Water Pollution

By
Dr Alok Sinha
Professor
Department of Environmental Science and
Engineering,
IIT (ISM), Dhanbad
 MAJOR WATER QUALITY ISSUES

Common issues of Surface and Ground water

• Pathogenic (Bacteriological) Pollution
• Salinity
• Toxicity (micro-pollutants and other industrial pollutants)

Ground Water
Surface Water
• Fluoride
• Eutrophication
• Nitrate
• Oxygen depletion
• Arsenic
• Ecological health
• Iron
• Sea water intrusion 2
PHYSICAL PROPERTIES
 COLOUR
Pure water as rain water is colourless.
But water is a universal solvent and is often
coloured by many substances.
Running water carries suspended solids which
cause apparent colour.
Water whose colour is due to suspended matter
is said to have apparent colour.
Apparent colour fades out when suspended
solids settle.
Colour contributed by dissolved solids is known
as true colour which remains permanently.
After contact with organic debris such as leaves,
weeds and wood, water picks up tannins, humic
acid and humates to take a yellowish brown hue.
Iron oxide causes reddish water and manganous
oxide gives brown or blackish water.
Fresh sanitary sewage is grey in colour and its
colour deepens with time.
Stale or septic sewage is dark in colour.
Colour is a visible pollutant.
Highly coloured water may not be accepted for
laundering, dyeing, papermaking, beverage
manufacturing, dairy production, food processing,
textile and plastic production.
 COLOR
Sources
Domestic and industrial wastes, natural decay
of organic materials.

Organic debris such as leaves, weeds or wood.

Industrial wastes from textile and dyeing

operations, pulp and paper production, food
processing, chemical production, and mining,
refining and slaughterhouse operations.
Impacts:
Color affects acceptability of water as both
domestic and industrial product.
Colored water is not aesthetically
acceptable to the general public.
Highly colored water is unsuitable for
laundering, dyeing, papermaking, beverage
manufacturing, dairy production and other
food processing, and textile and plastic
production.
True color exerts chlorine demand
While Chlorination the color causing
compounds can produce carcinogens
 COLOR

Measurement:
Color-comparison tubes containing a series of
standards (different colors) may be used for direct
comparison with the water sample.
Results are expressed as True Color Units (TCU)
where one unit is equivalent to the color produced
by 1 mg/L of platinum in the form of chlorplatinate
ions.
Special spectrophotometric techniques are
normally used for colored water originating from
industrial waste effluents.
Calibrated colored disks are used in field work.
Samples to be tested within 72 hrs
 TASTE AND ODOUR
 Minerals, metals and salts from the soil, end
products from biological reaction and
constituents of wastewater attribute taste and
odour to water.
 Odour is mainly caused because of gases of
decomposition of organic matter.
 Inorganic impurities mostly taste and not odour
 Fresh sanitary sewage has mild, earthy,
inoffensive odour or it may be even odourless.
Alkaline materials impart bitter taste; metallic
salts may give a salty or bitter taste.
Petroleum-based products are the primary
taste- and odor-producing organic chemicals
Biological decomposition of organics may
also result in taste- and odor-producing
liquids and gases. (e.g. ‘rotten egg’ taste and
odor of sulfur).
Certain species of algae secrete taste- and
odor-producing oily substance.
Because of anaerobic decomposition of proteins
and other organic matter rich in nitrogen, sulphur
and phosphorous, foul smelling and highly odourous
gases as ammonia, hydrogen sulphide, mercaptans
(Ca Hb Sc ) and skatol (Cx Hy Nz ) are produced.
IMPACTS:
 Offensive odours reduce appetite for food
 lower water consumption
 impair respiration, nausea, result in vomitting and
mental perturbation
 leads to deterioration of personal and community
pride
 interfere in human relations discouraging capital
investments
 lowering socio-economic status and deterring
growth and decline in value and sales.
Measurement:
Quantitative tests sometimes employ human
senses of taste and smell.
The Threshold Odor Number (TON) test uses
varying volumes of the sample diluted to 200mL
with distilled water.
5 to 10 people determine the mixture in which the
smell is just barely detectable. The TON is given
by, A B
TON 
A

where A = the volume of odorous water (mL), and

B = the volume (mL) of odor-free water required to
produce a 200-mL mixture.
 SOLIDS
Measurement:
Total Solids Test
 Measures all solids (suspended and dissolved,
inorganic and organic)

