Organic Matter

(Non-biodegradable and Biodegradable)

(TOC, COD, DO, BOD, BOD
u
and serial BOD)
Dr. Akepati S. Reddy
Thapar University
Patiala (PUNJAB) 147 004
INDIA
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 CO
2
and this in turn is measured
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 samples organic matter is measured as
Theoretical Oxygen Demand (ThOD): Oxygen demand (of the
samples 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 samples OC is
measured in terms of the amount of oxidizing agent consumed
Biological Oxygen Demand (BOD): microorganisms are made to use
the samples organic matter as food and aerobically oxidize into
inorganic end products, and oxygen utilized is measured as BOD
Theoretic Oxygen Demand
mpirical 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
3 2 2 2
2
3
2 4
3
2 4
cNH O H
c a
nCO O
c b a
n N O H C
c b a n
+

'
+

'

'
+

'

+ +
oxygen g requires e glu g of Oxidation
O H CO O O H C
192 cos 180
6 6 6
2 2 2 6 12 6
+ +
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 H
2
SO
4
reagent) at high temp. (150C 2
o
C) for 2 hrs., and can
be shown by:
C
n
H
a
O
b
N
c
+dCr
2
O
7
-2
+(8d+c)H
+
nCO
2
+ {(a+8d-3c)/2}H
2
O+cNH
4
+
+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
2 3 6 3
2 c b a n
d + =
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 (20/27C) 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
O
2
demand by acclimatized microorganisms and nitrification and
the O
2
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
Fate of organic matter of the sample in the BOD test
Organic Matter
(dissolved)
Non-biodegradable
& residual organic matter
Suspended & colloidal
organic matter
oxygen
CO
2
, H
2
O, NH
3
, Energy, etc.
New heterotrophic
Microbial biomass
Auto-oxidation
CO
2
, H
2
O, NH
3
, 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 debris
h
y
d
r
o
l
y
s
i
s
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
Ultimate BOD
BOD
t
is the samples oxygen demand when it is incubated for t
time (3 or 5 days) at XC temperature
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 (BOD
u
) is considered
important
For this the BOD
t
results are extrapolated through using BOD
kinetics model which assumes that the BOD exertion follows
first order decreasing rate of increase
Oxygen demand exertion pattern of a sample during incubation
BOD kinetics
Oxygen demand exertion pattern is first order decreasing rate of
increase and can be shown as
t t o u
L BOD L BOD
' '
+ = =
t time given any at
exp(-k.t)} - {1 L BOD
BOD
o t
t
=
as written be can
) 20
20 T
k k

=
T
J
T is temp. in C
J is constant - taken as 1.056 for
20-30C and as 1.135 for 4-20C
kL - dL/dt
L
0
=
+ =
t t
L BOD
exp(-k.t) L L
o t
=
dL/dt is rate of oxygen demand exertion
L
t
is oxygen demand that is yet to be exerted at
after incubation time t
L
0
is oxygen demand to be exerted by the sample
at incubation time zero (also known as BOD
u
)
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
Chemical Oxygen Demand (COD)
by Open and Closed (Titrimetry and
Spectrophotometry) Reflux Methods
COD
Measure of oxygen equivalent of organic matter content of sample
Oxidation of organic matter occurs under acidic conditions at
elevated temperature (1502C) 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
4 2 2
2
7 2
2 2 / 3 8 8
+ +
+ + + + + + + dCr cNH O H c d a nCO H c d O dCr N O H C
c b a n
2 3 6 3
2 c b a n
d + =
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
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
O H Cr Cl H O Cr Cl
2
3
2 7 2
7 2 3 14 6 + + + +
+ +
COD by Open reflux method
Sample and blank are refluxed in strongly acidic solution in the
presence of known excess of standard K
2
Cr
2
O
7
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 K
2
Cr
2
O
7
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
+ + + +
+ +
3 3 6 2
3 3 Cr Fe Cr Fe
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
used sample of ml
M B A
O as l mg COD

