How Is BOD Measured?


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Biochemical/biological oxygen demand (BOD) is measured using the following formula: BOD = (DO1 – DO2) * dilution factor/volume of the sample (if used). Dissolved oxygen (DO) is measured at the beginning (DO1) and then after a 5 day period (DO2) at 68°F (20°C). It is the difference between the two DO measurements that is used to calculate the BOD. The BOD indicates the amount of oxygen consumed by microorganisms during the 5 day test period, and is expressed in mg/L. 

Biochemical/biological oxygen demand (BOD) is an essential parameter for assessing the quality of water. The BOD level in water tells us the amount of oxygen consumption (mg O2 L 1) from aerobic microorganisms as they oxidize organic compounds. Sewage is a good example of a water body that has a high BOD, which can result in the death of some organisms due to a decrease in oxygen. Therefore, the consequences of water with a high BOD are the same as water with a low dissolved oxygen (DO) level – the aquatic organisms become stressed and can suffocate and die. 

In wastewater treatment plants, BOD is commonly an essential parameter to test to measure waste loads. It is also used to work out treatment efficiency and control plant processes before the water is discharged into the environment. 

Where Does BOD Come From?

BOD comes from various sources, such as:

  • Leaves
  • Woody debris
  • Decaying plants and animals
  • Animal manure waste
  • Wastewater treatment 
  • Poorly maintained septic systems or failing systems
  • Stormwater runoff (more common in urban areas)
  • Effluents (liquid or sewage discharge) from pulp and paper mills

Considerations When Measuring BOD

Before we look at how to measure BOD, it is important to understand the factors that can affect BOD measurements. 

BOD is affected by similar factors to DO. For example, aeration in water accelerated the decomposition of organic and inorganic materials. So, in areas where the water is shallow and has more movement from waterfalls, rapids, or other turbulent movements, the BOD rate will be accelerated. Whereas if water samples are collected in deeper waters with little to no movement, the BOD level will likely be higher.

Chlorine is another factor affecting BOD levels in water. This is due to chlorine inhibiting or killing the microorganisms that decompose the organic or inorganic material in the water sample. Therefore, if you are sampling the BOD level in chlorinated waters, you will need to neutralize the chlorine with sodium thiosulfate – Na2S2O3 (4mg of Na₂S₂O₃ per 50 ml of water). Chlorinated waters are most common below the effluent from sewage treatment plants. 

Equipment Used For Measuring BOD

Here’s a list of everything you will need to measure BOD:

  • Biochemical oxygen demand Incubator: This specialized equipment maintains precise temperature control throughout the experiment.
  • Burette/container and a stable stand: Utilized for precise liquid measurement and dispensing.
  • Bottles that hold 300ml: Explicitly designed for BOD experiments, these bottles feature glass stoppers for an airtight seal.
  • Dissolved Oxygen Meter: Instrument used for measuring dissolved oxygen content.
  • Wash bottle: Used for rinsing and cleaning equipment.
  • Conical flask: Employed for mixing and holding liquids.
  • Measuring cylinder: Ensures accurate liquid volume measurement.
  • Pipette that has an elongated tip: Enables precise transfer of small liquid volumes.
  • Gloves and seal starch: Personal protective gear for safety – gloves prevent any possible irritants from bacteria. 
  • Seal starch: Used for an airtight sealing. 

Chemicals you will need:

  • Manganese sulfate
  • Starch solution
  • Concentrated sulfuric acid
  • Iodide-azide solution – this is alkaline/a base
  • Sodium thiosulfate (0.025N)

BOD Formulation

Working out the biochemical oxygen demand (BOD) involves calculating the quantity of dissolved oxygen utilized by microorganisms throughout the testing duration.

To calculate BOD, you start by subtracting the initial dissolved oxygen concentration (DO1) from the final dissolved oxygen concentration (DO2). This difference is then multiplied by the dilution factor and divided by the sample volume. The resulting value, expressed in mg/L, indicates the oxygen consumed by microorganisms and indicates the level of pollution in the water sample.

Here is the BOD formula:

BOD = (DO1 – DO2) * (Dilution Factor) / (Volume of Sample)


BOD = Biochemical/Biological Oxygen Demand (milligrams per liter (mg/L)

DO1 = The first Dissolved Oxygen concentration (mg/L)

DO2 = The final Dissolved Oxygen concentration after the 5-day test period (mg/L)

The Dilution Factor represents the proportion of the sample volume to the volume of the diluted sample. This factor is utilized in the BOD formula when the sample undergoes dilution to reduce its volume. The “Volume of Sample” refers to the measurement of the undiluted water sample in milliliters (mL).

How To Measure BOD?

Determining the Biological Oxygen Demand (BOD) of water is a meticulous process involving various steps to ensure accurate results. Let’s take a look at each step involved!

Neutralizing the Sample:

Begin by transferring 50 ml of the sample into a 100 ml container or beaker. Utilize a calibrated pH meter to measure the sample’s pH, aiming for a value of 7.00 +/- 0.2. Adjust the pH accordingly with either 1N of sulfuric acid or 1N of sodium hydroxide. 

Make a note of the volume of the acid or base used to neutralize the water sample. Next, work out the amount required to neutralize a 1,000 ml sample and add it accordingly.

