Apparatus:

* Incubator

* Stoppard Bottles

* Burette

* Pipette

* Conical flask

Chemicals: Reagents

* MnSO₄ soln (364 gm MnSO₄.H₂O + 1 ltr boiling D.W.)

* CaCl₂ soln. (27.5gm anhydrous CaCl₂ + 1 Ltr D.W.)

* FeCl₃ soln. (0.25gm FeCl₃.6H₂O + 1 Ltr D.W.)

* Phosphate Buffer (8.5gm KH₂PO₄ +21.75 gm K₂HPO₄ +33.4gm Na₂HPO₄.7H₂O +1.7gm NH₄Cl + 1 Ltr D.W)

* MgSO₄ soln. (22.5 gm MgSO₄.7H₂O + 1 Ltr D.W.)

* Alkali Azide (a + b)

a. (500 gm NaOH + 135 gm NaI + 1 Ltr D.W)

b. (10gm NaN₃ + 40 ml D.W)

* Conc. H₂SO₄

* Starch indicator soln (2 gm starch +0.2 gm salicyclic acid +100 ml hot D.W.)

* 0.025 N Sodium Thiosulphate(6.205 gm Na₂S₂O₃.5H₂O + 1 Ltr D.W+ 0.4 gm NaOH )

Procedure:

1. Preparation of Dilution Water

* Wash 1 litre beaker in distilled water and take 1 litre of Distilled water in it.

*Add 1mL of MgSO₄, CaCl₂, FeCl₃ & Phosphate buffer.

* Aerate the sample for ½ hr to 2 hr.

2. Determination of Initial D.O.

* Take dilution water in 300 ml stoppered bottle

* Add 1mL of MnSO₄ Soln

* Add 1ml of “Alkali Azide” soln

* There is a formation of precipitate of Mn(OH)₂

* Add 1ml of concentrated sulphuric acid

* Shake the bottle till precipitate dissolves completely

* Transfer 201 ml of solution to a 500ml conical flask

* Titrate this solution against 0.025 N Sodium Thiosulphate (6.205 gm Na₂S₂O₃.5H₂O + 1 Ltr D.W. + 0.4 gm NaOH ) till colour becomes pale yellow.

* Add starch indicator till solution becomes blue

* Continue titrating with Thiosulphate till the solution becomes colourless

* The reading of the burette corresponds to the initial dissolved Oxygen in ppm

* One ml of 0.025 N- thiosulphate = 1 ppm of Dissolved Oxygen.Determination of final dissolved oxygen

* Take dilution water in 300 ml of stoppered bottle

* Add sample quantity according to the chart attached.

* Keep the bottles in incubator for 3 days at 27⁰C.

* Incubate a blank sample with each batch of samples

* After 3 days remove sample from the incubator

* Add 1ml of MnSO₄ followed by 1ml of Alkali azide reagent

* The precipitate of Mn (OH)₂ is dissolved by adding 1ml of concentrated sulphuric acid

* Transfer 201 ml of solution to a 500 ml conical flask and titrate with 0.025N

Thiosulphate

* The pale yellow colour is titrated further by adding starch till the blue colour is completely discharged & becomes Colourless.

* The burette reading obtained corresponds to the final dissolved oxygen.

Calculations: BOD ppm = (A – B) / P

Where

A = Initial Dissolved Oxygen

B = Final Dissolved Oxygen

P = Volumetric fraction of sample taken

Example: if 10 ml of sample is taken then P = (10 / 300)

Precision & Bias:There is no precision & bias for Biochemical Oxygen Demand.