 Water sample is heated to a temperature slightly

above boiling (104oC)

The residue is weighed and expressed as milligrams

per liter (mg/L) [‘dry-mass’ weight of residue (in mg)
per liter volume of sample].
Suspended Solids Test
Measures the mass of residue retained on a
filter (paper).
The water sample is filtered, and the filter
and residue is dried to a constant weight at
104oC (+/- 1oC).
The difference between weights of the filter
before and after filtration gives the weight of
residue (suspended solids).
Residue (dry-mass) is expressed as mg/L.
 SOLIDS
The Dissolved Solids passing through the filters is
the difference between the total-solids and
suspended-solids contents of the sample.

Figure: Filtration apparatus.

 SUSPENDED SOLIDS
 Solids suspended in water may consist of
inorganic or organic particles.

 Inorganic solids such as clay, silt and other soil

constituents are common in running surface
water as rivers and streams.

 Organic material such as plant fibers and

biological solids as algal cells and bacteria are
also common constituents of surface waters.
Impacts:
Aesthetically displeasing;

Provides adsorption sites (surfaces) for

chemical and biological agents;

Biologicaldegradation of organic solids

may produce objectionable by-products;

Biologically active solids may include

disease-causing organisms and toxin-
producing algae.
 TURBIDITY
 Turbidity is the property of absorption of light or its
scattering by suspended material in water.
 Both absorption and scattering are influenced by size
and surface characteristics of the suspended material.
 Colloidal material of clay, silt, rock fragments and metal
oxides from the soil, vegetable fibres and microorganism
cause turbidity.
 Also soaps, detergents and emulsifying agents produce
stable colloids that result in turbidity.
 Although turbidity measurements are not commonly run
on wastewater, discharges of wastewater may increase
the turbidity of natural bodies of water.
 IMPACTS:
Aesthetically displeasing opaqueness (‘milky’ coloration).
Turbidity-producing colloidal materials provide adsorption
sites for taste- and odor-producing chemicals, and harmful
organisms.
Disinfection of turbid waters is difficult because of the
adsorptive characteristics of the colloids and since the
solids may partially shield the organisms.
Turbid waters interfere with light penetration and
photosynthetic reactions in streams and lakes.
Turbidity-causing fine particles may deposit on porous
streambed and adversely affect the flora and fauna.
Suspended materials absorb heat from sunlight and raise
the water temperature.
Measurement

Jackson turbidity unit (JTU) (1 JTU = 1 mg/L SiO2)

Nephelometric turbidity unit (NTU) or Formazin

Turbidity Unit (FTU)

1 JTU = 25 NTU

Turbidity tests are commonly performed on natural

water bodies and potable (drinkable) water supplies.
Turbidity does not directly quantify the suspended
solids.
Turbidity
Measurement:

Nephlometer
Turbidimeter
 TEMPERATURE
Temperature is one of the most important
parameters.
It affects the self purification of streams.
Rise in temperature enhances toxicity of poisons
and intensity of odour besides changing the taste.
Also increase in temperature causes growth of
undesirable water plants and wastewater fungus.
It influences the biological species present and
their rates of biological activity.
Temperature has an effect on most chemical
reactions that occur in natural water systems.
Temperature also has a pronounced
effect on the solubilities of gases in water.
Aerobic digestion ceases at a temperature
greater than 50°C.
At less than 15°C anaerobic digestion is
affected as methane bacteria become
inactive.
Influences to a large extent the biological
species and their activity rates in water.
Impacts:
Cooler water usually allows wider diversity
of biological species.
Accelerated algal growth occurs in warm
water, and may cause problems by oil
secretion, thick ‘algal mat’ (cells joined
together) and decay products of the dead
algae cells.
Higher-order species, e.g. fish, are
drastically affected by change in
temperature, and by change in dissolved
oxygen (a function of temperature).
CHEMICAL PROPETRIES
 pH
 It is the measure of hydrogen ion
concentration
pH = −log[H+]
[H3O+][OH-] = 1.0 x 10-14 @ 25 degC
 Neutral water pH-7
 Acidic water has pH below 7
 Basic water has pH above 7
 Desirable limit 6.5-8.5
Let's assume that the concentration of
hydrogen ions is equal to 0.0001 mol/L.
Calculate pH

