PH,acidity and alkanity

Suspended Solids
(TSS, VSS, Biodegradable VSS, SVI
and Colloidal solids )

Suspended Solids
• Total solids (TS): Material residue left behind after
evaporation of a sample and its subsequent drying in a oven
at a defined temperature to constant weight
• Total dissolved solids (TDS): Portion of the material residue of
a sample that passes through a filter
• Total suspended solids (TDS): Portion of the material residue
of a sample retained by a filter
– Settlable solids: Material settling out within a defined period
• Fixed suspended solids: Residue of TSS left after ignition for a
specified time at a specified temperature
• Volatile suspended solids: weight loss on ignition of total
suspended solids
– Biodegradable volatile suspended solids: volatile suspended
solids lost through biodegradation
• Colloidal solids: cause turbidity and measured as turbidity
(NTU or JTU)

Suspended Solids
• Regulatory limits are imposed on TSS for sewage disposal
– Water with high suspended solids may be aesthetically
unsatisfactory (for bathing!)
• Removal of TSS is one of the sewage treatment objectives
– Primary treatment is mainly concerned with it
• Sludge generation calculations in biological treatment require
the knowledge of TSS, VSS and biodegradable VSS
– All VSS is not biodegradable, and biological treatment can
hydrolyze only the biodegradable VSS
• Biological treatment involves generation of suspended solids
(biosolids)
– These biosolids are monitored as MLSS (TSS) and MLVSS (VSS)
• Maintenance of higher levels of biosolids (activated sludge) is
important in biological treatment
– MLVSS is often used as a measure of active biomass/sludge
• SVI used in the design, operation and control of secondary
clarifiers require MLSS (TSS) monitoring

Total suspended solids (TSS)
TSS and MLSS are one and the same
Two alternate ways for TSS measurement
• Filter the sample through a weighed ash free filter paper, dry
the filter paper along with the residue retained on it to
constant weight at 103-105C, and gravimetrically find the TSS
– High measurement uncertainty values – in case of low TSS larger
volumes need sampling
– In case of samples with high TDS thoroughly wash the filter
paper with TDS free water to remove the dissolved material
• Find TS and TDS for the sample and take difference of TS and
TDS as TSS
– In case of the filter paper clogging and prolonged duration of
filtration this method is followed
Often settlable solids rather than TSS is measured as an
alternative
• Centrifugation for TSS measurement?

Volatile Suspended Solids
• VSS and MLVSS are one and the same
• Weight loss on ignition of the TSS represent the VSS
• Ash free filter paper leaves no residue on ignition
• Negative error is introduced from the loss of volatile matter
during drying
• Estimation of low concentrations of volatile solids in the
presence of high fixed solids concentration can be more
erroneous
• Dried residue left on the ash less filter paper is ignited to
constant weight at 550±50C in a muffle furnace to remove
volatile matter and obtain fixed or non-volatile matter
– Difference of TSS and NVSS (fixed solids) is taken as VSS

Solids in Samples with Solids > 20,000 mg/L
The methods used for samples with lower solids levels are not
used – can be associated with negative error
If the sample is a sludge, stir to homogenize and place it in a
evaporation dish, evaporate to dryness on a water bath, and
dry at 103-105C for 1 hour to find % solids
For finding fixed and volatile solids ignite the residue in muffle
furnace for one hour at 550±50C
– If the residue left in the evaporation dish contains large
amounts of organic matter then ignite it first over a gas burner
and then in the muffle furnace
 
BC
BA
solidstotal



1000
%
 
BA
DA
solidsvolatile



1000
%
 
BA
BD
solidsfixed



1000
%
A - weight of dish with residue
B - weight of the dish
C - weight of dish with wet sample
D - weight of dish with residue after ignition

Settlable Solids
• Determined on either volume (mL/L) or weight (mg/L) basis
• Measurement on volume basis requires an Imhoff cone
– Fill the cone to 1 L mark with sample and settle for 45 min.
– Gently stir sides of the cone with a rod by spinning and settle for
another 15 minutes
– Record volume of the settled solids in the Imhoff cone
• Measurement on weight basis
– Determine TSS of well mixed sample
– Pour >1-L of sample into a glass vessel of >9 cm dia. to depth
>20cm and let it stand quiescent for one hour
– Without disturbing the settled and floating material siphon out
water from the vessel center and determine TSS as non-
settlable TSS
Settlable solids = TSS – non-settlable TSS

Sludge Volume Index (SVI)
• Volume in mL occupied by 1 g of a suspension after 30 min.
settling
• Used to monitor settling characteristics of activated sludge
and other biological suspensions
– Determined for the mixed liquor of the aeration tank of the ASP
• Determine TSS concentration of a well mixed mixed-liquor
sample
• Use Imhoff cone for settling 1 L of well mixed mixed-liquor for
30 min. time and measure the settled sludge volume in mL
– Gently stir the sample during settling
• Calculate SVI as
)/(
1000)/(
Lgionconcentratsolidssuspended
LmLvolumesludgesettled
SVI



Colloidal Solids and Turbidity
• Colloidal matter causes turbidity
• Turbidity is an optical property caused by scattering of light,
and indicates clarity of water
• Biological treatment removes colloidal solids/turbidity
through bioflocculation
• Nephelometers are used for measurement and the results are
reported in Nephalometric Turbidity Units, NTU
– Intensity of light scattered by the sample is compared with the
standard reference suspension under the same conditions
• Formazin polymer suspension is used
• A light source and a photoelectric detector are used in the
measurement

Organic Matter
• TOC
• ThOD
• COD
• BOD
– DO
– BOD3 and BOD5
– BODu
• BOD kinetics
– Serial BOD test
– BOD kinetic parameters

Measurement of Organic Matter
Organic matter in wastewater is heterogeneous
– Suspended (VSS), colloidal (turbidity) and dissolved organic
matter
– Carbohydrates, proteins, fats, etc.
Organic matter is biodegradable and non-biodegradable
Single direct method for the measurement of organic matter is
not feasible – so indirect methods – these 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 inorganic carbon,
and then treated to convert organic carbon into carbon dioxide
and the amount of CO2 formed is measured

Measurement of Organic Matter
• Oxygen Demand (ThOD, COD and BOD)
– Organic matter is reduced substance and it can be completely
oxidized and transformed into inorganic end products and this
demands oxygen
– Amount of oxygen demanded is proportional to the organic
matter present – the oxygen demanded is measured and
related to organic matter
– Oxygen demand of the sample’s organic matter is measured as
• Theoretical Oxygen Demand (ThOD): If chemical formula of the
organic matter is known, oxygen demand of the sample’s organic
matter can be theoretically found through stoichiometry
• 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 organic matter as food and aerobically oxidize into
inorganic end products, and oxygen utilized is measured as BOD

Theoretic Oxygen Demand
Empirical formula of organic matter present in the sample is
used and a balanced equation of oxidation is written
Amount of oxygen required (for complete oxidation of one
unit mass of organic matter) is stoichiometrically
estimated
The oxygen demand equivalent to the organic matter
presented
3222
2
3
24
3
24
cNHOH
ca
nCOO
cba
nNOHC cban 












oxygengrequireseglugofOxidation
OHCOOOHC
192cos180
666 2226126 

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± 2oC) for 2
hrs., and can be shown by:
CnHaObNc+dCr2O7
-2+(8d+c)H+ nCO2+ {(a+8d-3c)/2}H2O+cNH4
++2dCr+3
d is moles of dichromate consumed
One mole of dichromate = 1.5 moles of COD/oxygen
• Not a good measure for biodegradable organic matter and not
capable of oxidizing all the organic matter
• Widely used because real time/reasonable time results are
possible
• In case of anaerobic treatment COD is preferred over BOD for
organic matter concentration measurement
2363
2 cban
d 

