Asr wastewater characteristics

Characterization of Sewage
UCE-601: Sewerage and Sewage
Treatment
Thapar University

Characterization of Sewage
• Flow rate and flow variations
• Solids (TSS, VSS and biodegradable VSS)
• Organic matter (TOC, COD and BOD)
• Nutrients (nitrogen and phosphorus)
– Nitrogen: TKN (Organic –N and Ammonical –N), Nitrate -N and
Nitrite –N
– Phosphorus (ortho and total phosphorus)
• Biological water quality (MPN or coliform count)
• Oil and grease
• pH, Acidity and Alkalinity
• Chlorides, Sulfates, Sulfides and Phenols
• VFA analysis

Why flow measurement?
• To quantify flows of
– water in streams in streams and rivers
– wastewater in sewers and wastewater drains
• To facilitate flow proportionated sampling of water
• To provide daily flow records required by regulatory agencies
• To determine
– Sizes of water and wastewater treatment plants and the
constituent treatment units
– Chemical dosage to the treatment units
• Interest may be to know instantaneous flow rates, cumulative
flows and variations in flow rates (peaking factor)
4

Flow proportionated sampling and
composite sample collection
Flow
Time
Base Flow
5

Basic requirements of flow meters
• Ability to calibrate
• Ability to integrate integrate flow fluctuations
• Ease of integration with piping system
• High accuracy
• High turn down ratio
• Low cost
• Low sensitivity to dust particles
• Low pressure loss
• Resistant to corrosion and erosion

Basic types of flow meters
• Differential pressure flow meters
• Velocity flow meters
• Positive displacement flow meters
• Mass flow meters
• Open channel flow measurement
• Miscellaneous type flow meters
7

Differential pressure flow meters
• Based on bernoullis equation
– Pressure drop over an obstruction inserted in the flow is used as
basis for flow measurement
• Used for flow measurement under pipeflow conditions
• Orifice meters, venturi meters and flow nozzles
– Orifice meter: a sharp edged orifice plate is introduced as
obstruction to flow – a simple and cheap but poorly accurate
specially at low flows - but can cause significant pressure drops
– Venturi meter: Flow cross section is gradually reduced to generate
pressure difference, and then increased for pressure recovery (low
pressure drops) – Preferred for accurate flow measurements and
for high turn-down rates (10:1)
– Flow nozzles: used usually for gas flow measurement - simple and
cheap - turn down rate and accuracy are comparable to orifice
plates - pressure drop across constricted area is maximum for
orifice plates & minimum for venturi tubes
8

Venturi meter
9
Consists of a conical contraction, a short cylindrical throat and a
conical expansion
P
1
P
2
V1 V2
Bernoulli equation between 1 and 2:
Continuity equation between 1 and 2: 2211 VAVAQ 
0
2
)VV()PP( 2
1
2
212





])/(A-[1
)(2
C 2
12
21
d,2
A
PP
V ideal


 Cd is discharge coefficient

Orifice Meter
A thin flat plate with a circular hole drilled in its center.
])/(A-[1
)(2
C 2
12
21
2
A
PP
V d


 Where Cd is the discharge
coefficient
P
1
P
2
A1, V1
1 2
Front view of
orifice plate
A2, V2

Nozzle Meter
P
1
P
2
])/(A-[1
)(2
C 2
12
21
2
A
PP
V d



• A Venturi meter without the diverging recovery section
• Less expensive than Venturi meter but higher head loss
• Accuracy: < ±1%; Range (turn-down ratio): 4:1
11
A1,V1 A2,V2

Velocity flow meters
• Flow is calculated by measuring flow velocity at one or more
points across the flow cross section
• Typical velocity flow meters
– Pitot tube
– Turbine flow meter (flow current meter)
– Electromagnetic flow meter
– Ultrasoic flow meter, etc.
• With the known flow velocity, flow rate is obtained using flow
cross sectional area

Turbine Flow meters
• Uses a multiple-bladed rotor
(turbine) mounted within a pipe,
perpendicular to flow
• The rotational speed is a direct
function of volume flow rate.
• The meter factor K is found by
direct calibration.
• Limited to pipes running full,
under pressure, and liquids low
in suspended solids
• Excellent accuracy (±0.25%)
and a good range of flows (turn
down ratio): 10:1

Measurement of flow rate
StageorDepth
Discharge, Q
Rating Curve
14

The Pitot Tube
P1 is a Static pressure: It is
measured by a device (static
tube) that causes no velocity
change to the flow. This is
usually accomplished by drilling
a small hole normal to a wall
along which the fluid is flowing.
P2 is a Stagnation pressure: It is
the pressure measured by an
open-ended tube facing the
flow direction. Such a device is
called a Pitot tube.
15
P1,V1 Stagnation
Point V2=0
1 2P2
2/1
12
1
)PP(2
V 















