Deals with the measurement of organic matter concentration in water and wastewater. BOD, BOD kinetics and COD tests are discussed at length. Further, as part of the ultimate BOD measurement, other associated tests like Dissolved Oxygen and Ammonical, Nitrate and Nitrite forms of nitrogen are also discussed.

1. Organic Matter
(Non-biodegradable and Biodegradable)
(TOC, COD, DO, BOD, BODu and serial BOD)
Dr. Akepati S. Reddy
Thapar University
Patiala (PUNJAB) – 147 004
INDIA

2. Measurement of organic matter
concentration
Organic matter in wastewater is heterogeneous
– Suspended colloidal and dissolved organic matter
– Carbohydrates, proteins and fats
Single direct method for the measurement of organic matter is not
feasible – so indirect methods are depended on
• Total organic carbon –TOC:
• Organic matter invariably has carbon, and the Organic Carbon (OC)
content is proportional to the Organic Matter (OM) content
• Samples also have inorganic carbon (carbonates, bicarbonates, etc.)
and these interfere in the measurement of organic carbon
• Samples are first treated for the removal of the inorganic carbon, and
then treated to convert the OC into CO2 and this in turn is measured

3. Measurement of organic matter
concentration
Oxygen Demand (ThOD, COD and BOD)
– Organic matter is a reduced substance
– OM can be completely oxidized and transformed into inorganic end
products and this demands oxygen
– The amount of oxygen demanded is proportional to the organic
matter concentration of the sample
Oxygen demand of the sample’s organic matter is measured as
– Theoretical Oxygen Demand (ThOD): Oxygen demand (of the
sample’s organic matter can be theoretically found through
stoichiometry, if chemical formula of the OC is known
– Chemical Oxygen Demand (COD): Organic matter of a sample is
chemically oxidized, and oxygen demand of the sample’s OC is
measured in terms of the amount of oxidizing agent consumed
– Biological Oxygen Demand (BOD): microorganisms are made to use
the sample’s organic matter as food and aerobically oxidize into
inorganic end products, and oxygen utilized is measured as BOD

4. Theoretic Oxygen Demand
Empirical formula of the organic matter present in the sample
should be known
Using this empirical formula a balanced equation should be
written
With the help of the balanced equation estimate stoichiometric
oxygen demand (for the complete oxidation of one unit mass of
the organic matter)
Estimate the oxygen demand equivalent to the organic matter
present in the sample
3222
2
3
24
3
24
cNHOH
ca
nCOO
cba
nNOHC cban +





−+→





−−++
oxygengrequireseglugofOxidation
OHCOOOHC
192cos180
666 2226126 +→+

5. Chemical Oxygen Demand (COD)
• Measures oxygen equivalent of organic matter provided the latter
is susceptible to oxidation by potassium dichromate
• Oxidation (wet) is brought about under acidic conditions (created
by H2SO4 reagent) at high temp. (150ºC± 2o
C) for 2 hrs., and can
be shown by:
Cn
Ha
Ob
Nc
+dCr2
O7
-2
+(8d+c)H+
→ nCO2
+ {(a+8d-3c)/2}H2
O+cNH4
+
+2dCr+3
d is moles of dichromate consumed
One mole dichromate = 1.5 moles of COD
• Not a good measure for biodegradable organic matter and not
capable of oxidizing all the organic matter
• Still widely used because real time and reasonable time results are
possible
• In case of anaerobic treatment COD is preferred over BOD
2363
2 cban
d −−+=

6. Biochemical Oxygen Demand (BOD)
• Microorganisms are used to oxidize the organic matter
aerobically under favourable conditions of pH, temperature,
osmotic pressure and nutrients
– Sample is incubated with acclimatized microorganisms at a
specific temperature (20/27°C) for specified period (5/3 d)
– Ensured by adding phosphate buffer, ferric chloride, calcium
chloride and magnesium sulfate salts
– For ensuring sufficient oxygen availability the sample is diluted
– O2 demand by acclimatized microorganisms and nitrification and
the O2 demand of the dilution water are found and corrected
• Organic matter is used by organisms as food and oxidize – only
the matter that can be consumed as food (biodegradable
fraction) can be measured
– COD on the other hand measures both biodegradable non-
biodegradable organic matter

7. Fate of organic matter of the sample in the BOD test
Organic Matter
(dissolved)
Non-biodegradable
& residual organic matter
Suspended & colloidal
organic matter
oxygen
CO2, H2O, NH3, Energy, etc.
New heterotrophic
Microbial biomass
Auto-oxidation
CO2, H2O, NH3, Energy, etc.
ammonia
oxygen
nitrite nitrate
oxygen
(Nitrogenous BOD)
BOD is sum of oxygen utilized during biooxidation of the organic matter
and during autooxidation of the microbial biomass
(Carbonaceous BOD)
oxygen
Nitrification
Residual biomass
Cell debrisBio-oxidation
Bio-synthesis
hydrolysis

8. Conclusions drawn from the analysis of the
fate of organic matter during BOD test
• Oxygen demand exerted is having
– Demand for biooxidation of organic matter and for autooxidation
of microbial biomass (carbonaceous BOD)
– Demand for the nitrification of the ammonia generated
(nitrogenous BOD) – chemical inhibition of nitrification
– Demand of the seed and of the dilution water used
• Because of non-biodegradable organic matter, residual organic
matter, and residual biomass, BOD is always lesser than ThOD
• Unless some of the biodegradable organic matter is resistant to
chemical oxidation BOD is lesser than COD
• Complete biodegradation of organic matter needs infinite time
• BOD includes two components: Carbonaceous BOD and
Nitrogenous BOD

9. Ultimate BOD
BODt is the sample’s oxygen demand when it is incubated for ‘t’
time (3 or 5 days) at X C temperatureᵒ
• Higher the temperature lower will be the time
Only a portion of the biodegradable organic matter is oxidized -
oxidation of total matter requires >25 d (60-90 days)
BODu test wherein the sample is aerated at regular interval and
incubated till daily demand becomes <1 or 2% of the
cumulative demand is used for finding
• Nitrification demand of oxygen is parallelly quantified and
subtracted from the BOD
Incubating and waiting for that long period for results is not
desirable but knowing ultimate BOD (BODu) is considered
important
For this the BODt results are extrapolated through using BOD
kinetics model which assumes that the BOD exertion follows
first order decreasing rate of increase

10. Oxygen demand exertion pattern of a sample during incubation

11. BOD kinetics
Oxygen demand exertion pattern is first order decreasing rate of
increase and can be shown as
ttou LBODLBOD
''
+==
ttimegivenanyat
exp(-k.t)}-{1LBOD
BOD
ot
t
=
aswrittenbecan
( )20
20T kk −
= T
φ
T is temp. in °C
φ is constant - taken as 1.056 for
20-30°C and as 1.135 for 4-20°C
kL-dL/dt
L0
=
+= tt LBOD
exp(-k.t)LL ot =
dL/dt is rate of oxygen demand exertion
Lt is oxygen demand that is yet to be exerted at
after incubation time ‘t’
L0 is oxygen demand to be exerted by the sample
at incubation time ‘zero’ (also known as BODu)
k is BOD reaction rate constant
K and L0 are known as BOD kinetics parameters
Use of BOD kinetic model requires knowledge of BOD kinetic parameters

12. Chemical Oxygen Demand (COD)
by Open and Closed (Titrimetry and
Spectrophotometry) Reflux Methods

13. COD
• Measure of oxygen equivalent of organic matter content of sample
• Oxidation of organic matter occurs under acidic conditions at
elevated temperature (150±2C) for about 2 hours
• Oxidation can be shown by
• Hexa-Cr is orange colored and Tri-Cr is greenish blue in color
– As a consequence of conversion of haxa-Cr into Tri-Cr, color of
digestion mixture changes from orange to greenish blue
• Amount of dichromate consumed is basis for COD estimation
(one mole dichromate consumption is equivalent to 1.5 moles
of COD)
• Oxidation is not complete - measures only the organic matter
susceptible to oxidation by potassium dichromate
( ) ( ){ } 3
422
2
72 22/388 ++−
++−++→+++ dCrcNHOHcdanCOHcdOdCrNOHC cban
2363
2 cban
d −−+=

14. COD
• Pyridine (and related compounds) and aromatic hydrocarbons are
not completely oxidized
• VOCs (originally present or formed durin oxidation) are oxidized
only to the extent of their contact with oxidant (at elevated temp.
may escape oxidation)
– Silver sulfate is used as catalyst for the effective oxidation of VOCs
– Halides of the sample form silver halides and make catalyst ineffective
– Mercuric sulfate is used at 10:1 ratio for preserving the effectiveness
(not appropriate when the halides level is >200 mg/l)
• Use of reflux condensers or closed reflux (or sealed digestion
containers), minimize escape of VOC from oxidation
• Oxidation at elevated temps, results in thermal decomposition of
the dichromate used and introduces positive error
– For estimating the error and making correction, a blank is digested
along with the sample
• Nitrite (NO2-), reduced inorganic species (like chloride, ferrous iron,
sulfide, manganous manganese) and ammonia (from organic mater
oxidation!) can also be oxidized and introduce positive error

