The Effects of Exposure Time and Concentration of Zequanox on the Mortality of

Dreissena polymorpha

Emma Carr and Abigail Gonzales

Macomb Mathematics Science Technology Center

Advanced Biology

Mrs. Dewey / Mr. Estapa / Mrs. Gravel

09 December 2016
 The Effects of Exposure Time and Concentration of Zequanox on the Mortality of
Dreissena polymorpha

In this experiment, the exposure time and concentration of Zequanox was tested

in order to determine if they have an effect on the mortality of Dreissena polymorpha, or

zebra mussels. Zequanox is a product derived from a naturally occurring strain of the

bacteria Pseudomonas fluorescens, making it an environmentally compatible

molluscicide, or a pesticide against molluscs. Collected zebra mussels were treated with

different exposure times of 3 hours, 8 hours, and 24 hours and different concentrations of

50 mg, 100 mg, and 150 mg. This was done by separating groups of thirty zebra mussels

into fifteen separate containers and treating them accordingly. After designated

treatments, the percentage of dead specimens was measured for each group over a

one-week period. Once the data was collected and measured, a two-factor design of

experiment was conducted to measure the effects and the interaction between the factors.

It was hypothesized that the highest concentration and exposure time of Zequanox would

yield the highest mortality of zebra mussels. After completing the DOE, the hypothesis

was accepted. As both concentration and exposure time were held high, the percentage of

the specimens dead increased. This experiment is valuable to the scientific community

because zebra mussels are an invasive species that pose a threat to native aquatic life.

Also, they cause numerous issues for many people and businesses.
 Table of Contents
Introduction......................................................................................................................... 1

Review of Literature........................................................................................................... 3

Problem Statement.............................................................................................................. 7

Experimental Design........................................................................................................... 8

Data and Observations...................................................................................................... 11

Data Analysis and Interpretation...................................................................................... 15

Conclusion........................................................................ 22

Appendix A: Collecting Zebra Mussels and Lake Water............................................. 25

Appendix B: TI-Nspire CX Trial Randomization............................................................ 26

Appendix C: Prediction Equation Sample Calculation..................................................... 27

Appendix D: Parsimonious Prediction Equation Sample Calculation.............................. 28

Works Cited...................................................................................................................... 29
 Carr - Gonzales 1

Introduction

In 2001, the total cost of controlling the zebra mussel invasion over the next 10

years, including impacts on industry, recreation, and fisheries, was estimated at $3.1

billion (United States General). This means that $31 million was projected to be spent on

average every year just on this species alone. Zebra mussels are an extremely invasive

species that not only negatively impact their aquatic environment, but people as well.

Because they cling to hard surfaces and are able to rapidly reproduce, they tend to clog

pipelines which creates issues for many industries and power plants. They are also an

inconvenience to people who enjoy spending time on the lake as they are known to attach

to boats, motors, and fishing equipment (Zebra Mussel Control). Additionally, zebra

mussels threaten their surrounding environment by reducing the food supply for native

aquatic lifeforms. (Zebra Mussel Fact Sheet). Also, due to them being filter feeders,

they increase water clarity which promotes the growth and spread of deadly algae blooms

(Invasive Mussels).

As zebra mussels continue to cause problems, methods for their removal are in

high demand. Though manual removal has already begun, it is quite costly and mostly

ineffective due to their rapid reproduction. Zequanox, a product derived from a naturally

Pseudomonas fluorescens, is a proposed alternative to

occurring strain of the bacteria

eradicating the zebra mussel population. Though it has not been formally put into

practice, Zequanox may be a potential method of combating the invasive zebra mussel

population. Experimentation with the product up to this point had only focused on

determining if it had any negative effects on other species or the surrounding

 Carr - Gonzales 2

environment (ZEQUANOX). The researchers decided to further experimentation

with the product to help determine at what levels the product was most effective.

This experiment attempted to aid in developing an effective method of removing

zebra mussels from the environment. Zequanox was researched in order to determine

whether its concentration and exposure time would have an effect on the mortality of

zebra mussels. If the mortality of zebra mussels is affected by the concentration and

exposure time of Zequanox, a more effective method of eradicating them can be

developed. It could also potentially help set guidelines for the use of Zequanox.