Biochemical Oxygen Demand (BOD)
• Acclimatized 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 days)
• Organic matter is used by organisms as food and oxidize –
only the matter that can be consumed as food (biodegradable
fraction) can be measured
• O2 is also demand by microorganisms for the nitrification of
ammonical-N into nitrite-N and Nitrate-N (introduces positive
error in the measurement)
• COD on the other hand measures both biodegradable non-
biodegradable organic matter

COD
• Measure of oxygen equivalent of organic matter content of a
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 

COD
• Pyridine (and related compounds) and aromatic hydrocarbons are
not completely oxidized
• VOCs (originally present or formed during 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
OHCrClHOCrCl 2
3
272 723146  

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

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, 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

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±2ᵒC.
• Digested samples are cooled and tested for COD by
• Titration with FAS (Titrimetric 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
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
Colorimetric method
• 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

Dissolved Oxygen (DO): Winkler Method
• Can be measured by either Winkler method (iodometric method!)
or 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 (forms 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 solubulized
• 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 also be used to measure the iodine liberated

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 g/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





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 procedure
• Permanganate modification
• Permanganate modification is needed if ferrous iron level is > 1.0
mg/L
• To the sample 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

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 oxygen utilization rates
– Fill aspirator bottle with the sample from the bottom by a tube
near the bottom while allowing overflow of 25-50% volume
– Add 10 ml of copper sulfate-sulfamic acid inhibitor solution to
1.0 L aspirator bottle with glass-stopper.
– 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

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

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

• 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

BOD Bottle Method for BOD Estimation
A BOD bottle filled with diluted sample with acclimatized
seed and stoppered is incubated at constant temperature
for a fixed duration
– Dilution of the sample
– Acclimatized seed
– Favourable nutrient and osmotic conditions
– No air bubble entrainment
– known initial DO
5 days incubation at 20°C (3 days at 27°C)
– only partial oxidation of the organic matter occurs
– 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

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 biooxidise 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, 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 20°C
• Experimental results to become acceptable
– Residual DO of the sample should be >1.0 mg/l
– DO difference between initial and final should be >2.0 mg/L

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

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































Factor
Dilution
ionnitrificat
byerror
-
aterdilution wand
seedbyerror
-
samplediluted
theofOD
BODt
)(
1000
sampledilutedofliteronepreparingforusedsampleofml
Df 
sfsi DODOOD 
DOsi & Dosf are initial & final DO of diluted
sample before & after ‘t’ days of incubation

F)DO-(DOaterdilution wseededofOD cfci
preparedcontrolseedofliterperseedofml
preparedsampledilutedofliterperseedofml
F 
f
f
cfcisfsi
o
t DF
D
DODODODOCXatBOD

















1
1)()(
cfci DO-DOseedofOD  DOci & DOcf are initial & final DO of
the seed control incubated for ‘t’ days
F
D
DODOwaterdilutionseededofOD
f
cfci 








1
1)(
Expression for BODt from test results
bottleBODinwaterdilutionseededoffractionvolumeis
Df









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 BODt 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 (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 BODu 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 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)

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.
• What can be used as 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

Aclimatized Seed
• Can be developed from
• Settled domestic sewage
• Suspension prepared from wastewater contaminated soil
• Prepared through continuously aerating for a few days and
adding small daily increments of the wastewater in question
• Preparation of acclimatized seed:
• Take mixed liquor or secondary sludge of a STP and start aeration
• While continuing aeration, gradually replace the mixed
liquor/secondary sludge with the wastewater in question over a
period of two days or more
• Settle the contents and use the supernatant as seed

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)

Dilution factor (Df)
• Diluted sample is aerated to rise DOi closer to DOS
• At 20°C, DO level can rise to about 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 >2 mg/L and residual DO
should be >1 mg/L
• A geometric progression of Df (1, 3, 9, 27, 81, …, 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 may not follow first order kinetics (BOD model assumption)
– 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)

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 sulfite 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 the dose

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

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 debris
hydrolysis

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 or already
present (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 (BODu)
BODt is the sample’s oxygen demand when it is incubated for ‘t’ time
(3 or 5 days) at XᵒC 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
Incubating and waiting for that long period for the 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

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
ttou LBODLBOD
''

ttimegivenanyat
exp(-k.t)}-{1LBOD
BOD
ot
t

aswrittenbecan
 20
20T kk 
 T

T is temp. in °C
 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 (per day units)
K and L0 are known as BOD kinetics parameters
Use of BOD kinetic model requires knowledge of BOD kinetic parameters

BOD Kinetics Parameters and their
Estimation
• K and L0 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 BOD kinetics parameters
• 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
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

Method of Moments for BOD kinetic parameters
• Moore’s diagram (a nomograph relating K with BOD/L0 and
BOD/(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 BOD and
BOD/ (BOD.t)
• BOD/(BOD.t) value is used to read k value and BOD/L0 value
from the Moore’s diagram
• From BOD/L0, since BOD 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

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

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
0
1
L
BOD
n

0
1
L
BOD
n

0
1
L
BOD
n

0
1
L
BOD
n

 

n
n
tBOD
BOD
1
1
.  

n
n
tBOD
BOD
1
1
.
 

n
n
tBOD
BOD
1
1
. 

n
n
tBOD
BOD
1
1
.

Methods for BOD Kinetic Parameters
Fujimoto method
• Serial BOD test results for n number of days are 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

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







• Nitrogen
– Kjeldahl nitrogen
• Ammonical nitrogen (NH3-N)
• Organic nitrogen (Organic-N)
– Nitrite nitrogen (NO2-N)
– Nitrate nitrogen (NO3-N)
– Total nitrogen
• Phosphorus
– Ortho phosphorus
– Total phosphorus
59

Total Kjeldahl Nitrogen
Organic-N
• Organically bound nitrogen is in the trinegative state
• Natural materials like proteins, peptides, nucleic acids and urea, and
many synthetic organic materials have organic-N
Ammonical-N
• Deamination of organic-N and hydrolysis of urea produce
ammonical-N
• Ammonical-N encountered in waters is <10 µg (in ground waters) to
>30 mg/l (in some wastewaters)
– Groundwater has low ammonical-N (soil absorbs and does not allow
leaching)
• Ammonia is often added to water in WTPs for forming combined
residual chlorine
Analytically organic-N and ammonical-N can be determined
together and referred to as Total Kjeldahl Nitrogen (TKN)
61

Methods of Analysis
Ammonical-N can be measured by:
– Nesslerization method (sensitive to 20 µg/l and used for <5 mg/l)
– Phenate method (sensitive to 10 µg/l and used <500 µg/l)
– Titrimetric method (preferred for higher levels, >5 mg/l)
– Ammonia selective electrode method (good for 0.03 to 1400
mg/l levels)
Usually samples need preliminary distillation
– When samples are turbid or coloured or having hydroxide
precipitates of calcium and magnesium (interfere with direct
methods)
– When samples are preserved with acid
When concentration is low, drinking water or clean surface
waters or good quality nitrified wastewater samples can be
tested by direct nesslerization or direct phenate methods - Still
for greater precision preliminary distillation is required 62