15. COD
• Interference caused by chloride ions can be shown by
– Oxidation of ammonia requires presence of significant levels of free
chloride ions
– Addition of excess mercuric sulfate prior to addition of other reagents
can eliminate chloride ion interference by making ions non-available
• Nitrite level is rarely >1-2 mg/l and hence insignificant interference
– Remove interference by adding 10 mg sulfamic acid per mg of nitrite
• Error introduced by other inorganic species, if significant, is
stoichiometrically estimated and necessary corrections are made
• Collect samples in glass bottles, and test preferably immediately
– If delay is unavoidable, acidify samples with H2SO4 to 2 pH and store
– If stored at room temperature, test within 7 days, and if stored at 4C,
then test within 28 days
– If sample has settlable solids, then homogenize the sample in a
blender prior to testing
• Two alternate methods (open reflux and closed reflux methods) are
used in the COD meaurement
OHCrClHOCrCl 2
3
272 723146 ++→++ ++−

16. COD by Open reflux method
• Sample and blank are refluxed in strongly acidic solution in the
presence of known excess of standard K2Cr2O7 solution for 2 hours
• A reflux apparatus, comprising of an Erlenmeyer flask, a vertical
condenser and a hot plate/heating mantle, is used for refluxing
• During refluxing
– Hexa-Cr of the K2Cr2O7 is reduced to tri-Cr and supplies oxygen
– Some fraction of the added dichromate is thermally decomposed
• Residual dichromate of the sample and of the blank are measured
by titrating against standard ferrous ammonium sulfate (FAS)
– Ferroin is used as indicator
– Titration involves conversion of residual hexa-Cr into tri-Cr
– Once all the Hexa-Cr is converted into Tri-Cr, Fe+2
ions of FAS form a
complex (of intense orange brown colour) with ferroin indicator
– Color change from greenish blue to orange brown is end point
– Redox potentiometer can also be used to detect the end point
++++
+→+ 3362
33 CrFeCrFe

17. COD by Open reflux method
• COD of the sample is calculated by:
• Open reflux method is associated with
– Consumption of costly and hazardous chemicals, like, silver sulfate,
mercuric sulfate etc.,
– Generation of hazardous waste with chromium, mercury, silver, etc.
• To reduce cost and minimize hazardous waste generation of,
instead of 50 ml, use smaller sample size (10 ml!)
– Smaller size samples demands proper homogenization of samples in
blender prior to use
• Refluxing time less than 2 hours can be employed provided the
results obtained are same as those obtained from 2 hour refluxing
8000
).(
/( 2
usedsampleofml
MBA
OaslmgCOD
−
=
‘A’ is ml FAS consumed in blank titration
‘B’ is ml FAS consumed in sample titration
‘M’ is molarity of FAS

18. COD by Open Reflux Method
Apparatus and reagents
• Reflux apparatus: digestion flask (capacity depends on sample
size - 125/250/500 ml) containing sample and reagents, glass
condenser with hoses (for cooling water), hot plate and stand
with clamps
• Sulfuric acid reagent: Add Ag2SO4 to conc. H2SO4, at 5.5 g/kg rate,
and allow the reagent to stand for 1-2 days
• Ferroin indicator: Dissolve 1.485 g 1,10-phenanthroline
monohydrate and 695 mg FeSO4.7H2O in distilled water and
adjust volume to 100 ml.
• Potassium hydrogen phthalate (KHP) standard (500 mg/L COD):
Dry crushed potassium hydrogen phthalate (HOOCC6H4COOK) to
constant weight at 120 C, dissolve 425 mg in distilled water andᵒ
adjust volume to 1.0 L (1 mg KHP = 1.176 mg COD)
• KHP standard, in the absence of visible biological growth, is stable for
3 months under refrigeration
• Mercuric sulfate (HgSO4) crystals or powder.

19. COD by Open Reflux Method
Apparatus and reagents
• Standard potassium dichromate (0.0417M): Dissolve 12.259 g
K2Cr2O7 (dried at 103 C for 2 hours) in water and adjust volumeᵒ
to 1.0 L
• Standard ferrous ammonium sulfate, FAS (0.25M): Dissolve 98 g
Fe(NH4)2(SO4)2.6H2O in distilled water, add 20 ml conc. H2SO4, cool
and adjust volume to 1.0 L
Standardization of FAS
• FAS tends to lose strength with age and requires standardization
prior to use
• Take 10 ml of standard dichromate solution, dilute to 100 ml, add
30 ml of concentrated sulfuric acid, cool, add 0.1 to 0.15 ml (2 to
3 drops) of ferroin indicator and titrate with FAS solution
• Using the volume of FAS solution consumed find strength of FAS
by
DichromateofMolarity
consumedFASofml
takenDichromateofml
FASofMolarity =

20. Open Reflex Method: Procedure
• Take 50 ml homogenized sample in 500 ml refluxing flask and add in
the same order 1 g mercuric sulfate, a few glass beads and 5.0 ml of
H2SO4 reagent while mixing and cooling the contents
• Parallel to the sample prepare a blank with distilled water as sample
and carry out the testing
• Add 25 ml of standard potassium dichromate solution (0.0417M)
• When smaller volume of sample is taken, smaller refluxing flask can be
taken and addition of mercuric sulfate, sulfuric acid reagent and
dichromate solution can be proportionately reduced
• When sample has low COD (< 50 mg/l), use diluted dichromate
solution (0.00417M)
• When having very low COD, take >50 ml sample (100 or 150 ml).
• Attach refluxing flask to condenser, turn on condenser cooling
water, add 70 ml H2SO4 reagent through the condenser opening,
and thoroughly mix the flask contents
• Volume of H2SO4 reagent is typically equal to combined volume of
sample and dichromate solution
• Whenever sample volume is >50 ml, disconnect condenser and boil to
reduce flask contents to about 150 ml and then connect the
condenser

21. Open Reflux Method: Procedure
• Continue running condenser cooling water, close condenser (by an
inverted beaker!) and start refluxing on a hot plate/heating mantle
• Refluxing for 2 hours, switch off the hot plate/heating mantle and
allow the set-up to cool down.
• Wash down the condenser into the refluxing flask by distilled water,
detach the flask and double its contents volume by distilled water
• After cooling, add 2/3 drops of ferroin indicator to the flask
contents, and titrate against 0.25 M FAS solution
• When sample has lower COD (50 mg/l) use 0.025 M strength FAS)
• Similar to the sample test the blank and record the volume of FAS
consumed for titrating both the sample and the blank
• Calculate COD of the sample by
( ) 8000
).(
/ 2
usedsampleofml
MBA
OaslmgCOD
−
=
‘A’ is ml FAS consumed to titrate blank
‘B’ is ml FAS consumed to titrate sample
‘M’ is molarity of FAS

22. COD by Closed reflux method
• Amount of sample used is small (2.5-10 ml) - for avoiding errors
from uneven distribution of suspended solids, the sample is
homogenized by a blender prior to testing
• Method has a cost advantage, generates minimum of hazardous
waste, and VOCs are more completely oxidized
• Sample and blank are digested for 2 hours in a closed system of
culture tubes with tight caps or of sealed ampules placed in a block
digester or in an oven preheated to 150±2 C.ᵒ
• Digested samples are cooled and tested for COD by
• Titration with FAS (Titrimetirc closed reflux method)
• Measuring color change (Colorimetric closed reflux method)
• Basis for the colorimetric method
• Hexa-Cr is orange colored and Tri-Cr is greenish blue in color
• As a consequence of conversion of haxa-Cr into Tri-Cr, color of
digestion mixture changes from orange to greenish blue
• Fading of orange color (at 400 nm) or appearance of greenish blue
color (at 600 or 620 nm) is measured and compared against
standards

23. COD by Closed Reflux Method
Apparatus, Glassware and Chemicals
• Screw capped culture tubes of (16/20/25 mm) dia. and (100/150
mm) height or Standard ampules (10 ml) and ampule sealer
• Cast aluminum heating block (with 45 to 50 mm deep holes sized for
the close fit of culture tubes or ampules) or block heater or oven
Titrimetry
• Standard potassium dichromate digestion (0.0167M): Dissolve 4.913
g K2Cr2O7 (dried at 103 C for 2 hr.) in 500 ml distilled water, add 167ᵒ
ml conc. H2SO4 and 33.3 g HgSO4, cool and adjust volume to 1.0 L.
• Standard ferrous ammonium sulfate, FAS (0.10M): Dissolve 39.2 g
FAS in distilled water, add 20 ml conc. H2SO4, cool and adjusting
volume to 1.0 L
Colourimetry
• Spectrophotometer for use at 600/620 nm or 400 nm wavelength
with adapter for culture tubes/ampules
• (Standard digestion solution (0.0347 M): Dissolve 10.216 g K2Cr2O7
(dried at 103 C for 2 hr), 167 ml conc. Hᵒ 2SO4 and 33.3 g HgSO4 in 500
ml distilled water, cool and adjust volume to 1.0 L