Hopefully, after an effective method for removing zebra mussels is developed, zebra

mussels will be considered less threatening to both industry and the environment and

save many people a lot of money.

In the future, this research may be valuable when eradicating the invasive zebra

mussel population. Specific concentrations and exposure times of Zequanox to zebra

mussels in crucial areas of the environment could be used to aid their removal. These

concentrations and exposure times will hopefully be used to prevent zebra mussels from

clogging important pipelines and threatening indigenous species in their environment.

 Carr - Gonzales 3

Review of Literature

The purpose of this experiment was to determine which concentration and

exposure time of Zequanox would cause the greatest deterioration to the stomach lining

of zebra mussels. Zequanox is a molluscicide, or pesticide used against molluscs, that

specifically targets the Dreissena species. It is created from dead cells of a natural

microbe, which allows the mussels to perceive it as a reliable food source. As the mussels

filter the Zequanox, the substance deteriorates the lining of their stomach, often resulting

in fatality. It has been looked into as a potential controlling agent for both zebra and

quagga mussels due to the fact that it does not appear to damage industrial equipment or

the surrounding aquatic environment (ZEQUANOX).

Zebra mussels are a highly invasive species that belong to the class Bivalvia, as

do most types of mussels. Bivalves are composed of two outer shells that are connected

by a ligament. Between these shells, bivalves shield their softer tissues and more

vulnerable parts. Included in these vulnerable parts is the stomach, which is what the

zebra mussels use to filter their food (The Delaware Geological Survey). A zebra

mussels diet consists of large amounts of plankton and detritus; detritus is also known as

organic matter produced by the decomposition of organisms (Zebra Mussel Fact

Sheet). Because zebra mussels already filter feed dead organic matter of organisms,

which is what Zequanox is, the product is able to pass through the mussels digestive

system undetected. The zebra mussels filter the Zequanox through their stomach, and

their stomach slowly begins to deteriorate, turning a dark color due to necrosis, which is

the death of cells in a tissue.

 Carr - Gonzales 4

Figure 1. Mussel Interior

Figure 1 displays a mussel broken open. As shown, beneath the shell of the

mussel is all of its softer tissue. The small white circle is the mussels stomach, which is

the primary concern in this experiment.

Zebra mussels thrive in water that is nutrient-rich, supports healthy populations of

plankton, and is slightly alkaline. They live best in temperatures between 68-77 F, but

even though these are the preferred temperatures for zebra mussels to live in, they can

survive in a much greater range. Also, a considerable amount of calcium is needed for

their survival; this is used in the production of their shells. Hard surfaces are needed for

their byssal fibers, or thread-like strands tipped with a strong, sticky substance, to attach

to hard surfaces (Zebra Mussel Fact Sheet).

Zebra mussels only live about 2-5 years. A mature female zebra mussel can

release up to one million eggs every year and mature males release almost two hundred
 Carr - Gonzales 5

million sperm into the water. Approximately two days after an egg has been fertilized, it

develops into a veliger, or a free-swimming larvae. Around 2-3 weeks, they start to feel

the weight of their forming shells and begin to attach themselves to firm, underwater

surfaces (Zebra Mussel Fact Sheet).

In another experiments attempt to control the zebra mussel population,

professional researchers Eric Davis, Hing Wai Wong, and Willard Harman tested the

zebra mussels ability to filter three different concentrations of sodium chloride. All

concentrations of the sodium chloride resulted in mortality of the zebra mussels, because

the mussel is unable to filter sodium chloride through its stomach (Davis, Harman,

Wong). This is similar to the setup of the experiment because the researchers tested the

zebra mussels ability to filter three different concentrations of Zequanox. The

researchers did not run the experiment with sodium chloride because unlike Zequanox,

sodium chloride may cause damage to the surrounding aquatic environment.

Marrone Bio Innovations, Illinois Department of Natural Resources, SIU, the

District, and PLM Lake and Land Management (PLM) conducted a study on the

mortality of zebra mussels when Zequanox was introduced into a natural lake setting. The

results were that treated sites had an average mussel mortality of 97.1%, fish mortality

was not observed at the treated sites after 24 hours, and there seemed to be no lasting

effects on water quality (Weber, Roberts, Rackl). This is similar to the experiment

because they were testing the effects of Zequanox on zebra mussels. Unlike the previous

experiment in which they were testing to see if the product worked and if it had any other
 Carr - Gonzales 6

effects on the environment, this experiment tested to see what concentration and what

exposure time of the product was the most effective in killing zebra mussels.