Organic-N of the sample can be measured from
– The residual left after preliminary distillation of the sample for
ammonical-N measurement or
– Sample after the removal of ammonical-N from it
• Measurement of organic-N involves
– Conversion of organic-N into ammonical-N through digestion
– Estimation of ammonical-N by one of the Ammonical-N
estimation methods
• Depending on the concentration, either macro-kjeldahl or
semi-micro-kjeldahl method is used for organic-N analysis
A sample is directly tested, without the preliminary distillation,
for TKN (ammonical-N plus organic-N) measurement
Methods of Analysis
63

Sampling and analysis for ammonical-N and organic-N or TKN
involves
• Sample collection, preservation and storage
– If residual chlorine is present, immediately after sample collection
destroy it (for preventing ammonical –N oxidation)
– As far as possible analyze fresh samples
– Preserve samples by acidifying with conc. H2SO4 to 1.5 to 2.0 pH, and
store at 4°C – neutralize to 7 pH with NaOH /KOH prior to testing
• Preliminary distillation and collection of the distillate in boric
acid or sulfuric acid solutions
– Estimation of ammonical-N by any of the methods
• Kjeldahl digestion to convert organic-N into ammonical-N
• Kjeldahl distillation and collection of the distillate in boric
acid or sulfuric acid solutions
– Estimation of organic-N as equivalent to ammonical-N
Method of Analysis
64

Preliminary distillation: interferences
Glycine, urea, glutamic acid, cyanates and acetamide if present in
samples can hydrolyze on standing and introduce + error
– Sample is buffered at 9.5 pH with borate buffer to decrease
hydrolysis of cyanates and organic nitrogen compounds
Volatile alkaline compounds like hydrazines and amines
influence titrimetric results
Some organic compounds, ketones, aldehydes, alcohols and some
amines, cause yellowish/greenish colour even after distillation
– Glycine, hydrazine and some amines give characteristic yellow
colour on nesslerization
– Boiling the distillate at low pH before nesslerization can remove
formaldehyde like interferences
65

• Steam out the distillation apparatus
– Take water into distillation flask, add borate buffer, adjust pH
to 9.5 with NaOH and steam out
• Distillation of the sample
– Take 500 ml sample, or a fraction of it diluted to 500 ml, or 1 L
if ammonical-N is <100 µg/l, into the distillation flask, adjust pH
to 9.5 with 6N NaOH and add 25 ml borate buffer solution
– Disconnect steaming out flask and connect sample distillation
flask and distill at 6-10 ml/min. rate
– Collect distillate in 500 erlenmeyer flask into 50 ml of boric acid
or sulfuric acid solution - submerge condenser outlet tip in acid
– After collecting 200 ml distillate, free condenser outlet tip from
absorbent acid and continue distillation for 1-2 min to clean
condenser and its delivery tube
• Analyse the distillate for ammonical-N
Preliminary distillation
67

Kjeldahl digestion
Meant to convert organic-N into ammonical-N while not
affecting the other forms of nitrogen
– Fails to influence azide, azine, azo, hydrazone, nitrate, nitrite,
nitrile, nitro, nitroso, oxime and semi-carbazone nitrogens
Macro or semi micro kjeldahl digestion method is used
– Macro-kjeldahl method for samples with low organic-N
– Semi-micro-kjeldahl method for samples with high organic-N
In the presence of H2SO4, K2SO4 and (mercuric sulfate) catalyst
(all present in the digestion reagent) organic-N is converted
into ammonium sulfate
– During digestion ammonium complex is formed with mercury
and this is decomposed by sodium thiosulfate
– Even the free ammonia of the sample is converted into
ammonium sulfate
68
Hands on Training Program on Water and Wastewater Analysis (24-29th June, 2013)

Nitrate can prove both a + and a - interference
– At >10 mg/l, it can oxidize some fraction of the ammonical-N
during digestion
– In the presence of sufficient organic matter, nitrate can be
reduced to ammonical-N
The acid and the salt of the digestion reagent are meant for
producing 360-370°C temperature for digestion
– Higher salt concentration can raise the temp. to >400°C during
digestion and this can result in the pyrolytic loss of nitrogen
– Higher salt levels demand more acid for maintaining the desired
acid-salt balance (1 mL H2SO4 per gram of salt is needed)
– Too much acid can reduce digestion temp. to <360°C and this
can lead to incomplete digestion
– Higher levels of organic matter in the sample can consume more
acid – this can increase salt to acid ratio and the digestion
temperature (every 3 grams of COD requires 10 mL of acid)
Kjeldahl digestion: Interferences
69

Digestion reagent:
• Dissolve 134 g K2SO4 in 650 ml water and 200 ml of conc. H2SO4.
• While stirring add 25 ml mercuric sulfate solution (8 g of mercuric
oxide in 100 ml of 6N H2SO4)
• Makeup the volume to one liter and keep the reagent at 20°C
– Toxicity and residues disposal are problems when mercuric sulfate is
used as a catalyst
– 10 ml of copper sulfate solution (25.115 g/L of CuSO4) per 50 ml
digestion reagent can be used in place of mercuric sulfate
– Selenium can also be a catalyst (but it is highly toxic and also acts as an
interference)
Sodium hydroxide-sodium thiosulfate reagent:
• Dissolve 500 g NaOH and 25 g Na2S2O3.5H2O in water and dilute to
one liter
Kjeldahl digestion
70

• Take measured volume of sample in 800 ml capacity digestion
flask and diluted to 500 ml
Volume of the sample should be such that it has 0.2 to 2 mg of
TKN in it
• 500 ml when organic-N is 0.1-1 mg/l
• 250 ml when organic-N is 1-10 mg/l
• Take 1 L sample when organic –N is <0.1 mg/L and use bigger
Kjeldahl flask
• Remove ammonia by distillation after adding 25 ml borate
buffer and adjusting pH to 9.5 with 6N NaOH
– Distillate can be collected into boric acid or sulfuric acid for
determining ammonical-N of the sample
– Residue left behind after preliminary distillation of sample for
ammonical-N can be used for organic-N measurement
Kjeldahl digestion and distillation
71

• Cool the sample after distillation removal of ammonical-N,
add 50 ml digestion reagent and glass beads, and mix contents
• Heat the digestion flask under hood with suitable ejection
equipment to briskly boil until the volume is reduced to 25-50
ml and release of copious white fumes
• Continue digestion for another 30 min. till the sample turns
clear or straw-coloured
• Cool the flask contents, dilute to about 300 ml, and add 50 ml
of hydroxide-thiosulfate reagent along the walls so as it forms
an alkaline layer at the flask bottom
• Connect the flask (with diluted digested sample and bottom
alkaline layer) to a steamed out distillation system
• Mix the contents and distillate (similar to the preliminary
distillation) and collect distillate into boric acid/ sulfuric acid
Run reagent blank parallel to the sample through all the steps and
apply necessary corrections to the results on the basis of the
blank results
Kjeldahl digestion and distillation
72

Semi-micro Kjeldahl method
• Take measured volume of the sample, adjust to 50 mL, add 3
ml borate buffer and adjust pH to 9.5 with 6N NaOH
– 50 ml for 4-40 mg/l concentration
– 25 ml for 8-80 mg/l
– 10 ml for 20-200 mg/l
– 5 ml for 40-400 mg/l
• Transfer the contents to 100 mL semi-micro kjeldahl flask and
boil off 30 mL of the contents for remove the ammonical-N
• Add 10 ml digestion reagent and a few glass beads, heat till the
sample becomes clears and copious fumes come out, and
continue heating, at maximum heating, for 30 minutes more.
• Cool the contents and transfer into a micro-kjeldahl distillation
apparatus while ensuring the total volume <30 mL
• Add 10 mL hydroxide-thiosulfate reagent, turn on distillation,
and collect 30-40 ml distillate in 10 ml H3BO3/H2SO4 solution
73

