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Biodegradable Materials and Their Effect on Dissolved Oxygen Levels
Introduction
In aquatic systems, aerobic microorganisms will consume biodegradable material for energy, and in
doing so will also take up oxygen from the environment as part of the cellular respiration process.
Scientists use dissolved oxygen levels as an indication of contamination by such pollutants as sewage,
agricultural runoff, organic industrial effluents, etc.
If partially treated sewage and other wastes are placed into water, they serve as food for
microorganisms. The population of decomposers increases as the quantity of nutrients increases. The
increase in microorganisms will change water in ways you will discover by doing this lab. When
microorganisms consume organic matter in a water solution, they also use oxygen in the water, just as
people use oxygen to metabolize their nutrition. The amount of oxygen needed for all of the living
things in an ecosystem is called the Biological Oxygen Demand, or BOD. As the number of
organisms in a system increases so does the BOD.
Oxygen-rich environments are said to be "oxidizing." Oxidizing chemicals, such as oxygen, strip
electrons away from atoms of other elements. Chemical elements that have such a strong attraction for
electrons that they can "rip" electrons away from other types of atoms are known as oxidizing agents,
and are very useful in chemistry. Methylene blue indicates the presence of oxidizing agents because it
is oxidized itself by these compounds. When electrons are stripped from methylene blue, the resulting
molecule imparts a blue color to the solution--giving a clear sign of a chemical change. Methylene
Blue is clear in the absence of oxygen and blue in the presence of oxygen. During respiration, as the
yeast consumes the available food, oxygen is also consumed and carbon dioxide is produced. The blue
color disappears as the oxygen concentration drops and the carbon dioxide level rises. Carbon dioxide
combines with water to form carbonic acid which changes the indicator color.
Pre-lab questions
 List dissolved organic materials and possible sources that may decrease oxygen levels as
a result of decomposition when present in water.
 Explain what a serial dilution is and its importance in determining toxicity
 Why is the chemical methylene blue clear in the absence of oxygen and blue in the
presence of oxygen?
 List possible organisms that may act as decomposers of dissolved organic material in
aquatic systems.
 Develop a hypothesis for the effect of biodegradable materials on dissolved oxygen
levels. What is your independent variable? Your dependent variable? Label and sketch
a graph of the data you would expect based on your hypothesis.
Background research information links:
 http://www.epa.gov/ebtpages/water.html
 http://www.forsea.org/PSP9.HTML
 http://itsligontime.com APES Water Pollution ppt
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Materials
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Whole milk
Six test tubes with screw cap or cork – at least 10 mL capacity
10-mL graduated cylinder
Test tube rack (see-through)
Six 1- mL pipettes & syringes
Yeast
Methylene Blue (in dropper bottle)
Permanent marker
Safety Measures
 Methylene Blue may be harmful if swallowed, inhaled, and/or in contact with skin. It
may cause severe eye irritation. Wear an apron and goggles for the duration of the
experiment.
Procedure
1) Label the test tubes #1 through #6. Place them in the test tube rack.
2)
Using a graduated cylinder, add 5 mL of tap water to tubes #2 through #6.
3)
Add 10 mL of milk to tube #1.
4) Using a graduated cylinder, remove 5 mL of milk from tube #1 and place it in tube #2.
5) Mix the contents well.
6) Remove 5 mL from tube #2 and place it in tube #3. Mix the contents well.
7) Remove 5 mL from tube #3 and place it in tube #4. Mix the contents well.
8) Remove 5 mL from tube #4 and place it in tube #5. Mix the contents well, and then
discard 5 mL of the solution.
9) Add 3 drops of methylene blue to each tube. Be careful to hold the dropper bottle
upright so that the drops are uniform. Mix each tube well.
10) The contents of each tube should now be as below:
10) Noting the time, quickly add 1 mL of yeast mixture to each test tube. Invert each test
tube 4 times and place in the test tube rack. Do not move the tubes until any color change is
complete.
11) Carefully observe each tube and record the exact time at which the color change from
blue to white is complete (no more color change). The surface of each test tube will remain
blue.
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13) BEFORE YOU CLEAN UP, SHAKE ONE OF THE TEST TUBES WHICH HAS
TURNED WHITE. Record your observations.
Data
Include your raw data and a graph (using an Excel or comparable spreadsheet) of the data in your
submitted report.
X axis - milk concentration
Y axis - length of time for color change
TEST
Methylene Blue
% Milk
Dilution
TUBE #
mL Milk
and/or
H20
1
5
3 drops
100%
1:1
2
5
3 drops
50%
1:2
3
5
3 drops
25%
1:4
4
5
3 drops
12.5%
1:8
5
5
3 drops
6.25%
1:16
6
5
3 drops
0%
0
Start time
(A)
End time
(B)
Time
(B-A)
Observations
Analysis and Conclusions (Type on separate paper)
1) What is the gas taken in by the microorganisms?
2) What is the gas given off by the microorganisms?
3) Where do microorganisms living in water get the oxygen that they use in decomposition?
4) Where do the green plants living in water get the carbon dioxide that they need?
5) Which part of your experiment represents the decomposers?
6) Which part of your experiment represents sewage or some other form of biodegradable waste
dumped into water?
7) What was the dependent variable? The independent variable? What were the constants?
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8) Write a conclusion as to what you learned by performing this lab. Be sure to include the
following:
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Summary of the experiment
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Was the hypothesis supported or refuted? Why or why not?
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What were the sources of error in this experiment?
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What might be done differently to improve the quality of the data?