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BOD Test – Biological Oxygen Demand Test

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  • All aquatic organisms depend on the oxygen in the water (dissolved oxygen) for survival. As a food supply, aquatic microbes consume the organic matter discharged into the water.
  • Plant decomposition and leaf fall are common sources of organic debris. Using the dissolved oxygen in the water, bacteria will decompose this organic stuff and form less complicated organic molecules.
  • With increased discharge of waste items (including organic compounds), microbes will make more use of dissolved oxygen. Therefore, the water becomes oxygen-depleted.
  • In an anaerobic environment, microbes will produce noxious byproducts, which may lead to unfavourable outcomes such as fish suffocation.
  • Consequently, the amount of dissolved oxygen in water is an indicator of water quality.
  • The biological oxygen demand is a common method for expressing the concentration of organic materials in waste water samples. It quantifies the amount of dissolved oxygen utilised by bacteria in water.
  • The greater the amount of organic matter in sewage, the greater the oxygen consumption by microorganisms to decompose sewage with a high BOD value.
  • In neutral ecosystems such as lakes, rivers, etc., the digestion of these organic molecules can deplete available oxygen, resulting in fish suffocation.
  • Typically, the BOD of a water sample is determined by incubating the sample at 20 degrees Celsius in the dark for five days under aerobic conditions (in BOD incubator).
  • In water samples where more than 70% of the initial oxygen has been consumed, it is important to aerate or oxygenate and dilute the sample with BOD-free water (de ionised glass distilled water) in order to avoid O2 stress.

Objectives of BOD Test

  • To determine the concentration of organic matter in samples of wastewater.
  • To evaluate the water quality of surface waters.
  • To comprehend the properties of DO found in water and wastewater.

Principle of BOD Test

Under alkaline circumstances (by adding Alkaline-iodide-azide), manganese sulphate precipitates manganese hydroxide as a white precipitate. This combines with the sample’s dissolved oxygen to generate a brown precipitation. On an acidic environment, manganese changes to its divalent form and releases iodine. This released iodine is titrated using starch as an indicator against Sodium thiosulphate.

Materials required

  • Alkaline-iodide-azide solution
  • Manganese sulphate
  • Con. Sulphuric acid
  • Starch solution
  • 0.025N sodium thiosulphate
  • BOD bottle
  • Water bottle
  • Pipettes
  • Measuring cylinders
  • BOD Incubator
  • Burette and burette stand
  • Standard flask
  • Magnetic stirrer
  • Stir bar
  • Glass funnel
  • Gloves
  • Glove box
  • Tissue paper

Procedure of BOD Test

Neutralization of Sample

The accuracy of the BOD test is entirely dependent on the presence of the correct bacterial growth in the water sample. For accurate findings, the pH of the diluted sample must be adjusted to 7.00 0.2 before to incubation for five days. The sample should be neutralised as described below.

  1. Take 50 ml of the sample and place it in a 100 ml beaker.
  2. Utilizing the calibrated pH metre, determine the solution’s pH.
  3. Adjust the pH with 1N sulfuric acid if it is greater than 7.00 and 1N sodium hydroxide if it is less than 7.00.
  4. Notate the volume of sulfuric acid or sodium hydroxide used to alter the pH of a 50-milliliter sample to 7,00 0.0.
  5. Calculate the amount of sulfuric acid or sodium hydroxide necessary to neutralise the 1000 ml sample.
  6. To neutralise a sample, add the specified volume of sulfuric acid or sodium hydroxide.
  7. Using 2.1 ml of 1N sulfuric acid or sodium hydroxide to neutralise 50 ml of sample to a pH of 7.00 0.0 is an example.
  8. 2. Calculate the volume of 1N sulfuric acid or sodium hydroxide that must be added to the 1000 ml sample to neutralise it as follows: 1N sulfuric acid or sodium hydroxide required = (2.1 ml x 1000 ml)/50 ml = 2100/50 = 42 ml.

Note: Hydrochloric acid or any other acid containing chlorine should not be used to neutralise the sample, as chlorine interferes with the BOD results of wastewater.


Removal of Chlorine Content

As chlorine is a powerful oxidising agent that can limit microbial growth during wastewater BOD measurement, it must be removed from the sample prior to beginning the analysis. Sodium sulfite can be used to eliminate chlorine from a sample in the following manner:

  1. Take a water sample of 50 millilitres in a conical flask for analysis.
  2. Add 2.5 ml of 50 percent diluted acetic acid.
  3. Add 2.5 ml of a 10% w/v potassium iodide solution.
  4. Add 1 ml of indicator for starch and titrate with 0.025N sodium sulfite solution.
  5. Note the volume and calculate to add 1000 ml of the sample as mentioned previously in the section on Neutralization of Sample.
  6. To neutralise the chlorine, add the determined volume of sodium sulfite solution to the sample and mix thoroughly.