74

Nesslerization method
• Undistilled samples
– Add 1 ml ZnSO4 solution (100 g ZnSO4.7H2O in 1 liter) to 100
mL of sample, mix, adjust pH to about 10.5 with 6N NaOH and
allow the sample to stand
– Clarify the supernatant by centrifuging or filtering prior to
nesslerization
• Can remove calcium, iron, magnesium, etc. (which form turbidity
on nesslerization) and suspended solids & colour
• Samples with >10 mg/l of NH3-N may loose some ammonia from
higher pH
– To 50 ml of the filtered/centrifuged (or a portion of it diluted to
50 ml) sample add a drop of EDTA reagent or 1 or 2 drops of
Rochelle salt solution, mix and then nesslerize
• Addition of EDTA or Rochelle salt solution inhibits precipitation of
calcium, iron, magnesium, etc., when nesslerized (but EDTA
demands additional nessler reagent)
75

• Distilled samples
– Prepare standard solution (1 mL = 10 µg NH3-N) from stock
ammonium solution ((1 mL = 1 mg of NH3-N)
– Distill samples, standards and reagent blanks and collect distillate for
nesslerization
– Dilute the distillate plus boric acid solution to 500 mL volume and take
50 mL for nesslerization
• Nesslerize the sample with 2 mL Nessler reagent (if the sample
is already neutralized with NaOH use only 1 mL)
– For the reaction to occur allow at least 10 min. (when NH3-N is very
low use 30 min. reaction time)
– Keep temperature and reaction time same for samples, blanks
and standards
76

• Measure transmittance or absorbance of samples and standards
against reagent blank by spectrophotometer
– For low NH3-N levels (0.4 to 5.0 mg/l) measure colour at 400-
425 nm and use light path of 1 cm (5 cm light path allows
measurements as low as 5-60 µg/L)
– For NH3-N levels approaching 10 mg/l use 450-500 nm
wavelength
– Measurements for standards are used for calibration
• Visual comparison against standards can be alternative to
spectrophotometer
– Temporary standards prepared from standard NH4Cl in the range
of 0-6 ml in 50 mL water and nesslerized by adding 1 ml of
Nessler reagent can be used
– Permanent standards prepared from potassium chloroplatinate
and cobaltous chloride solutions and calibrated against
temporary standards can also be used
77

– EDTA reagent: dissolve 50 g of ethylene diamine tetra
acetate dihydrate in 60 ml water containing 10 g NaOH
(heat to dissolve if needed and cool to room temp.) and
dilute to 100 mL
– Rochelle salt solution: dissolve 50 g of potassium sodium
tartrate tetra hydrate in 100 ml water, boil out to reduce
volume to 30 ml, cool and dilute 100 ml
– Stock ammonium solution: dissolve 3.819 g anhydrous
NH4Cl (dried at 100°C) in water and adjust volume to 1
liter (1 mL = 1 mg of NH3-N)
– Nessler reagent: dissolve 160 g NaOH in water, cool,
slowly add mixer of 100 g of mercuric iodide (HgI2) and 70
g potassium iodide (KI) dissolved in water, and adjust
volume to 1 liter
78

Titrimetric method
• Distillate collected into boric acid solution is used
– Sample size: 250 ml for 5-10 mg/l of NH3-N; 100 ml for 10-20
mg/l; 50 ml for 20-50 mg/l and 25 ml for 50-100 mg/l
– Indicating boric acid: dissolve 20 g of H3BO3 in water, add 10 ml
of mixed indicator and adjust volume to 1 liter
– Mixed indicator: dissolve 200 mg of methyl red in 100 mL of
95% ethyl or isopropyl alcohol and 100 mg of methylene blue in
50 mL of 95% ethyl or isopropyl alcohol and mix the two
• Titrate the distillate with 0.02N H2SO4 to pale lavender colour
end point (1ml titrant used = 280 µg of NH3-N)
• Run blank through all the steps and correct results
79

Phenate method
• Method is good for 10 to 500 µg/l
• Preliminary distillation of sample and collection of distillate
• Alkalinity >500 mg/l, acidity >100 mg/l and turbidity can
interfere with direct phenate method
• Distillate is collected into 0.04N H2SO4
• Ammonia is made to react with hypochlorite and phenol in
the presence of manganous salt catalyst to form indophenol
(an intensely blue coloured compound)
• Concentration of indophenol is measured by
spectrophotometer at 630 nm at path length of 1cm
80

Ammonia selective electrode method
Uses hydrophobic gas permeable membrane to separate sample
from an electrode internal solution (NH4Cl)
• By raising pH to 11 NH3-N is converted into gaseous form
• Gaseous NH3 diffuses through membrane and changes pH of the
internal solution
• This changes the millivolt reading of the meter proportional to NH3-
N concentration
Measurement
• 100 ml sample is taken, and ammonia selective electrode is
immersed in it
• While mixing with magnetic stirrer pH of the sample is adjusted to
11 by adding 10N NaOH
• After stabilization take millivolt reading for the sample
81

Ammonia selective electrode method
Calibration
• Prepare standards with 1000, 100, 10, 1 and 0.1 mg/l levels
• Take millivolt reading for each of the standards in a way similar to
that of sample
• Plot readings on semi-log plot (take concentrations on the log axis
and millivolt readings on linear axis)
Method is applicable for measurement of 0.03 to 1400 mg/l
The sample does not require distillation
Interference
• High concentration of dissolved ions affect the measurement but
color and turbidity do not
• Amines introduce positive error
• Mercury & silver through complexing introduce negative error
82

Nitrite nitrogen and Nitrate
nitrogen

Nitrite and Nitrate Nitrogen
• Oxidized Nitrogen may be present in water mainly in two
forms: nitrite and nitrate
• Nitrite
• Represents an intermediate oxidation state and present
usually in very low concentrations
• Often used as corrosion inhibitor in industrial process water
• Nitrate
• Occurs in trace quantities in surface water (however,
wastewaters of biological nitrifying treatment plants can
have upto 30 mg/L), but ground waters have higher levels
• High levels of nitrate in water can be problematic
– thought to be toxic to humans, particularly to babies –
contributes to methemoglobinemia
– oxidized nitrogen is a factor in the eutrophication of waters
• All forms of nitrogen (reduced and oxidized) can be digested
and converted into nitrate for measuring as total nitrogen 84

Sample preservation and storage
• Samples for nitrate
– Samples should be promptly analyzed
– Store at 40C up to 2 days (24 hr.!)
– Unchlorinated samples can be preserved with 2 mL/L conc
H2SO4 and stored at 40C
• Samples for nitrite
– Analyse promptly, if not nitrite can be converted into
nitrate/ammonia by bacteria
– Freeze sample at –20°C for preservation or store at 4°C for
short-term preservation (1 to 24 hrs.)
• For acid preserved samples nitrate and nitrite can not be
determined as individual species
85

Methods of analysis
• Nitrite
– Colorimetric method – suitable for 5 to 1000 µg/L – acid
preservation for samples should not be used
– Ion-chromatography
• Nitrate
– UV Spectrophotometric Method – used for screening
uncontaminated water low in organic matter
– Cd-reduction Method (range 0.01 – 1.0 mg/L)
– Ion Chromatography or capillary ion electrophoresis
– Nitrate electrode method (0.14 – 1400 mg/L)
• Total nitrogen
– Measured through conversion of all (reduced and oxidized)
forms of nitrogen into nitrate and estimation of nitrate
– Persulfate/UV digestion or persulfate digestion is used
• Not effective for wastes with high (suspended) organic loadings
• Recovery of some industrial nitrogen containing compounds is low86

