BOD (3 DAYS, 27°C)

Methode : BOTTLE INCUBATION FOR 3-DAYS AT 27°C
Apparatus
a. BOD bottles, 300 mL, narrow mouth, flared lip, with tapered and pointed ground
glass stoppers.
b. Air incubator or water bath, thermostatically controlled at 27 ± 1°C. Light entry
must beprevented in order to avoid photosynthetic oxygen production
c. Accessories: plastic tube, screw-pin and a 5-10 L water container.

Reagents
a. Phosphate buffer solution. Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g
Na2HPO4.7H2O and 1.7 g NH4Cl in 1L distilled water.
b. Magnesium sulphate solution. Dissolve 22.5 g MgSO4.7H2O in 1L distilled water.
c. Calcium chloride solution. Dissolve 27.5 g CaCl2 in 1L distilled water.
d. Ferric chloride solution. Dissolve 0.25 g FeCl3.6H2O in 1L distilled water.
e. Acid and alkali solution. 1N NaOH and 1N H2SO4. Use for neutralising samples.
f. Glucose-glutamic acid solution (prepare fresh). Dissolve 150 mg dry reagent grade
glucose and 150 mg dry reagent grade glutamic acid in 1L distilled water
g. Sample dilution water. Add 1 mL each of phosphate buffer, MgSO4, CaCl2 and FeCl3
solutions per litre distilled water.

Procedure
a. Prepare required amount of dilution water at the rate of 1000 to 1200 mL per
sample per dilution. Bring the diluted water temperature to 27°C. Saturate with
air by shaking in apartially filled bottle, by bubbling with organic free
filtered air or by storing in cotton-plugged bottles for a day.
b. Some samples do not contain sufficient microbial population (for example, some
industrial wastes, high temperature wastes, or wastes with extreme pH values).
For such wastes, the dilution water is seeded using effluent from a biological
treatment system processing the waste. Where this is not available, use
supernatant from domestic wastewater after settling for at least 1 h but not more
than 36 h. Seed from a surface water body receiving the waste may also be
suitable. Add enough seed volume such that the DO uptake of the seeded dilution
water is between 0.6 and 1.0 mg/L. For domestic wastewater seed, usually 4 to 6
mL seed / L of dilution water is required. Surface water samples usually do not
require seeding.Laboratory Manual ID: 1.2 Version: 1 Page: 2/3
c. Dilution of sample. Dilutions must result in a sample with a residual DO (after 3
days of incubation) of at least 1 mg/L and a DO uptake of at least 2 mgl/L. Make
several dilutions using the Table and experience with the particular sample
source. Polluted surface waters may have 5 to 25 mg/L BOD
Using percent mixture By direct pipetting into 300mL bottles



Table Dilutions for varying BOD values
For preparing dilution in graduated cylinders, siphon dilution water, seeded if necessary,into a 1 to 2 L capacity cylinder. Siphoning should always be done slowly without bubbling,use a screw-pin on the tube to regulate the flow. Keep the tip of the tube just below the water surface as it rises. Fill cylinder half full, add desired quantity of sample and dilute to appropriate level, mix with plunger type mixing rod. Siphon mixed diluted sample in three BOD bottles, stopper without entraining any air. Determine initial DO (method 1.9) on one bottle and incubate the other two at 27°C. Determine final DO (method 1.9) in duplicate after 3days.
For direct pipetting, siphon the desired sample volume to individual bottles and fill with enough dilution water. Complete the test as in the earlier case.

d. Dilution water blank. Find the DO consumption of unseeded dilution water by
determining initial and final DO as in c above. It should not be more than 0.2
mg/L
e. Seed control. Determine the DO uptake by seeding material according to the
procedure in above.

Calculation
a. When dilution water is seeded:


b. When dilution water is not seeded:


where:
D0 = DO of diluted sample initially, mg/L
DT = DO of diluted sample after 3 day incubation at 27°C, mg/L
P = decimal volumetric fraction of sample used
Laboratory Manual ID: 1.2 Version: 1 Page: 3/3
B0 = DO of seed control initially, mg/L
BT = DO of seed control after incubation, mg/L
f = ratio of %seed in diluted sample to %seed in seed control
Notes:
• Report average results of duplicates if both dilutions are correct.
• Formula does not correct for BOD of dilution water which is only valid for
dilution water meeting the criteria. BOD of dilution water should not be more than
0.2 mg/L, preferably lower than 0.1 mg/L.
• The standard glucose-glutamic acid should have BOD of198 ± 37 mg/L (BIS3025 (part
44): 1993). Check procedure otherwise.
• Report BOD values lower than 0.5mg/L or 2 times the measured BOD of the dilution
water (whichever is lower) as lower than detection limit.