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Running head: INDEPENDENT STUDY 1

Can Aegagropila linnaei (Marimo Balls) Filter Phosphates from Water: Implications for Water

Filtering

Sara E. Jastrzembski

Introduction to Ecology

April 3, 2018
INDEPENDENT STUDY 2

Introduction:

In light of the recent political concentrations on pollution, and announcements by the

Environmental Protection Agency (E.P.A.) for repeal of the clean power plan, it has become

crucial for biologists as a community to enhance efforts in clean power research. The burden of

supporting clean energy claims, as well as the efficacy and cost reduction, have landed on the

scientific community’s shoulders. It is no secret that factories, power plants, and construction

have polluted our Earth and it is easy to understand, you can see it with your own eyes, what

effects point pollution causes. Point pollution is when the source of contaminants to bodies of

water, air, and/or soil are directly known. More importantly, it is necessary to understand

nonpoint pollution, or the pollution of bodies of water, air, and/or soil that has an unseen cause.

Nonpoint pollution forms from fertilizers, insecticides, the manure of livestock, cities and

residential areas, and many more. One of the contaminants of nonpoint solution, is the buildup of

the natural nutrients that are usually found in healthy ecosystems, such as nitrogen and

phosphorus (Carpenter et al., 1998).

Phosphorus, the nutrient focused on in this experiment, is a necessary nutrient for

organisms but in substantial amounts, will begin to negatively affect the environment (Minnesota

Pollution Control Agency, 2008). Phosphorus in nature is a limiting nutrient, meaning the

amount of phosphorus available dictates the growth of organisms in that ecosystem. When

phosphorus leaches out of the soil near nonpoint pollution sources, it is called runoff. Runoff

carries the nutrients to ecosystems farther away, affecting plant and wild life, such as when

runoff ends up in lakes, rivers, and streams (Carpenter et al, 1998). When lakes, rivers, and

streams become packed with high amounts of phosphorus, plant life, such as algae, grow

abundantly and use up a majority of the oxygen and sunlight available to other organisms in the
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water (Krishnaswamy, Muthuchamy, Perumalsamy, 2011).The buildup of nutrients and

following repercussions cause what is known as eutrophication, or a relatively unlivable state in

reference to other organisms. Furthermore, when this algal bloom dies, the decomposers found in

water start to decompose the algae and use up copious amounts of oxygen, producing carbon

dioxide. This process causes what is known as a dead zone. As a result, fish, among many other

organisms that inhabit the ecosystem, are killed (Minnesota Pollution Control Agency, 2008).

Human beings are producing phosphates in amounts that exceed the input necessary,

causing a disruption in its natural cycling. This coupled with water purification processes that do

not efficiently remove phosphates, are causing increased amounts of phosphorus in our drinking

waters (Lenntech B. V., Part III of “Matter Cycles”: The phosphorus cycle). Negative effects on

human health can occur from consuming too much phosphorus, such as kidney damage and

osteoporosis (Lenntech B. V., Chemical properties of phosphorus - Health effects of phosphorus

- Environmental effects of phosphorus). The effects of eutrophication due to phosphate nutrient

enrichment are vast, especially regarding cost associations (Lenntech B. V., Phosphorus removal

from wastewater). The E.P.A. (2015), published that effects of nutrient pollution caused losses in

tourism, recreation, commercial fishing, and property values. The E.PA. (2015), also cited not

only these losses in economics, but cost associations for control of nutrient pollution, such as

drinking water treatments, mitigation treatments (e.g., aeration systems, alum treatment, barley

straw application, bio-manipulation, dredging, herbicide/copper sulfate treatment, hypo-limnetic

withdrawal), restoration costs, and costs to administer nutrient trading and offset programs.

In response to increasing environmental, health, and economic concerns, biologists began

research on more efficient and naturally occurring treatments. One such study was published by

Azarpira, Behdarvand, Dhumal, and Pondhe (2014), in which blue green algae, specifically
INDEPENDENT STUDY 4

Oscillatoria limosa and Nostoc commune, were used in what is known as phycoreremediation.