Nitrite -N: Colorimetric method
Good for 10 to 1000 g/L levels (light path of 5 cm allows
measurement in the 5-50 g/L range)
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)
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
Use nitrite free water during sample analysis for nitrite
87

Nitrite -N: Colorimetric method
• Filter the sample through 0.45 m pore membrane filter and adjust
pH to 5-9 with HCl or NH4OH
• Take 50 ml or a portion diluted to 50 ml (dilution when conc. is >1.0
mg/L) and add 2 ml colour reagent and mix
• After 10 min but before 2 hrs measure absorbance at 543 nm
• Treat standards also with colour reagent and measure absorbance
– Plot absorbance of standards against NO2
- concentration for obtaining
a standard/calibration curve
• Read sample’s nitrite concentration from the standard curve
Colour reagent: add 100 ml of 85% phosphoric acid to 800 ml water,
dissolve 10 g of sulfanilamide, then dissolve 1 g of N-(1-naphthyl)-
ethylenediamine dihydrochloride, and adjust volume to 1 liter – can
be stored upto a month in dark bottle in refrigerator
Standard stock solution : dissolve 1.232 g NaNO2 in water and dilute to
1000ml: 1 mL = 250µg Nitrite -N
88

Nitrate: Cd reduction method
• Range: 0.01 to 1 mg/L Nitrate-N
• Nitrate-N is almost quantitatively reduced to Nitrite-N in the
presence of cadmium (Cd).
• Nitrite thus produced is diazotized with sulfanilamide and
coupled with N-(1–naphthyl)-ethylene diamine dihydro
chloride to form colored azo dye
• The colour intensity is measured spectrophotometrically
• Correction is needed for the nitrite-N originally present in the
sample
– Testing the sample for nitrite without subjecting it to nitrate
reduction step is used for the correction needed
89

Handling interferences
• Turbid samples need filtering through 0.45 µm pore (nitrate
free) membrane filter
– Suspended solids will restrict sample flow so pre filtration is
needed
• EDTA is added to remove interference from iron, copper or
other metals
• Residual chlorine if present is removed by dechlorination with
sodium thiosulfate
• If oil and grease are present the sample is pre-extracted with
organic solvent.
• Chloride ions can significantly decrease the rate of reduction
90

Cd reduction column 91

Cd reduction column
Cd reduction column
• Constructed from two pieces of tubing (3.5 mm ID and 2 mm ID
tubing) joined end to end
• 3 cm ID and 10 cm long tube is fused on the top of 25 cm long and
3.5 mm ID tubing
• Stopcock arrangement is made to allow control of flow rate
Activation
• Wash the column with 200 mL dilute NH4Cl-EDTA solution
• Activate the column by passing >100 mL of a solution (of 25% 1.0
mg/L nitrate standard and 75% NH4Cl-EDTA solution) through the
column at 7 to 10 mL/min, rate.
Ammonium chloride-EDTA solution: dissolve 13 g NH4Cl and 1.7 g
disodium ethylene diamine tetra acetate (EDTA) in 900 mL water,
adjust pH to 8.5 with NH4OH and dilute to 1L.
92

• Screen the sample and adjust the pH between 7 and 9.
• To 25.0 mL sample (or a portion diluted to 25.0 mL), add 75
mL NH4Cl- EDTA solution, mix and pass through the column
at 7 to 10 mL/min. rate - discard the first 25 mL, and collect
the rest in original sample flask.
• Within 15 min after reduction, add 2.0 mL color reagent to 50
mL sample and mix, and within 10 min. to 2 hours measure
absorbance at 543 nm
• From the stock solution, prepare (100 mL) standards in the
range 0.05 to 1.0 mg/L nitrate-N
• Carry out cadmium reduction of the standards exactly as has
been done for the sample.
Stock nitrate solution (1.00mL = 100µg NO3
- -N): dissolve 0.7218 g
dry potassium nitrate in water and dilute to 1000 mL – preserve the
stock solution with 2mL CHCl3 /L.
– Intermediate stock nitrate solution (of 1.0 mL = 10 µg NO3
- -N
strength) is prepared from this stock for routine use 93

Nitrate: Ion electrode method
Interferences
• Chloride and bicarbonate ions interfere when their weight
ratios to nitrate-N are >10 and >5, respectively
• NO2–, CN–, S2–, Br–, I–, ClO3–, and ClO4– are also
potential interferences (but do not normally occur at
significant levels in potable waters)
• Electrodes function satisfactorily in buffers over 3 to 9 pH
range – but for avoiding erratic responses pH is held constant
• Since the electrode responds to nitrate activity, ionic strength
must be constant in all the samples and the standards
• A buffer solution containing
a) Ag2SO4 to remove Cl–, Br–, I–, S2–, and CN–,
b) sulfamic acid to remove NO2–,
c) a buffer at pH 3 to eliminate HCO3– and to maintain a constant
pH and ionic strength, and
d) Al2(SO4)3 to complex organic acids is used 94

Preparation of calibration curve
• Transfer 10 mL of 1 mg/L nitrate -N standard to a 50-mL
beaker, add 10 mL buffer, and stir with a magnetic stirrer
– Immerse the electrode tip and record millivolt reading when
stable (after about 1 min)
– Remove the electrode, rinse, and blot dry
• Repeat this for 10 mg/L and 50 mg/L nitrate-N standards
• Plot potential measurements against nitrate -N concentration
on semilog graph paper (nitrate-N on the log axis and potential
on the linear axis)
– A straight line with a slope of +57 ±3 mV/decade at 25°C should
result
• Recalibrate electrodes several times daily (check potential
reading for 10 mg/L nitrate-N standard and adjust the
calibration control until the reading plotted on the calibration
curve is displayed again 95

Measurement of sample:
• Transfer 10 mL sample to a 50-mL beaker, add 10 mL buffer
solution, and stir (for about 1 min) with a magnetic stirrer
• Immerse electrode tip in sample and record potential reading when
stable (after about 1 min).
• Measure standards and samples at about the same temperature.
• Read concentration from calibration curve.
The electrode responds to nitrate ion activity corresponding to
0.14 to 1400 mg/L nitrate –N
Buffer solution: Dissolve 17.32 g Al2(SO4)318H2O, 3.43 g
Ag2SO4, 1.28 g H3BO3, and 2.52 g sulfamic acid (H2NSO3H),
in 800 mL water. adjust to pH 3.0 by 0.10N NaOH, makeup
volume to 1000 mL and store in a dark glass bottle
96

Nitrate: UV Spectrophotometric Method
• Used for samples having low organic matter
• Nitrate ion and organic matter absorb at 220 nm and only
organic matter absorbs at 275 nm
• Interferences
– Dissolved organic matter, surfactants and Cr6+
– Acidification with 1N HCl can prevent the interference from
hydroxide or carbonate concentration
• Procedure
• Filter the sample and add 1 mL of 1 N HCl to 50 mL sample.
• Prepare 50 mL each of NO3
- calibration standards in the range
from 0 to 7 mg/L NO3
- -N from the stock
• Read absorbance at 220 nm and 275 nm
• Construct a standard/calibration curve by plotting concentration
against corrected absorbance.
• Discard the method if correction value is more than 10% of the
reading at 220nm 97