pH = -log(0.0001) = 4

Now, you can also easily determine pOH

and a concentration of hydroxide ions:
pOH = 14 - 4 = 10

[OH-] = 10-10 = 0.0000000001

 ELECTRICAL CONDUCTIVITY
 Conductivity of a substance is defined as 'the ability
or power to conduct or transmit heat, electricity, or
sound'.
 Its units are Siemens per meter [S/m] in SI and
millimhos per centimeter [mmho/cm] in U.S.
customary units.
 An electrical current results from the motion of
electrically charged particles in response to forces that
act on them from an applied electric field.
 Within most solid materials a current arise from the
flow of electrons, which is called electronic conduction.
In water and ionic materials or fluids a net motion of
charged ions can occur. This phenomenon produce an
electric current and is called ionic conduction.

Specific conductance is measured by conductivity

meter which works on the principle of wheatstone bridge

Typical conductivity of waters:

Ultra pure water = 5.5 x 10-6 S/m
Drinking water = 0.005 – 0.05 S/m
Sea water = 5 S/m

TDS (mg/L) = (0.055-0.09) Conductance (mS)

Anions (meq/L) = Cations (meq/L)

ION BALANCE ±10%
 ACIDITY
 The acidity of water is a measure of its capacity
to neutralise bases.
 Acidity of water may be caused by the
presence of uncombined carbon dioxide,
mineral acids and salts of strong acids and
weak bases.
 It is expressed as mg/L in terms of calcium
carbonate.
 Acidity is nothing but representation of carbon
dioxide or carbonic acids.
 Carbon dioxide causes corrosion in public
water supply systems.
 Alkalinity
 Alkalinity is the ability of water to neutralize
acids.
 CO32– , HCO3– , OH– , H SiO3– , H2–BO3– , HPO
4– and NH3 which are quite common in
atmosphere and soil contribute to alkalinity.
 These compounds result from the dissolution of
mineral substances in the soil and atmosphere.
 Phosphates from detergents and fertilizers and
insecticides of agricultural land may also cause
alkalinity.
Alkalinity is classified as
(i) hydroxide alkalinity or caustic alkalinity
(ii) carbonate alkalinity and
(iii) bicarbonate alkalinity.
Hydroxide alkalinity occurring at a pH
greater than 8.3 (generally above 10) causes
bitter taste, affects the lacrimal fluid around
the eye ball of swimmers,.
whereas bicarbonate alkalinity occurring
below a pH of 8.3 (but above 4.5) mainly
causes scale formation in boilers and
incrustations in pipe lines.
 ALKALINITY
Impacts:
In large quantities, alkalinity imparts a bitter taste
to water.
A major problem with alkaline water is the
reaction that may occur between alkalinity
species and certain cations. Resultant
precipitates may damage pipes and other
appurtenances.

Use:
Alkalinity indicates the buffering capacity (acid
neutralizing capacity) of natural waters.
Also used as process control variable in water and
wastewater treatment.
 OH + ½ CO3
HCO3 + ½ CO3

P M

P=M all OH
P = M/2 all CO3
P=0 all HCO3
 HARDNESS
 Waters which readily give lather with soap are soft
waters.
 Those which do not readily give lather are hard waters.
 Hardness is due to dissolved divalent or multivalent
metallic cations as Ca++, Mg++, Fe ++, Mn++ and Sr+
+ and Al3+ out of which the most abundant in natural
waters are Calcium and Magnesium.
 In association with anions as bicarbonates, chlorides
and sulphates
 Carbonate hardness is due to bicarbonates of Calcium
and Magnesium which can be easily removed by simple
means as boiling and hence is called temporary
hardness.
 Alkalinity alone causes carbonate hardness.
 Non-carbonate hardness due to chlorides and sulphates
of Calcium and Magnesium cannot be removed that
easily and hence is called permanent hardness.
 Concentration of multivalent metallic cations in solution.
In supersaturated condition, the hardness cations react
with anions to form a solid precipitate.
 Hardness can be classified as carbonate hardness and
noncarbonate hardness depending on the anions they
are associated with. Hardness equivalent to the
alkalinity is called carbonate hardness. Any remaining
hardness is called noncarbonate hardness. Carbonate
hardness is sensitive to heat and precipitates readily at
high temperatures.
CLASSIFICATION OF HARDNESS
 HARDNESS

