Mylan Laboratories Limited

HSF - Hosur Steriles Facility

Hosur, Tamil Nadu, India
Standard Operating Procedure

PROCEDURE FOR ESTIMATION OF BIO CHEMICAL OXYGEN DEMAND (BOD)

MLLHSF-SOP-EHS-XXXX

1.0 PURPOSE

1.1 To lay down procedure for measurement of Bio Chemical Oxygen Demand of
effluent samples in Zero Liquid Discharge Plant (ZLD).

2.0 SCOPE

2.1 This procedure is applicable for Effluent Treatment Plant (ETP), Membrane
Bio Reactor (MBR), Reverse Osmosis (RO) & Forced Evaporation Reactor (FER)
available at Mylan Laboratories Limited, Hosur Steriles Facility (MLLHSF).

3.0 RESPONSIBILITY

3.1 Operators and above of EHS department - to carry out the activity as per the
SOP.

3.2 Associate and above of EHS - to ensure training on SOP to Zero Liquid
Discharge Plant (ZLD) operators.

3.3 Manager/ Team leader - ENG to ensure the periodical maintenance for BOD
incubator.

3.4 DH-EHS/DH-QA or Designee - to ensure the compliance to the SOP.

4.0 REFERENCES

4.1 Indian Standard Procedure – IS 3025.

4.2 Refer instrumentation manual - LABTHERM

4.3 MLLHSF-SOP-QA-QMS-0002 for Incident report.

4.4 MLLHSF-SOP-QA-QMS-0003 for Investigation.

4.5 MLLHSF-SOP-ENG-MAN-0002 for Breakdown maintenance.

 Mylan Laboratories Limited
HSF - Hosur Steriles Facility
Hosur, Tamil Nadu, India
Standard Operating Procedure

PROCEDURE FOR ESTIMATION OF BIO CHEMICAL OXYGEN DEMAND (BOD)

MLLHSF-SOP-EHS-XXXX

5.0 DEFENITION
5.1 BOD: It is the amount of oxygen utilized during a specified incubation
period for biochemical degradation of organic materials and other
inorganic such as sulfides / ferrous ion / to oxidize reduced forms of
nitrogen compounds contained in unit volume of effluent by maintaining
specific requirements like pH, temperature, nutrients for the survival of
microorganisms. Micro organisms are responsible for degradation. BOD is
expressed in mg/L which is corresponding to specific temperature &
incubation period.
5.2 DO: Dissolved Oxygen
5.3 mL: milliliter
5.4 g: gram
5.5 L: Liter
0
5.6 C: Degree Centigrade
5.7 N: Normality
5.8 BC : Blank Correction

6.0 PROCEDURE
6.1 Principle
6.1.1 The method consists of filling diluted and seeded samples into
airtight bottles, to over flow, of specified size and incubating them at
the specified temperature for 3 days at 270C. Dissolved oxygen
shall be measured before and after incubation, and BOD is
computed from difference in initial and final DO.
6.2 Apparatus
6.2.1 Incubation Bottles 300 mL, conical flasks, pippets
6.2.2 Air incubator
6.3 Reagents preparation:
 Mylan Laboratories Limited
HSF - Hosur Steriles Facility
Hosur, Tamil Nadu, India
Standard Operating Procedure

PROCEDURE FOR ESTIMATION OF BIO CHEMICAL OXYGEN DEMAND (BOD)

MLLHSF-SOP-EHS-XXXX

6.3.1 General
Prepare reagents in advance. Discard the reagents if any sign of
microbial growth or precipitation is observed in stock bottles. Used
distilled water, sterilized preferably, to prepare the reagents. All
dilutions are made in volumetric flasks.
6.3.2 Phosphate Buffer solution:
Dissolve 8.5g of Potassium hydrogen phosphate (KH 2PO4), 21.75 g
of Dipotassium Hydrogen Phosphate (K 2HPO4), 33.4 g of Disodium
Hydrogen phosphate Hepta Hydrate (Na 2HPO4.7H2O) and 1.7 g of
Ammonium Chloride NH4Cl in about 500 mL distilled water and
dilute to 1 L
6.3.3 Magnesium Sulphate solution:
Dissolve 22.5 g of Magnesium Sulphate Hepta Hydrate
(MgSO4.7H2O) in distilled water and dilute to 1 L
6.3.4 Calcium Chloride (CaCl2) solution:
Dissolve 27.5 g CaCl2 in distilled water and dilute to 1 L
6.3.5 Ferric Chloride solution:
Dissolve 0.25 g of Ferric Chloride in distilled water and dilute to 1 L
6.3.6 Sodium sulfite solution (0.025 N):
Dissolve 1.575 g Na2SO3 and dilute to 1 L. Solution should be
prepared freshly.
6.3.7 Acid and Alkali Solutions:
Acid: Slowly while stirring, add 28 mL conc. Sulfuric acid to distilled
water. Dilute to 1 L.
Alkali: Dissolve 40 g NaOH (C.S flakes) in distilled water. Dilute to 1
L.
6.3.8 Ammonium Chloride Solution:
Dissolve 1.15 g NH4Cl in about 500 mL distilled water, adjust pH to
7.2 with NaOH solution, and dilute to 1 L. Solution contains 300 mg
N/ L.
6.3.9 Manganous Sulphate solution:
 Mylan Laboratories Limited
HSF - Hosur Steriles Facility
Hosur, Tamil Nadu, India
Standard Operating Procedure

