School Science/Winkler test for dissolved oxygen

The Winkler test is used to determine the level of dissolved oxygen in fresh water samples. What follows is a set of instructions on how to perform the test.


Stage 1Edit

  • Manganese(II) sulfate 48%
  • Potassium iodide 15% in potassium hydroxide 70%
  • Sample of fresh water
  • Latex gloves
  • Safety glasses

Stage 2Edit

  • Sulfuric acid 50%
  • Sodium thiosulfate 0.31%
  • Starch making up solutions 0.1%
  • Burette
  • Burette stand
  • 10 ml pipette and pipette filler
  • 100 ml conical flasks
  • Filter paper
  • Latex gloves
  • Safety glasses


Stage 1Edit

Collect and label water samples in 25 ml stoppered bottles. (Two samples per location are required for Biological Oxygen Demand testing.)
Add 0.1 ml of manganese(II) sulfate solution, and mix carefully, without letting in air. Add 0.2 ml of alkaline potassium iodide, and again mix without letting in air. A pinky-brown precipitate should appear.
At this point the sample may be stored for later analysis in the laboratory.

Stage 2Edit

Add 0.3 ml sulfuric acid to each sample, and mix. Allow the sample to stand for two minutes. If the precipitate does not dissolve into iodine solution, add a further 0.1 ml acid. Fill the burette with thiosulfate solution and adjust to zero (or note the burette reading). Transfer 10 ml of the sample to a conical flask, and add a few drops of starch solution. The subsample should turn blue. Titrate the subsample with thiosulfate until it turns clear. (You may find the endpoint easier to see if the conical flask is stood on a sheet of filter paper.) Record and repeat the titration.


Each milliliter of thiosulfate titer is equivalent to 0.1 mg of oxygen in the 10 ml subsample. Thus 1 ml of thiosulfate is equivalent to 1 mg oxygen per 100 ml fresh water.

To determine five-day Biological Oxygen Demand (BOD5), several dilutions of a sample are analyzed for dissolved oxygen before and after a five-day incubation at 20 degrees Celsius (68 degrees Fahrenheit) in the dark. In some cases the sample must be provided a source of oxygen-using bacteria, called "seed", in order to obtain results. The difference in DO and the dilution factor are used to calculated BOD5. The resulting number (usually reported in parts per million or milligrams per Liter) is useful in determining the relative organic strength of sewage or other polluted waters.

The BOD5 test is an example of analysis that determines classes of materials in a sample.

Instrumental methods for measurement of Dissolved Oxygen have widely supplanted the routine use of the Winkler test, although it is still widely used to check instrument calibration.


  • Moran, Joseph M.; Morgan, Michael D., & Wiersma, James H. (1980). Introduction to Environmental Science (2nd ed.). W.H. Freeman and Company, New York, NY ISBN 071671020X
  • Standard Methods for the Examination of Water and Wastewater - 20th Edition ISBN 0-87553-235-7. This is also available on CD-ROM and online by subscription