Determination of Biological Oxygen Demand (BOD) in Waste Water : Pharmaceutical Guidelines

Determination of Biological Oxygen Demand (BOD) in Waste Water

Determination of the Biochemical Oxygen Demand (BOD) in polluted waste water as a pollution indicator by incubating the BOD bottles for five days at 20 degree C..

Biological Oxygen Demand (BOD):

The amount of oxygen, taken up by the microorganisms that decompose the organic waste matter in waste water is known as biological oxygen demand or biochemical oxygen demand.
Therefore it is used to measure the amount of certain type of organic water pollution  BOD is calculated by keeping a sample of water containing a known amount of oxygen for five days at 20 °C. The oxygen content is measured again and BOD is calculated. A high BOD indicates the presence of a large number of microorganisms which indicates a high level of pollution in wastewater.

BOD determination

1.  Neutralization of Sample:

Accuracy of BOD test totally depends upon the proper bacterial growth, present in the water sample. pH of the diluted sample should be adjusted 7.00 ±0.2 before the incubation for five days for proper results. Sample should be neutralized in following manner.
1.1  Take 50 ml of sample in a 100 ml beaker.
1.2  Measure the pH of the solution by using the calibrated pH meter.
1.3  Add the 1N sulfuric acid to adjust the pH if it is higher than 7.00 and 1N sodium hydroxide if pH is lower than 7.00.
1.4  Note down the volume of sulfuric acid or sodium hydroxide used to adjust the pH of 50 ml sample to 7.00 ±0.2.
1.5  Calculate the volume of sulfuric acid or sodium hydroxide required to neutralize the 1000 ml sample.
1.6  Add the calculated volume of sulfuric acid or sodium hydroxide to the sample to neutralize.
For example, if 2.1 ml of 1N sulfuric acid or sodium hydroxide are used to neutralize 50 ml of sample to pH 7.00 ±0.2.  Calculate the volume of 1N sulfuric acid or sodium hydroxide to be added to neutralize the 1000 ml sample 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 other acid containing chlorine should not be used to neutralize the sample because chlorine interfere the results of waste water BOD.

2.0  Removal of Chlorine Content:

Chlorine is a strong oxidizing agent and it can inhibit the microbial growth during wastewater BOD analysis, so it should be removed from sample before start the analysis. Chlorine can be removed by adding the sodium sulfite to the sample in following manner.
2.1  Take 50 ml of water sample to be tested in a conical flask.
2.2  Add 2.5 ml of acetic acid diluted to 50% with water.
2.3  Add 2.5 ml of 10% w/v solution of potassium iodide.
2.4  Add 1 ml of starch indicator and titrate with 0.025N sodium sulfite solution.
2.5  Note down the volume and calculate to add in 1000 ml of sample as described above in Neutralization of Sample section.
2.6  Add the calculated volume of sodium sulfite solution to the sample and mix well to neutralize the chlorine.

3.0  Preparation of Phosphate Buffer Solution:

Dissolve accurate weighed 8.5 gm of potassium di hydrogen phosphate (KH2P04), 21.75 gm of Di potassium hydrogen phosphate (K2HP04), 33.4 gm of Di sodium hydrogen phosphate (Na2HP04.7H20) and 1.7 gm of ammonium chloride (NH4Cl) in 500 ml distilled water. Dilute the solution up to 1000 ml.

4.0  Preparation of Alkali-Iodide-Azide Reagent:

Dissolve 500 gm of sodium hydroxide (NaOH) and 135 gm of sodium iodide (NaI) in distilled water. Make up the solution to 1000 ml with distilled water. Now dissolve 10 gm of sodium azide in this solution.

5.0  Preparation of Dilution Water:

The dilution water for wastewater BOD analysis must be free from organic content. Dilution water can be prepared by following method.
5.1  Take five liters of double distilled water in a glass container  
5.2  Aerate the water with the clean compressed air for not less than 12 hours.
5.3  Allow to stable for at least 6 hours at 20 °C.
5.4  Add 5 ml of 27.5% w/v solution of calcium carbonate.
5.5  Add 5 ml of 22.5 % w/v solution of magnesium sulfate.
5.6  Add 5 ml of 0.15% w/v solution of ferric chloride.
5.7  Add 5 ml of phosphate buffer solution.
5.8  Mix well and allow to stand for 2 hours.

6.0  Procedure to Determine the Biological Oxygen Demand of Water:

6.1  Take four 300 ml BOD bottles and add 10 ml of samples to two bottles and fill the remaining volume with dilution water.
6.2  Fill remaining two BOD bottle only with dilution water for blank.
6.3  Immediately close the bottles when filled and there should not be any air bubble in the bottle.
6.4  Mark the bottles as blank and sample.
6.5  Incubate one sample and one blank bottle at 20 °C for 5 days.
6.6  Analyze immediately remaining one blank and one sample bottle for dissolve oxygen (DO).
6.7  Analyze incubated bottles for DO after 5 days.

Recommended sample volume for BOD determination
BOD range (mg/liter)
Sample volume (ml)
Dilution water (ml)

7.0  Test for Dissolved Oxygen (DO):

7.1  Add 2 ml of 36.4% of manganous sulfate (MnSO4.H2O) solution inserting the tip of pipette tip into the sample because the drops of solution can allow to insert the oxygen into the solution.
7.2  Add 2 ml of alkali-iodide-azide reagent by above method.
7.3  Allow to react the solutions with the oxygen present in sample.
7.4  When precipitates are settled down at the bottom add 2 ml of concentrated sulfuric acid by placing the pipette tip very near to sample surface.
7.5  Mix well to dissolve the precipitates.
7.6  Take 203 ml of sample from BOD bottle into an Erlenmeyer flask.
7.7  Titrate immediately with 0.025N sodium thiosulfate solution using starch indicator until blue color disappears and note down the burette reading.
7.8  Determine the burette reading for blank in the same manner.


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

Also see: Purified Water System Validation

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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