Impacts:
 Sodium-based soaps react with the hardness cations
to form a precipitate, thereby losing their surfactant
properties.
 Soap consumption by hard water incurs economic
loss to the water user.
 Precipitates formed by hardness and soap adhere to
appurtenance surfaces and may stain clothing and
utensils.
 Use of hard water may result in rough, uncomfortable
skin.
 Scale formation in boilers
 Certain soaps do not react with hardness.
 FLOURIDE
 It is an ingredient of igneous and sedimentary rocks.
 Fluoride is rarely found in appreciable quantities in surface
waters but appears in certain ground waters.
 Concentrations of approximately 1 mg/l in drinking water
help to prevent dental cavities in children (dental carries).
 During the formation of permanent teeth, fluoride
combines chemically with tooth enamel resulting in harder
and stronger teeth that are more resistant to decay.
 Excessive intakes of fluoride can result in discolouration of
enamel of teeth called mottling (Dental Fluorosis).
 Excessive dosages of fluoride can also result in fluorosis
of bones and other skeletal abnormalities (Skeletal
Fluorosis).
Dental Fluorosis
 CHLORIDES
 Chloride ion may be present in combination with one or more
of the cations of calcium, magnesium, iron and sodium.
 Chlorides of these minerals are present in water because of
their high solubility in water.
 Each human being consumes about six to eight grams of
sodium chloride per day, a part of which is discharged
through urine and night soil.
 Thus, excessive presence of chloride in water indicates
sewage pollution.
 Chlorides in natural water result from the leaching of chloride
containing rocks and soils with which the water comes in
contact and in coastal areas from sea water intrusion.
 In addition, agricultural, industrial and domestic wastewaters
discharged into surface waters are a source of chlorides.
 DISSOLVED OXYGEN
 The living organisms are dependent upon
oxygen in one form or another to maintain the
metabolic processes that produce energy for
growth and reproduction.
 All the gases of atmosphere dissolve in water to
some degree.
 Both nitrogen and oxygen are poorly soluble.
 The solubility of atmospheric oxygen in fresh
waters ranges from 14.6 mg/l at 0°C to about
7.6 mg/l at 30°C at 1 atmospheric pressure.
 Dissolved salts of water reduce the solubility of
oxygen so also impurities in water.
 AMMONICAL NITROGEN AND TKN
• Nitrogen is present in water, wastewater and industrial
wastewater/effluent samples in various concentrations
and forms.
• In surface water samples, nitrogen is present mainly as
nitrate (which is the most oxidised form of nitrogen).
• In groundwater samples nitrogen may be present as
both nitrate and nitrite.
• In raw wastewater samples, and in water contaminated
with domestic and industrial wastes, nitrogen is mainly
present as ammonical nitrogen (NH4/NH3-N), and
organic nitrogen (Org-N).
• Two types of nitrogen measurements are performed on
wastewater samples, (a). Ammonical Nitrogen (NH4-N)
and (b). Total Kjeldahl Nitrogen (TKN).
 METALS

 All metals are soluble to some extent in water.

 Metals that are harmful in relatively small
amounts are considered toxic.
 Heavy metals (density > 5 g/cm3)
 Metals in natural waters originate from
dissolution of natural deposits, and discharges of
domestic, industrial or agricultural wastewaters.
 Measurement usually made by atomic absorption
spectrophotometer (AAS).
 METALS
Nontoxic Metals:
 Commonly found in water; include sodium, iron, manganese,
aluminum, copper and zinc.
 Sodium is the most common nontoxic metal and highly reactive
to other elements; excessive concentrations of sodium salts
impart a bitter taste, and are hazardous to cardiac and kidney
patients; also corrosive to metal surfaces and toxic to plants.
 Iron and manganese frequently occur together in natural
waters and present no health hazard. May produce color
problems and bacterial growth causing taste and odor.
 Significant quantities of iron are usually found in systems
devoid of oxygen such as groundwater and bottom layers of
stratified lakes.
 Copper and zinc, if both present, may be toxic to many
biological species.
 METALS
Toxic Metals:
Harmful to human and other organisms in small
quantities.
Toxic metals that may be dissolved in water include
arsenic, barium, cadmium, chromium, lead,
mercury and silver. Arsenic, cadmium, lead and
mercury are particularly hazardous.
These metals are concentrated by the food chain.
Biomagnification
Toxic metals usually originate from natural,
industrial, or agricultural sources.
 ORGANICS
Biodegradable Organics:
Organics that are used up by microorganisms for food;
usually consist of starches, fats, proteins, alcohols,
aldehydes and esters.
Microbial decomposition of dissolved organics normally
takes place accompanied by oxidation (addition of
oxygen or reduction of hydrogen), or by reduction
(addition of hydrogen or reduction of oxygen).

