OBJECTIVE:

To use the Winkler Titration Method to determine the BOD value of water sample.

APPARATUS:
a) Beakers
b) Conical Flasks
c) Burette
d) Graduated Cylinder
e) Pipette
f) Volumetric Flasks
g) BOD bottles
h) Reagent bottles
i) Incubator
j) Distilled Water
k) Dilution Water
l) Starch solution
m) Sulfuric acid
n) Manganous Sulphate Solution
o) Alkali-iodize-azide reagent
p) Concentrated Sulphuric acid
q) Natrium Thiosulphate Titrant

PROCEDURE:
A. Preparation of sample

1. Prepare 3 litres of Dilution water by adding with per litre of distilled water a 1 ml
of prepared solutions of phosphate buffer, magnesium sulphate (MgSO4), Calcium
Chloride (CaCl2), Ferric Chloride (FeCl3) and Alitiourea (ATU).
2. Aerated the dilution water by shaking it in a partially filled bottle.
3. Label two 1 Litre of volumetric flasks with ‘sample’ and ‘blank’ respectively. The
flask water labelled ‘blank’ is used as control to make sure that the dilution water
is not polluted.
4. Transfer 200 ml of water sample into the flask labelled ‘sample’ and add dilution
water until it reaches the 1 Litre level, which will give a dilution 1/5 of the
sample. Shake the flask thoroughly.
5. To the ‘blank’ flask, transfer 1 Litre of Dilution water.
6. Label 4 clean BOD bottles as 1-4.
7. Fill bottle 1 & 2 with solution from ‘blank’ flask and bottle 3 & 4 with solution
from ‘sample’ flask.
8. Cover the bottles carefully.
9. Determine the initial (day zero) dissolved oxygen value for solution in bottle 1 &
3 using Winkler Titration Method.
10. Incubate bottle 2 & 4 for 5 days ± 1 hour at temperature 20 ± 1˚C.
 11. Determine the dissolved oxygen values of solutions in bottle 2 & 4 after 5 days.

B. Dissolved Oxygen (DO)

1. To the sample collected in a 250 – 300 ml reagent bottle, add 1 ml of Manganous

Sulphate (MnSO4) solution, followed by 1 ml Alkali-iodide-azide reagent. Make
sure to rinse the pipettes using distilled water before returning them to reagent
bottles, if they are dipped into sample.
2. Stopper carefully to exclude air bubbles and mix by inverting bottle few times.
Allow to settle. Shake the bottle once again and allow to settle.
3. When precipitate has settled successfully to approximately half the bottle volume,
add 1 ml concentrated sulphuric acid.
4. Restopper and mix by inverting several times until dessolution is complete.
5. Transfer 100 ml of the solution into a 250 ml conical flask.
6. Titrate with 0.0125M natrium thiosulphate (Na2S2O3) solution to a pale straw
colour.
7. Add 5 drops of starch solution and continue titration to first disappearance of blue
colour.
8. Record the reading and repeat the Titration process.
9. Calculation: 1ml 0.0125M Na2S2O3 = 1 mg/1DO.

RESULT:
Sample type: ___________________________
Dilution Ratio: __________________________

Day 0 Dissolved Oxygen

Sample

Burette Reading ‘Blank’ (bottle 1) ‘Sample’ (bottle 3)

(i) (ii) (i) (ii)

Initial
Final
Volume
Average
DO (mg/l)
Day 5 Dissolved Oxygen

Sample

Burette Reading ‘Blank’ (bottle 2) ‘Sample’ (bottle 4)

(i) (ii) (i) (ii)

Initial
Final
Volume
Average
DO (mg/l)

(𝐷1 −𝐷2 )−(𝐵1 −𝐵2 )𝑓

BOD5 (mg/l) =
𝑃

Where, D1 = DO of Diluted sample immediately after preparation (mg/l).

D2 = DO of diluted sample after 5 days incubation at 20 ˚C (mg/l).
B1 = DO of seeded blank just before incubation (mg/l).
B2 = DO of seeded blank after incubation (mg/l).
P = Decimal Volumetric fraction of sample used.
f = Ratio of seed in diluted sample to seed in seeded blank.