24. Closed Reflux Method: Procedure
• Take measured amount of homogenized sample in a culture tube or
ampule
• Add measured quantity of standard dichromate digestion solution
• Digestion solution used in the colorimetric method is slightly different
from that used in titrimetric method
• Run H2SO4 reagent into the culture tube along the walls to form a
distinct acid layer underneath the sample
• Sample, dichromate digestion solution and H2SO4 reagent are
usually added in the volume ratio of 5:3:7
• In case of the titrimetric method, an additional culture tube (blank)
of distilled water (as sample) is maintained along with the sample
• In case of the colorimetric method, maintain 5 or 6 culture tubes of
standards (synthetic samples) of 0 to 900 mg/l COD strength along
with the sample
• Tightly cap the culture tubes, mix contents (through inverting),
place the tubes in block digester (preheated to 150±2C), and reflux
• Reflux for 2 hours switch off the block digester and cool the tubes

25. COD by closed reflux method
Titrimetric method
• Remove caps of the culture tube and transfer contents into a
conical flask
• Add 1 or 2 drops of ferroin indicator and titrate against FAS.
• Record the amount of FAS consumed
• Calculate the sample’s COD from the results by
Colorimetricmethod
• Invert the cooled culture tubes for thoroughly mixing the
contents and allow proper settling of suspended solids
• Read absorbance (color intensity) either at 400 nm or at 600 nm
with the help of a spectrophotometer
• Through using the readings obtained for the standards, construct
a calibration curve
• Through using the calibration curve find COD of the sample
corresponding to its absorbance
8000
).(
/( 2
usedsampleofml
MBA
OaslmgCOD
−
=
‘A’ is ml FAS consumed in blank titration
‘B’ is ml FAS consumed in sample titration
‘M’ is molarity of FAS

26. Precautions
• Mercuric sulfate and dichromate are highly toxic , sulfuric acid is
corrosive
• Addition of water to acid and refluxing at elevated temp are explosive
and unsafe
• Mixing acid with water generates heat
• Avoid swallowing, inhalation and contact with skin, eyes and clothing
• Handle the chemicals under a chemical hood
• Thoroughly mix the contents of reflux flask prior to heating to
prevent localized heating to avoid super heating and blow out from
the top of the condenser.
• Protect hands from heat, specially, while mixing the flask or the
capped culture tube contents
• Wash the glassware with 20% H2SO4 before using
• Use ground glass joints, rather than greased, for setting up the reflux
apparatus
• Avoid using scratched or blemished glassware, specially in the
colorimetric closed reflux method

27. Precautions
• Vial caps temperature should be low enough to avoid cap damage
(a potential source of sample contamination)
• In case of colorimetric method a blank must be run with each lot of
samples.
• In cases of turbid or highly colored samples, prefer titrimetric than
colorimetric closed reflux method.
• There can be transmittance differences between hot and cold
samples.
• Precision and accuracy of the method and quality of the reagents
are evaluated through testing synthetic samples
• Add a series of known amounts of COD standard to the sample, run
COD test on all, and examine final results for the recovery of the
added COD standard

28. Dissolved Oxygen (DO)
by Winkler and
Membrane Electrode methods

29. Dissolved Oxygen (DO): Winkler Method
• Can be measured by either Winkler method (iodometric method!)
and Membrane electrode method
• BOD bottle containing the sample is added with Manganous sulfate
and alkaline potassium iodide solutions
• DO present in the sample oxidizes an equivalent amount of divalent
manganese ions to higher valency states (form oxides)
• Rest of the manganese ions form divalent hydroxide precipitate
• On acidification with sulfuric acid, the higher valency manganese
ions are reduced into divalent ions (by iodide ions), and iodine,
equivalent to the sample’s DO content, is liberated
• All precipitates formed (both oxides and hydroxides) get solubilized
• Amount of iodine liberated is measured by titrating with standard
sodium thiosulfate solution, while using starch as indicator
• For detecting end point more precisely, in place of using starch
indicator, electrometric method can also be used
• If interferences (suspended solids, color and chemicals) are absent,
spectrophotometer can be used to measure the iodine liberated

30. Winkler method for DO
NaIOSNaIOSNa
OHMnHOHMnb
OHMnIHIMnOa
OHMnOHMnc
OHMnOOOHMnb
OHMnOOOHMna
22.3
22)(.2
242.2
)(2.1
5.0)(.1
5.02.1
6422322
2
2
2
2
2
22
2
2222
222
2
+→+
+→+↓
++→++↓
↓→+
+↓→+
+↓→++
++
++−
−+
−+
• Reactions involved in the Winkler method of DO testing are
• Sources of error:
• Presence of Nitrite (more than 50 µg/L as N) introduces positive error
• Nitrite can oxidize the iodide ions back into iodine and introduce the
error (a chain reaction)
– Biologically treated effluents, incubated BOD bottle samples, and
stream samples may have nitrite interference
– For eliminating, instead of alkaline-iodide solution, alkaline-iodide-
azide solution is used – the azide added reacts with NO2¯ and removes
it as N2 and N2O gases
+−
+−−
+→++
++→++
HNOOHOON
OHONIHINO
225.0
422
22222
22222
OHONNHNOHN
NaHNHNaN
22223
33
++→++
+→+
+−
++

31. Winkler Method for DO
• For avoiding errors, the sample should not come in contact with air
during sampling and testing (at least till the sample’s DO is fixed)
• Samples with iodine demand can be preserved for 4-8 hours by
adding 0.7 mL conc. H2SO4 and 1.0 mL of 2% azide (NaN3) prior to
actual analysis by usual procesdure
• Permanganate modification
• Permanganate modification is needed if ferrous iron level is > 1.0
mg/L
• To the suample collected add 0.7 mL conc. H2SO4, 1.0 mL KMnO4 and
1.0 ml of KF below the surface, and stopper and mix the contents
• KMnO4 addition may be increased if the resulting violet tinge do not
persist for at least 5 minutes
• Decolourize the sample by adding 0.5 to 1.0 mL of potassium oxalate
(K2C2O4) and mixing the contents

32. Winkler Method for DO
• Ferric iron interference can be overcome by addition of 1 ml of KF
and Azide provided titration is done immediately after acidification
• Addition of 1.0 mL of KF solution prior to acidification is needed for
samples with 100-200 mg/L of ferric iron (acidified sample should be
immediately titrated)
• Copper sulfate-sulfamic acid flocculation modification
– Used for biological flocs having high O2 utilization rates
– Add 10 ml of copper sulfate-sulfamic acid inhibitor solution to 1.0 L aspirator
bottole with glass-stopper.
– Fill the bottle with the sample from the bottom by a tube near the bottom
while allowing overflow of 25-50% volume
– Stopper the bottle, mix the contents by inverting the bottle and allow the
bottle to stand and siphon out sample into the BOD bottle for DO
measurement

33. Reagents for DO testing
• Manganous sulfate solution: Dissolve 480 g MnSO4.4H2O in
distilled water, filter and make up volume to one liter
• Should not give blue color when added to acidified KI solution with
starch indicator
• Alkali-iodide-azide reagent: Dissolve 700 g KOH or 500 g NaOH,
and 150 g KI or 135 g NaI in water and adjust volume to one liter.
Dissolve 10 g sodium azide (NaN3) in 40 ml water and add to alkali-
iodide solution
• The resultant reagent should not contain free iodine – check through
diluting and acidifying and adding starch indicator and observe for
blue color
• Concentrated sulfuric acid
• Aqueous solution of starch indicator: Dissolve 2 grams of soluble
starch and 0.2 grams of salicylic acid in 100 ml of hot distilled water
• Standard sodium thiosulfate solution (0.025M): dissolve 6.205 g
Na2S2O3.5H2O in distilled water, add 1.5 ml 6N NaOH, and adjust
volume to one liter.

34. Reagents for DO testing
• Standard potassium bi-iodate solution: Dissolve 812.4 mg of
potassium bi-iodate, KH(IO3)2, in distilled water and adjust volume
to one liter
• Potassium permanganate (KMnO4): Dissolve 6.3 g KMnO4 in
distilled water and adjust volume to one liter
• Potassium oxalate (K2C2O4.H2O): Dissolve 2 g K2C2O4 in 100 mL
distilled water
• Potassium fluoride (KF.2H2O): Dissolve 40 g KF.2H2O in 100 mL
distilled water
• Copper sulfate-sulfamic acid inhibitor solution: Dissolve 32 g
NH2SO2OH in 475 mL water, dissolve 50 g CuSO4.5H2O in 500 mL
water, combine the two solutions and add 25 mL conc. acetic acid.
• 6N sulfuric acid solution
• 6N sodium hydroxide solution

35. Procedure for DO measurement
• Pour off the additional sample present in the funnel mouth of the
sample containing BOD bottle
• Open stopper, add 1-2 ml of MnSO4 and alkali KI reagents in the
same order at mid-depth with pipettes, and stopper.
• Mix BOD bottle contents (by gentle & repeated bottle inversions)
and allow settling of the formed precipitates to about 1/3rd depth
• Open stopper, add 1-2 ml of concentrated H2SO4 at the top along
the walls without disturbing the settled precipitate, and stopper
• Pour off the liquid from the funnel mouth, mix bottle contents (by
gentle & repeated bottle inversions) till the precipitates disappear
• Take measured volume of clear acidified sample (202 mL) in a
conical flask and titrate against standard sodium thiosulfate
solution, while using starch as indicator.
here x is total volume (in ml) of manganous sulfate and alkali-
iodide-azide reagents added to the BOD bottle
• Add starch indicator after titrating the flask contents to light yellow
color and record ml of titrant consumed as mg/l of sample’s DO
200
300
300
)(
x
mLvolumeSample
−
=