 

f
fm
XgV


21
ρm and ρf are fluid and manometic fluid densities
ΔX is manometric fluid level difference

Electromagnetic flow meter
Faraday’s law: Voltage produced by a
conducting fluid through a magnetic
field is proportional to fluid flow
velocity
• Advantages: Turn down ratio is quite
large (10:1); No head loss; and
Unaffected by temperature,
conductivity, viscosity, turbulence, &
suspended solids
• Problems: High initial cost and need
of trained personnel to handle
routine O&M
16
E=BDVx10-8
E=voltage, volts
B=magnetic flux density, gauss
D= length of the conductor, cm
V=velocity of the conductor, cm/sec

Ultrasonic flow meters
Ultrasonic Doppler flow meter:
• Frequency of a reflected signal is
modified by the velocity and
direction of the fluid flow
– If the fluid is moving towards a
transducer frequency of the
returning signal is increased and
otherwise it is decreased
– Frequency difference (reflected
frequency minus originating
frequency), known as ‘Doppler
effect’, is used to find flow velocity







C
V
FF SourceDoppler
‘V’ Flow velocity between source and receiver
‘C’ Speed of sound
‘Fsource‘ Transmitted frequency.

18
Time-of-travel flow meters
Have 2 transducers mounted on each
side of the pipe
The transducers function as both as
sound wave transmitters and
receivers – operate alternatively as
transmitters and receivers
Sound wave is transmitted in the direction of the fluid flow and in the
opposite direction of flow and time of flight is measured
Differential in the time of flight is used to know the flow velocity and flow
measurement
Ultrasonic Flow meters

Positive displacement flow meters
Devices that isolate fixed volumes of fluid flowing into them in
sealed compartments and discharge to the outlet.
These may be passive (operate on the power from flowing fluid) or
active (metering pumps - driven by external power source)
Leakage and pressure loss are two problems associated with the PD
flow meters
Volume flow rate is calculated from the size and number of
compartments delivered per unit time
PD meters can be classified as rotary, reciprocating, or nutating
PD meters for liquids: Nutating disk meters, reciprocating-piston
meters, rotary-piston meters, rotary-vane meters, rotor meters
PD meters for gases: Roots-type meters, diaphragm-type meters,
liquid-sealed drum-type meters

Nutating disk meters
(a disk nutates in a dual conical housing)
Reciprocating – single piston meters
Plunger or piston is driven by a cam

Rotary-vane flow meters
Flat vanes are inserted into matching
slots around the perimeter of a
cylindrical drum.
Cylindrical drum is located eccentrically
within the housing
Rotary-(oscillating) piston flow meters
A cylindrical drum mounted
eccentrically inside a cylindrical
housing

Rotor meters: Oval Gear Meters
Rotor meters: Gear flow meters

Rotor meters: Helical gear flow meter
Roots-type flow meters
Lobe Rotary Piston

Diaphragm-type meters
Liquid-sealed wet gas flow meters (liquid provides sealing action)
Liquid ring pump

Mass flow meters
• These are also known as inertial flow meters
• If density is variable (temperature, pressure and fluid
composition influence density) mass flow rate can not be
obtained from volumetric flow rate
• Includes
– Coriolis flow meters
– Thermal flow meters
• Capillary tube type thermal mass flow meter
• Constant temperature differential method
• Constant current method
densityfluid
rateflowmass
rateflowvolumetric 

Coriolis mass flow meters
• with the help of an actuator the
inlet arm and the outlet arm are
vibrated at the same frequency
• when there is fluid flow, the inlet
arm and the outlet arm vibrate
differently and a phase shift
occurs
• The (measured) degree of phase
shift is proportional to the mass
flow in the tube


2
2
2Kd
IK
Q uu
m


Qm is fluid mass flow rate
Ku is temperature dependent tube stiffness
K is shape dependent factor
‘d’ is width, τ is time lag
ω is vibration frequency
Iu is inertia of the tube