Phycoreremediation is the low cost process of reducing the amount of pollutants in water using

the natural occurrence of filtration by algae. This study yielded decreases, between 95 to 98%, in

many parameters of wastewater causing an increase in its quality (Azarpira et al., 2014). Another

research experiment by Khuantrairong and Traichaiyaporn (2012), concerned the genus

Cladophora, or green algae. It is concluded along with their main focus (on enhancement of

carotenoids and chlorophylls) that, when algae grows in wastewater composed of additive

phosphates, water quality is improved by decreasing the amount of toxicity in the water

(Khuantrairong & Traichaiyaporn, 2012). Although, algae blooms can be harmful to ecosystems

as discussed, it can also be cultivated and used as a resource instead of a hindrance. Algae

cultivation can provide many benefits, such as toxin/pollution reduction, biofuel production, and

low costs of implementation (Dalgleish, 2017).

In this experiment, we set out to investigate the claim that Aegagropila linnaei can filter

phosphates from water. A. linnaei are organisms of the genus Cladophora (Van den Hoek, 1963).

This algae is a popular method of aquarium filtration advertised in pet stores, who claim that

among other pollutants, A. linnaei can filter out phosphates (Rob & Anthony, 2017).

Implications for filtration of wastewater exists in the close relationship to A. linnaei of algae that

has been researched and supported. Of substantial interest, it has been documented that A. linnaei

commonly grow in lakes that have gone through substantial contamination, or even

eutrophication (Togashi, Saski, & Yoshimura, 2014). This algae is becoming rare in the wild, but

the algae sold is primarily manufactured by humans. The popularity, resilience to eutrophic

water, low cost, and easy manufacturing qualities of A. linnaei as a potential component of future

phycoreremediation treatments. Our general hypothesis states, A. linnaei in phosphate


INDEPENDENT STUDY 5

contaminated freshwater, will cause a greater decrease in phosphate concentration when

compared to contaminated freshwater treatments with no A. linnaei present. The null statistical

hypothesis is, there is no significant difference between the mean final concentrations of

phosphorus. Subsequently, the alternate statistical hypothesis states, there is a significant

difference between the mean final concentrations of phosphorus.

Materials & Methods:

Independent Variable –

The independent variable in this experiment was the presence/absence of A. linnaei. A.

linnaei were obtained through purchase on Amazon.

Dependent Variable –

Phosphorus concentration in water was the dependent variable measured. Phosphates

were obtained by purchase at a gardening store as fertilizer, in the form of triple phosphate.

There was no nitrate or potassium in the phosphate fertilizer to ensure no other nutrient was

affecting A. linnaei. Water was obtained in our laboratory from the distilled water jugs to ensure

the water was uniform in all treatments.

Treatment Levels –

Three treatment levels were evaluated, which consisted of:

 The lowest treatment level – 0.82 g/L of phosphorus.

 The middle treatment level – 1.09 g/L of phosphorus.

 The highest treatment level – 1.73 g/L of phosphorus.

Control Treatment –
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In this experiment, 1 control treatment was used per experimental treatment level. The

controls consisted of three treatment levels of phosphorus concentration without A. linnaei

present: lowest treatment level concentration (0.82 g/L), the middle treatment level concentration

(1.09 g/L), and the highest treatment level concentration (1.73 g/L).

Experimental Treatment –

The experimental treatment consisted of 5 replications per experimental treatment level,

to increase precision and accuracy in our results. The experimental replications corresponded to

the same three levels of phosphorus concentrations as the controls but with the presence of A.

linnaei (15 A. linnaei overall): the lowest treatment level concentration (0.82 g/L) + 1 A. linnaei,

the middle treatment level concentration (1.09 g/L) + 1 A. linnaei, and the highest treatment level

concentration (1.73 g/L) + 1 A. linnaei.

