Biochemical Oxygen Demand (BOD) IS 3025

Principle

Biological oxygen demand is defined as the amount of oxygen required bacteria in decomposing organic material in a sample under aerobic condition at 20 °C over a period of 5 days.

C₆H₁₂O₆ + 6O₂ microbes CO₂ + H₂O + new microbe

Since, the test is mainly a bioassay procedure, involving measurement of O2 consumed by bacteria while stabilizing organic matter under aerobic condition, it is necessary to provide standard conditions of nutrient supply pH, absence of microbial growth inhibiting substances and temperature. Because of low solubility of O2 in water, strong wastes are always diluted to ensure that the demand does not increase the available oxygen present in the sample. A mixed group of microorganisms should be present in the sample if not, sample has to be seeded artificially temperature is controlled at 20ºC.The test is conducted for 5 days as 70-80 % of the waste is oxidized during this period. In the waste, there are two major biodegradable compounds; the BOD could be divided into Decomposition of carbonaceous compounds (CBOD) and Decomposition of Nitrogenous Compound.

Apparatus & Glasswares

1. Incubation BOD bottles, 300 mL capacity.

2. Incubator: - Thermostatically controlled at 20ºC

3. Volumetric Flask

4. Pipette

5. Burette

Reagent

• Ferric chloride (FeCl3)

• Magnesium sulphate (MgSO4)

• Calcium chloride (CaCl2 )

• Phosphate buffer solution

• Standard sodium thiosulphate 0.025 N

• Standard potassium dichromate (0.25 N )

• Starch indicator

• Alkali Azide solution

• Manganous sulphate

• Nitrification inhibitor

• Glucose-glutamic acid solution

Preparation of Reagents

1. Ferric chloride (FeCl3):

   Dissolve 0.25 g FeCl3.6H2O and dilute to 1000 mL.

2. Magnesium sulphate (MgSO4):

   Dissolve 22.5 g MgSO4 .7H2O in 1000 mL. Dissolve 100 mg metacresol purple in 100 mL water.

3. Calcium chloride (CaCl2 ):

   Dissolve 27.5 g anhydrous CaCl2 in 1L.

4. Phosphate buffer solution:

   Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4, 33.40 g Na2HPO4.7H2O and 1.7 g NH4Cl in distilled water and dilute 1000 mL. The pH should be 7.2 without further adjustment.

5. Standard sodium thiosulphate 0.025 N :

   Dissolve 6.2045 g sodium thiosulphate (Na2S2O3.5H2O) and add 0.4 g NaOH in distilled water and dilute up to 1000 mL .Store in brown bottle. This solution will have to be standardized against potassium dichromate solution for each set of titration.

6. Standard potassium dichromate (0.25 N ):

   Dry K2Cr2O7 at 103 ºC for 2 hours and take accurately 12.259 g and dilute 1000 mL of distilled water.

   Standardization of sodium thiosulphate:

   • Add 2 g KI and 150 mL Distilled water in a 500 mL conical flask.

   • Add few drops of con. H2SO4 and 10 mL of 0.25 N K2Cr2O7.

   • Dilute to 200 mL with distilled water. Keep in dark for 5 minutes.

   • The liberated iodine is titrated with Sodium thiosulphate solution, adding starch indicator towards the end of titration when a pale straw color is reached.

   Normality of sodium thiosulphate = $\frac{N of K2Cr2O7 \times 10}{mL of Na2S2O3}$

7. Starch indicator:

   Take 5 g soluble starch and dissolve in 800 mL boiling water by continuous stirring. Again dilute to 1L and keep boiling for few minutes and then cool it and store in stopper bottle.

8. Alkali Azide solution:

   Dissolve 500 g of NaOH and 135 g of NaI and dilute to 950 mL add 10 g Azide (NaN2) dissolved in 40 mL of distilled water. Cool the solution and make up the volume to 1000mL. Store it in cool and dark and stoppered bottle.

9. Manganous sulphate:

   Dissolve 480 g MnSO4.4H2O / 400g MnSO4.2H2O or 364g MnSO4.H2O in distilled water and dilute to 1000 mL.

10. Nitrification inhibitor:

    2-chloro-6-( trochloromethyl )pyridine (Nitrification inhibitor 2570-24).

11. Glucose-glutamic acid solution:

    Dry reagent grade glucose and glutamic acid at 103°C for 1 h. Dissolve 0.150 g glucose and 0.150 g glucose acid in distilled water and dilute to 1000 mL. Prepare fresh immediately before use.

Procedure

• Dilution water

• DO measurement

  1. After incubation period or titration. Add 1 mL MnSO4 solution + 1 mL of Alkali iodide azide solution.

  2. Re-stopper with care to exclude air bubbles and mix by repeatedly inverting the bottle 2-3 times. Settle down precipitate up to 50%.

  3. Add 1 mL Con. Sulphuric acid. The bottle is re-stoppered and mixed by inverting until the suspension is completely dissolved and yellow color is uniform throughout the bottle.

  4. A volume of 200mL is taken into the conical flask and titrated with 0.025N sodium thiosulphate solution by using starch as indicator till color disappear.

  5. Note down reading and calculate value.

Calculation

BOD mg/L =$\frac{(D0-D3)-(C0-C3)\times 300}{mL of sample }$

D0= Initial DO of diluted water

D3 = DO at the end of 3 days for the diluted water

C0= Initial DO of distilled water

C3= DO at the end of 3 days for the distilled water