Non Biodegradable organics

Industrial and agricultural discharges (pesticides,
pharmaceuticals etc.)
Toxic to microbes
The decomposition may occur in aerobic (oxygen-
present) or anaerobic (oxygen-absent)
environment.
Anaerobic decomposition results in unstable and
objectionable end products.
When oxygen utilization occurs more rapidly than
oxygen is replenished, anaerobic conditions occur
that severely affect the ecology of the system.
The amount of oxygen consumed during microbial
utilization of organics is called Biochemical
Oxygen Demand (BOD).
 BIOCHEMICAL OXYGEN DEMAND (BOD)
 Biochemical oxygen demand (BOD) is defined as the
amount of oxygen required by microorganisms to
stabilize decomposable organic matter at a particular
time and temperature.
 BOD test is widely used to determine the pollutional
strength of domestic and industrial wastes in terms of
the oxygen that they require to deliver end products as
CO2 and H2O.
 The BOD test is essentially a bioassay procedure
involving the measurement of oxygen consumed by
living organisms (mainly bacteria) while utilizing the
organic matter present in the waste as carbohydrates,
proteins and fats.
It is standardized at 20°C the usual peak
temperature of summer of London where the test
originated.
Theoretically infinite time is required for complete
biological oxidation of organic matter of domestic
sewage but for all practical purposes, the reaction
may be considered to be completed in about (90–
95%) 20 days.
In case of domestic wastewaters, it has been
found that the 5 day BOD value is about 70 to 80%
of the ultimate (I stage – carbonaceous) BOD.
This is fairly a higher percentage and hence 5 day
(at 20°C) values are used for many considerations
and unless otherwise mentioned BOD means only 5
day 20°C value only.
 Nitrifying bacteria is the bacteria which oxidize
proteinous matter for energy. The nitrifying bacteria
are usually present in relatively small numbers in
untreated domestic wastewater.
 Their reproductive rate at 20°C is such that their
populations do not become sufficiently large to exert
an appreciable demand for oxygen until about 8 to 10
days.
 Once the organisms become established, they oxidize
nitrogen in the form of ammonia to nitries and nitrates
in amounts that induce serious error in BOD
estimation.
 BOD
Procedure
 Saturate overhead tank water with oxygen by bubbling air through it
by using a compressor. Measure dissolved oxygen (DO)
concentration in this water. It should be at least 9 mg/L at 20 deg C.
This is known as dilution water.
 Prepare a blank sample (using 300 mL of dilution water only) in a
BOD bottle. Incubate for 5 days at 20oC. The DO of the dilution
water should not be much different from the initial value. Call this
value DOI. Minimum 2 mg/L and maximum 7mg/L consumption.
 Prepare three samples by adding 5 mL of the wastewater in the BOD
bottle, and making up to 300 mL with dilution water. Incubate for 5
days at 20oC. Measure DO in each sample after 5 days. Call this
value DOF.
Calculations
BOD 5 (mg/l) = (DOI-DOF)/P
Where,
P = Decimal volumetric fraction of sample used
y t = L0 – Lt
yt = L0 – L0e-kt
yt = L0(1-e-kt)

K values range from 0.1-0.5 d-1

Easily degradable organics like sugars and starches have high k

values whereas complex organics like phenols have low k values
K values depends upon temperature
At higher temperature k values are higher
Vant-Hoff Arrhenius equation
kT = k20ϴT-20
ϴ = 1.047
Closed reflux method
Open reflux method
• In COD vials, 3.5 mL of sulphuric acid reagent, 1.5 mL potassium
dichromate solution and 2.5 mL of sample are added and then
refluxed for 2 hours.
• Samples are titrated with FAS solution
• The volume of FAS required is noted and then calculation is done to
estimate COD in terms of mg/L.
• Same procedure is repeated for blank, with distilled water.

