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Indian Institute of Technology Delhi

CEL212 Environmental Engineering
Second Semester 2012-13
(Dr. Arun Kumar; [email protected])
Laboratory 7: Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD)

7A: Biochemical Oxygen Demand (BOD)

Objective : To determine BOD value for determining biodegradability of solution.

Background:
The most widely used test indicating organic pollution of both wastewater and surface
water is the 5-day BOD (BOD5). This determination involves the measurement of the dissolved
oxygen used by microorganisms in the biochemical oxidation of organic matter. BOD5 is the
total amount of oxygen consumed by microorganisms during the first five days of
biodegradation. Oxygen demand is associated with the biodegradation of the carbonaceous
portion of wastes and oxidation of nitrogen compounds such as ammonia. The following
equations simplify the process of biodegradation:
Organic matter + O2 + microorganisms
CO2 + H2O + new microbial cells
Ammonia + O2 + microorganisms
NO3 + H2O + new microbial cells

Procedure:
Apparatus: Incubation bottle 300mL volume; Air compressor, 20°C incubator
Reagents for DO measurement:
1. Manganese sulfate solution: Dissolve 480 g MnSO4.4H2O, 400 g MnSO4.2H2O or 364 g
MnSO4.H2O in distilled water, filter, and dilute to 1L. The MnSO4 solution should not
give a color with starch when added to an acidified potassium iodide (KI) solution.
2. Alkali-iodide-azide reagent
3. Sulfuric acid: One mL is equivalent to ~ 3mL alkali-iodide-azide reagent.
4. Starch solution: Dissolve 2 g laboratory-grade soluble starch and 0.2 g salicyclic acid as
preservative in 100 mL hot distilled water.
5. Standard sodium thiosulfate titrant: Dissolve 6.205 g Na2S2O3 .5H2O in distiller water and
add 1.5 mL 6N NaOH or 0.4 g solid NaOH and dilute to 1000 mL. Standardize with bi-
iodate solution.
6. Standard potassium bi-iodate solution (0.0021M): Dissolve 812.4 mg KH(IO3) in distilled
water and dilute to 1000 mL.
7. Standardization: Dissolve e ~ 2 g KI, free from iodate in an Erlenmeyer flask with 100 to
150 mL distilled water; add 1 mL 6N H2SO4 or a few drops of conc. H2SO4 and 20.00
mL standard bi-iodate solution. Dilute to 200 mL and titrate librated iodine with
thiosulfate titrant, adding starch toward end of titration, when a pale straw color is
reached. When the solution is of equal, 20.00 mL 0.025M Na2S2O3 should be required. If
not, adjust the Na2S2O3 solution to 0.025M.
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Steps:
DO measurement:
1. Make dilution water by adding 2mL/L of following reagents in distilled water:
a. Phosphate buffer solution
b. Magnesium sulfate solution
c. Calcium chloride solution
d. Ferric chloride solution
e. Sodium Sulfite solution
2. For a given sample bottle, add 1 mL of alkali azide and then 1 mL manganous sulfate
solution. Shake well the bottle and keep it open for 5 minutes to settle the precipitate.
Add 2 mL concentrated H2SO4 and place the cap on the bottle. Shake well the bottle till
all the precipitate is dissolved.
3. Take 203 mL of sample in conical flask and titrate with standard sodium thiosulfate
solution (0.025N) till the colour changes from dark yellow to light yellow. Then add few
drops of starch indicator and continue to titrate till the color of the solution becomes
either colorless or changes to its original sample colour. Note down volume of 0.025N
sodium thiosulfate consumed.
4. Calculate DO value of the sample. Remember that in 200 mL sample, 1 mL of sodium
thiosulfate of 0.025N equals to 1 mg/L dissolved oxygen:
=>Dissolved oxygen (DO) (in mg/L) = mL of sodium thiosulfate (0.025N) consumed.