Thermal mass flow meters
• Thermal dispersion or immersible mass flow meters
– Fluid mass flow rate is measured through measuring the heat
convected from a heated surface to the flowing fluid
– Commonly used for the gas flow measurement
– Heat is introduced into the flow stream and the heat dissipated is
measured by sensors
– Heat dissipated depends on the sensor design and the thermal
properties of the fluid
• Constant temperature differential method: two sensors, a heated
sensor and a gas temperature sensor are used - Power required for
maintaining constant temperature difference between the two sensors
is measured and used
• Constant current method: also have two sensors – power used to heat
the sensor is kept constant – temperature difference between the two
sensors is measured and used for flow measurement
• Capillary tube type of thermal mass flow meter
– Heat is transferred to the flowing fluid from a small heated
capillary tube carrying fluid
– Used for measuring smaller flows of cleaner gases and liquids

Rotameter, variable area flow meter
Fluid flowing moves the float/bob
upwards and maintains in a equilibrium
position when
 










tan2
1
.
.Re
min
2
22
22
hmor
D
DD
mwhere
gm
RnoRuppel
DU
Rnoynold
gVgVgVF
flowturbulentforUDCF
flowarlaforUDCF
buoyancyBobweightBobforceDrag
b
b
b
b
u
bin
e
bbbbbd
bTd
bLd
















Fd is drag force
ρb and ρ are bob density and fluid density
Vb is volume of the bob
Db is maximum bob diameter
D is tube diameter at the bob height
U is flow velocity at the annular gap around the bob

 
 
 
 
 
4
4
4
4
4
44
2
2
2
2
2
222
b
T
bb
b
bTbb
L
bbb
b
bLbb
b
bb
mD
C
gV
Q
mD
Q
DCgV
C
mDgV
Q
mD
Q
DCgV
mD
Q
U
UmDUDDQ
























 Q is volumetric flow rate
--- for laminar flow conditions
--- for turbulent flow conditions
Rotameter, variable area flow meter

Flow meters for open channel flow
Weirs and flumes: used as flow meters for open channel flow
Weirs:
• Elevated structures in open channels used for flow
measurement
• Can be sharp crested weirs (thin plates set vertically across
the width of the channel) and board crested weirs
• Can be contracted weirs or suppressed weirs
– Contracted weirs: Nappe is open to atmosphere at the edges; Nappe
width is slightly lesser than the weir width
– Suppressed weirs: Channels sides are also the sides of the weir
opening; Nappe is not open to atmosphere, but usually some type of
air vent is provided beneath the nappe)
• Weirs for flow measurement
– Rectangular weirs
– Cipolletti weirs
– Triangular (V-notch) weirs

Rectangular weirs
  2
3
2
3
2
3
2.083.1
83.1
075.0611.0
2
3
2
HHbQ
bHQ
H
H
C
HbgCQ
w
d
d




Discharge for suppressed rectangular weir
Discharge for contracted rectangular weir
Applicable for H/Hw is <5
‘b’ is width at the weir crest
H is water depth above the crest at 4H to 5H
distance upstream side
Cd according to Rouse (1946) & Bievins (1984)
Hw is weir crest height from channel bottom
For H/Hw <0.4 Cd is 0.62 & Q is
Acceptable for b≥3H

Cipoletti weir and V-notch weir
Cipoletti or trapezoidal weir
• Side slope is 1:4 (H:V)
• Corrections for end contractions not needed
• Can be used when the H is >6 mm (for <6
mm the nappe does not spring free of crest)
V-Notch weir
• Has V shaped opening with θ = 10° to 90°
• Cd value decreases with increasing angle
• Minimum Cd value is 0.581
• 0.58 can be used as Cd for θ = 20° to 100°
2
3
859.1 HbQ  ‘b’ is bottom weir width
2
5
2
tan2
15
8
HgCQ d 







Weirs cause high head losses and suspended
solids tend to accumulate behind the weirs

  













3
2
2
3
2
97.4
5.0
5.0
a
hgabCQ
a
hbaQ
d














 
5.0
1
tan
2
1
a
y
bx

.max
.max
.min
5.0
.max
.min5.1
.max
.max
262.0
H
Q
Q
a
Q
Q
gH
Q
b








Cd value is 0.6 to 0.65
b is taken as ‘channel width – 150 mm’!
Proportional weir (Sutro weir)

Broad crested weirs
• Very robust flow measurement device used in rivers/canals
• A broad rectangular weir with a level crest and rounded edges
• Works on the principle that the flow over the weir occurs at
critical depth
– Flow at critical depth occurs when the weir height is above a
specific value
– Uptill critical depth occurs, raising the crest level will not affect
the upstream water level
– Once critical depth is achieved, any further rise of crest height
also rises the upstream water level
• For a proper broad crested weir used for flow measurement
– Upstream flow is sub-critical
– Flow over the crust is critical flow
– Super critical on the downstream side
– On the downstream side a super critical flow turns back to a sub-
critical flow after a hydraulic jump
• Hydraulic jump in the downstream side is in fact an evidence
for critical flow on the crest