36. • Precautions
• Rinse the pipettes with distilled water whenever used for transferring
reagents into the sample (specially if dipped in the sample) prior to
returning back into the reagents.
• Identify end point by the first decolorization during titration and
disregard subsequent recolorization
• If standard thiosulfate solution is overrun (beyond end point), use
standard bi-iodate solution for back titrating and making necessary
correction to the thiosulfate consumed
• Standardization of sodium thiosulfate solution
• Take 2 grams KI in 100-150 ml of distilled water plus 1 ml of 6N H2SO4
and 20 ml of standard potassium bi-iodate solution dilute to 200 ml
• Titrate the solution with standard sodium thiosulfate solution while
using starch as indicator
• Consumption of 20 ml of the standard sodium thiosulfate solution
indicates that its strength is 0.025M
DO by Winkler Method

37. Membrane Electrode Method for DO
• Membrane electrode is composed of two solid metal electrodes and an
electrolyte solution forming a bridge between them
• The electrodes and the electrolyte solution are separated from the
sample by a molecular oxygen permeable membrane
• The membrane electrode system (DO probe) is either a polarographic
system or a galvanic system
• Because of the permeable nature, a dynamic equilibrium is established
(through oxygen diffusion) between the DO of the electrolyte solution
and that of the sample
• Oxygen present in the electrolyte is reduced at the cathode and electrons
required are produced at the anode and transported to the cathode
• Current resulting from the required electron transport is proportional to
the DO concentration in the electrolyte solution (indirectly in the sample)
• Current in the circuit is measured and related with the DO of the sample

38. Membrane Electrode Method for DO
Calibration
• Establishing relationship between DO of the sample and current in
the circuit
• Calibration of membrane electrode system involves use samples of
known DO
• Samples with known DO can be prepared by aeration, bubbling
nitrogen gas, addition of sodium sulfite and traces of cobalt chloride
• The membrane electrode (DO probe) is placed in water saturated
air, and current generated in the circuit is taken as proportional to
the DOs at that temperature and pressure
• When calibrated in saturated air, necessary compensation for altitude
(or atmospheric pressure) should be made (Manufacturer provides a
standard table for altitude correction)
• Distilled water (or unpolluted water with known conductivity/
salinity/ chlorinity) saturated with DO can also be used for calibration
• Samples with known DO can also be used for the calibration
• Winkler method is used for knowing DO with precision and accuracy
• Manufacturer of DO probe and DO meter provides a written
calibration procedure and it should be strictly followed

39. Membrane Electrode Method for DO
• Membrane permeability is both temp. and salt conc. Sensitive.
– Temp and salt conc. of the sample should be monitored and necessary
corrections be made to the probe sensitivity
– Nomographic charts available from the manufacturer can be used
– Certain DO meters may include facilities for automatic temp. and salt
conc. compensation
– For confirming the corrections made by nomographic charts,
sensitivity of the DO probe is frequently cross-checked at one or two
temp. and salt conc.
• With time membrane looses its properties, and hence, it is
frequently changed and the electrode system is calibrated afresh
• Precision and accuracy of membrane electrode method (± 0.1 mg/l
and ± 0.05 mg/l) is not very good
• Precision of Winkler method is ± 50 µg/l, but being a destructive
test, can not be used for continuous DO monitoring in samples

40. Membrane electrode method for DO
Procedure
• Carefully insert a calibrated DO probe into the sample while not
allowing air entrapment in vicinity of exposed membrane portion
• Read temp. and salt conc. of the sample and make temp. and salt
conc. compensation
• Measure DO, while ensuring sufficient flow of sample across the
membrane surface, through stirring, and report the result in mg/l
Precautions
• Strictly follow the manufacturer’s procedure for cleaning
electrodes, and for changing membrane and electrolyte solution
• Use high quality electrolyte solution (either prepared as per
manufacturer’s specifications or supplied by manufacturer)
• Use unpunctured and clean membranes, and avoid entrapment of
even minute air bubbles while changing the membrane
• Use of the membrane electrode system can result in plating/
etching and/or contamination of electrodes
• Manufacturer’s instructions for storing the DO probe when not in
use (short term and long term storage) should be strictly followed
• Give enough time for DO probe to reach thermal and DO equilibria

41. Biological Oxygen Demand
(5 or 3 day BOD)
by BOD Bottle Method

42. BOD Bottle Method for BOD Estimation
A BOD bottle filled with diluted sample with seed and
stoppered is incubated at constant temperature for a
fixed duration
– Dilution of the sample
– Acclimated seed
– Favourable nutrient and osmotic conditions
– No air bubble entrainment
– known initial DO
5 days incubation at 20C (3 days at 27C)
– only partial oxidation of the organic matter
– complete oxidation needs incubation for longer time (60 to 90
days)
Measurement of final DO
– Difference between initial and final DO is oxygen demand of the
diluted sample during the incubation period

43. Sources of Error
Seed added is organic matter and undergoes bio-oxidation exerting
oxygen demand during incubation
– Positive error introduced is measured through incubating a blank
containing seed in dilution water but no sample
– Measured error is then subtracted from the overall oxygen demand
for obtaining oxygen demand of the sample
Oxygen demand is denoted as BODt at X°C (BOD5 at 20°C, BOD3 at 27°C,
etc.)
– Units for BODt at X°C are mg/L (BODt is oxygen demand when the
sample is incubated for ‘t’ days at X°C
Testing gives oxygen demand of diluted sample - multiplication of this
with dilution factor gives sample’s oxygen demand
NH3-N added (as nutrient supplement) and NH3-N released during
incubation are prone to nitrification and introducing positive error
• To eliminate this error, either inhibit the nitrification or quantify
and subtract from the measurement
– In 5-day BOD test, use of nitrification inhibitor chemical is preferred
– In BODu test quntification and subtraction of error is preferred

44. Expression for BODt from test results
BODt at X°C of a sample can be written as
Dilution Factor ‘Df’ is the factor by which original sample is
diluted for obtaining diluted sample - can be defined as:
OD of diluted sample:
Error introduced by the seed
– Oxygen demand of dilution water is almost negligible
– But, seeded dilution water has significant oxygen demand
– Add known volume of seed (5 times or more to that added to diluted
sample) to dilution water to raise the OD to > 2 mg/l
– Test the seed control for OD through incubating parallel with the
diluted sample for the same duration






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
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
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
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ionnitrificat
byerror
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aterdilution wand
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sampledilutedofliteronepreparingforusedsampleofml
Df =
sfsi DODOOD −=
DOsi & Dosf are initial & final DO of diluted
sample before & after ‘t’ days of incubation

45. F)DO-(DOaterdilution wseededofOD cfci=
preparedcontrolseedofliterperseedofml
preparedsampledilutedofliterperseedofml
F =
f
f
cfcisfsi
o
t DF
D
DODODODOCXatBOD
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−−−−=
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seed control incubated for ‘t’ days
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f
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Expression for BODt from test results
bottleBODinwaterdilutionseededoffractionvolumeis
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Error by nitrification: Nitrification reaction is inhibited by adding
nitrification inhibition chemical and hence no correction needed.

46. Incubation conditions
• Favourable pH conditions
– Micro-organisms are pH sensitive - 7.2 is considered as
optimum
– pH of incubated sample can change from production of CO2
– Phosphate buffer is used to adjust the pH to optimum and to
maintain pH during incubation
• Favourable nutrient conditions
– Bio-oxidation of organic matter involves synthesis of new
microbial biomass
– This synthesis requires nitrogen (NH3-N or NO3-N), phosphorus
(orthro) and other inorganic nutrients
– Insufficient nutrients make bio-oxidation nutrient limiting
– The sample is supplemented with nutrient formulations
(phosphate buffer has KH2PO4, K2HPO4, Na2HPO4 and NH4Cl)
– Salts added for maintaining osmotic conditions (FeCl3, CaCl2
and MgSO4) may also contribute
• Favourable osmotic conditions:
– Maintaining osmotic conditions is important for ensuring this
FeCl3, CaCl2 and MgSO4 salts are added

47. Incubation conditions: Constant
temperature throughout
• 5/3 day incubation bio-oxidizes only a fraction of organic matter
(OM)– total oxidation requires infinite time – BOD kinetics model is
used estimating the total OM by extrapolating BODtresults
– BOD kinetics model involves a reaction rate constant (K) which is
temp. sensitive
– BOD kinetics model can not be applied to the results obtained from a
test where the sample is not incubated at constant temperature
• The BOD test results are always reported along with temperature
and period of incubation (BOD5 at 20°C).
• By conviction incubated for 5 days at 20°C (annual average temp. of
UK and time taken by the Thames to reach the ocean) – CPCB
recommends 3 days at 27°C (annual average temp. of India!)
• 5 days incubation has an advantage - nitrogenous BOD in many
cases will not interfere with carbonaceous BOD measurement
– One can adapt any temp. within the range that will not affect the
microbial metabolic activity
– Incubation period giving BODt = 60-70% of BODucan be adapted
• For ensuring incubation at constant temp., samples are incubated
either in BOD incubators or in water baths set at desired temp.