=
A is ml FAS consumed in blank titration
B is ml FAS consumed in sample titration
M is molarity of FAS
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 Ag
2
SO
4
to conc. H
2
SO
4
, 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 FeSO
4
.7H
2
O in distilled water and adjust
volume to 100 ml.
Potassium hydrogen phthalate (KHP) standard (500 mg/L COD): Dry
crushed potassium hydrogen phthalate (HOOCC
6
H
4
COOK) to
constant weight at 120C, dissolve 425 mg in distilled water and
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 (HgSO
4
) crystals or powder.
Sulfamic acid
COD by Open Reflux Method
Apparatus and reagents
Standard potassium dichromate (0.0417M): Dissolve 12.259 g
K
2
Cr
2
O
7
(dried at 103C for 2 hours) in water and adjust volume
to 1.0 L
Standard ferrous ammonium sulfate, FAS (0.25M): Dissolve 98 g
Fe(NH
4
)
2
(SO
4
)
2
.6H
2
O in distilled water, add 20 ml conc. H
2
SO
4
,
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
Dichromate of Molarity
consumed FAS of ml
taken Dichromate of ml
FAS of Molarity =
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
H
2
SO
4
reagent while mixing and cooling the contents
Parallel to the sample preparae 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 H
2
SO
4
reagent through the condenser opening, and
thoroughly mix the flask contents
Volume of H
2
SO
4
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
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
used sample of ml
M B A
O as l mg COD

=
A is ml FAS consumed to titrate blank
B is ml FAS consumed to titrate sample
M is molarity of FAS
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 1502C.
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
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 K
2
Cr
2
O
7
(dried at 103C for 2 hr.) in 500 ml distilled water, add 167
ml conc. H
2
SO
4
and 33.3 g HgSO
4
, 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. H
2
SO
4
, 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 K
2
Cr
2
O
7
(dried at 103C for 2 hr), 167 ml conc. H
2
SO
4
and 33.3 g HgSO
4
in
500 ml distilled water, cool and adjust volume to 1.0 L
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 H
2
SO
4
reagent into the culture tube along the walls to form a
distinct acid layer underneath the sample
Sample, dichromate digestion solution and H
2
SO
4
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 1502C), and reflux
Reflux for 2 hours switch off the block digester and cool the tubes
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 samples 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
used sample of ml
M B A
O as l mg COD

=
A is ml FAS consumed in blank titration
B is ml FAS consumed in sample titration
M is molarity of FAS
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% H
2
SO
4
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
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
Dissolved Oxygen (DO)
by Winkler and
Membrane Electrode methods
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 samples 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
Winkler method for DO
NaI O S Na I O S Na
O H Mn H OH Mn b
O H Mn I H I MnO a
OH Mn OH Mn c
O H MnO O OH Mn b
O H MnO O OH Mn a
2 2 . 3
2 2 ) ( . 2
2 4 2 . 2
) ( 2 . 1
5 . 0 ) ( . 1
5 . 0 2 . 1
6 4 2 2 3 2 2
2
2
2
2
2
2 2
2
2 2 2 2
2 2 2
2
+ +
+ + +
+ + + + +
+ +
+ + +
+ + + +
+ +
+ +
+
+
Reactions involved in the Winkler method of DO testing are
Sources of error:
Presence of Nitrite (more than 50 Qg/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 NO
2
and removes
it as N
2
and N
2
O gases
+
+
+ + +
+ + + +
H NO O H O O N
O H O N I H I NO
2 2 5 . 0
4 2 2
2 2 2 2 2
2 2 2 2 2
O H O N N H NO HN
Na HN H NaN
2 2 2 2 3
3 3
+ + + +
+ +
+
+ +
Winkler Method for DO
For avoiding errors, the sample should not come in contact with air
during sampling and testing (at least till the samples 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
(K
2
C
2
O
4
) and mixing the contents
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 bottole 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
Reagents for DO testing
Manganous sulfate solution: Dissolve 480 g MnSO
4
.4H
2
O 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 (NaN
3
) 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
Na
2
S
2
O
3
.5H
2
O in distilled water, add 1.5 ml 6N NaOH, and adjust
volume to one liter.
Reagents for DO testing
Standard potassium bi-iodate solution: Dissolve 812.4 mg of
potassium bi-iodate, KH(IO
3
)
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
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 MnSO
4
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 H
2
SO
4
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 samples DO
200
300
300
) (
x
mL volume Sample