BOD:
1. Prepare BOD dilutions. Use dilution water (it contains nutrients, the exact contents are
described in Standard Methods):Blank (only dilution water);5 mL sample in 300 mL
BOD bottle, fill up with dilution water;15 mL sample in 300 mL BOD bottle, fill up with
dilution water;20 mL sample in 300 mL BOD bottle, fill up with dilution water
2. Take 300 mL sample in BOD bottle. Prepare two sets of this sample. Keep one set for
DO analysis for day 0 (i.e., Sample0Day) and another sample in BOD incubator for 5
days at 20°C (Sample5Day).
3. Measure DO in different samples at t=0.
4. Incubate samples in 20oC for 5 days.
5. Come back in the lab after 5 days and record dissolved oxygen.
6. Record data in following manner.
Bottle no. Wastewater sample (mL) Initial DO (mg/L) DO at 5-day (mL)
(DO0) (DO5)
1
2
3
4
Calculate 5-day BOD value of the sample at 20°C:
t-day BOD= [DOt-DO0]/(P) (1)
where P= Dilution factor = 300mL/(sample volume in mL)

Questions to Answer:
1. If 5-day BOD at 20°C=150mg/L (k=0.23/day), calculate 5-day BOD at 15°C and 22°C?
Use following dependence of (k) with temperature: kT=k20 {θ}(T-20)
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Reference:
AWWA, WEF, APHA, 1998, Standard Methods for the Examination of Water and Wastewater
(Method: 4500-O. C. Azide Modification)

AWWA, WEF, APHA, 1998, Standard Methods for the Examination of Water and Wastewater
(Method: 5210B,5-day BOD)

Sawyer, C.N., McCarty, P.L., and Parkin, G.F. 2000. Chemistry for Environmental Engineering
4th Edition. Tata McGraw-Hill Publishing Company Limited.

++++++++++++Additional questions (not to submit)+++++++++++++

Q: Calculate oxygen consumption rates of samples with given data and comment on
biodegradability
time(d) sample 1(BOD(mg/L) at sample 2(BOD(mg/L) at
20degC) 20degC)
1 5 10
4 10 20
5 70 40
8 90 50
30 120 80
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7B:Chemical Oxygen Demand (COD) (closed reflux method)

Objective 2: To determine COD value for determining organic strength of solution (Closed
Reflux Method)

Background:
Chemical oxygen demand (COD) is termed as the amount of a specific oxidizing agent
that reacts with sample under controlled conditions and it is expressed as oxygen equivalence.
This parameter indicates the extent of organic matter contamination of water and is always
higher than the biochemical oxygen demand (BOD). It is used to indicate organic matter
contamination and it helps in knowing overall organic load to the receiving body.

Selection of Method
There are two methods for COD determination. The first method: open reflux method is
suitable for a wide range of wastes where large volume of sample is required (for samples with
COD= 50 mg O2/L). In the second method: closed reflux methods, small quantities of metallic
salt reagents are required and small quantities of hazardous waste is produced (for samples with
COD= 5 to 50 mg O2/L). In the closed reflux method, ampules and culture tubes with
premeasured reagents are used and then samples is placed in the tube and COD is determined.
In this experiment, closed reflux method is used and samples with COD < 50 mg O2/L
are tested.

Reaction with dichromate solution of sample:

Potassium dichromate is a strong oxidizing agent and it can be used to prepare solution of exact
normality.
CnHaObNc+d Cr2O72- + (8d+c) H+ => nCO2 +[(a+8d-3c)/2]H2O+c NH4+ +2dCr3+ (1)
Here d=(2n/3)+(a/6)-(b/3)-(c/2)

During experiment, excess dichromate concentration is determined by titrating it with ferrous

ammonium sulfate (FAS). The reaction is given by:
6Fe2+ Cr2O72- + 14 H+ => 6Fe3++ 2Cr3+ +7H2O (2)
Here d=(2n/3)+(a/6)-(b/3)-(c/2)

Ferroin (ferrous 1,10-phenanthroline sulfate): It is used to indicate change in oxidation-reduction

potential of the solution and it indicates the condition when all dichromate has been reduced by
ferrous ion. It gives a very sharp brown color change which can be seen in spite of blue color
generated by the Cr3+ ions formed on reduction of the dichromate.