Broad crested weirs
• Problems associated with broad crested weirs
– Accumulation of silt and debris in the region of dead water on the
upstream side
– Loss of energy from the downstream side hydraulic jump formation
• A solid weir has no hydraulic jump (!)
• Crump weir can to a great extent solve the above problems
– Crump weirs have an upstream slope of 1 in 2 and a
downstream slope of 1 in 5 to reduce the region of dead water
on the upstream side
• For critical depth of flow over the crest of the weir, unique
relationship exists between the head above the crest and the
flow rate/discharge

Broad crested weir
Discharge equation/formula
5.1
5.1
5.1
705.1
6.1
LHQ
LHQ
CLHQ



L is weir length
H is head over the crest
H is actually height of the total energy line
from the crest of the weir
It is measured usually in a stilling chamber a
few meters upstream the weir where the
water level is affected by draw-down
C is weir coefficient, its value is taken as 1.6
C is estimated from the total energy or bernouli’s equation as 1.705
From this the coefficient of discharge can be calculated as 0.94
Critical depth of flow should occur on the crest for the discharge
formula to work
The discharge formula is based on the critical flow on the crest and
does not be influenced by the weir shape
Value of ‘C’ however can be influenced by the weir shape

Flumes
• Flumes are specially shaped fixed hydraulic structures that
force flow to accelerate through in such a way that the flow
rate becomes related to the liquid level
– Converging side walls or raising bottom or both are used in
shaping the special hydraulic structures
• Flumes usually have 3 sections: converging section, throat
section and diverging section
– All the sections do not necessarily be present in all the flumes -
Cutthroat flume has no throat
• Compared to weirs, head loss for flumes is lesser (it is just
1/4th of a sharp crested weir)
• Flumes have no dead zones on the upstream side where
sediment and debris can accumulate
• Types of flumes commonly used:
– Parshall flumes
– Palmer-Bolus flumes

Parshall flume
• Consists of a converging section, a throat section and a
diverging section
– Crest of the throat section is tilted to the downstream side
– In channels of < 2.44 m width, inlet of the converging section
may be rounded
• Parshall flumes are constructed for standard dimensions
defined by the width of the constriction
• Parshall flumes operate on the venturi principle
– Narrow throat causes water level to raise on the upstream side
• Flow rate is obtained by measuring water depth in the
converging section of the parshall flume
n
KHQ 
H is water depth at point h1
K is a constant (function of the constriction and of the
units chosen for the measurement – value increases with
the increasing flume width)
‘n’ is a constant of exponent (function of the constriction’s
dimensions – value is between 1.522 - 1.607

Standard dimensions
W 305±0.8
A 1372
2/3A 914
B 1343
C 610
D 845
E 914
F 610
G 914
H ----
K 76
M 381
N 229
P 1492
R 508
X 51
Y 76Parshall flume of standard dimensions

Parshall flume (submerged conditions)
• When downstream water depth is higher than the crest level
of the flume (floor level of the converging section), a second
water depth measurement (h2) is needed for the flow
measurement
• If h2/h1 is crossing 50% to 80% (50% for smaller flumes and
80% for larger flumes) then flow is said to be submerged
• Flow measurement for submerged flow conditions is possible
when h2/h1 is <0.95
 
2
1
1
2
211
log
n
n
h
h
hhC
Q









C1 is a constant – its value increases with the
increasing width of the flume
‘n1’ and ‘n2’ are constants – their values also
increase with increasing flume width
‘h1’ and ‘h2’ are water depths against a
reference level in the converging section and
at the downstream of the throat

Parshall flume
• Parshall flume must be located in the straight section of the
channel for flow measurement
• Crest level of the flume must be higher than the channel
bottom
– The crest level is raised at 1 in 4 slope from the channel
• Parshall flume is extremely effective for flow measurement
when the water contains suspended solids
• Parshall flume creates very little head loss
• Turndown ratio is >100
– A feet wide standard parshall flume can measure a minimum
flow of 0.00439 m3/sec. (h1 is 31 mm) and a maximum flow of
0.4568 m3/sec. (h1 is 762 mm)
• Margin of error is ±3%

Palmer-Bolus flume
• It is a venturi type flume
– High velocity critical flow is produced in the throat by flow
constriction
• Usually prefabricated - designed to install in existing channels
– Installed in sewers or in manholes or in open, round or
rectangular bottom channels
• Advantages
– Easy to install
– Minimum restriction to flow, less energy loss, less cost and low
maintenance
– Less sensitive to upstream disturbances
– Can be used in submerged flow conditions (80-90%
submergence is no problem
– Does not require upstream or downstream crest differential
– Water containing solids can be measured