PROCEDURE FOR ESTIMATION OF BIO CHEMICAL OXYGEN DEMAND (BOD)

MLLHSF-SOP-EHS-XXXX

Weigh 480 g of MnSO4. 4 H2O or 400 g of MnSO4. 2 H2O or 364 g of

MnSO4. H2O of and dissolve in distilled water and dilute to 1000 ml
in a volumetric flask. Filter if necessary.
6.3.10 Alkali Iodide-azide reagent:
Dissolve 500 g NaOH (or 700 g KOH) and 135 g NaI (or 150 g KI)
in distilled water and dilute to 1 L. Add 10 g NaN 3 dissolved in 40
mL distilled water. Potassium and sodium salts maybe used
interchangeably.
6.3.11 Sulfuric Acid:
H2SO4 Conc. 1 ml is equivalent to about 3 ml alkali-iodide-azide
reagent.
6.3.12 Starch Indicator:
Dissolve 2 g laboratory grade soluble starch and 0.2 g salicylic
acid, as a preservative, in 100 mL hot distilled water. Cool the
solution and store it for use.
5.3.13 Standard Sodium thiosulphate titrant (0.025 M):
Dissolve 6.205 g Na2S2O3. 5H2O in distilled water. Add 1.5 mL 6N
NaOH or 0.4 g solid NaOH and dilute to 1000 mL. Standardize the
this solution as per point no. 5.3 of SOP/cEHS/ENV/STP/006/00
6.4 Testing Procedure
6.4.1 Sampling, Storage and pretreatment:
If analysis begins with in 2 h of sample collection, no cold storage is
required. Store at or below 4 0C for analysis with in 6 h. For higher
times, report the time and temperature of storage. Do not start the
analysis after 24 h in any case. Check pH. If it is not between 6 and
8, adjust temperature to 20 ± 3 0C and adjust pH to 7 using
solutions of H2SO4 and/or NaOH solutions. If samples contain
residual chlorine, it has to be dechlorinated before testing.
Generally all residual chlorine dissipates in 1-2 h of standing in
light. If chlorine does not dissipate in short period sodium sulfite
solution is added to destroy chlorides.
6.4.2 Preparation of Dilution water:
 Mylan Laboratories Limited
HSF - Hosur Steriles Facility
Hosur, Tamil Nadu, India
Standard Operating Procedure

PROCEDURE FOR ESTIMATION OF BIO CHEMICAL OXYGEN DEMAND (BOD)

MLLHSF-SOP-EHS-XXXX

Check to ensure dilution water DO concentration is at least 7.5

mg/L before using for BOD tests. If not, aerate distilled water with
organic free filtered air by taking into a large container for 8 hrs to
24 hrs and ensue the DO is 7.0 – 7.5 mg/L. Use this dilution water
for sample preparation. Add 1 mL each of phosphate buffer,
Magnesium Sulphate, Calcium Chloride and Ferric Chloride
solutions each one liter of prepared distilled water. Mix thoroughly
and bring temperature to 20 ± 3 0C. Prepare dilution water
immediately before use.
6.4.3 Seeding Suspension:
The seed can be collected from an existing biological treatment
system. Use supernatant from settled mixed liquor from aeration
basin.
6.4.4 Preparation of Dilutions:
Using the above dilution water, prepare at least 5 dilutions ranging
between 0.01 and 1 %. Dilution of the sample must be such that
there is minimum DO uptake of 2 mg/L and residual DO of 1 mg/L
after incubation. The actual sample volume for each kind of effluent
must be determined by experience.
Dilutions can be either made in volumetric flask or directly in the
BOD bottles. For dilutions greater than 1:100 make primary dilution
before making final dilution in another flask. After filling the
volumetric flask up to 2/3rd of volume, add 1-3 mL of seeding
suspension and dilute to final volume. Transfer the sample into
BOD bottles.
6.4.5 Sealing of bottles:
Complete filling of each bottle such that on insertion of stopper no
air bubbles are left in the bottle. Seal the cap with plastic sheet to
prevent loss of water due to evaporation.
 Mylan Laboratories Limited
HSF - Hosur Steriles Facility
Hosur, Tamil Nadu, India
Standard Operating Procedure