=
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 H
2
SO
4
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
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
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 DO
s
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
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 g/l, but being a destructive
test, can not be used for continuous DO monitoring in samples
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 manufacturers procedure for cleaning
electrodes, and for changing membrane and electrolyte solution
Use high quality electrolyte solution (either prepared as per
manufacturers 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
Manufacturers 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
Biological Oxygen Demand
(5 or 3 day BOD)
by BOD Bottle Method
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
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 BOD
t
at XC (BOD
5
at 20C, BOD
3
at
27C, etc.)
Units for BOD
t
at XC are mg/L (BOD
t
is oxygen demand when the
sample is incubated for t days at XC
Testing gives oxygen demand of diluted sample - multiplication of this
with dilution factor gives samples oxygen demand
NH
3
-N added (as nutrient supplement) and NH
3
-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
Expression for BOD
t
from test results
BOD
t
at XC of a sample can be written as
Dilution Factor D
f
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

'
+

'

'
+

'

'
+

'

'
+

'

=
Factor
Dilution
ion nitrificat
by error
-
ater dilution w and
seed by error
-
sample diluted
the of OD
BOD
t
) (
1000
sample diluted of liter one preparing for used sample of ml
D
f
=
sf si
DO DO OD =
DO
si
& Do
sf
are initial & final DO of diluted
sample before & after t days of incubation
F ) DO - (DO ater dilution w seeded of OD
cf ci
=
prepared control seed of liter per seed of ml
prepared sample diluted of liter per seed of ml
F =
f
f
cf ci sf si
o
t
D F
D
DO DO DO DO C X at BOD

'
+

'

=
1
1 ) ( ) (
cf ci
DO - DO seed of OD =
DO
ci
& DO
cf
are initial & final DO of
the seed control incubated for t days
F
D
DO DO water dilution seeded of OD
f
cf ci

'
+

'

=
1
1 ) (
Expression for BOD
t
from test results
bottle BOD in water dilution seeded of fraction volume is
D
f

'
+

'

1
1
Error by nitrification: Nitrification reaction is inhibited by adding
nitrification inhibition chemical and hence no correction needed.
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
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 BOD
t
results
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 (BOD
5
at 20C).
By conviction incubated for 5 days at 20rC (annual average temp. of
UK and time taken by the Thames to reach the ocean) CPCB
recommends 3 days at 27C (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 BOD
t
= 60-70% of BOD
u
can be adapted
For ensuring incubation at constant temp., samples are incubated
either in BOD incubators or in water baths set at desired temp.
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 BOD
5
measurement, and in the
BOD
u
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)
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
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
Dilution factor (D
f
)
Oxygen is sparingly soluble in water and depends on altitude,
temperature and salinity
Altitude (in
meter)
Saturated
DO (in
mg/l)
Temperat
ure (in
rC)
Saturated
DO (in
mg/l)
Chlorini
ty
Saturated DO
(in mg/l)
sea level 9.2 0.0 14.62 0.0 9.09 (20rC)
305 8.9 5.0 12.77 7.56 (30rC)
610 8.6 10.0 11.29 6.41 (40rC)
914 8.2 15.0 10.08 5.0 8.62 (20rC)
1219 7.9 20.0 9.09 .. 7.19 (30rC)
1524 7.6 25.0 8.26 .. 6.12 (40rC)
1829 7.4 30.0 7.56 10.0 8.17 (20rC)
2134 7.1 35.0 6.95 .. 6.85 (30rC)
2438 6.8 40.0 6.41 .. 5.84 (40rC)
2743 6.5 45.0 5.93 15.0 6.51 (30rC)
3048 6.3 50.0 5.48 20.0 6.20 (30rC)