Sample
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
Nitrate: UV Spectrophotometric Method
Discard the method if correction value is more than 10% of the
reading at 220nm
98

Total Nitrogen
Chemicals
• Borate buffer solution: Dissolve 61.8 g boric acid, H3BO3, and
8.0 g NaOH in water and dilute to 1000 mL.
• Copper sulfate solution: Dissolve 2.0 g CuSO4˜5H2O in 90 mL
water and dilute to 100 mL.
• Ammonium chloride solution: Dissolve 10.0 g NH4Cl in
water, adjust to pH 8.5 by adding NaOH pellets or NaOH
solution and make up volume to 1 L (stable for 2 weeks when
refrigerated)
• Color reagent: Combine 1500 mL water, 200.0 mL conc.
H3PO4, 20.0 g sulfanilamide, and 1.0 g N-(1-naphthyl)-
ethylene diamine dihydro chloride, dilute to 2000 mL, add 2.0
mL polyoxyethylene 23 lauryl ether and store at 4°C in the
dark (stable for 6 weeks)
100

Total Nitrogen
• Calibration standards: Prepare nitrate calibration standards
(100 mL) in 0 to 2.9 mg/L range, and treat the standards in the
same manner as samples.
• Digestion check standard: Prepare glutamic acid digestion
check standard of 2.9 mg N/L by diluting the stock, and treat
the digestion check standard in the same manner as samples.
• Blank: Carry a reagent blank through all steps of the procedure
and apply necessary corrections to the results
Stock glutamic acid solution: Dry glutamic acid,
C3H5NH2(COOH)2, in an oven at 105°C for 24 h. Dissolve
1.051 g in water and dilute to 1000 mL; 1.00 mL = 100 Pg N.
Preserve with 2 mL CHCl3/L.
– Intermediate glutamic acid solution (1.00 mL = 10.0 Pg N)
101

Total Nitrogen
Digestion:
• Samples should not be preserved with acid for digestion
• To a culture tube (20 mm OD and 150 mm long), add 10.0 mL
sample (or a portion diluted to 10.0 mL) or standard, add 5.0 mL
digestion reagent, cap tightly, mix by inverting twice
– In case of reagent blank, 10 mL water is taken in place of sample
• Heat for 30 min in autoclave/ pressure cooker at 100 to 110°C
• Slowly cool to room temperature, add 1.0 mL borate buffer solution,
mix by inverting twice
Nitrate measurement: Determine by cadmium reduction
Digestion reagent: Dissolve 20.1 g low nitrogen (<0.001% N)
potassium persulfate, K2S2O8, and 3.0 g NaOH in water and
dilute to 1000 mL just before use
Borate buffer solution: Dissolve 61.8 g boric acid, H3BO3, and
8.0 g NaOH in water and dilute to 1000 mL.
102

Chemicals
• Colour reagent: add 100 ml of 85% phosphoric acid to 800 ml
water, dissolve 10 g of sulfanilamide, then dissolve 1 g of N-(1-
naphthyl)-ethylenediamine dihydrochloride, and adjust volume to 1
liter – can be stored upto a month in dark bottle in refrigerator
• Standard stock solution : dissolve 1.232 g NaNO2 in water and
dilute to 1000ml: 1 mL = 250µg Nitrite -N
• Ammonium chloride-EDTA solution: dissolve 13 g NH4Cl and 1.7 g
disodium ethylene diamine tetra acetate (EDTA) in 900 mL water,
adjust pH to 8.5 with NH4OH and dilute to 1L.
• Stock nitrate solution (1.00mL = 100µg NO3
- -N): dissolve 0.7218 g
dry potassium nitrate in water and dilute to 1000 mL – preserve the
stock solution with 2mL CHCl3 /L.
– Intermediate stock nitrate solution of 1.0 mL = 10 µg NO3
- -N
strength is prepared from it used
103

Nitrite free water
• Add a small crystal of KMnO4 and Ba(OH)2 or Ca(OH)2 to
distilled water and redistill in all borosilicate glass apparatus to
obtain nitrite free water
– Initial 50 mL of the redistillate and final distillate with permangamage
(giving red colour with DPD reagent) should be discarded
• Add 1 mL/L of conc. H2SO4 and 0.2 mL/L of MnSO4 solution
(36.4 g of MnSO4.H2O in distilled water and 1 liter final
volume), make the water pink by adding 1 to 3 ml of KMnO4
solution and redistill
104

Importance
• Used extensively in the treatment of boiler water (tri-sodium
phosphate) to control scaling
– At higher temperatures polyphosphates are hydrolyzed into
orthophosphates
• Essential for growth of organisms
– Limiting & important nutrient for primary productivity of water
bodies
– applied in agriculture as fertilizers (orthophosphates)
– microbes of wastewater treatment plants require phosphorus -
domestic effluents have enough of it
– Biological sludge is rich (1%, in case heat dried ASP sludge it is
1.5%) – has good fertilizer value
• Excess in water bodies causes eutrophication
– 0.005 mg/l of available phosphorus is critical for algal blooms to
occur
106

Sources
Domestic waste, prior to synthetic detergents, contains 2-3 mg/l of
inorganic form and 0.5-1.0 mg/l of organic form
– Polyphosphates added to water supplies (to control corrosion), soft water (to
stabilize CaCO3) and to water (during laundering or other cleaning
processes) find their way into sewage
– Synthetic detergents use increased inorganic form by 2-3 times (have
polyphosphates as builders, 12-13% or more)
– Body wastes and food residues contribute organic form – liberated during
metabolic breakdown of proteins and comes out in urine (1.5 g/day per
capita)
Industrial effluents – mostly inorganic forms
– Boiler blowdown water is important source - at higher temperatures even the
poly forms are hydrolyzed into ortho form
Agricultural run off - fertilizer applied (orthophosphates) and organic phosphorus
are found
Poly forms of water bodies get gradually hydrolyzed into ortho forms
– high temperature and low pH increases the hydrolysis rates
– Enzymes of microorganisms also bring about hydrolysis
107

Classification and forms
Present in water and wastewater mostly as phosphates
Classified as
– Orthophosphates – mono, di and trisodium phosphates and
diammonium phosphate
– Poly (condensed) phosphates (pyro, meta and other polyphosphates)
– sodium hexameta phosphate, sodium tripolyphosphate,
tetrasodium pyrophosphate
– Organically bound phosphates - formed primarily by biological
processes – occurs both in dissolved and suspended forms
Can be present in water as
– soluble phosphates
– particulate phosphates in particles or detritus
• precipitated inorganic forms in the bottom sediments
• incorporated into organic compounds in the biological
sludge/debris
– In the bodies of the aquatic organisms
108

• Filtering through 0.45 m pore size membrane filter is believed to
separate dissolved form of phosphorus from suspended form
• Analytically phosphorus of a sample can be divided into three
chemical types
– Reactive phosphorus
– Acid-hydrolysable phosphorus (polyphosphates)
– Organic phosphorus
• Reactive phosphorus: Phosphorus that respond to colorimetric
tests without preliminary hydrolysis or oxidative digestion
– Can include both dissolved and suspended forms
– Largely a measure of orthophosphate
109

• Acid-hydrolysable phosphorus: phosphorus that is converted into
into dissolved orthophosphate on acid hydrolysis at boiling water
temperature
– Mostly condensed phosphate and can be both suspended and
dissolved condensed phosphate
– Some fraction of the organic phosphate can also be hydrolyzed
– Appropriate selection of acid strength, hydrolysis time and
temperature can minimize hydrolysis of organic phosphate
• Organic or organically bound phosphorus: phosphate fraction that
is converted to orthophosphate only by oxidative destruction of
organic matter
– Can be in both soluble and particulate forms
110