Experimental Unit –

The experimental units for our experiment consisted of 18 – 16 ounce clear plastic cups,

1 cup was used per control treatment and 5 per experimental treatment level (5 replicates per

each of 3 treatment levels). The experimental unit either had no A. linnaei (control treatments) or

1 A. linnaei (experimental treatments) present.

Samples –

Our samples were, indirectly, the uptake of phosphorus by A. linnaei. Directly, the samples

measured were the concentrations of phosphates in the water of each cup. First, samples were

prepared in 3 – 1 G glass jugs, using 2.40 L of distilled water (provided 300 mL/cup and an extra

600 mL incase the mixtures spilled during distribution to cups). Then, phosphorus was grinded

into powder and mass of phosphorus necessary was calculated: lowest concentration used .002 g,

middle concentration used .003 g, and the highest concentration level used .004 g. A. linnaei
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were prepared before use, to make sure they were still living, following instructions provided by

the seller. Each ball like organism was rinsed with distilled water and squeezed slightly a few

times. They were then put into cups of distilled water and placed under a grow light, we waited

approximately 15 minutes, and as the instructions said, the A. linnaei started to fizz (produce

bubbles). If the organism had started to fizz, they were assumed to be alive and were used in this

experiment. If the organism had not started to fizz, they were assumed to be dead and were

discarded.

The sample measurements were observed and recorded by using a colorimeter to read

absorbance of light through our fluid medium, using a calibration chart (provided below) to

interpret the absorbance in terms of phosphorus concentration. The colorimeter used was made

by LaMotte, model PAL – code 3121-01.

Using the Colorimeter –

The LaMotte model colorimeter used was specifically made for measuring low-range

phosphate concentrations from 0 to 3.0 ppm. The procedures outlined in LaMotte’s instructions

for this model are as followed:

1. Rinse and clean colorimeter tube (0967) with sample water. Fill to the 10mL line with

sample.

2. Select setting 6 on “Select Wavelength” knob and press “30 Second Read” button.

3. Insert tube into colorimeter chamber and adjust to 100%T with “Set Blank” knob.

This is the 100%T blank.

4. Remove tube from colorimeter. Use 1.0 mL pipet (0354) to add 1.0 mL of

*Phosphate Acid Reagent (V-6283).


INDEPENDENT STUDY 8

5. Use the 0.1 g spoon (0699) to add one measure of *Phosphate Reducing Agent (V-

6283). Cap and shake until powder dissolves. Wait 5 minutes for full color

development. Solution will turn blue if phosphates are present.

6. At end of 5 minute waiting period, press the “30 Second Read” button and insert tube

into colorimeter chamber. Record %T as soon as reading stabilizes.

7. Consult calibration chart to determine the phosphate concentration in parts per

million (ppm).

Confounding Variables –

Saran wrap was used to cover each experimental unit and holes were poked into it for

aeration. The saran wrap helped to control for variables that could have influenced our results

such as debris contamination and/or spillage of water. The pH level of the water and other

various nutrients commonly in water were controlled for by using distilled water instead of tab or

bottled water, which may have additive nutrients. Temperature and availability of sunlight for

photosynthesis were controlled for by keeping each unit stored in the same place, directly under

a grow light, this ensured that all units were not only the same temperature as one another

throughout the experiment but also ensured that each unit had equal availability to sunlight.
INDEPENDENT STUDY 9

The balance used to measure our phosphate powder to mix into concentration levels was

not as accurate as we would have hoped. The most precise balance in our resources was accurate

to +/- 0.001 g. This confounding variable is a substantial source of error for our experiment. One

other substantial confounding variable was observed, some of the A. linnaei could have died

throughout the experiment. The A. linnaei had broken up in a few replicates, although the algae

can survive when their spherical form is separated by trauma, there was no trauma present. This

leads us to believe that some of the replicate’s A. linnaei may have experienced mortality

between the start and end of the experiment 4 weeks later.

Statistical Analysis –

Three separate T-tests were conducted using the final concentration for the control

treatment against each experimental data point of its respective treatment level (see Table 2, 3,

and 4 of the Results section).