48. Acclimatized seed
• For the bio-oxidation of OM, the incubated sample should
have appropriate microbial populations
• During initial period of incubation, selection among the
populations and their size increase occurs – this results in
initial lag in oxygen demand pattern and consequently
• Cumulative demand may not follow first order kinetics
• Negative error may be made in BOD5 measurement, and in the
BODu estimation
• Municipal sewage, biologically treated effluents and samples
collected from receiving water bodies are supposed to have
these populations
• Many industrial wastewaters may not have (w/w generated at
elevated temp. and w/w containing toxicants above the
threshold limits)

49. Acclimatized seed
• Microbes have preferences as to the OM they can bio-oxidize
• seed added may not have appropriate microbial populations in
significant size
• W/w not having appropriate microbial populations require
addition of these populations as seed
• The initial lag can be eliminated through use of acclimated
seed.
• Preparation of acclimatized seed:
• Take mixed liquor or secondary of a STP and start aeration
• While continuing aeration, gradually replace the mixed
liquor/secondary sludge with the wastewater wastewater in
question over a a period of two days or more
• Settle the contents and use the supernatant as seed

50. Aclamatized Seed
• Samples of domestic wastewater, undisinfected effluents from
biological treatment units, and of receiving waters usually have
appropriate and adapted microbial populations - require no seed
• Many untreated industrial wastewaters (disinfected wastewaters,
high temperature wastewaters and wastewaters with extreme pH
value) require addition aclamitized seed
• What can be seed
– Settled domestic sewage, clarified and undisinfected effluents of
biological treatment units, and clear water from receiving waters
– Effluent from the biological treatment plant, treating the wastewater
being sampled (most appropriate)
– Clear water collected from the water body, which is receiving the
wastewater in question, at a point 3 to 8 KM down stream
– Seed, specially, developed in laboratory
• Developed from settled domestic sewage or suspension prepared
from wastewater contaminated soil, through continuously aerating
for a few days and adding small daily increments of the wastewater
in question

51. Dilution factor (Df)
• Oxygen is sparingly soluble in water and depends on altitude,
temperature and salinity
Altitude (in
meter)
Saturated
DO (in
mg/l)
Temperat
ure (in
°C)
Saturated
DO (in
mg/l)
Chlorini
ty
Saturated DO
(in mg/l)
sea level 9.2 0.0 14.62 0.0 9.09 (20°C)
305 8.9 5.0 12.77 7.56 (30°C)
610 8.6 10.0 11.29 6.41 (40°C)
914 8.2 15.0 10.08 5.0 8.62 (20°C)
1219 7.9 20.0 9.09 .. 7.19 (30°C)
1524 7.6 25.0 8.26 .. 6.12 (40°C)
1829 7.4 30.0 7.56 10.0 8.17 (20°C)
2134 7.1 35.0 6.95 .. 6.85 (30°C)
2438 6.8 40.0 6.41 .. 5.84 (40°C)
2743 6.5 45.0 5.93 15.0 6.51 (30°C)
3048 6.3 50.0 5.48 20.0 6.20 (30°C)

52. Dilution factor (Df)
• Diluted sample is aerated to rise DOi closer to DOS
• At 20°C, DO level can rise to aboit 8 mg/l level - diluted sample’s
initial DO: about 8 mg/l
• At ≤ 0.5 mg/l DO, bio-oxidation rates are influenced by DO and
assumption of first order kinetics (BOD kinetics) becomes invalid
• DO in incubated samples should be >1.0 mg/L – final DO should be
>1.0 mg/L
• DO available for bio-oxidation can be about 7 mg/L
• Sample needs dilution so as its cumulative OD is ≤ 7 mg/L.
• For finding Df, an idea of range of expected BOD for the sample
should be known (Published literature or past experience can help)
• COD of the sample can also help
• Take upper limit of the range and divide by 7 mg/l to get Df.
• If no idea on expected BOD range, then test at a series of dilutions
• For acceptable results, OD should be >1 mg/L and residual DO
should be >1 mg/L
• A geometric progression of Df (1, 5, 25, 125, 625, …, so on) can be
used in the test

53. Standard BOD Bottle Method: Limitations
• Sample dilution introduces error in measurement and affect
reproducibility
• Can not be successfully used for the measurement of BOD
contributed by suspended organic matter
– Must first undergo hydrolysis - takes time (2 to 3 days or more), BOD
exertion not follow first order kinetics (BOD model assumes)
– Very difficult to ensure uniform distribution of the TSS among the BOD
bottles - Consequence is erroneous BOD measurement.
• Testing requires long time (5 days) - results become less relevant
(for operation and control of, specially, biological treatment units)
– Attempt to reduce the time required: increase the incubation
temperature (to 27°C to reduce time to 3 days).
• Dilution of sample with nutrient rich buffer solution may not reflect
the conditions existing in the treatment processes
• Inaccuracy of BODt measurement: 15 to 50% (18% SD)

54. 5-day BOD Test by BOD Bottle Method
• BOD is a bioassay test used to measure biodegradable organic
matter concentration
– Amount of oxygen required to bio-oxide organic matter of the sample
is measured
• Diluted sample is incubated with appropriate microbial populations
for 5 days at 20ºC
– Distilled water (or tap water or water collected from receiving water s,
if having negligible BOD) is used for diluting the sample
– Water should not have bio-inhibitory substances like chlorine, heavy
metals etc.
• Aerobic bio-oxidation of biodegradable organic matter consumes
DO of the sample
• Change in DO of the incubated sample is measured and reported as
BOD5 at 20C
• Experimental results to become acceptable
– Residual DO of the sample should be >1.0 mg/l
– DO difference between initial and final should eb >1.0 mg/L

55. Interferences
• Secondary effluent samples and samples seeded with secondary
effluents, and polluted water samples collected from surface water
bodies show significant nitrification rates
• Nitrification inhibitor chemicals: TCMP (2-chloro, 6-trichloro methyl
pyridine)
– Whenever nitrification inhibitor chemical is used, results are reported
as CBOD5 (not as BOD5)
• Dilution water used can also introduce positive error
– Good quality dilution water exerts < 0.1 or 0.2 mg/l of oxygen demand
during 5-day incubation at 20°C.
• Sulfides and ferrous iron can be oxidized during incubation and
introduce positive error
• Residual chlorine if present can inhibit biological activity and bio-
oxidation of organic matter
• Samples with residual chlorine are first dechlorinated
– Keeping under light for 1 to 2 hours can dechlorinate the sample
– Addition of predetermined quantity of sodium sulfite can dechlorinate
• Dose of sodium sulfate required: Take 200 ml sample, add 2 ml of
1:1 acetic acid or 1:50 H2SO4 and 2 ml of 1% KI, and titrate against
Na2SO3, use starch as indicator - Na2SO3 consumed is dose

56. Apparatus, Glassware and Chemicals
Apparatus and glassware
• BOD incubator or Water Bath set at 20±1°C or 27±1°C.
• Diaphragm aerator delivering organic free-filtered air and
diffused aeration system for aerating the dilution water
• BOD Bottles (funnel mouth, 300-330 mL volume, unique number,
grounded neck, heavy stopper with ground surface and conical
tip).
• Aspirator bottles of 3 L capacity, beakers of 3 to 5 capacity,
measuring cylinders, burette, pipette, etc. glassware
Chemicals
• Phosphate buffer solution: 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g
Na2HPO4.7H2O and 1.7 g NH4Cl in 500 ml water and final volume
adjusted to one liter (discard if biological growth observed).
• Magnesium sulfate solution: 22.5 g MgSO4.7H2O in water and
adjust final volume to one liter.
• Calcium chloride solution: 27.5 g CaCl2 in water and adjust final
volume to one liter.
• Ferric chloride solution: 0.25 g FeCl3.6H2O in water and adjust
final volume to one liter.