Overall, zebra mussels are a fairly simple species, but there is still a lot of

information to be learned about them. They are bivalve mussels that filter feed only on

plankton and detritus. Zebra mussels require certain nutrients, such as calcium, in their

environment and can survive in a wide range of temperatures. They reproduce and

develop rapidly and live for only a few years. Zequanox is a product derived of dead

bacteria that degenerates the stomach lining of zebra mussels. From previous

experiments, the researchers were able to derive a similar, yet unique, experimental

design using Zequanox intended to help control the zebra mussel population.
 Carr - Gonzales 7

Problem Statement

Problem:

Which concentration of Zequanox, in milligrams, and which exposure time, in

hours, will have the greatest effect on the mortality of Dreissena polymorpha.

Hypothesis:

The greatest concentration and the longest exposure time to Zequanox will have

the greatest effect on the mortality of Dreissena polymorpha.

Data Measured:

The independent variables in this experiment were the concentrations of

Zequanox and the exposure times. The concentrations used were 50 mg, 100 mg, and 150

mg. The exposure times used were 3 hours, 8 hours, and 24 hours. The dependent

variable was the mortality of the Dreissena polymorpha which was measured in

percentage of dead specimens. A two-factor design of experiment was conducted to

measure the interaction between factors.

 Carr - Gonzales 8

Experimental Design

Materials:

(8) 200 mg Samples of Zequanox (6) 300 mg Samples of Zequanox

(6) 100 mg Samples of Zequanox (15) 1L Containers
(450) Zebra Mussels 7.57L Bucket
18.93L Fish tank Paint scraper
Freshwater source (2) 56.78L Plastic bin
TI-Nspire CX Calculator Gloves
Scientific Collectors License API Freshwater Master Test Kit
2L Pitcher Hand net
3.8754L Bleach Dust/Mist Filtering Respirator
Goggles (15) 100 mL beakers
Masking tape Permanent marker
Strainer 500 mL Beaker
Tetra Whisper Internal Power Filter 10i
Darice Stiff Plastic Canvas Clear 30.48 x 45.72 cm
Thermolyne Maxi Mix II Type 36700 Mixer Vortexer

Procedure:

Preparation:

1. Apply for and obtain a Scientific Collectors permit in order to legally collect the
zebra mussels.

2. Collect zebra mussels and lake water (see Appendix A).

3. Set up 18.93L tank and filter where the mussels will be kept for the duration
of the experiment.

4. Remove any mussels that may have died in the transportation process.

5. When Zequanox is obtained, put into and keep stored in a refrigerator.

 Carr - Gonzales 9

Data Collection:

6. Set up and label the 15 containers using tape and the permanent marker. Then, fill
them with 450 mL of freshwater from the bin using the 500 mL beaker. Label the
15 100 mL beakers as well. Transfer 30 zebra mussels into each container.

7. Randomize trials using TI-Nspire CX Calculator (see Appendix B).

8. Set up data tables.

9. Put on all safety equipment including gloves, respirator, and goggles.

10. Depending on the trial, remove either one of the 100 mg, 200 mg, or 300 mg
samples of Zequanox from the refrigerator. Add 25 mL of
freshwater to each tube. Reseal the caps. Invert each tube and tap sharply against
a hard surface. Using the Thermolyne Maxi Mix II Type 36700 Mixer Vortexer,
vortex samples on the highest setting until no clumps are observed. Add solution
into appropriate jar. Rinse out the tube into the jar at least three times with
25 mL of freshwater.

11. Allow zebra mussels to be exposed for the amount of time indicated in that trial.

13. After mussels have been treated for the appropriate amount of time, pour them
into the strainer over the container they were in. Rinse off the mussels with more
untreated freshwater from the bin. Place mussel group into its correctly allocated
beaker in the tank. Treat the water left in the container with bleach before pouring
down the drain to ensure any veliger are dead.

14. Every day after treatment, record the amount of mussels that are alive and that
have died in each section and remove from tank using hand net. Dead mussels
tend to gape open or they do not close if disturbed.

