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Puget Sound Project
Grades 9-12
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Where Does All the O2 Go?
Key Concepts
  1. Oxygen levels in Puget Sound waters affect the kinds and numbers of living plants and animals.

  2. Dissolved oxygen concentrations are affected by factors such as temperature, salinity and dissolved organic matter.

Biochemical Oxygen Demand
While oxygen is seldom in short supply for terrestrial organisms, the same is not true for aquatic organisms. Living things and the natural processes of decomposition compete for oxygen. Aquatic systems can decompose reasonable amounts of organic wastes without harmful effects. Excessive amounts of sewage wastes, on the other hand, reduce oxygen levels and productivity.

Biochemical oxygen demand (B.O.D.)is a measure of the organic matter in water. The concentration of oxygen in the water sample is tested when the sample is taken and then again after five days of incubation in the dark at 20° C. The B.O.D. is calculated by subtracting the dissolved oxygen concentration in the sample after incubation from the dissolved oxygen concentration before incubation.

In the sealed, darkened sample, organic matter is decomposed by the bacteria normally found in the water. The bacteria require oxygen for decomposition. Thus, the more organic matter present in the water sample, the more dissolved oxygen will be consumed by the bacteria over the five-day test period.

The B.O.D. test is easy to conduct and provides your students with a first hand glimpse of the impact of organic pollution. As your students transfer water samples, recall that mixing increases the D.O. concentration and instead of pouring the sample, siphon the water whenever it is transferred into or out of the sample bottle.

“Part One – Using a Model System to Study the Oxygen Demand of Sewage”employs a simple model using yeast, powdered milk, and methylene blue, instead of actual sewage, to study the effect of sewage on dissolved oxygen concentrations. Yeast represent the decomposers present in sewage and natural waters. Powdered milk contains sugar represents the nutrient-rich wastes of sewage. Methylene blue is an indicator dye, blue when oxygen is present and colorless when oxygen is absent.

The equipment used to determine oxygen concentration in “Measuring Water Quality” is used in this activity as well. Seal the dark sample and cover it with foil or black electrical tape.

  1. Look at the list of… An increase in any of the sources for dissolved organic matter tends to decrease the amount of oxygen the water can hold. (The bold face word correctly completes the sentence.)

  2. Two sources of dissolved organic matter in runoff from urban areas may be selected from:
    • pet wastes from streets and sidewalks,
    • nutrients from lawn fertilizers,
    • leaves,
    • grass clippings,
    • and paper from residential areas.
  3. Two sources of dissolved organic matter in runoff from agricultural areas may be selected from:
    • soil from eroded fields,
    • manure from farms,
    • failing septic systems,
    • and agricultural fertilizers.

Part 1
  1. The arrows shown connect the model elements with the components they represent.

  1. a.  A completed TABLE 1 showing the percent of milk contained in each of the three test tubes is shown below:
Table 1
Contents of Test Tubes
Test Tube
Milk (ml)
Water (ml)
Yeast Solution (ml)
% Concentration of Milk
b.  Test tube #3 contained the most food for the decay organisms(decomposers).

c.  Test tube #1 had the least food.

  1. A ring of blue color at the surface of the test tubes, even after the remainder of the liquid had changed color, might be due to oxygen entering the sample at the boundary between the solution and the air.

  2. Usually, the change occurs most rapidly in tube #3. Expectations may vary.

  3. Explanations that account for the differences in time needed for the change to occur in each of the three tubes are likely to say that the time for the change is decreased by an increase in food for the yeast. The yeast can more readily find and consume food particles.

  4. Shaking the tubes rapidly so that air is mixed with the liquid re-oxygenates the solution causing the indicator to change color again.
Problems for Furthur Investigation
  1. Up to a point, an increase in temperature would likely increase the speed of the reaction on these mini-systems. However, since the yeast cells are living a dramatic increase in temperature will cause their death. Your students could find out by duplicating the experiment with a higher temperature, something you might wish to encourage.

  2. If more decomposers (yeast) had been present at the beginning of your experiment the time needed to see the color change would be decreased.

  3. If the decay organisms began to multiply the dissolved oxygen in Puget Sound would decrease.

  4. Encourage interested students to try some of the variations suggested.

  5. A visit to a nearby sewage plant can be enlightening in terms of what we do with sewage. While the feasibility of such a visit depends on local availability of a wastewater facility, you should not overlook this opportunity to dramatically demonstrate that “out of sight, should not be out of mind.”

  6. Answers will vary depending upon experimental results.

Biochemical Oxygen Demand (BOD) Testing
Biochemical Oxygen Demand (BOD) is an important measure of the health of Puget Sound waters. The procedure presents a standard protocol which will enable your students to measure BOD in water they sample and compare the results with samples from other areas of the Sound.

Key Words
  • decomposers
  • dissolved organic matter
  • dissolved oxygen
  • microorganisms
  • methylene blue
  • nutrients
  • sewage

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