Palmer-Bolus flume
• Size may range from 100 mm to 1000 mm
– Dimensions of the flume depend on the diameter or size of the
channel in which installed
• Throat is trapezoidal in shape
– Has a flat bottom and inclined sides (20°)
– Length of the throat is usually equal to the diameter
• The flume is elevated from the channel bottom by D/6
• Inclined section from channel to flume has 1 in 3 slope
• Length of the base of the flume is D+2P where P is length of
the inclined section (D/2)
• Turndown ratio is relatively small (9 or 10:1)
– Difference between the minimum flow and the maximum flow
that can be measured is relatively small
– For a 12’’ flume (D=12’’) the minimum and the maximum flow
measured is 0.0056 m3/Sec. and 0.0158 m3/Sec. respectively

t = D/6
B = W = D/2 or 5D/12
m = D/4
mm

PALMER-BOWLUS FLUME – STANDARD DIMENSIONS

Palmer-Bolus flume
 
 mz
mzgz
DQ
8.41
4.21
12
5
33
2
5



Q is flow rate
D is diameter of the channel
‘g’ is acceleration due to gravity
‘z’ is dc/D where ‘dc’ is depth of flow
‘m’ is vertical constrictions base projection (D/4)
Flow measurement equation
Flow rate is determined by measuring water depth upstream
from the flume
Liquid depth is measured at a point D/2 distance from the
flume on the upstream side
Within the normal range of flow (10% to 90% of the flume
capacity) error in flow measurement is <3%

Acoustic Meter
• Use sound waves to measure
the flow rates
• Sonic meter or ultrasonic meter
depending on whether the
sound waves are in or above
audible frequency range
• Determine the liquid levels,
area, and actual velocity
• Advantages: low head loss,
excellent accuracy (2~3%),
usable in any pipe size, no
fouling with solids, and wide
flow ranges (10:1)
• Disadvantages: High initial cost
and need for trained personnel
to handle routine O&M 50

Miscellaneous Flow
Measurement Devices
• Depth Measurement
– Need to measure the flow depth and sewer slope and use
Manning equation for flow estimation
– Frequently used for interceptor flow estimation
• Open Flow Nozzle
– Crude devices used to measure flow at the end of freely
discharging pipes.
– Must have a section of pipe that has a length of at least six
times the diameter with a flat slope preceding the discharge.
– Examples: Kennison nozzle and the California pipe
51

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

• 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
63

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)
65

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 66

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
67

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
68

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
69

• 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
71

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
72
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
73

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
74

• 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
75

• 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
76

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
77

78

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)
79

• 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
80

• 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
81

– 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
82

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
83

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
84

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
85

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
86

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 88

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
89

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 low90

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
91

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
92

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
93

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
94

Cd reduction column 95

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.
96

• 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 97

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 98

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 99

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
100

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 101

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
102

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)
104

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)
105

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.
106

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
107

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
108

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
110

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
111

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
112

• 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
113

• 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
114

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
115

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
116

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
117

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
118

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
119

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
120

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
121

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
122

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
123

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
124

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
125

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  
126

• 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
127

• 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
128

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
129

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
130

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)
131

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
132

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)
133

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)
134

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
135

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
136

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
137

Biological Water Quality
(coliform count, MPN test)

Biological water quality testing
Interest is to know about presence of waterborne pathogens
– Too many varieties to test and not feasible for direct methods
Presence and density of indicator organisms is established
Fecal contamination of water is established through testing for the
presence and density of an indicator organism
– Fecal matter of the infected is source for pathogens
– Fecal contamination indicates higher probability of pathogen presence
Coliform bacteria (Escherichia coli), specifically fecal coliform is the
indicator organism
– It is present in water, whenever fecal contamination is there, in larger
numbers than any of the water borne pathogens
– Testing for its presence and density is cheaper, easier and faster
– Working with it does not produce serious health threats to laboratory
workers

• Actually tested for Total Coliform Count
– Since coliform can also be contributed by sources other
than fecal contamination, waters may also be tested for
Fecal Coliform Count
– Incubation temperatures are different (35C for total
coliform and 44.5C for fecal coliform)
• Two techniques are used to test waters for coliform
count
– Multiple tube fermentation technique
– Membrane filtration technique
Biological water quality testing