Procedure:
Apparatus: Digestion vessels; block heater; microburet; ampule sealer. Borosilicate culture tubes
(16mm*100 mm or 20 mm*150mm) with TFE lined-screw caps are used. The block heater is
required to operate at 150±2°C with holes to accommodated digestion vessels. Do not use an
oven because of the possibility of leaking samples generating corrosive and explosive
atmosphere.

Reagents:
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a. Standard potassium dichromate digestion solution, 0.01667M: Add to about 500 mL distilled
water 4.903 g K2Cr2O7, primary standard grade, previously dried at 150°C for 2 h, 167 mL conc.
H2SO4, and 33.3 g HgSO4. Dissolve, cool to room temperature, and dilute to 1000 mL.
b. Sulfuric acid reagent:
c. Ferroin indicator solution: Dilute it by a factor of 5 as required. This indicator is used to
indicate change in oxidation-reduction potential of the solution.
d. Standard ferrous ammonium sulfate titrant (FAS), approximately 0.10M: Dissolve 39.2 g
Fe(NH4)2(SO4)2.6H2O in distilled water. Add 20 mL conc H2SO4, cool, and dilute to 1000 mL.
Standardize solution daily against standard K2Cr2O7 digestion solution as follows: Pipet 5.00
mL digestion solution into a small beaker. Add 10 mL reagent water to substitute for sample.
Cool to room temperature. Add 1 to 2 drops diluted ferroin indicator and titrate with FAS titrant.

Molarity of FAS solution = [VK2Cr2O7 ×0.1] / (VFAS) (3)

Where: VK2Cr2O7 = volume of K2Cr2O7 (mL); VFAS = volume of FAS (mL)
e. Sulfamic acid:
f. Potassium hydrogen phthalate standard:

Steps:
1. Wash culture tubes and caps with 20% H2SO4 before using to prevent contamination.
2. Place sample in culture tube or ampule and add digestion solution. Carefully run sulfuric
acid reagent down inside of vessel so an acid layer is formed under the sample-digestion
solution layer and tightly cap tubes or seal ampules, and invert each several times to mix
completely.
3. Place tubes or ampules in block digester preheated to 150°C and reflux for 2 h behind a
protective shield. CAUTION: These sealed vessels may be under pressure from gases
generated during digestion. Wear face and hand protection when handling and dangerous
pressures will be generated at 150°C.
4. Cool to room temperature and place vessels in test tube rack. Some mercuric sulfate may
precipitate out but this will not affect the analysis.
5. Remove culture tube caps and add small TFE-covered magnetic stirring bar. If ampules
are used, transfer contents to a larger container for titrating.
6. Add 0.05 to 0.10 mL (1 to 2 drops) ferroin indicator and stir rapidly on magnetic stirrer
while titrating with standardized 0.10M FAS. The end point is a sharp color change from
blue-green to reddish brown, although the bluegreen may reappear within minutes. In the
same manner reflux and titrate a blank containing the reagents and a volume of distilled
water equal to that of the sample.
7. COD is given by
COD as mg/L O2/L = [(A-B)× M ×8000) / (Vsample) (4)
Where: A = volume of FAS used for blank (mL);
B= volume of FAS used for sample (mL);
M=molarity of FAS; 8000= miliquivalent weight of oxygen ×1000 mL/L
Analyze samples in duplicates because of small sample size.

Questions to Answer:
1. Why do the COD analysis and BOD analysis give different results for the same waste?
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2. What could be inferred from the following samples concerning the relative ease of
biodegradability: Sample A (5-d BOD/COD=24/30) and Sample B (5-d
BOD/COD=10/50)?

Reference:
AWWA, WEF, APHA, 1998, Standard Methods for the Examination of Water and Wastewater
(Methods: 5220 C. Closed Reflux Titrimetric Method)

Sawyer, C.N., McCarty, P.L., and Parkin, G.F. 2000. Chemistry for Environmental Engineering
4th Edition. Tata McGraw-Hill Publishing Company Limited.