57. Apparatus, Glassware and Chemicals
Chemicals
• 1N acid or alkali solution (28 ml of conc. H2SO4 to one liter; or 40
g NaOH in one liter final volume).
• Sodium sulfite solution: 1.575 g Na2SO3 in 1000 ml distilled water
(not stable – prepare afresh every time).
• Nitrification inhibition chemical: 2.2% solution of 2-Chloro-6-
(trichloro methyl) pyridine
• Glucose-glutamic acid solution: Dry reagent grade glucose and
glutamic acid at 103ºC for one hour, take 150 mg each, dissolve
in water and adjust to one liter final volume (not stable and
prepare afresh every time)
• Ammonium chloride: 1.15 g NH4Cl in 500 ml water, adjust pH to
7.2 with NaOH solution and adjust final volume to one liter

58. Procedure for 5-day BOD test
• Take dilution water, add 1 mL/L each of phosphate buffer, ferric
chloride, calcium chloride and magnesium sulfate solutions;
Adjust temp. to 20C by keeping in an incubator and oxygenate
the water through bubbling organic free filtered air
• Identify appropriate source for seed material - ensure that the
seed is sufficiently clear and devoid of visible suspended solids –
add the seed to the water at 1 mL/L rate
• Added seed should not increase oxygen demand of dilution
water (over 5-day period at 20C) beyond 1.0 mg/l.
• If nitrification inhibitor chemical is to be used, store the seeded
dilution water in the incubator at 20C until its oxygen demand
(over 5-days) falls below 0.2 mg/l.
• Addition of seed is not required if the sample is having
appropriate and adapted microbial populations
• If residual chlorine is suspected dechlorinate the sample.
• If temp. is <20C, adjust sample’s temp. to 20C (partially fill the
sample in a bottle and vigorously agitate)

59. Procedure for 5-day BOD test
• Adjust sample’s pH to 6.5-7.5 by 1N H2SO4 or NaOH
• Decide on Df and find out sample volume for preparing 2 or 3 L of
diluted sample
• Take required volume of the sample, add oxygenated (and
seeded!) dilution water and adjust volume to 2 or 3 liters
• If nitrification inhibition is desired, add 20 or 30 mg of TCMP to
the 2 or 3 liters of the prepared diluted sample
• If DO is suspected as insufficient, then aerate the sample
• Transfer sample to aspirator bottle and fill 4 or 6 BOD bottles
• Incubate 2 or 3 of the bottles at 20±1C in incubator/water bath
• Use rest of the bottles to measure DOi of the incubated sample
• For measuring the DO use either the Winkler method (azide
modified) or the Membrane electrode method
• Prepare seed control by taking measured volume of seed (5 times
more than that used in preparing the diluted sample) and adjust
volume to 2 or 3 liters with oxygenated (unseeded) dilution
water

60. Procedure for 5-day BOD test
• Aerate the prepared seed control, transfer into aspirator bottle
and fill 4 or 6 BOD bottles
• Incubate 2 or 3 of the bottles at 20C and use rest of the bottles
for measuring DOi of the incubated seed control
• Testing seed control is not required if the sample is not seeded
• If nitrification inhibitor chemical is added to the sample then also
add the inhibitor chemical to the seed control as well
• For evaluating the dilution water quality, and for assessing the
effectiveness of the seed and the analytical technique on the
whole, perform synthetic glucose-glutamic acid sample testing
• Take 40 or 60 ml of glucose-glutamic acid solution into a
measuring cylinder and adjust volume to 2 or 3 L with seeded
and oxygenated dilution water
• Aerate the prepared synthetic sample, transfer to an aspirator
bottle and fill 4 or 6 BOD bottles
• Incubate 2 or 3 of the bottles and use the rest for measuring DOi
of the incubated synthetic sample
• Running glucose-glutamic acid check is optional

61. Procedure for 5-day BOD test
• After incubating for 5 days ± 1 hour, take out the incubated BOD
bottles and measure their DOf either by Winkler method (azide
modification) or by Membrane electrode method
• Discord the results if DOf is <1.0 mg/L, or if DOi-DOf is <2.0 mg/L
• Record results and find out BOD5 at 20ºC for the sample (and also
for the synthetic glucose-glutamic acid check)
• Use the results of the glucose-glutamic acid check for establishing
the Laboratory Control Limit (LCL)
• Use results of around 25 of glucose-glutamic acid checks to find
out the LCL (LCL = Mean ± 3.Standard Deviation)
• LCL should be within the 198±30.5 mg/l - If not, something is
wrong with the technique, or with the dilution water, or with the
seed employed – investigate and correct the problem
• Results obtained for the sample are considered precise if the
results for the glucose-glutamic acid check lie within 204±10.4
mg/L range

62. Format for recording the BOD test results
• Sample: Date:
• Dilution factor:
• Incubation period (days): Incubation temp. (°C):
• Volume of in the diluted sample (mL/L):
• Volume of seed in the seed control (mL/L):
Diluted Sample Seed control
Bottle
No.
Initial DO
(DOsi)
Bottle
No.
Final DO
(DOsf)
Bottle
No.
Initial DO
(DOci)
Bottle
No.
Final DO
(DOcf)
Average Average Average Average

63. Remarks and Precautions
• Method is appropriate, precise and accurate for measuring soluble
bio-degradable organic matter concentration
• Particulate suspended, floating or settleable organic matter affects
accuracy and precision of BOD measurement
• For checking effectiveness of seed material, very often, instead of
glucose-glutamic acid check, perform a check on the pure organic
compound, which is major constituent of the sample in question
• 5-day BOD test by BOD bottle method is not appropriate for testing
sample with BOD5 at 20 C <2 mg/l.ᵒ
• CPCB has suggested 3-days incubation at 27 C temp., in place of 5ᵒ
days incubation at 20 C temp.ᵒ

64. Remarks and Precautions
• Ensure that the incubated BOD bottles have water seals and avoid
their drying up by placing paper/plastic/foil caps over the BOD
bottle mouths
• Pour off the sample acting as water seal prior to proceeding with
the DOf measurement
• Protect incubated BOD bottles from light – algal photosynthesis can
introduce negative error
• Avoid volume errors, by not preparing dilutions directly in the BOD
bottle (BOD bottles volume is not constant, but varies)
• To know oxygen demand of unseeded dilution water, conduct test
on seed control at different seed concentrations, plot oxygen
demand against seed concentration and read intercept on oxygen
demand axis as oxygen demand of the unseeded sample

65. Ultimate BOD (BODu)

66. Ultimate BOD (BODu) by BOD Bottle Method
• 5-day BOD test fails to measure BODu - Kinetic descriptors (BOD
kinetic parameters) can relate BOD5 with BODu and estimate BODu
• Oxidation is not complete in 5 days of incubation - 60 to 90 days
incubation needed for complete oxidation and BODu measurement
• For the measurement of BODu, incubate the sample till its weekly
oxygen demand drops to <1-2% of the cumulative demand -
further, use appropriate statistical extrapolation technique for
estimating BODu from the measured cumulative oxygen demand
• This method usually includes measurement of DO of the incubated
sample at regular intervals (for acceptable results, >2 mg/l of DO
depletion should occur between two successive DO measurements)
• The sample is diluted with dilution water to the range of 20 to 30
mg/l of oxygen demand (BODu)
• For ensuring availability of sufficient DO, the incubated sample is
reaerated at regular intervals (DO measurement, reaeration and DO
measurement sequence of steps)
• Oxygen sensitive membrane electrode method, rather than Winkler
method, is used for the DO measurement - Winkler method is a
destructive method and not suitable for use.

67. Ultimate BOD (BODu) by BOD Bottle Method
• Concentrations of NO3-N and NO2-N of the incubated sample are
measured on day 0 and on the last day of incubation (if interested
in nitrification rates then measuring at regular intervals is needed) -
oxygen equivalency of nitrification is computed and subtracted
from the exerted oxygen demand
• Oxygen equivalency of nitrification of NH3¯N to NO3¯N and NO2¯N to
NO3¯N are 3.43 and 1.14 mg/mg respectively
• When intended to know in-stream oxygen demand rates, then the
sample is as far as possible not diluted, and it is not supplemented
with any nutrients or buffer formulations or with any seed
• Samples should be drawn from the incubated bottle contents for
NO3-N and NO2-N measurement
• The sample bottle is subjected to frequent insertion of membrane
electrode into it and stoppering
• These involve loss of the incubated sample and can introduce
errors
• For minimizing the errors, bottles of 2-L or more capacity are used.
• For making up the sample losses, additional sample is maintained in
a reservoir bottle in the incubator along with the sample bottle

68. Procedure for BODu measurement
• Additional apparatus and glassware
– BOD bottles of 2L or more capacity (with all the features of 300 ml
capacity bottles)
– Reservoir bottles of 2L or more capacity (2 L BOD bottles can be used
reservoir bottles, through partial filling and unstoppering
– Oxygen sensitive membrane electrode and DO meter for measuring
DO of the incubated sample
– Magnetic stirrer and magnetic bits for facilitating mixing of the
incubated sample
• Preparation of unseeded dilution water (similar to that done for the
5 day BOD bottle method)
– Collecting conservatively estimated volume of water
– addition of phosphate buffer, ferric chloride, calcium chloride and
magnesium sulfate solutions at 1 mL/L rate
– conditioning to 20C
– oxygenation to saturation
• If required seed material is added to the dilution water (volume of
seed added should not significantly increase oxygen demand of the
diluted sample, <1 mg/l )

69. Procedure for BODu measurement
• If residual chlorine is suspected, dechlorinate the sample
• Adjust sample’s temperature to 20ºC and pH to 6.5 to 7.5
• Decide on the need for seed addition and on the extent of dilution
required for reducing strength to 20-30 mg/l of BODu
• Prepare diluted sample, if required aerate, fill 2 or 3 (2 L) BOD
bottles, and insert clean magnetic bits
• Shift bottles into incubator set at 20C, place over magnetic stirrer,
and stir the contents all through the incubation
• After 5 minutes of stirring, measure initial DO through inserting a
pre-calibrated DO probe and oxygen meter, remove the probe,
make up the sample loss and stopper the bottle
• Transfer diluted sample into a reservoir bottle while leaving some
empty space within, insert a magnetic bit, shift into an incubator,
place it over a magnetic stirrer, loosely cover the bottle with a cap
and continuously stir the contents
• Collect desired quantity of diluted sample adjust pH to <2 with
H2SO4 and preserve under refrigeration for nitrogen estimation
• For knowing BODu of the dilution water, fill both seeded and
unseeded dilution water in BOD bottles and incubate, and measure
their initial and final DO.