15. Test the pH and ammonia levels of both the water in the tank where the mussels
are being held, and of the extra freshwater being contained in the 56.78L plastic
bin. If any ammonia is observed or if the pH falls below 7.8 or above 8.4, change
up to half of the water per day until levels return back to normal.

16. Using the pitcher, replace about 1L of water from the main tank every day.

17. Repeat steps 10-13 for each trial.

 Carr - Gonzales 10

18. Repeat steps 14-16 every day for two weeks.

19. After experiment is done, treat all water and any remaining muscles used in the
experiment with bleach before disposal.

Figure 2. Materials

Figure 2 displays the materials used in the experiment. Not all materials are

displayed in this image. However, all materials that were vital to the experiment are

shown above.
 Carr - Gonzales 11

Data and Observations

Table 1
DOE 1 Data
Number of Dead Mussels (Out of 30)
Trial
Day 1 Day 2 Day 3 Day 6 Day 7

Standard 1 1 1 1 1

+,+ 0 1 3 26 27

-,- 0 0 0 0 0

Standard 0 3 3 5 5

+,- 0 1 2 2 2

-,+ 0 0 0 6 9

Standard 2 2 2 2 2

Table 1 shows the data from the first DOE conducted. Each column shows

the number of dead zebra mussels (out of thirty) corresponding to the day the data

was collected. Data was collected over a period of seven days with the omission of

days four and five, due to a lack of access.

 Carr - Gonzales 12

Table 2
DOE 2 Data
Number of Dead Mussels (Out of 30)
Trial
Day 1 Day 2 Day 3 Day 6 Day 7

Standard 1 1 1 1 1

+,+ 1 3 3 15 22

-,- 2 2 2 2 2

Standard 0 0 0 1 3

+,- 0 0 0 2 2

-,+ 0 0 0 2 5

Standard 0 0 0 4 5

Control 0 0 0 0 0

Table 2 shows the data from the second DOE conducted. Each column

shows the number of dead zebra mussels (out of thirty) corresponding to the day

the data was collected. Data for this DOE was also collected over a period of seven

days with the omission of days four and five, due to accessibility.
 Carr - Gonzales 13

Table 3
Percentage Dead
Percentage Dead
Trial
DOE 1 DOE 2

Standard 3.3333 3.3333

+,+ 90.000 73.3333

-,- 0.0000 6.6667

Standard 16.6667 10.000

+,- 6.6667 6.6667

-,+ 30.000 16.6667

Standard 6.6667 16.6667

Control 0.0000 0.0000

Table 3 shows the total percent dead for each trial in each DOE. The

percent was found by taking the number dead, dividing by the original number of

thirty, and multiplying by 100.

 Carr - Gonzales 14

Table 4
Observations
Day Observations

1 pH: 7.4
Ammonia: 0.5ppm
Necrosis is apparent in dead specimens
Control group is attaching to container while other groups are not

2 pH: 7.4
Ammonia: 0.25ppm
Shells appear to be peeling off of dead specimens
Specimens are beginning to attach to each other

3 pH: 7.4
Ammonia: 0.25ppm
Specimens appear to have brittle shells

6 pH: 7.4
Ammonia: 0.5ppm
Necrosis is apparent in dead specimens
Less specimens are attached to their container

7 pH: 7.4
Ammonia: 0.5ppm
Necrosis is apparent in dead specimens
Control group is attaching to container while other groups are not

Table 4 shows the observations recorded throughout the duration of the

experiment. In each set of observations, the pH and ammonia levels of the tank

were recorded.
 Carr - Gonzales 15

Data Analysis and Interpretation

Table 5
Factors Used in Experiment
Concentration (mg) Time (hours)

- Standard + - Standard +

100 200 300 3 8 24

Table 5 shows the factors that were used in the experiment. For concentration, the

low amount used was 100 mg, the standard amount was 200 mg and the high amount was

300 mg. For time, the low was 3 hours, the standard was 8 hours, and the high was 24

hours. The standard factors were based off of company instructions on how much product

should be used and for how long. Then, the high and low concentrations were chosen by

adding and subtracting 100 mg to the recommended amount; the high and low times were

chosen based off of the researchers schedules.