INDEPENDENT STUDY 10

Results:

Table 1: Data at each Concentration (g/L)

Initial
0.82 1.09 1.73
Concentrations
Final
Concentrations: Lowest Middle Highest
Replicate 1 0.36 0.92 1.79
Replicate 2 0.44 0.75 1.37
Replicate 3 0.24 0.82 1.37
Replicate 4 0.55 0.88 1.32
Replicate 5 0.47 0.98 1.32

Average
0.412 0.87 1.434
Standard Deviation
0.117771 0.0888819 0.2005742
Control Final
0.82 0.79 1.5
Concentrations
Table 1:
Shows the initial values, final raw data, average values, and standard deviation for the concentration of
phosphorous in each treatment level. The lowest treatment level was 0.82 g/L, the middle was 1.09 g/L, and the
highest was 1.73 g/L. The lowest and highest treatment levels both have average final concentration lower than the
respective final control concentration; however, the middle concentration’s final value was higher than that of its
control. It is important to point out that the final concentration of replicate 1 for the highest concentration had a
higher value than the initial concentration.
INDEPENDENT STUDY 11

Figure 1:
Shows the initial concentration of phosphorus, treatment level final concentrations for replicates 1-5, and control
treatment level final concentration values. This line chart is a visual representation showing how much each level
compared between replicates and to their control regarding loss of phosphorus.
INDEPENDENT STUDY 12

Figure 2:
Shows a visual representation of the average final concentrations, found in Table 1, for each level’s experimental
and control treatments. Standard deviation bars are represented by black vertical lines above and below the
specified datum point. It is important to point out that the highest level’s control standard deviation almost overlaps
with its respective experimental treatment level. It is also important to mention that the standard deviation bars here
represent the standard deviation from the mean of the experimental replicate values and were applied to the control
treatment’s bar value for a visual reference only, not to be used in analyzing or interpreting this data.

Table 2: T-Test - Paired Two Sample for Means (Lowest Concentration)


Column1 Variable 1 Variable 2
Mean 0.82 0.412
Variance 0 0.01387
Observations 5 5
Pearson Correlation #DIV/0!
Hypothesized Mean Difference 0
df 4
t Stat 7.746525141
P(T<=t) one-tail 0.000748036
t Critical one-tail 2.131846786
P(T<=t) two-tail 0.001496072
t Critical two-tail 2.776445105
Table 2:
Shows a statistical analysis of the data from Table 1, the initial and final concentrations phosphorous, for the lowest
treatment level (0.82 g/L), in cups with (replicates 1-5) and without (control) Aegagropila linnaei. A T-test was
conducted on excel to compare the control treatment’s concentrations to their respective experimental treatment
levels. Pearson’s correlation coefficient (r) resulted in an error, “#DIV/0!”, because the control final value was
used in comparison to each replicate’s final value (control value was used as data value points 1-5 for variable 1),
which yields a 0 value for standard deviation. Standard alpha value was used, significance level 0.05, in comparison
to our P-value, one tail, of 0.0000748036. Our P-value for the lowest treatment concentration was less than our
alpha, leading to the conclusion to reject the null hypothesis.

Table 3: T-Test - Paired Two Sample for Means (Middle Concentration)


Column1 Variable 1 Variable 2
Mean 0.79 0.87
Variance 0 0.0079
Observations 5 5
Pearson Correlation #DIV/0!
Hypothesized Mean Difference 0
df 4
t Stat -2.012618422
P(T<=t) one-tail 0.057228333
t Critical one-tail 2.131846786
P(T<=t) two-tail 0.114456666
t Critical two-tail 2.776445105
INDEPENDENT STUDY 13

Table 3:
Shows a statistical analysis of the data from Table 1, the initial and final concentrations phosphorous, for the
middle treatment level (1.09 g/L), in cups with (replicates 1-5) and without (control) Aegagropila linnaei. A T-test
was conducted on excel to compare the control treatment’s concentrations to their respective experimental
treatment levels. Pearson’s correlation coefficient (r) resulted in an error, “#DIV/0!”, because the control final
value was used in comparison to each replicate’s final value (control value was used as data value points 1-5 for
variable 1), which yields a 0 value for standard deviation. Standard alpha value was used, significance level 0.05, in
comparison to our P-value, one tail, of 0.057228333. Our P-value for the middle treatment concentration was
slightly greater than our alpha, leading to the conclusion that we fail to reject our null hypothesis.