70. Procedure for BODu measurement
• As per the predecided schedule carry out the following
– Insert DO probe and test for DO of the (2L) BOD bottle contents
– Reaerate bottle contents and collect samples for NO3 & NO2 testing
– Stir the bottle for 5 minutes, insert DO probe and measure DO
– Remove the probe, makeup sample loss, stopper the bottle and leave
for incubation
• Fix schedule for reaeration in such a way that at no time the
sample’s DO falls below 2 mg/l
– Interval between two successive reaerations can be 2 to 5 days or
more and can increase with the cumulative incubation period
– For reaeration, transfer about 1/4th of the BOD bottle contents into a
clean glass beaker, stopper the bottle and vigorously agitate the
bottle contents – repeat this process for 10 to 15 times
– Collect sample for testing NO3-N and NO2-N from the beaker, adjust pH
to <2 by adding H2SO4and preserved through refrigeration
– Transfer rest of the contents of the beaker back into the BOD, fill the
bottle through using the reservoir bottle contents
• Continue measurement of DO, reaeration and measurement of DO
as per the schedule until the oxygen demand during the interval
falls below 1 to 2% of the cumulative oxygen demand

71. Procedure for BODu measurement
• Analyse the samples collected for NO3-N and NO2-N
• Record results of the testing
– Time of start of incubation
– Time of measurement of initial DO and of final DO for an interval
– Time beginning of aeration of an interval
– DO measured (both initial and final DO) for an interval
– NO3-N and NO2-N measured at the end of the interval
– Time elapsed between DOi and Dof measurements for an interval
– Duration of an interval (time gap between 2 successive reaerations)
– Cumulative time of incubation of the sample
• Process the data for estimating sample’s overall oxygen demand
(OD); nitrogenous OD and carbonaceous OD for each of the
intervals, and estimate cumulative carbonaceous oxygen demand





−
=
sreaerationsuccesive
twobetweenerval
DOandDObetweenreval
DODO
demandoxygenOverall
fi
fi int
int
)(
[ ] [ ]( )
[ ] [ ]( ) 









−−−
+
−−−
=





29.222
43.333
NNOfinalNNOfinal
NNOfinalNNOinitial
demandoxygen
sNitrogenou

72. Serial BOD test
and BOD kinetic parameters

73. Serial BOD test by BOD bottle method
• Needed for finding out BOD kinetics parameters
• Involves measurement of BOD1, BOD2, …, BODi, …, BODn
• Similar to 5 day or 3 day BOD test, but daily BOD is measured
• Large number of diluted sample bottles are incubated and daily 2
or 3 bottles are taken out for measuring DO and BODi estimation
• For acceptable results, the conditions, DOf >1.0 mg/L and DOi-Dof >2.0
mg/L should be satisfied in all the cases
• For ensuring this, the sample may be incubated at different dilutions
(shorter the incubation period lesser will be the dilution)
• If X is dilution factor for 5 day BOD, the following dilution factors
may be used in the serial BOD test
– X/4 dilution factor for BOD1, and BOD2 measurement
– X/2 dilution factor for BOD2, BOD3 and BOD4 measurement
– X dilution factor for BOD4, BOD5 and BOD6 measurement
– 2X dilution factor for BOD6, BOD7 and BOD8

74. BOD Kinetics Parameters and their
Estimation
• K and Lo are BOD kinetics parameters
• Use of BOD kinetics model requires values of these parameters
• Results of a serial BOD test for n days can be used for finding
the BOD kinetic parameter values
• Methods used to determine the BOD kinetics parameters are
• Method of least squares
• Method of moments (Moore et al. 1950)
• Log difference method (Fair, 1936)
• Fugimoto method (Fujimoto, 1961)
• Daily difference method (Tsivoglou, 1958)
• Rapid ratio method (sheehy, 1960)
• Thomas method (Thomas, 1950)

75. Method of least squares for BOD kinetics
parameters
( )
n
BOD
Kn
dt
BODd
BOD
BODBODn
dt
BODd
BODBOD
dt
BODd
n
K
tt
BODBOD
dt
BODd
BODKLKLK
n
i i
n
i
i
u
n
i i
n
i i
n
i
n
i
i
n
i ii
i
ii
ii
∑∑
∑∑
∑ ∑∑
=
=
==
= ==
−+
−+
+=
−






−





−=
−
−
=−==
1
1
2
11
2
1 11
11
11
0
.
)(
.
)(
..
)(
.
)(
...
dt
d(BOD)
Time (day) BOD BOD2
dBOD/dt (dBOD/dt).BOD
1
2
…
I
…
n
Results of serial BOD test for n days are needed

76. Method of Moments for BOD kinetic parameters
• Moore’s diagram (a nomograph relating K with ΣBOD/L0 and
ΣBOD/Σ(BOD.t)) is needed
– Moore’s diagram is different for different n value
• Results of serial BOD test for n days are used to find ΣBOD and
ΣBOD/ Σ(BOD.t)
• ΣBOD/Σ(BOD.t) value is used to read k value and ΣBOD/L0 value
from the Moore’s diagram
• From ΣBOD/L0, since ΣBOD is known, L0 is found
• Using the following formulae Moore’s diagram can be constructed
( )( )
( )
( )
( )( )
( )
( )[ ]∑∑∑
∑
∑
−
−
−−
−
−−
−






−
−
−
=






−
−
−=
n Kin
K
KnK
n
n
K
KnK
n
ii
n
tBOD
BOD
n
L
BOD
1
.
1
.
1
1
.
0
1
exp.
1exp
1expexp
.
1exp
1expexp

77. k 4 days 5 days 6 days 7 days 8 days
value ∑Y/L0 ∑Y/∑tY ∑Y/L0 ∑Y/∑tY ∑Y/L0 ∑Y/∑tY ∑Y/L0 ∑Y/∑tY ∑Y/L0 ∑Y/∑tY
X- axis Y1-axis Y2-axis Y1-axis Y2-axis Y1-axis Y2-axis Y1-axis Y2-axis Y1-axis Y2-axis
0.001 0.01 0.333 0.01 0.273 0.02 0.231 0.03 0.200 0.04 0.177
0.01 0.10 0.334 0.15 0.273 0.21 0.231 0.27 0.201 0.35 0.177
0.025 0.24 0.335 0.36 0.274 0.50 0.232 0.66 0.201 0.84 0.178
0.05 0.46 0.336 0.69 0.276 0.94 0.234 1.24 0.203 1.57 0.179
0.1 0.86 0.339 1.26 0.278 1.71 0.237 2.21 0.206 2.76 0.182
0.15 1.21 0.341 1.74 0.281 2.33 0.239 2.98 0.209 3.68 0.185
0.2 1.51 0.344 2.14 0.284 2.84 0.242 3.60 0.211 4.40 0.188
0.25 1.77 0.347 2.49 0.286 3.26 0.245 4.09 0.214 4.96 0.190
0.3 2.00 0.349 2.78 0.289 3.61 0.247 4.49 0.216 5.40 0.193
0.35 2.20 0.351 3.03 0.291 3.91 0.249 4.82 0.218 5.76 0.195
0.4 2.38 0.354 3.24 0.294 4.15 0.251 5.09 0.221 6.05 0.197
0.45 2.53 0.356 3.43 0.296 4.36 0.254 5.32 0.223 6.29 0.199
0.5 2.67 0.358 3.59 0.298 4.54 0.256 5.51 0.224 6.49 0.200
0.55 2.79 0.360 3.72 0.300 4.69 0.258 5.67 0.226 6.65 0.202
0.6 2.89 0.362 3.84 0.302 4.82 0.259 5.80 0.228 6.79 0.203
0.7 3.07 0.366 4.04 0.305 5.03 0.262 6.02 0.231 7.02 0.206
0.8 3.22 0.369 4.20 0.308 5.19 0.265 6.19 0.233 7.19 0.208
0.9 3.33 0.372 4.32 0.311 5.32 0.268 6.32 0.235 7.32 0.210
1 3.43 0.375 4.42 0.313 5.42 0.270 6.42 0.237 7.42 0.211
Method of Moments for BOD kinetic parameters