Phosphate estimation
Analysis involves two steps
– Conversion of the phosphorus form of interest to dissolved
orthophosphate
– Colorimetric determination of dissolved orthophosphate
Digestion should oxidize the organic matter and release phosphorus as
orthophosphate – There are three methods
– Perchloric acid method (very drastic and time consuming method – used for
difficult samples such as sediments
– Nitric acid – sulfuric acid method – recommended for most samples
– Persulfate oxidation method – simplest method – prior to adopting make
comparison with the two drastic methods
Gravimetric, volumetric and colorimetric methods can be used for
estimating ortho forms
– Gravimetric is suitable for very high concentrations
– For >50 mg/l volumetric is appropriate (boiler blowdown water and
anaerobic digester supernatant)
– For usually encountered levels colorimetric is preferred
111

Colorimetric: After digestion the liberated orthophosphate is
determined by
– Vanadomolybdophosphoric acid colorimetric method – good for
concentration range of 1 to 20 mg/l
– Stannous chloride method – good for 0.01 to 6 mg/l
– Ascorbic acid method
Different forms of phosphorus
Poly-P = acid hydrolysable-P – ortho-P
Organic-P = digested-P – acid hydrolysable-P
112

Selection of method depends largely on concentration range of the
orthophosphate
– In case of lower concentrations in order to overcome interferences an
extraction step may be added
For finding different forms of phosphorus, subject the sample to
– Direct colorimetric – gives reactive phosphorus
– Acid hydrolysis and then colorimetric – gives both reactive phosphorus and
acid hydrolysable phosphorus
– Digestion and then colorimetric – gives total phosphorus (reactive, acid
hydrolysable and organic phosphorus)
For getting the dissolved fractions of different forms of phosphorus filter
the sample and test the filtrate
113

Sample reservation and storage and other
precautions
For preserving, freeze the sample at or below –10C
For storing the sample for longer periods add 40 mg/l of HgCl2 (a
hazardous substance) to the sample
If interest is to estimate different forms of phosphorous avoid adding acid
or CHCl3 as a preservative
In case of estimation of total phosphorus 1 ml HCl/liter of sample can be
added for preservation – in case of freezing there is no need to add any
acid
Samples with low phosphorus concentration should not be stored in plastic
bottles because walls of the bottles adsorb phosphorus
Prior to use all glass containers should be first rinsed with hot dilute HCl
Commercial detergents containing phosphorus should not be used for
cleaning
114

Sample preparation (including digestion)
Depending on the need filter the sample through 0.45 um membrane
filter (in case of hard to filter samples filter through a glass fiber
filter)
– Before use, wash the membrane filter by soaking in distilled water
(change the distilled water at least once) or by filtering several
batches of 100 ml distilled water samples through the membrane
filter
Acid hydrolysable phosphorus:
– Taken as the difference between the phosphorus measured in the
untreated sample and that measured in acid hydrolyzed sample
– Includes condensed phosphates (pyro, tripoly and higher molecular
weight phosphates like hexametaphosphate)
– Some organo phosphate compounds natural water samples may also
get hydrolyzed and contribute
115

Acid hydrolysis procedure
1. Acidify known volume of sample (add 1/2 drops
phenolphthalein, discharge colour by drop wise addition of
strong acid solution (SAS), and add SAS (1:100)
– Prepare strong acid solution by slowly adding concentrated 300 ml of
H2SO4 to 600 ml distilled water, cool and add 4 ml of concentrated HNO3
and then making up volume to one liter
2. Carry out hydrolysis by either of the following
– Gently boiling acidified sample for > 90 min. (do not allow sample volume
to drop below 25% of the original - add distilled water
– autoclave acidified sample at 98-137 kPa for 30 minutes
3. Cool, neutralize hydrolyzed sample with 6N NaOH to faint pink
& adjust to original volume with distilled water
Use a calibration curve constructed from the acid hydrolyzed series of
standards in the colorimetric measurement
116

Perchloric acid digestion
Heated mixtures of HClO4 and organic matter can explode violently
– Do not add HClO4 to hot solutions containing organic matter
– Initiate digestion with HNO3 and complete digestion using mixture
of HNO3 and HClO4
– Use hoods specially constructed for HClO4 fuming (connected to a
water pump)
– Do not allow the sample to evaporate to dryness during dryness
117

Digestion process
– Take measured volume of sample (containing desired quantity of
phosphorus) in a conical flask, acidify to methyl orange with con.
HNO3 and then add 5 ml of con. HNO3
– Evaporate acidified sample on hotplate/steam bath to 15-20 ml
volume
– Cool, add 10 ml of con. HNO3, cool and add 10 ml of HClO4
– Add few boiling chips and gently evaporate on hot plate until dense
white fumes of HClO4 appear
– if the contents are not clear cover the flask with watch glass and keep
them barely boiling till they become clear – if needed add 10 ml more
of HNO3
– Cool the contents, add phenolphthalein and neutralize to pink colour
with 6N NaOH - If needed filter the sample (wash the filter with
distilled water)
– Makeup the volume to 100 ml
Use a calibration curve constructed from the perchloric acid digested
series of standards in the colorimetric measurement
Perchloric acid digestion
118

Sulfuric acid-nitric acid digestion
• Take measured volume of sample containing desired amount of
phosphate into micro-kjeldahl flask, and add I ml of conc. H2SO4
and 5 ml of conc. HNO3
• Digest the sample on a digestion rack with provision for fumes
withdrawal to 1 ml volume and continue till the sample becomes
colourless (HNO3 removed)
• Cool and add about 20 ml distilled water, add phenolphthalein
indicator and neutralize with 1N NaOH to pink stinge, and if
needed filter the solution to remove suspended matter and
turbidity
• Makeup the final volume to 100 ml
Use a calibration curve constructed from the sulfuric acid-nitric acid
digested series of standards in the colorimetric measurement
119

Persulfate digestion method
Take measured volume of sample (50 ml of less), add
phenolphthalein indicator and discharge colour with drop-wise
addition of H2SO4 solution
– Prepare H2SO4 solution by slowly adding 300 ml of conc. H2SO4 to 600 ml
distilled water and then making up volume to one liter
Add additional 1 ml acid solution and 0.4 g of solid ammonium
persulfate or 0.5 g of solid potassium persulfate
Boil the sample on hotplate for 30-40 min. till volume is reduced to
10 ml (certain organophosphorus compounds may require 1.5 to 2
hours digestion) or
Autoclave the sample at 98-137 kPa for 30 minutes
Cool the digested contents, add phenolphthalein indicator and
neutralize to faint pink colour with 1 N NaOH
120

Makeup the volume to 100 ml
do not worry if precipitate is formed – shake well if the sample is
subdivided – acidic conditions of colorimetric testing may re-dissolve
the precipitate
Use calibration curve constructed from persulfate digested series of
standards in the colorimetric measurement
Persulfate digestion method
121