Removing the Chlorine Content:

In a conical flask, take 50 ml of the water sample and put in 2.5 ml of the 50% diluted acetic acid and a 10% potassium iodide solution. 

Introduce 1 ml of starch indicator and titrate the mixture with 0.025N sodium sulfite solution. Calculate and add the set volume of sodium sulfite solution to offset the chlorine in the sample.

Preparing the Phosphate Buffer Solution:

Dissolve specified amounts of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and ammonium chloride in 500 ml of distilled water. Dilute the solution to reach a final volume of 1,000ml/1 liter.

Preparation of Alkali-Iodide-Azide Reagent:

In distilled water, dissolve sodium hydroxide and sodium iodide to prepare a 1,000 ml solution. Add sodium azide to complete the preparation.

Preparing the Dilution Water:

Start with 5 liters of distilled water and condition it with clean compressed air for at least 12 hours. Stabilize the water for 6 hours at 20°C. Incorporate specified amounts of calcium carbonate solution, magnesium sulfate solution, and ferric chloride solution. Add 5 ml of the phosphate buffer solution and let the mixture sit for 2 hours.

How to Determine the BOD of Water?

Pour 10 ml of the sample into 2 x 300 ml BOD bottles and fill the rest of the bottle with dilution water until the bottle is full, but not overflowing. 

Prepare 2 additional bottles with dilution water for the blank sample. 

Seal all bottles immediately, ensuring no air bubbles are trapped.

Incubate one sample and one blank bottle at 20°C for 5 days. Analyze the remaining sample and blank vial for dissolved oxygen (DO) immediately. After five days, analyze the incubated bottles for DO.

Testing Dissolved Oxygen (DO):

To the water sample, add 2 ml of a 36.4% manganous sulfate solution followed by 2 ml of iodide-azide reagent. Allow the solutions to mix and react with the dissolved oxygen molecules, and then add 2 ml of sulfuric acid and mix to allow the precipitates to dissolve.

Transfer 203 ml of the (BOD) sample to an Erlenmeyer flask and immediately titrate with the 0.025N Sodium Thiosulfate solution using a Starch indicator. Make a note of the burette readings for both the sample and the control/blank.

Equation & Calculations For Working Out BOD:

To calculate BOD, we use the same formula as above:

BOD = (DO1 – DO2) * Dilution Factor / Volume of Sample

Finally, report the BOD value in mg/L, representing the organic pollution level of the water sample. This value signifies the amount of oxygen consumed by microorganisms during the test period, ensuring a comprehensive assessment of water quality.

Alternative Methods for Measuring BOD

Another prevalent approach to assessing BOD is the 3-day BOD test, akin to the 5-day version but shorter, evaluating dissolved oxygen levels over three days.

Emerging methods gaining traction include respirometry and online monitoring. 

Respirometry tracks oxygen consumption by microorganisms in water samples, aiding in gauging carbon dioxide and nitrogenous waste production, crucial for assessing aquatic ecosystem health. 

Online monitoring, a novel technology, employs sensors for continuous Dissolved Oxygen level tracking, offering real-time insights into water quality shifts or pollution occurrences.

The Pros & Cons Of Using Different BOD Measurement Methods

The Closed Bottle Test:


  • Shorter incubation period (2-3 days)
  • Less time-consuming


  • Less accurate compared to the SP method

The Respirometer Method:


  • Higher accuracy than the closed bottle test
  • Provides detailed insights into oxygen consumption by microorganisms


  • More expensive
  • More time-consuming

The Dilution Method:


  • Cost-effective
  • Less time-consuming


  • Lower accuracy compared to other methods
  • May not provide as detailed insights into oxygen consumption

BOD Measurement Troubleshooting

BOD measurements are susceptible to various challenges, potentially impacting result accuracy and reliability. Here are some common issues and how to deal with them:

Sample Preservation: Maintaining water sample integrity from collection to lab is critical. Improper storage or delayed analysis can alter samples, affecting BOD results.

Fluctuations in Temperature: Precise temperature control during incubation is vital. Temperature changes can affect microbial activity, leading to inaccurate BOD readings.

Contamination: External sources during collection, handling, or analysis can introduce organic matter or microorganisms, skewing BOD values.

Oxygenation: Exposure to atmospheric oxygen during testing can transfer oxygen, affecting measured dissolved oxygen levels. Sealing BOD bottles tightly is crucial.

Partial Metabolism: Some complex organic compounds may not fully metabolize within standard incubation, leading to underestimated BOD values.

Toxic/Harmful Substances: The presence of pollutants like heavy metals or pesticides can inhibit microbial activity, reducing BOD values.

Microbial Interference: High microbial activity can inflate BOD values, overestimating organic pollution levels.


Biochemical/biological oxygen demand (BOD) is an essential parameter for assessing the quality of water. Dissolved oxygen (DO) is measured at the beginning (DO1) and then after 5 days (DO2) at 68°F (20°C). It is the difference between the two DO measurements that is used to calculate the BOD. The BOD indicates the amount of oxygen consumed by microorganisms during the 5-day test period and is expressed in mg/L.

If you want to know more about measuring BOD or would like to know about our range of water quality testing kits, reach out to the world-class team at Atlas Scientific

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