78. Moore's Diagram for n = 5 days
2.779476
0.295758
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 0.2 0.4 0.6 0.8 1
'k' value
CumulativeBOD
0.27
0.275
0.28
0.285
0.29
0.295
0.3
0.305
0.31
0.315
CumulativeBOD.t
Moore's Diagram (for n = 8 days)
4.955678
0.198616
0
1
2
3
4
5
6
7
8
0 0.2 0.4 0.6 0.8 1
k value
CumulativeBOD
0.175
0.18
0.185
0.19
0.195
0.2
0.205
0.21
0.215
CumulativeBOD.t
Moore's Digram (for n = 7 days)
4.491721
0.224454
0
1
2
3
4
5
6
7
0 0.2 0.4 0.6 0.8 1
'k' value
CumulativeBOD
0.2
0.205
0.21
0.215
0.22
0.225
0.23
0.235
0.24
CumulativeBOD.t
Moore's Diagram (for n = 6 days)
3.264788 0.251606
0
1
2
3
4
5
6
0 0.2 0.4 0.6 0.8 1
'k' value
cumulativeBOD
0.23
0.235
0.24
0.245
0.25
0.255
0.26
0.265
0.27
CumulativeBOD.t
Method of Moments for BOD kinetic parameters

79. Methods for BOD Kinetic Parameters
Fujimoto method
• Serial BOD test results for n number of days is used
• BODt+1 is plotted against BODt in a graph
– On the same graph another plot with slope=1 is plotted
– Point of intersection of the two plots is taken as BODu
• Using the BODu obtained, with the help of BOD kinetics model K
value is found
Rapid ratio method
• Serial BOD test results for n number of days is used
• Ratio of BODt+1 to BODt is plotted against BODt+1 in a graph
– On the same graph another plot with slope=1 is plotted
– Point of intersection of the two plots is taken as BODu
• Using the BODu obtained, with the help of BOD kinetics model K
value is found

80. Methods for BOD Kinetic Parameters
Thomas method
• Serial BOD test results are needed
• The kinetic parameters determination is based on the following
equation (Thomas equation)
• (t/BOD)1/3
is plotted against t
• (KL0)1/3
is obtained as intercept and K2/3
/6L1/3
as slope
• Form the slope and intercept K and L are calculated
( ) t
L
K
LK
BOD
t
.
6
.
3
1
0
3
2
3
1
0
3
1
+=






81. Nitrate and Nitrite Nitrogen

82. Nitrite and Nitrate Nitrogen
• Nitrate and nitrite concentrations are needed to know N-BOD and
finding out C-BOD, specially when samples are analyzed for BODu
• Analyze the samples promptly to avoid conversion of nitrite into
nitrate/ammonia and denitrification of nitrate
• Nitrite samples after adjusting pH to <2 with H2SO4, can be freezed
and stored at -20°C (for 1 to 24 hr preservation store at 4°C)
• Nitrate samples can be store at 40
C upto 24 h after adding 2 mL
conc H2SO4/L
• Nitrite analysis is by colorimetric method
• Nitrate can be analyzed by
– UV Spectrometric Method,
– Cd-reduction Method
– Ion Chromatography

83. Nitrite-N: Colorimetric Method
• Nitrite forms reddish purple azo dye at 2-2.5 pH by coupling
diazotized sulfanilamide with N-1(1-naphthyl)-ethylene diamine
dihydro chloride (NED dihydrochloride)
• Good for 10 to 1000 µg/L levels (with the light path of 5 cm, 5-50
µg/L can also be measured)
• Use nitrite free water in analysis of samples for nitrite –
• Interferences
– NCl3 imparts false red colour
– Sb3+
, Au3+
,Bi3+
,Fe3+
,Pb2+
,Hg3+
,Ag3+
, chloroplatinate (PtCl6
2-
) and
metavanadate can precipitate under test conditions and interfere
– Cupric ion can catalyze decomposition of the diazonium salt and
introduce negative error
– Colored ions and suspended solids can also interfere

84. • Filter the sample through 0.45 µm pore membrane filter and adjust
pH to 5-9 with HCl or NH4OH
• Take 50 ml (or a portion diluted to 50 ml) add 2 ml colour reagent
and mix
– Colour reagent: add 100 ml of 85% phosphoric acid to 800 ml water,
dissolve 10 g sulfanilamide and 1 g N-(1-naphthyl)-ethylene diamine
dihydro chloride, and adjust volume to 1 liter
– Can be stored upto one month in a dark bottle in refrigerator
• After 10 min but before 2 hours measure absorbance at 543 nm
• Treat standards also with colour reagent and measure absorbance
• Standard stock solution : Dissolve 1.232 g NaNO2 in water and dilute
to one liter (gives 1 mL = 250µgN stock solution)
• Plot absorbance of standards against NO2
-
conc. for calibration curve
• Read concentration in the sample by the standard calibration curve
Nitrite Nitrogen: Colorimetric Method

85. Nitrite free water
For preparing nitrite free water
• Add a small crystal of KMnO4and Ba(OH)2 or Ca(OH)2 to distilled
water and redistill in borosilicate glassware
• Discard initial 50 mL of the redistillate and also the final distillate
giving red colour with DPD reagent
• Add 1 mL/L of conc. H2SO4 and 0.2 mL/L of MnSO4 (36.4 g MnSO4.H2O
in 1 L distilled water), make the water pink by adding 1 to 3 ml
KMnO4 solution and redistill to get nitrite free water
• DPD reagent (N,N-Diethyl-p-phenylenediamine indicator solution):
dissolve 1 g DPD oxalate or 1.5 g DPD sulfate pentahydrate or 1.1 g
anhydrous DPD sulfate in chlorine free distilled water containing 8
ml of 1+3 H2SO4 and 200 mg disodium EDTA and makeup volume
to 1 liter.
– Store in brown glass-stoppered bottle in the dark – discard when
discoloured or when its absorbance exceeds 0.002/cm at 515 nm

86. Nitrate: UV Spectrophotometer Method
Interferences
• Dissolved organic matter, hydroxide and carbonate ions,
surfactants and Cr6+
interfere with this method of measurement
• Acidification with 1N HCl prevents interference OH-
and CO-2
ions
• Nitrate and organic matter absorb at the same wavelength (220nm)
- organic matter also shows absorbs at 275 nm but not nitrate
• Interference by organic matter is taken care of by measuring
absorbance at both 220 and 275 nm and absorbance correction by
• If correction value is >10% of the reading at 220nm then the
method is discarded
• Samples with significant organic matter levels are not analyzed
U = S – 2T
S = Absorbance at 220 nm
T = Absorbance at 275 nm

87. • Filter sample and add 1 mL 1N HCl to 50 mL sample.
• Prepare NO3
-
calibration standards in the 0 to 7 mg/L
range from the stock nitrate solution.
• Stock nitrate solution: Dissolve 0.7218 g dry potassium nitrate in
water and dilute to 1.0 L. (1.0 mL = 100 µg NO3
-
-N)
• Preserve the stock with 2mL CHCl3 /L.
• Read absorbance at both 220 nm and 275 nm
• Make correction to the absorbance at 220 nm and
construct calibration curve (conc. versus corrected abs.)
Nitrate-N: UV Spectrophotometer
Method
Standards
NO3
-
-N/L
Absorbace at
220 nm (R )
Absorbance at
275 nm (S)
T = 2S U=R-T
0.2
0.4
0.8
1.4
2
7

88. Cd reduction method
Approach
• NO3
¯ is reduced to nitrite (NO2
¯) by passing the sample through
a cadmium (Cd) reduction column
• Nitrite (NO2
¯) is determined colorimetrically
• Correction can be made for any NO2
¯ present in the sample by
analyzing without the reduction step
• Method can be used for nitrate concentrations 0.01-1mg/L
Interference
• Suspended solids will restrict flow through Cd reduction column
and hence the sample requires pre-filtration
• Iron, copper or other metals can cause interference (EDTA is
added to remove the interference)
• Residual chlorine can be an interference (dechlorinate the
sample with sodium thiosulfate to remove the interference)
• If oil and grease are present remove them by pre-extracting the
sample with an organic solvent

89. • Constructed from end to end joining of two pieces of tubing (10-cm
length of 3-cm-ID tubing and 25-cm length of 3.5-mm-ID tubing)
• Add a TFE stopcock with metering valveto control flow rate.
Cd reduction column

90. • Wash column with 200 mL dilute NH4
Cl-EDTA solution
• Activate the column by passing through 100 ml of solution (made
by mxing 25 mL of 1.0 mg/L nitrate-N and 75 mL of NH4Cl-EDTA) at
7 to 10 mL/min rate
• Ammonium chloride-EDTA (NH4
Cl-EDTA) solution: Dissolve 13 g NH4Cl
and 1.7 g disodium ethylene diamine tetra acetate in 900 mL water,
adjust pH to 8.5 with NH4OH and makeup volume to 1L.
• Screen the sample, adjust pH to 7-9, take 25 mL sample (or a
portion made to 25 mL), add 75 mL NH4
Cl- EDTA solution, mix, and
pass through the column a 7 to 10 mL/min rate
• Discard initial 25 mL and collect in the original sample flask.
• Within 15 min., add 2.0 mL color reagent to 50 mL sample, mix and
measure absorbance at 543 nm within another 10 min. to 2 hours
• Prepare nitrate standards in 0.05 – 1.0 mg/L range in the similar
manner, run for reduction from nitrate to nitrite, add color reagent
and measure the absorbance, and construct a calibration curve
Cd reduction method