Table 6
Table of Results
Order Runs Result (Percentage Dead) Order Runs Result (Percentage Dead)

1 Stand 3.3333 1 Stand 3.3333

6 ++ 90.0000 6 ++ 73.3333

5 - - 0.0000 3 - - 6.6667

4 Stand 16.6667 4 Stand 10.0000

3 + - 6.6667 2 + - 6.6667

2 - + 30.0000 5 - + 16.6667

7 Stand 6.6667 7 Stand 16.6667

 Carr - Gonzales 16

Table 6 shows the results of the experiment. The trials were randomized in order

to prevent bias and minimize lurking variables. For each trial, the percentage was found

by dividing the total amount of zebra mussels found dead in the group by the amount of

mussels the group originally started with, which was thirty.

Table 7
Table of Standards
Standards

3.3333% 16.6667% 6.6667% 3.3333% 10.0000% 16.6667%

Table 7 shows the values of all of the standards from the experiment.

Figure 3. Nine Standard Runs

Figure 3 shows a dot plot of the standards from the experiment. Even though there

are two values that are repeated (3.3333% and 6.6667%), there seems to be no noticeable

pattern. The range of these standards is found by subtracting the lowest value from the

highest value. In this case, it would be 3.3333% subtracted from 16.6667%, which results
 Carr - Gonzales 17

in a range of 13.3333%. With no rapidly increasing or decreasing trends, it can be

inferred that the data is consistent.

Table 8
Averages
Runs
First DOE Second DOE Average
Concentration Time

+ + 90.0000% 73.3333% 81.6667%

- - 0.00000% 6.6667% 3.3333%

+ - 6.66667% 6.66667% 6.6667%

- + 30.0000% 16.6667% 23.3333%

Table 8 shows the averages of all the trials that were not standards in the

experiment. They were found by adding the results found for one trial from each DOE

together and then dividing by two. The grand average is 28.75%. This was found by

adding all of the averages from table 8 together and then dividing by four.

Table 9
Effect of Concentration
Concentration

(-) 100 mg (+) 300 mg

3.3333% 81.6667%

23.3333% 6.6667%

Avg = 13.3333% Avg = 44.1667%

Figure 4. Effect of Concentration

 Carr - Gonzales 18

Both table 9 and figure 4 show the effect that concentration had on the mortality

of Dreissena polymorpha. Table 9 shows the averages for when concentration was held

low and held high. The effect of concentration is about 30.8333%. This was found by

subtracting the low average from the high average. On average, as concentration

increases, the mortality increases by 30.8333%.

Table 10
Effect of Time
Time

(-) 3 Hours (+) 24 Hours

3.3333% 81.6667%

6.6667% 23.3333%

Avg = 5.0000% Avg = 52.500%

Figure 5. Effect of Time

Both table 10 and figure 5 show the effect that time had on the mortality of

Dreissena polymorpha. Table 10 shows the averages for when time was held low and

held high. The effect of time is 47.5%. This was found by subtracting the low average

from the high average. On average, as time increases, the mortality increases by 47.5%.

Table 11
Interaction Effect
Concentration

(-) 100 mg (+) 300 mg

(+) 24 23.3333% 81.6667%

Solid Segment
Hours
Time
(-) 3 3.3333% 6.6667%
Dotted Segment
Hours
 Carr - Gonzales 19

Table 11 shows the averages for the four different trials that are found in a DOE.

With these averages, the interaction effect can be found. This is done by first, subtracting

the low average from the high average, and then dividing by two in order to find the

slope. For example, the slope for the solid segment will be 23.3333% subtracted from

81.6667% and then divided by two; the result is about 29.1667%. The process is then

repeated to calculate the slope of the dotted segment which ends up being about 1.6667%.

The slope of the lower value is then subtracted from the higher value to calculate the

interaction effect, which happens to be 27.5% in this experiment.

Figure 6. Interaction of Concentration and Time

Figure 6 shows the interaction of the two factors. Again, the slope of the solid

segment, or 29.1667%, minus the slope of the dotted segment, or 1.6667%, gives the

effect value of 27.5%. It can be noted that the segments are not parallel and that the
 Carr - Gonzales 20

difference in their slopes is relatively large. This suggests that there may be an interaction

and that the rate of change of the outcome as concentration goes from low to high does

depend on whether time is held low or high.