Table 4: T-Test - Paired Two Sample for Means (Highest Concentration)


Column1 Variable 1 Variable 2
Mean 1.5 1.434
Variance 0 0.04023
Observations 5 5
Pearson Correlation #DIV/0!
Hypothesized Mean Difference 0
df 4
t Stat 0.735790068
P(T<=t) one-tail 0.251337097
t Critical one-tail 2.131846786
P(T<=t) two-tail 0.502674195
t Critical two-tail 2.776445105
Table 4:
Shows a statistical analysis of the data from Table 1, the initial and final concentrations phosphorous, for the
highest treatment level (1.73 g/L), in cups with (replicates 1-5) and without (control) Aegagropila linnaei. A T-test
was conducted on excel to compare the control treatment’s concentrations to their respective experimental
treatment levels. Pearson’s correlation coefficient (r) resulted in an error, “#DIV/0!”, because the control final
value was used in comparison to each replicate’s final value (control value was used as data value points 1-5 for
variable 1), which yields a 0 value for standard deviation. Standard alpha value was used, significance level 0.05, in
comparison to our P-value, one tail, of 0.251337097. Our P-value for the highest treatment concentration was
greater than our alpha, leading to the conclusion that we fail to reject our null hypothesis.

Discussion:

Our statistical analysis (Tables 2, 3, and 4) of our raw data (Table 1) consisted of a T-test.

The tests conducted used the respective treatment level’s final concentrations of phosphates for

the control treatment versus the experimental treatment’s final concentration of phosphates.
INDEPENDENT STUDY 14

Standard alpha value was used, significance level 0.05, in a comparison to our P-value, which

was one tail. The results of this statistical analysis are as follows:

The P-value for our lowest treatment level (0.82 g/L) was 0.0000748036, which is less

than our alpha, led to the conclusion to reject the null hypothesis; our lowest concentration had a

significant difference in the means of our data, µControl ≠ µExperimental, and that any difference is

most likely not due to chance.

The P-value of our middle treatment level (1.09 g/L) was 0.057228333. Our P-value for

the middle treatment concentration was slightly greater than our alpha, leading to the conclusion

that we fail to reject our null hypothesis; our middle concentration had no significant difference

in the means of our data, µControl = µExperimental, and that any difference is most likely due to

chance.

The P-value of our highest treatment level (1.73 g/L) was 0.251337097. Our P-value for

the highest treatment concentration was greater than our alpha, leading to the conclusion that we

fail to reject our null hypothesis; our highest concentration had no significant difference in the

means of our data any µControl = µExperimental, and that any difference is most likely due to chance.

The data for the lowest concentration supports our general hypothesis and in contrast, the

data for the middle and highest concentrations does not. This data is well explained through the

study by Boedeker and Immers (2009), which aimed to conclude the exact reason why the

Earth’s populations of A. linnaei are declining so rapidly. The study mentions that, though

previous works have supported the favorability of eutrophic water by A. linnaei, there are

conflicting views with little knowledge on nutrient level requirements of this species. In fact,

views are so conflicted that articles have been published that believe mat colonies (There are
INDEPENDENT STUDY 15

implications that the preference of eutrophic water may not be the case. The preferred habitat, as

claimed by Boedeker, Eggert, Immers, and Smets (2010), is likely to be oligo-mesotrophic, or

lakes with medium to low concentrations of nutrients, with the exception of calcium. Boedeker et

al. (2010) stated, A. linnaei thrive in water with high concentrations of calcium.

The confounding variable of conflicting views about preferred growing conditions is a

substantial source of error in our experiment. The eutrophic state of our water and the lack of

calcium (usage of distilled water to ensure no influence from other nutrients) may have interfered

with A. linnaei’s ability to survive and/or take up copious amounts of phosphates. Although, the

higher concentrated treatment levels did not support our general hypothesis, the lowest

concentration treatment level did. This explanation is further strengthened by the low

concentration level’s support of our general hypothesis. This would also explain the confounding

variable of A. linnaei found broken up in their cups after four weeks. There is a strong possibility

that too much phosphate with little to no calcium was unfavorable to the algae and in the higher

concentration levels A. linnaei died off. For further research, it should be suggested to

incorporating low, medium, and high concentrations of calcium as well. Other confounding

variables existed, mostly through lack of research on the exact characteristics of A. linnaei.
INDEPENDENT STUDY 16

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