Preparation of Phosphate Buffer Solution:

In 500 ml of distilled water, dissolve 8.5 grammes of potassium dihydrogen phosphate (KH2P04), 21.75 grammes of dipotassium hydrogen phosphate (K2HP04), 33.4 grammes of disodium hydrogen phosphate (Na2HP04.7H20), and 1.7 grammes of ammonium chloride (NH4Cl). Dilute the solution to a volume of 1 litre.

Preparation of Alkali-Iodide-Azide Reagent

In distilled water, dissolve 500 grammes of sodium hydroxide (NaOH) and 135 grammes of sodium iodide (NaI). Bring the solution to 1000 millilitres of distilled water. In this solution, dissolve ten grammes of sodium azide.

Preparation of Dilution Water

The dilution water for wastewater BOD analysis must be free from organic content. The following is a method for creating dilution water.

  1. Take five litres of double-distilled water and place it in a glass jar.
  2. Air-condition the water using clean compressed air for a minimum of 12 hours.
  3. Allow to stabilise for a minimum of six hours at 20 °C.
  4. Add 5 ml of a 27.5% w/v calcium carbonate solution.
  5. Add 5 ml of 22.5 % w/v solution of magnesium sulfate.
  6. Add 5 ml of a ferric chloride 0.15% w/v solution.
  7. Add five millilitres of phosphate buffer solution.
  8. Mix thoroughly and let stand for two hours.

Procedure to Determine the Biological Oxygen Demand of Water

  1. Add 10 ml of sample to two of the 300 ml BOD bottles and fill the remaining space with dilution water.
  2. Only fill the remaining two BOD bottles with dilution water for the blank sample.
  3. When bottles are filled, they should be sealed immediately, and there should be no air bubbles within.
  4. Mark the bottles as empty and collect samples.
  5. At 20 °C, incubate one sample and one blank bottle for five days.
  6. Immediately analyse the remaining one sample and one blank vial of dissolved oxygen (DO).
  7. Analyze bottles incubated for 5 days for DO.

Recommended sample volume for BOD determination

BOD range (mg/liter)Sample volume (ml)Dilution water (ml)

Test for Dissolved Oxygen (DO)

  1. Add 2 ml of a 36.4% manganous sulphate (MnSO4.H2O) solution to the sample by putting the tip of the pipette into the sample, since the droplets of solution will allow oxygen to enter the solution.
  2. Add 2 ml of the alkali-iodide-azide reagent using the technique described above.
  3. Permit the solutions to react with the oxygen present in the sample.
  4. When precipitates have settled to the bottom, add 2 ml of strong sulfuric acid very close to the surface of the sample.
  5. Blend thoroughly to dissolve the precipitates.
  6. Transfer 203 ml of the BOD sample to an Erlenmeyer flask.
  7. Note the burette reading after promptly titrating with 0.025N sodium thiosulfate solution using a starch indicator until the blue hue fades.
  8. Method for determining the burette reading for blank.


Blank correction = B.R. for blank at D0  – B.R. for blank at D5 

BOD mg/l = [(B.R. for sample at D0 –D5)– blank correction] x dilution factor

Dilution factor = Bottle volume (300 ml)/Sample volume


  • B.R. = burette reading
  • D0 = Initial
  • D5 = Day five after incubation


  • https://vlab.amrita.edu/?sub=3&brch=272&sim=1430&cnt=1
  • https://www.pharmaguideline.com/2013/06/determination-of-biological-oxygen.html
  • https://www.ebsbiowizard.com/articles/running-the-biochemical-oxygen-demand-bod-test/
  • https://www.climate-policy-watcher.org/wastewater-treatment-3/biochemical-oxygen-demand-testing.html
  • https://sitefiles.camlab.co.uk/BOD%20Oxitop%20Determination.pdf
  • https://www.umces.edu/sites/default/files/BOD%20ANALYSIS%20METHOD.pdf
  • http://www.lagoonsonline.com/laboratory-articles/bodtest.htm
  • https://www.owp.csus.edu/glossary/biochemical-oxygen-demand-bod-test.php
  • https://extension.uga.edu/publications/detail.html?number=C992


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