Dilution factor (D
f
)
Diluted sample is aerated to rise DO
i
closer to DO
S
At 20C, DO level can rise to aboit 8 mg/l level - diluted samples
initial DO: about 8 mg/l
At e 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 e 7 mg/L.
For finding D
f
, 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 D
f
.
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 D
f
(1, 5, 25, 125, 625, , so on) can be
used in the test
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 27C 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 BOD
t
measurement: 15 to 50% (18% SD)
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 20C
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
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 20C.
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
Apparatus, Glassware and Chemicals
Apparatus and glassware
BOD incubator or Water Bath set at 201C or 271C.
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 KH
2
PO
4
, 21.75 g K
2
HPO
4
, 33.4 g
Na
2
HPO
4
.7H
2
O and 1.7 g NH
4
Cl in 500 ml water and final volume
adjusted to one liter (discard if biological growth observed).
Magnesium sulfate solution: 22.5 g MgSO
4
.7H
2
O in water and
adjust final volume to one liter.
Calcium chloride solution: 27.5 g CaCl
2
in water and adjust final
volume to one liter.
Ferric chloride solution: 0.25 g FeCl
3
.6H
2
O in water and adjust
final volume to one liter.
Apparatus, Glassware and Chemicals
Chemicals
1N acid or alkali solution (28 ml of conc. H
2
SO
4
to one liter; or 40
g NaOH in one liter final volume).
Sodium sulfite solution: 1.575 g Na
2
SO
3
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 103C 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 NH
4
Cl in 500 ml water, adjust pH to
7.2 with NaOH solution and adjust final volume to one liter
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 samples temp. to 20C (partially fill the
sample in a bottle and vigorously agitate)
Procedure for 5-day BOD test
Adjust samples pH to 6.5-7.5 by 1N H
2
SO
4
or NaOH
Decide on D
f
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 201C in incubator/water bath
Use rest of the bottles to measure DO
i
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
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 DO
i
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 DO
i
of the incubated synthetic sample
Running glucose-glutamic acid check is optional
Procedure for 5-day BOD test
After incubating for 5 days 1 hour, take out the incubated BOD
bottles and measure their DO
f
either by Winkler method (azide
modification) or by Membrane electrode method
Discord the results if DO
f
is <1.0 mg/L, or if DO
i
-DO
f
is <2.0 mg/L
Record results and find out BOD
5
at 20C 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 19830.5 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 20410.4
mg/L range
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
(DO
si
)
Bottle
No.
Final DO
(DO
sf
)
Bottle
No.
Initial DO
(DO
ci
)
Bottle
No.
Final DO
(DO
cf
)



Average Average Average Average

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 BOD
5
at 20C <2 mg/l.
CPCB has suggested 3-days incubation at 27C temp., in place of 5
days incubation at 20C temp.
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 DO
f
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
Ultimate BOD (BOD
u
)
Ultimate BOD (BOD
u
) by BOD Bottle Method
5-day BOD test fails to measure BOD
u
- Kinetic descriptors (BOD
kinetic parameters) can relate BOD
5
with BOD
u
and estimate BOD
u
Oxidation is not complete in 5 days of incubation - 60 to 90 days
incubation needed for complete oxidation and BOD
u
measurement
For the measurement of BOD
u
, 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.
Ultimate BOD (BOD
u
) by BOD Bottle Method
Concentrations of NO
3
-N and NO
2
-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 NH
3
N to NO
3
N and NO
2
N
to NO
3
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
NO
3
-N and NO
2
-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
Procedure for BOD
u
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 )
Procedure for BOD
u
measurement
If residual chlorine is suspected, dechlorinate the sample
Adjust samples temperature to 20C 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 BOD
u
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
H
2
SO
4
and preserve under refrigeration for nitrogen estimation
For knowing BOD
u
of the dilution water, fill both seeded and
unseeded dilution water in BOD bottles and incubate, and measure
their initial and final DO.
Procedure for BOD
u
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 NO
3
& NO
2
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
samples 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 NO
3
-N and NO
2
-N from the beaker, adjust pH
to <2 by adding H
2
SO
4
and 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
Procedure for BOD
u
measurement
Analyse the samples collected for NO
3
-N and NO
2
-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
NO
3
-N and NO
2
-N measured at the end of the interval
Time elapsed between DO
i
and Do
f
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 samples overall oxygen demand
(OD); nitrogenous OD and carbonaceous OD for each of the
intervals, and estimate cumulative carbonaceous oxygen demand