PROCEDURE FOR ESTIMATION OF BIO CHEMICAL OXYGEN DEMAND (BOD)

MLLHSF-SOP-EHS-XXXX

6.5 Determination of BOD:

6.5.1 Take two set of 300 mL glass stoppered BOD bottles.
6.5.2 Add 10 mL of the sample to each of the two BOD bottles and the fill
the remaining quantity with the dilution water. i.e., we have
diluted the sample 30 times.
6.5.3 The remaining two BOD bottles are for blank, to these bottles add
dilution water alone.
6.5.4 After the addition immediately place the glass stopper over the BOD
bottles and note down the numbers of the bottle for
identification.
6.5.5 Now preserve one blank solution bottle and one sample solution
bottle in a BOD incubator at 20ºC for five days.
..The other two bottles (one blank and one sample) needs to be
analysed immediately.
6.5.7 Avoid any kind of bubbling and trapping of air bubbles. Remember –
no bubbles!
6.5.8 Add 2mL of manganese sulfate to the BOD bottle by inserting the
calibrated pipette just below the surface of the liquid.
6.5.9 Add 2 mL of alkali-iodide-azide reagent in the same manner.
(The pipette should be dipped inside the sample while adding the
above two reagents. If the reagent is added above the sample
surface, you will introduce oxygen into the sample.)
6.5.10 Allow it to settle for sufficient time in order to react completely with
oxygen.
6.5.11 When this floc has settled to the bottom, shake the contents
thoroughly by turning it upside down.
6.5.12 Add 2 mL of concentrated sulfuric acid via a pipette held just above
the surface of the sample.
 Mylan Laboratories Limited
HSF - Hosur Steriles Facility
Hosur, Tamil Nadu, India
Standard Operating Procedure

PROCEDURE FOR ESTIMATION OF BIO CHEMICAL OXYGEN DEMAND (BOD)

MLLHSF-SOP-EHS-XXXX

6.5.13 Carefully stopper and invert several times to dissolve the floc.
 Mylan Laboratories Limited
HSF - Hosur Steriles Facility
Hosur, Tamil Nadu, India
Standard Operating Procedure

PROCEDURE FOR ESTIMATION OF BIO CHEMICAL OXYGEN DEMAND (BOD)

MLLHSF-SOP-EHS-XXXX

6.5.14 Titration needs to be started immediately after the transfer of the

contents to Erlenmeyer flask.
6.5.15 Rinse the burette with sodium thiosulphate and then fill it with
sodium thiosulphate. Fix the burette to the stand.
6.5.16 Measure out 203 mL of the solution from the bottle and transfer to
an Erlenmeyer flask.
6.5.17 Titrate the solution with standard sodium thiosulphate solution until
the yellow color of liberated Iodine is almost faded out. (Pale
yellow color)
6.5.18 Add 1 mL of starch solution and continue the titration until the blue
color disappears to colourless.
6.5.19 Note down the volume of sodium thiosulphate solution added ,
which gives the D.O. in mg/L. Repeat the titration for
concordant values.
6.5.20 After five days, take out the bottles from the BOD incubator and
analyse the sample and the blank for DO.
6.5.21 Repeat the steps 6.5.8 to 6.5.19

6.6 Calculation

Initial dissolve oxygen value for blank B1 (day 1) = ------- ml

Final dissolve oxygen value for bkank B2 (day 5) = ----- ml
Initial dissolve oxygen value for sample D0 (day 1) = ------ ml
Final dissolveoxygen value for sample D5 (day 5) = ------ ml
Blank Correction (BC) = B1 – B2
 Mylan Laboratories Limited
HSF - Hosur Steriles Facility
Hosur, Tamil Nadu, India
Standard Operating Procedure

PROCEDURE FOR ESTIMATION OF BIO CHEMICAL OXYGEN DEMAND (BOD)

MLLHSF-SOP-EHS-XXXX

(D0-D5-BC)× Volume of diluted sample

Bod in mg/l = --------------------------------------------------------
Volume of sample taken
 Mylan Laboratories Limited
HSF - Hosur Steriles Facility
Hosur, Tamil Nadu, India
Standard Operating Procedure

PROCEDURE FOR ESTIMATION OF BIO CHEMICAL OXYGEN DEMAND (BOD)

MLLHSF-SOP-EHS-XXXX

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