Sample collection,
preservation and storage
Cleaned, rinsed (final rinse with distilled water) and sterilized
(either by dry or wet heat) sampling bottles are used
For collecting samples with residual chlorine, to prevent
continued bactericidal action, sodium thiosulfate is added to
sample bottles prior to sample collection
– 100 mg/l in case of wastewater samples
– 18 mg/l in case of drinking water
For collecting samples with high copper or zinc or high heavy
metals add chetaling agent EDTA to the bottle prior to
sterilization to give 372 mg/l in the sample

Sample collection,
Sample collection
– Use aseptic conditions
– Do not contaminate inner surface of stopper and bottle’s neck
and keep bottle closed untill to be filled with sample
– Fill without rinsing and replace stopper immediately
– Leave ample space (2.5 cm) to facilitate mixing by shaking
Sample collection from a tap
– Run the tap full for 2 to 3 min. to clear the pipeline, reduce
water flow to permit sample collection without splashing
– Avoid sampling from leaking taps
– Remove tap attachments (screen/splash guard!)
– If you desire clean tap tip with hypochlorite (100 mg/l), and run
it fully opened for 5-6 min prior to sample collection

Sample collection,
Sample collection from other sources
• In case of hand pump, run it for 5 min. prior to sampling
• In case of a well sterilized bottle can be fitted with weight at
the base and used
– Avoid contact with bed
• Avoid taking sample too near to banks or far from water draw
off point in case of river/lake/spring/shallow well
– If collecting from boat collect from upstream side
– Hold bottle near base, plunge it below water surface with neck
downward, turn it until its neck points slightly upwards and
mouth directed towards water current and collect sample (if no
current push bottle forward to create)
– Special apparatus can be used to mechanically remove stopper
under the water surface

Start testing promptly
– If not to be started within 1 hr. ice cool the sample
Transport sample within 6 hr while holding temperature <10C
– Use ice cooler for sample storage during transport
If testing not started within 2 hrs of receipt refrigerate
– Time elapsed between collection and testing should be <24 hrs
Record time elapsed and temperature of storage for each of the
samples analysed
Sample collection,

Multiple Tube Fermentation Test
Also known as MPN test (Most Probable Number)
• An estimate of mean density of coliforms - reported as MPN/100 ml
• Poisson distribution (random dispersion) of coliforms is assumed
Defintion of coliform bacteria for MPN test: All aerobic and
facultative anaerobic gram negative, non-spore forming, rod
shaped bacteria that ferment lactose with gas and acid
formation within 48 hrs at 35C

Multiple-tube fermentation technique
Conducted in 3 phases
• Presumptive test
– Serial dilutions of a sample (to extinction) are incubated in
multiple tubes of lauryl tryptose broth at 35°C for 48 hrs
– Positive results (production of gas/acid) is an indication for the
presence of coliforms
• Confirmed test
– Sample from positive tubes of presumptive test are incubated in
tubes of Brilliant Green Lactose Bile (BGLB)/MacConkey Broth at
35°C or in tubes of EC/A1 broth at 44.5°C
– Positive result confirms presence of coliforms in case of BGLB
tubes and presence of fecal coliforms in case of EC broth tubes

Multiple-tube fermentation technique
• Completed test
– Involves streaking of LES Endo agar plates with inoculum from
positive BGLB/MaCB or EC/A1 broth tubes for obtaining isolated
colonies
– Gram stain the cells from isolated colonies and examine under
microscope
– Gram negative, non-spore forming, rod shaped bacteria are
coliforms – completion test
• Calculation of MPN is
– Directly from Poisson distribution
– From the MPN tables
– By Thomas equation

Presumptive phase of MPN test
Lauryl tryptose broth or alternatively lactose broth is used as
medium
Dehydrated medium is mixed in distilled water, and heated to
dissolve the ingredients after pH adjustment
– Bromocresol purple (0.01 g/L) can be added for indicating acid
production
– Double strength medium is also required
– Quantity required depends on number of samples and number
of decimal dilutions

Medium is dispensed into fermentation tubes with inverted vials
(Derham tubes)
– Dispense double strength medium into the tubes that will be
inoculated with 10 ml sample to avoid dilution of ingredients
below the standard medium level
– Ensure that the medium level in the tubes is sufficient to totally
submerge the inverted vials
– 9 or 10 ml medium is usually dispensed into each tube
Close fermentation tubes with heat resistant caps and sterilize in
autoclave