EC ET ECT
Y = GA + 2 * C oncentration + 2 * T ime + 2 * (Concentration * T ime) + noise

Figure 7. Prediction Equation

Figure 7 shows the prediction equation. The equation includes the grand average

plus all the effects of the factors divided by two and multiplied by the factor. Noise is

also included to account for anything else that may have had an effect on the data. This

equation can be used to make predictions about future experiments (see Appendix C for a

sample calculation).

Figure 8. Dot Plot of Effects

Figure 8 shows a dot plot of all of the effect values of the different factors. Effects

are considered significant if they are greater than double the range of standards, which for
 Carr - Gonzales 21

this experiment is about 26.6667%; this is represented by the two red lines in figure 8. All

three effects, or the effect of concentration (30.8333%), the effect of time (47.5%), and

the interaction effect (27.5%) are greater than double the range of standards. Therefore,

all three effects can be deemed significant.

EC ET ECT
Y = GA + 2 * C oncentration + 2 * T ime + 2 * (Concentration * T ime) + noise

Figure 9. Parsimonious Prediction Equation

Figure 9 shows the parsimonious prediction equation. This equation uses only the

effects that are found to be significant. Since all of the effects in this experiment were

found to be significant, it is the same as the prediction equation in figure 7. This equation

also aids in making predictions for future experiments (see Appendix D for a sample

calculation).

Overall, it can be determined that both the factors of concentration and exposure

time had a significant effect on the mortality of zebra mussels. There was also an

interaction effect between these variables because the rate of change of the outcome as

concentration goes from low to high depends on whether time was held low or high.

Also, it appeared that as both factors increased, the mortality rate did as well. Therefore,

the higher the concentration and exposure time, the more dead specimens there were.
 Carr - Gonzales 22

Conclusion

The purpose of this experiment was to determine the effects of concentration and

exposure time of Zequanox on the mortality of Dreissena polymorpha (zebra mussels).

The initial hypothesis was that the highest concentration and exposure time of Zequanox

would yield the greatest mortality of zebra mussels. This hypothesis was accepted. To

determine this, a two-factor design of experiment (DOE) was conducted. The

concentrations of Zequanox used were 50 mg for low, 100 mg for standard, and 150 mg

for high. The exposure times used were 3 hours for low, 8 hours for standard, and 24

hours for high. The two-factor DOE was used to determine the significance and the

interaction effect of the two factors.

After conducting this experiment, it can be determined that both the factors of

concentration and exposure time are significant, and there is an interaction effect between

the two factors. The rate of change of the outcome as concentration goes from low to

high depends on whether time is held low or high. Also, it appeared that as both of the

factors increased, the mortality of the zebra mussels increased as well. These results lead

to the acceptance of the hypothesis that the highest concentration and exposure time of

Zequanox would yield the greatest mortality of zebra mussels.

As the zebra mussels are exposed to the Zequanox, they begin to filter it through

their digestive system. The Zequanox is derived from a naturally occurring strain of the

bacteria Psuedomonas fluorescens, making it undetectable to the zebra mussels. As they

filter feed the Zequanox through their digestive system, the Zequanox causes necrosis, or

the death of cells in a tissue, and decays the stomach lining within the zebra mussel. This
 Carr - Gonzales 23

eventually destroys the zebra mussels digestive system, ultimately resulting in their

death.

As the concentration of the Zequanox is increased, more of the active ingredient

of Zequanox was filtered through the zebra mussels digestive system. A greater

concentration of Zequanox ensures its filtration through the zebra mussels, meaning that

more zebra mussels exposed to a higher concentration of Zequanox will die. If the

concentration of Zequanox is lower, it is not easily ensured that there will be an effect on

the zebra mussels digestive system, or the effect may not be severe enough to result in

death. This is displayed in the results because as the concentration of Zequanox increases,

the mortality of the zebra mussels does as well.

When zebra mussels are exposed to the Zequanox for longer periods of time, they

are given a longer period of time to filter it through their digestive system. With lower

exposure times, the zebra mussels may not be given a chance to equilibrate to the changes

in their environment, meaning that they may not begin filtering the Zequanox in the

window of time that it is present. In this experiment, the zebra mussels in groups where

exposure time is held low did not appear to have filtered the Zequanox, due to the fact

that they had not adapted to their environment.