'
+

'

=
s reaeration succesive
two between erval
DO and DO between reval
DO DO
demand oxygen Overall
f i
f i
int
int
) (
? A ? A )
? A ? A )

+

=

'
+

'

29 . 2 2 2
43 . 3 3 3
N NO final N NO final
N NO final N NO initial
demand oxygen
s Nitrogenou
Serial BOD test
and BOD kinetic parameters
Serial BOD test by BOD bottle method
Needed for finding out BOD kinetics parameters
Involves measurement of BOD
1
, BOD
2
, , BOD
i
, , BOD
n
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 BOD
i
estimation
For acceptable results, the conditions, DO
f
>1.0 mg/L and DO
i
-Do
f
>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
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)
Method of least squares for BOD kinetics parameters
)
n
BOD
K n
dt
BOD d
BOD
BOD BOD n
dt
BOD d
BOD BOD
dt
BOD d
n
K
t t
BOD BOD
dt
BOD d
BOD K L K L K
n
i
i
n
i
i
u
n
i
i
n
i
i
n
i
n
i
i
n
i
i i
i
i i
i i

=
=
= =
= = =
+
+
+ =

'
+

'

'
+

'

= = =
1
1
2
1 1
2
1 1 1
1 1
1 1
0
.
) (
.
) (
. .
) (
.
) (
. . .
dt
d(BOD)
Time (day) BOD BOD
2
dBOD/dt (dBOD/dt).BOD
1
2

n
Results of serial BOD test for n days are needed
Method of Moments for BOD kinetic parameters
Moores diagram (a nomograph relating K with 7BOD/L
0
and
7BOD/7(BOD.t)) is needed
Moores diagram is different for different n value
Results of serial BOD test for n days are used to find 7BOD and
7BOD/ 7(BOD.t)
7BOD/7(BOD.t) value is used to read k value and 7BOD/L
0
value
from the Moores diagram
From 7BOD/L
0,
since 7BOD is known, L
0
is found
Using the following formulae Moores diagram can be constructed
) )
)
)
) )
)
) ? A

=
n
K i
n
K
K n K
n
n
K
K n K
n
i i
n
t BOD
BOD
n
L
BOD
1
.
1
.
1
1
.
0
1
exp .
1 exp
1 exp exp
.
1 exp
1 exp exp
k 4 days 5 days 6 days 7 days 8 days
value Y/L
0
Y/tY Y/L
0
Y/tY Y/L
0
Y/tY Y/L
0
Y/tY Y/L
0
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
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
C
u
m
u
l
a
t
i
v
e

B
O
D
0.27
0.275
0.28
0.285
0.29
0.295
0.3
0.305
0.31
0.315
C
u
m
u
l
a
t
i
v
e

B
O
D
.
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
C
u
m
u
l
a
t
i
v
e

B
O
D
0.175
0.18
0.185
0.19
0.195
0.2
0.205
0.21
0.215
C
u
m
u
l
a
t
i
v
e

B
O
D
.
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
C
u
m
u
l
a
t
i
v
e

B
O
D
0.2
0.205
0.21
0.215
0.22
0.225
0.23
0.235
0.24
C
u
m
u
l
a
t
i
v
e

B
O
D
.
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
c
u
m
u
l
a
t
i
v
e