Decimal dilution and inoculation of fermentation tubes
• Done in inoculation chambers aseptically and requires
– Sterilized dilution tubes each with 9 ml of dilution water
– Sterilized 1 ml and 10 ml capacity pipettes
Sterilized fermentation tubes with contamination free medium
and air bubble free inverted vials are used
– 3 or 5 fermentation tubes at each of the decimal dilutions
– One set of 3 or 5 tubes will be of double strength medium

Thoroughly mix the sample in sample bottle and aseptically
transfer 10 ml into each of the set of fermentation tubes with
double strength medium
– transfer 1 ml of the sample into a sterilized dilution tube with 9
ml of dilution water
Thoroughly mix dilution tube contents and transfer 1 ml into
each of the 3-tube set with single strength medium
– transfer 1 ml of diluted sample from the dilution bottle into the
next dilution tube
Repeat the dilution and inoculation process till the desired level
of dilution is reached
– Dilution to extinction is the concept behind the decision
– Use a separate sterile pipette for each of the dilution
– Shake vigorously (samples & dilutions) while preparing
– Sample volumes used are 10, 1, 0.1, 0.01, 0.001, …

Mix fermentation tube contents after inoculation (through gentle
agitation) and incubate at 35±0.5C
After 24±2 hours of incubation shake each of the tubes gently and
examine for gas in the inverted vials or acidic growth
– If no gas or no acidic growth, reincubate and reexamine at the
end of 48±3 hours for gas or acidic growth
Record results (number of positive tubes for each dilution) and submit
positive tubes for confirmation phase of the test
– From recorded results read MPN value from MPN table
– If a positive tube of presumptive test gives negative result in the
confirmation phase accordingly adjust the results

Confirmed phase of the test
Conducted on only the positive presumptive tubes
– If all tubes are positive at 2 or more dilutions, then conduct the
test on all the tubes of the highest dilution of positive reaction
and on all positive tubes of subsequent dilutions
Can be conducted simultaneously for both total coliforms and fecal
coliforms
– Fermentation tubes with Brilliant Green Lactose Bile Broth
(BGLB)/MaCB for total coliforms
– Fermentation tubes with EC/A1 medium for fecal coliforms
Inoculate one BGLB/MaCB tube (and/or one EC/A1 broth tube) from
each of the positive presumptive tubes
– Gently shake or rotate the positive tube of presumptive test to
resuspend microorganisms
– Transfer a loop full of the culture into the BGLB/MaCB and/or
EC/A1 tube with a 3 mm diameter sterile metal loop

Confirmed phase of the test
Incubate inoculated BGLB/MaCB tubes at 35±0.5°C
– Gas production within 48±3 hours of incubation is taken as
positive confirmed total coliform reaction
Incubate EC/A1 broth tubes within 30 minutes of inoculation in water
bath at 44.5±0.2°C
– Immersed in the bath till medium level in the tubes is below the
water level in the water bath
– Gas production within 24±2 hours of incubation is taken as a
positive confirmed fecal coliform reaction
Adjust recorded results of the presumptive test if any of the positive
presumptive tubes gave negative reaction
– The results adjusted on the basis of negative results with
BGLB/MaCB tubes give total coliform count
– Results adjusted on the basis of negative results with EC/A1
medium tubes give fecal coliform count

Completed test
Meant to definitively establish presence of coliform bacteria in the
positive confirmed tubes
Positive confirmed tubes of EC/A1 broth at elevated temperature do
not require completed test
– Positive confirmed tubes are taken as positive completed test
responses
Completed test involves
• Streaking one LES endo agar petriplate from each of the positive
BGLB/MaCB confirmed tube to obtain discrete colonies

Completed test
• Picking up a typical colony (or atypical colony) that is most likely
consist of coliform bacteria and transfering to
– A lauryl tryptose broth fermentation tube to check for gas
production on incubation at 35±0.5C for 24±2 hours
– A nutrient agar slant for incubating for 24 hours and obtaining
bacterial culture for Gram staining and microscopic examination
• Microscopic examination of bacterial culture of the nutrient agar
slant after gram staining
Production of gas in the lauryl tryptose broth and demonstration of
gram negative, non-spore forming rod shaped bacteria are taken as
positive results
If the result is negative accordingly adjust the results recorded during
presumptive test

Liquify sterile LES endo agar, aseptically pour into sterile petri
plates and allow the poured medium to solidify
Gently shake or rotate the positive confirmed tube to resuspend
the organisms, take a loopful of the culture and streak an LES
endo agar plate
– Avoid picking up of any scum or floating membrane by the
inoculation loop
– Do streaking in such a way that isolated colonies obtained
Incubate the streaked plates at 35±0.5C for 24±2 hours
Completed test