The results of this experiment allowed the hypothesis that the highest

concentrations and exposure times of Zequanox would result in the greatest mortality to

be accepted. Although the results of this experiment were deemed valid, some further

improvements could be made. A more effective method of separating the zebra mussels

after treatment may provide more accurate data, and another DOE would help to solidify
 Carr - Gonzales 24

the results gathered. Potential errors in the experiment may be attributed to discrepancies

when classifying a zebra mussel as dead and potential miscounting of living zebra

mussels. It can sometimes be difficult to determine if a zebra mussel is dead. Healthy

mussels will quickly close to protect themselves if disturbed, whereas unhealthy or dead

mussels will remain open or close very slowly. Even so, there is still discrepancy in this

method of determining whether or not a mussel is dead, and the researchers were not

always confident in classifying a mussel as living. Also, some empty mussel shells get

dirt trapped in them which gives them the appearance of being an alive mussel. They only

remain shut, though, because of the dirt inside (Faulkner; Hsieh). Further research with

Zequanox may be conducted to ensure it does not threaten the environment. Also, it could

be studied whether or not zebra mussels can become resistant to the product and make it

lose its effectiveness. Other methods of eradicating zebra mussels may also be discovered

in further research.

The results of this experiment are valuable to the scientific community

responsible for creating and determining agents to help kill of zebra mussels. The results

are also valuable to companies struggling with zebra mussels clogging crucial pipelines.
 Carr - Gonzales 25

Appendix A: Collecting Zebra Mussels and Lake Water

Materials:

Bucket Scientific Collectors License

Freshwater Source Paint scraper
18.93L Fish tank 56.78L Plastic bin

Procedure:

1. Locate a freshwater source with a sea wall. Fill the bucket halfway with water.

2. Search along the sea wall for attached, living zebra mussels.

3. Remove the mussels from the sea wall using the paint scraper or hands. After
removal, place them in the bucket of water.

4. Repeat Step 2 until 420 zebra mussels have been collected.

5. Pour bucket of water and zebra mussels into the 18.93L fish tank.

6. Fill the bucket with water and pour it into the 56.78L plastic bin. Repeat this
until the bin is full of water.
 Carr - Gonzales 26

Appendix B: TI-Nspire CX Trial Randomization

Materials:

Ti-Nspire CX Calculator

Procedure:

1. Open a new Calculator page.

2. Press the menu button.

3. Select option five, or Probability, in the drop down menu.

4. Select option four, or Random, in the drop down section that appears.

5. Select option two, or the Integer function. The words randInt() will appear
on the calculator page. Type (1, 2) and press enter.

6. Type (1,10) and press enter. Repeat this until all trials are randomized.
 Carr - Gonzales 27

Appendix C: Prediction Equation Sample Calculation

Y = 28.75 + 30.8333
2
47.5 27.5
* C oncentration + 2 * T ime + 2 * (Concentration * T ime) + noise

Figure 10. Prediction Equation Sample Calculation

Figure 10 shows a sample calculation of the prediction equation shown in figure

7. The variables were replaced with the effects that were found in the DOE. So, 30.8333

was substituted for EC, or the effect of concentration, 47.5 was substituted for ET, or the

effect of time, and 27.5 was substituted for ECT, or the interaction effect of concentration

and time. This equation could be used to make predictions if the experiment were to be

tested again, based off of the results from this one.

 Carr - Gonzales 28

Appendix D: Parsimonious Prediction Equation Sample Calculation

Y = 28.75 + 30.8333
2
47.5 27.5
* C oncentration + 2 * T ime + 2 * (Concentration * T ime) + noise

Figure 11. Parsimonious Prediction Equation Sample Calculation

Figure 11 shows a sample calculation of the parsimonious prediction equation

shown in figure 9. Only effects found significant are used in it, and since all of the effects

of this experiment were found significant, it is the same as the prediction equation. The

variables were replaced with the effects that were found in the DOE. So, 30.8333 was

substituted for EC, or the effect of concentration, 47.5 was substituted for ET, or the

effect of time, and 27.5 was substituted for ECT, or the interaction effect of concentration

and time. This equation could also be used to make predictions if the experiment were to

be tested again. based off of the results from this one.

 Carr - Gonzales 29

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