COD (mg/l) = (A-B) x 8000 x M

V
Where.
M = Strength of FAS
V = Volume of sample used (2.5 ml).
A = Amount of FAS used for titration of Blank.
B = Amount of FAS used for titration of sample.
BOD/COD ratio

> 0.5 than easily biodegradable

0.4 – 0.5 average biodegradable
0.2 – 0.4 slowly biodegradable
< 0.2 not biodegradable
 Total Organic Carbon (TOC)
 The European drinking water directive includes the category
of indicator parameter value specifications. These are not
directly linked to health problems but have an indicator
function
 TOC is not been assigned a limiting value or criterion but can
be considered as cautionary warning for action under unusual
circumstances
 Lead to undesirable disinfection by-products
 PATHOGENS
Communicable diseases are caused by
bacteria, viruses, protozoa or helminths
(parasitic worms).
Bacterial diseases include typhoid,
paratyphoid, salmonellosis, shigellosis,
bacillary dysentery, and cholera.
Viraldiseases include hepatitis, poliomyelitis,
and some varieties of gastroenteritis.
Protozoasuch as Giardia and Cryptosporidium
may produce gastroenteritis.
Certainfungi such as Aspergillus also produce
diseases in human bodies.
 PATHOGEN INDICATORS

Testing for all possible microorganisms is

infeasible or very expensive.
Possibility
of water contamination is usually
assessed by determining the number of coliform
bacteria.
Escherachia coli is excerted up to 4X1010
organisms per person per day.
Presence of coliform is not a proof that water is
contaminated; but absence indicates that the
water is free of pathogens.
 Two types of MPN tests are generally
performed, the total coliform (TC) and
the fecal coliform (FC) test.

 The FC test is generally more crucial,

since the presence of fecal coliforms in
water indicates mammalian fecal
contamination of the water.

 This enhances the possibility of

pathogens being present in the water
sample.
 TOTAL COLIFORM TEST
Procedure for Presumptive Test
 Prepare lauryl-tryptose broth as per direction given
on the label of the bottle containing the nutrient.
 Using the wastewater sample prepare three dilutions
using the serial dilution procedure.
 10 mL of the broth in each test tube.
 Put a durham tube in each of these tubes, ensuring
that no air bubble is present in the durham tubes.
 Seal each tube with cotton wool. Then sterilize the
tubes for 30 minutes in the sterilizer.
 Cool the tubes and add 1 mL portions of wastewater
(or diluted portions) in six tubes (three dilutions in
duplicate).
 Add 1 mL of a sterilized blank solution to the
seventh tube.
 Incubate at 35oC for 24 hours. After this time visually
examine for turbidity and gas production in the durham
tube.
 Tubes exhibiting gas production are assumed to have
given positive results indicating the presence of
coliform organisms.
5.24.1 Theory
The statistical procedure used for MPN determination is described below. Let "n" be the
number of organisms in a sample of volume "V" mL. Therefore the average number of
n
λ
organisms per mL of sample is V . Note that this does not mean that every 1 mL aliquot from
the original V mL samples must have λ organisms. In fact, any particular 1 mL aliquot may
have 0, 1, 2, …n organisms, or may not have any organism. The probability that any 1 mL
aliquot will have x organisms is given by poission distribution:
x . exp()
p( x ) 
x!
Thus the probability of any 1 mL sample having no organism and having one or more organism
is given by : p( x  0)  exp() ; and p( x  0)  1  exp() respectively. The probability
theory described above is used to calculated the value of most probable number (MPN) of
organisms.
The results of the presumptive MPN test for total coliforms (TC) were done with sample
volumes of 1, 0.1, and 0.01 mL. Each dilution was done in triplicate. The results were as
follows:

1 mL + + +
0.1 mL + - -
0.01 mL - - -

The probability distribution for the above result is:

p( )  (1  exp( )) 3 .(exp( 0.1 )).(1  exp(0.1)) 2 .(exp( 0.03 ))
The MPN value for the above sample is defined as that value of λ for which the value of p(λ)
is maximum. The solution can be obtained analytically, or by plotting the value of p(λ) vs λ
using a spreadsheet software and observing the value of λ corresponding to the maximum value
of p(λ).

Alternatively, the MPN value may be estimated using Thomas formula:

no. of positive tubes x 100

(mL sample in negative tubes ) x (mL sample in all tubes )
MPN/100 mL =