B
O
D
0.23
0.235
0.24
0.245
0.25
0.255
0.26
0.265
0.27
C
u
m
u
l
a
t
i
v
e

B
O
D
.
t
Method of Moments for BOD kinetic parameters
Methods for BOD Kinetic Parameters
Fujimoto method
Serial BOD test results for n number of days is used
BOD
t+1
is plotted against BOD
t
in a graph
On the same graph another plot with slope=1 is plotted
Point of intersection of the two plots is taken as BOD
u
Using the BOD
u
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 BOD
u
Using the BOD
u
obtained, with the help of BOD kinetics model K
value is found
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
(KL
0
)
1/3
is obtained as intercept and K
2/3
/6L
1/3
as slope
Form the slope and intercept K and L are calculated
) t
L
K
L K
BOD
t
.
6
.
3
1
0
3
2
3
1
0
3
1
+ =

'
+

'

Nitrate and Nitrite Nitrogen

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 BOD
u
Analyze the samples promptly to avoid conversion of nitrite into
nitrate/ammonia and denitrification of nitrate
Nitrite samples after adjusting pH to <2 with H
2
SO
4
, can be freezed
and stored at -20C (for 1 to 24 hr preservation store at 4C)
Nitrate samples can be store at 4
0
C upto 24 h after adding 2 mL
conc H
2
SO
4
/L
Nitrite analysis is by colorimetric method
Nitrate can be analyzed by
UV Spectrometric Method,
Cd-reduction Method
Ion Chromatography
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 Qg/L levels (with the light path of 5 cm, 5-50 Qg/L
can also be measured)
Use nitrite free water in analysis of samples for nitrite
Interferences
NCl
3
imparts false red colour
Sb
3+
, Au
3+
,Bi
3+
,Fe
3+
,Pb
2+
,Hg
3+
,Ag
3+
, chloroplatinate (PtCl
6
2-
) and
metavanadate can precipitate under test conditions and interfere
Cupric ion can catalyze decomposition of the diazoniumsalt and
introduce negative error
Colored ions and suspended solids can also interfere
Filter the sample through 0.45 Qm pore membrane filter and adjust
pH to 5-9 with HCl or NH
4
OH
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 NaNO
2
in water and dilute
to one liter (gives 1 mL = 250gN stock solution)
Plot absorbance of standards against NO
2
-
conc. for calibration curve
Read concentration in the sample by the standard calibration curve
Nitrite Nitrogen: Colorimetric Method
Nitrite free water
For preparing nitrite free water
Add a small crystal of KMnO
4
and 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. H
2
SO
4
and 0.2 mL/L of MnSO
4
(36.4 g
MnSO
4
.H
2
O in 1 L distilled water), make the water pink by adding 1
to 3 ml KMnO
4
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
Nitrate: UV Spectrophotometer Method
Interferences
Dissolved organic matter, hydroxide and carbonate ions, surfactants
and Cr
6+
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
Filter sample and add 1 mL 1N HCl to 50 mL sample.
Prepare NO
3
-
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 g NO
3
-
-N)
Preserve the stock with 2mL CHCl
3
/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
NO
3
-
-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
Cd reduction method
Approach
NO
3
is reduced to nitrite (NO
2
) by passing the sample through
a cadmium(Cd) reduction column
Nitrite (NO
2
) is determined colorimetrically
Correction can be made for any NO
2
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 sodiumthiosulfate to remove the interference)
If oil and grease are present remove them by pre-extracting the
sample with an organic solvent
Constructed from end to end joining of two pieces of tubing (10-cm
length of 3-cm-ID tubing and 25-cmlength of 3.5-mm-ID tubing)
Add a TFE stopcock with metering valve to control flow rate.
Cd reduction column
Wash column with 200 mL dilute NH
4
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 NH
4
Cl-EDTA) at
7 to 10 mL/min rate
Ammonium chloride-EDTA (NH
4
Cl-EDTA) solution: Dissolve 13 g NH
4
Cl
and 1.7 g disodium ethylene diamine tetra acetate in 900 mL water,
adjust pH to 8.5 with NH
4
OH 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 NH
4
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 nmwithin 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