Bacterial colonies developed on the plate are divisible into
• Typical colonies: pink to dark red colonies with a green metallic
surface sheen (covering the entire colony, or appearing only in a
central area or on the periphery)
• Atypical colonies: pink, red, white or colourless colonies without
green metallic surface sheen
• Other colonies: non-coliform colonies
Pick up one or more typical colonies for inoculating the
secondary lauryl tryptose broth tubes and the nutrient agar
slants
– in the absence of typical colonies pick up the colonies that are likely to
contain coliforms
Completed test

• Place a loopful of dilution water in the center of microscopic slide
and add to the water drop a loopful of the bacterial culture of the
nutrient agar slant
– Also maintain separate gram positive and gram negative control
cultures on the same microscopic slide for comparison
• Spread the culture in the water drop to make uniform dispersion
over an area of the slide, and then air dry & heat fix
• Stain the heat fixed smear with ammonium oxalate – crystal violet
solution for 1 min., rinse with tap water and drain off
– Ammonium oxalate – crystal violet solution: mix 2 g of crystal violet,
in 20 ml 95% ethyl alcohol, and 0.8 g ammonium oxalate, in 80 ml
distilled water, age for 24 hrs and filter
Completed test

• Apply iodine solution for one min., rinse with tap water and allow
acetone alcohol solvent to flow across the smear till colourless
solvent starts flowing off from the slide
– Lugol’s solution (Iodine solution): Grind 1 g iodine crystals and 2 g KI in
a mortar first dry then with distilled water till solution is formed, and
rinse the solution into amber bottle with 300 ml distilled water
– Acetone-alcohol solvent: 1:1 mixer of 95% alcohol and acetone
• Counterstain the smear with safranin for 15 sec., rinse with tap
water, blot day and then examine microscopically
– Counterstain: dissolve 2.5 g safranin dye in 100 ml of 95% ethyl alcohol
and then add 10 to 100 ml distilled water
Completed test

Estimation of bacterial density
Estimated from the results of the presumptive phase of the test, after
necessary adjustments made consequent to the negative results of
confirmed phase and completed phase
Bacterial density is read from MPN index table corresponding to the
number of positive tubes for 3 consecutive dilutions
– MPN index table for 5 tubes per dilution and the table for 3 tubes per
dilution are different
– MPN index table relates the number of positive tubes at 10, 1 and 0.1
ml sample volumes to MPN/100 mL
– When dilutions considered are different from 10, 1 and 0.1 ml, for
calculating MPN (from the index table reading) use
considereddilutionlowesttheatsampleofmL
tablereadingMPN
mlMPNMPN
10
)100/(



Estimation of Bacterial Density
When tested at sample volumes beyond 10, 1 and 0.1 ml, choose the
results of highest dilution (at which all the tubes are positive) and
the next two dilutions
5/5-5/5-2/5-0/5 ..-5-2-0
5/5-4/5-2/5-0/5 5-4-2-..
Of all the dilutions tested if only one gave positive results then
consider results of that dilution and of one dilution below and one
dilution above it
0/5-0/5-1/5-0/5-0/5 ..-0-1-0-..
If positive results are obtained even at a dilution beyond the series of
dilutions considered then add that positive result to the results of
the highest dilution considered
5/5-3/5-2/5-1/5 5-3-2-..
5/5-3/5-2/5-0/5 5-3-2-..

Estimation of bacterial density
MPN index table do not include the unlikely combination of results
(the combination whose probability is <1%)
– Obtaining the unlikely combination of results usually indicates faulty
multiple tube fermentation technique
The MPN index table can also include 95% confidence limits
For estimating MPN from the unlikely combination of results and from
the results of a test where decimal dilutions are not used, use the
following (Thomas) equation:
Precision of multiple tube fermentation test is low because of random
distribution and clustering of the coliform bacteria














tubestheall
insampleofmL
tubesnegative
insampleofmL
tubespositiveofNumber
mlMPN
100
100/

MPN test for fecal coliforms
Elevated incubation temperature is used for the separation
of coliforms into those of coliform origin and those of
non-coliform origin
Two approaches can be followed
• Use of EC broth and incubation at 44.5±0.2C in the
confirmation phase of the test
• Use of a single step method with A-1 medium in place of the
three phase total coliform test
– EC medium is not recommended in place of A-1 medium – prior
enrichment in the presumptive medium is needed
– Inoculated tubes of A-1 broth need incubation first at 35±0.5C
for 3 hours and then at 44.5±0.2C for 21±2 hours in a water
both
– Gas production within 24 hours of incubation is a positive
reaction for fecal coliform

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