number 0.4602. This number
times two is 0.9204. Data that lies outside of the fences are considered statistically significant
and did not happen by chance alone. The data above shows that the three values lie inside of the
fences. No effects clearly stand out, or are considered significant. Because of this, the
parsimonious prediction equation will only consist of the grand average plus noise.

Y = 0.5228975
Figure 9. Parsimonious Prediction Equation
The figure above shows the parsimonious prediction equation. The parsimonious
prediction equation is used to predict the outcome of any given variable using any statistically
significant data from the set. Because there were no effect values that were statistically
significant, the prediction equation is equal to the grand average. The grand average is the

Schmitz Orlando 19
change in mass that is expected if the pH and salinity are set inside the boundaries of the low and
high values that were chosen for the factors in this experiment. If pH was set to 7.5 and the
salinity was set to 40 ppt, the expected outcome would still be equal to the grand average, about
0.52 grams. This is because the range of the factors is too little for anything to be significant.

The data collected in this experiment is useful to the scientific community. Even though
none of the effect values that were determined in this paper are statistically significant, the
experiment overall was a success. The factors that were used are found where Bryopsis is grown,
and the experiment shows that it is not completely resistant to change. The Bryopsis seemed to
grow less when the pH and salinity of the water was reduced. The first effect value implies that
salinity had the greatest overall effect in the experiment. The effect value of salinity was
0.216205. The effect value of pH implies that pH had somewhat of an effect, but not as great as
salinity. The effect value of pH was 0.126195. The interaction effect value of the two variables
implies that the two variables have a moderate interaction, but nothing significant. The
interaction effect value is -0.086195. The graph of standards in this paper shows how close the
standards are to each other. Unfortunately, due to restraints, there were not enough standards
tested in this experiment to ensure proper data collection. One of the pieces of data can be
assumed to be an outlier, but with only three standards, it cannot be determined. This standard
was used in calculations in this paper, which has a chance to cause errors in this research. The
dot plot of effects shows the effect values relative to each other. This visually shows that salinity
had the greatest effect. The test of significance shows if there are any significant pieces of data.
With this data, there is nothing significant about this research. This could be at fault, because the
standard that is believed to be an outlier could have ruined the range of standards. The

Schmitz Orlando 20
parsimonious prediction equation in this paper is equal to the grand average, because there are no
significant pieces of data.

Schmitz Orlando 21

Conclusion
It was hypothesized that if the salinity is set to 25 parts per thousand and pH is set to 7 in
a controlled environment, then the growth of Bryopsis will be reduced. This hypothesis was not
accepted because there was not enough data to prove it.
Bryopsis is a problem in fish tanks, lakes, and oceans all around the world. This
experiment was designed to end those troubles and for people to keep their fish tanks healthy.
Salinity and pH were the two variables used. The purpose of salinity was to test if Bryopsis could
survive in different types of water. For example, lakes have a minimal amount of salt in the
water. Oceans and seas have much higher amounts of salt. To test this salt was weighed in a
cylinder. Then added to the testing water and stirred for 2 minutes. This changed the pH to a
certain level. Three different levels were used, 25, 35, and 45 parts per thousand. Next, pH was
used to test the acidic levels that Bryopsis could survive in. Changing the pH level can
dramatically affect marine ecosystems because of its critical role (Wootton,Pfister,Forester). This
was tested by adding citric acid to lower the pH and baking soda to raise it. The three levels used
were 7, 8, and 9. The pH level of seawaters is usually constant around 8.2 (Hansen). In the
experiment, Bryopsis grew in a cup set to a specific salinity and pH level. The standards had the
greatest growth change in trial one (0.6070), in trial two +,- had the greatest growth change
(1.0310), and in trial three +,+ had the greatest growth change (0.6349). None of these trials were
similar. But, it was observed that the standards and +s held the greatest growth change in the
three trials. Salinity had the greatest effect on the Bryopsis. pH had a smaller effect. This most
likely is true because the levels were set too close together.

Schmitz Orlando 22
There were some major flaws with the experiment. One flaw was that there were only
three standards. This made it difficult to find significant data. In addition to this, the salinity
levels were not set correctly. Third, the last test was held over a weekend which did not allow the
pH of each cup to be set back to its original level. This is important because the pH starts to
increase after about two days (Nyholm). Finally, the pH levels were set by looking at a shade of
the color on the strip compared to the shade on the box. This is not the most consistent way of
doing this. A pH pen could have been used to get more exact levels and to keep the tests as
consistent as possible. Finally, the algae that was used was not all the same size or weight. This
may have had a dramatic effect on the growth change.
Even with these issues, there was still important data collected. The most important thing
learned through this research is how to properly set up an experiment and conduct research. This
can be applied in the real world by saying do not use the pH or salinity levels in an actual fish
tank because it is hazardous to the other living organisms in the fish tank. Plants normally can
withstand more diverse levels of pH and salinity than other organisms can. In conclusion, this
experience provides the information to follow through with a more accurate experiment in the
future.

Schmitz Orlando 23
Works Cited
Hansen, P.J. Effect of High pH on the Growth and Survival of Marine Phytoplankton:
Implications for Species Succession. Vol. 28. n.p., 2002. 279-88. Marine Biological
Laboratory, University of Copenhagen, Strandpromenaden 5, 3000 Helsingr, Denmark.
Web. 3 Feb. 2016.
Hargreaves, J. W., and B. A. Whitton. Effect of pH on Growth of Acid Stream Algae n.p.,
1976. Web. 15 Mar. 2016.
Latala, Adam. Effects of Salinity, Temperature and Light on the Growth and Morphology of
Green Planktonie Algae. 1991. University of Gdask. n.p., Web. 12 May 2016.
Nyholm, Niels, and Torsten Kallqvist. Methods for Growth Inhibition Toxicity Tests with
Freshwater Algae. n.p.: Wiley Online Library, 1989. N. pag. Web. 5 Feb. 2016.
Wootton, Timothy J., Catheron A. Pfister, and James D. Forester. Dynamic Patterns and
Ecological Impacts of Declining Ocean pH in a High-Resolution Multi-Year Dataset.
Vol. 105. Chicago: PNAS, 2008. n. pag. 48 vols. Department of Ecology and Evolution,
University of Chicago, 1101 East 57th Street, Chicago, IL 60637. Web. 5 Feb. 2016.