Vanadomolybdophosphoric acid
colorimetric method
Under acidic conditions sample’s orthophosphate reacts with
ammonium molybdate and forms molybdophosphoric acid
– In the presence of vanadium, molybdophosphoric acid produces
yellow colour (proportional to con. of phosphate)
– Colour intensity is measured as absorbance at 400-490 nm
Take 50 ml sample, adjust pH by discharging phenolphthalein colour
with 1:1 HCl and makeup volume to 100 ml
– HNO3 or H2SO4 or HClO4 can be substitute for HCl
– If sample is coloured shake 50 ml of the sample with 200 mg of
activated carbon for 5 min and filter to remove carbon
– Take care activated carbon itself is having any phosphate
    OHNHMoOPONHHMoONHPO 243434424
3
4 122112.2412  
122

• Take 35 ml sample or less containing 0.05 to 1.0 mg/l of
phosphate into 50 ml volumetric flask
• Add 10 ml of vanadate-molybdate reagent and then makeup
volume to the mark with distilled water
– Dissolve 1.25 g of ammonium metavanadate, NH4VO3, in 300 ml of
distilled water by heating to boiling; cool and add 330 ml of conc.
HCl; cool and add 25 g of ammonium molybdate
(NH4)6Mo7O24.4H2O dissolved in 300 ml distilled water; and
makeup final volume to one liter
– Room temperature variations affect colour intensity
• After 10 minutes or more measure absorbance of the sample at
400-490 nm
• Maintain blank also
colorimetric method
123

• Prepare calibration curve by using suitable volumes of standard
phosphate solutions parallel with the sample and the blank
– Prepare stock standard phosphate solution by dissolving 219.5 mg of
anhydrous KH2PO4 in one liter solution to get 1ml=0.05 mg
phosphate
– calibration curves may be constructed at various wavelengths
between 400-490 nm
colorimetric method
124

Unless heated silica and arsenate will not cause positive interference
Arsenate, fluoride, thorium, bismuth, sulfide, thiosulfate, thiocyanate
and excess of molybdate can cause negative interferences
– Sulfide interference can be removed by oxidation with bromine water
If HNO3 is used in the test chloride concentration >75 mg/l can
cause interference
– Below 100 mg/l ferrous iron may not affect the results
– Below 1000 mg/l many ions do not cause interfere
The method is most suitable for a range 1 to 20 mg/l
– Minimum detectable concentration is 200 g/liter in 1-cm light path
of the spectrophotometer cells
colorimetric method: interferences
125

Stannous chloride method
Under acidic conditions sample’s orthophosphate reacts with
ammonium molybdate and forms molybdophosphoric acid
– Stannous chloride reduces the molybdophosphoric acid to intensely
coloured molybdenum blue
– Colour intensity is measured as absorbance at 690 nm
Method is more sensitive – by increasing light path length
concentration as low as 0.007 mg/l can be measured
– When concentration is <0.1 mg/l an extraction step can enhance
reliability and lessen interference (with extraction step minimum
detectable limit is 0.003 mg/l)
– Concentration range for which suitable is 0.01 to 6 mg/l
126

Take 100 ml sample and discharge phenolphthalein pink colour by
drop wise addition of strong acid solution
– When phosphorus level is >2 mg/l take sample volume with <0.2
mg of phosphorus makeup volume to 100 ml
– If strong acid solution consumed is more than 5 drops then also
dilute the sample
While keeping all the samples’ temperature in 20-30C range and
constant (all samples temperature within 2 C range) add 4 ml of
molybdate reagent, mix and then add 10 drops (0.5 ml) of
stannous chloride solution and mix
– Molybdate reagent: cautiously add 280 ml of conc. H2SO4 in 400 ml,
cool, add 25 g ammonium molybdate dissolved in 175 ml distilled
water, makeup the final volume to 1 liter
– Stannous chloride reagent: dissolve 2.5 g of stannous chloride
(SnCl2.2H2O) in 100 ml glycerol (heat in water bath for dissolution)
127

Measure colour after 10 min but before 12 min photometrically at
690 nm and read concentration from calibration curve and adjust
to the sample dilution made
– Chose light path length suitably (0.5 cm for 0.3 – 2 mg/l, 2 cm for
0.1 – 1.0 mg/l and 10 cm for 0.007 – 0.2 mg/l)
– The calibration curve may deviate from a straight line at higher
concentrations range (0.3 to 2 mg/l)
Always run blank (distilled water) on reagents
Prepare at least one standard with each set of samples or once a day
128

Needed for overcoming interferences
• Take 40 ml sample (or diluted sample) into a 125 ml separating
funnel, add 50 ml of benzene-isobutanol and 15 ml of molybdate
reagent-E
• Close the funnel immediately and shake vigorously for 15 sec.,
remove stopper and transfer 25 ml of the separated organic layer
into 50 ml volumetric flask
• Add 15-16 ml of alcoholic H2SO4, swirl, add 0.5 ml of stannous
chloride-E reagent, swirl and dilute to mark with alcoholic H2SO4
• After 10 min. but before 30 min measure colour at 625 nm against
a blank (40 ml distilled water) and read concentration from a
calibration curve
Stannous chloride method (Extraction)
129

Reagents
– Benzene isobutanol solvent: mix equal volumes of benzene
and isobutanol (highly flammable)
– Molybdate reagent-E: dissolve 40.1 g of ammonium molybdate
in 500 ml distilled water and slowly add 396 ml of molybdate
reagent, cool and makeup final volume to 1 liter
– Alcoholic sulfuric acid solution: cautiously add 20 ml of conc.
H2SO4 to 980 ml of methyl alcohol while continuously mixing
– Stannous chloride reagent-E: mix 8 ml of stannous chloride
reagent with 50 ml of glycerol
Stannous chloride method (Extraction)
130

Ascorbic acid method
Under acidic conditions, ammonium molybdate and potassium
antimonyl tartrate react with orthophosphate to form a
heteropoly acid-phosphomolybdic acid, and ascorbic acid reduces
the resultant acid to intensely coloured molybdenum blue
Detectable ranges are 0.3 to 2 mg/l for 0.5 cm light path length, 0.15
to 1.3 mg/l for 1 cm path and 0.01 to 0.25 mg/l for 5 cm path
Interferences include arsenates, hexavalent chromium, nitrites, sulfide
and silicate
– Arsenates: at conc. as low as 0.1 mg/l, react with molybdate to
produce blue colour similar to that formed with phosphate
– Hexavalent chromium and nitrite can introduce negative error of 3%
at 1 mg/l of phosphate conc. and 10-15% at 10 mg/l conc.
– Sulfides and silicates cause no interference at <1 mg/l and 10 mg/l
respectively
131

Pipette out 50 ml of sample into a 125 ml dry Erlenmeyer flask and
discharge pink colour of phenolphthalein indicator by drop wise
addition of 5N H2SO4 solution
Add 8 ml combined reagent, mix thoroughly and then measure colour
at 880 nm after 10 min. but within 30 min.
In case of highly coloured or turbid waters prepare a blank by adding
all reagents except ascorbic acid and subtract its colour
measurement from that of each of the samples
132

Combined reagent: mix the following reagents in the same order
in the following proportions:
– 50 ml of 5N H2SO4
– 5 ml of potassium antimonyl tartrate (dissolve 1.3715 g of potassium
antimonyl tartrate in distilled water and adjust final volume to 500
ml)
– 15 ml of ammonium molybdate (dissolve 20 g of ammonium
molybdate in 500 ml distilled water)
– 30 ml of 0.01M ascorbic acid (dissolve 1.76 g of ascorbic acid in 100
ml distilled water and store at 4C for one week
– mix after addition of each of the reagent and cool to room
temperature - if turbidity appears shake well and let the reagent stand
until it disappears
– Reagent is stable for 4 hours
133

Biological Water Quality
(coliform count, MPN test)

PH,acidity and alkanity

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