Experiment No.

10
Determination of Biochemical Oxygen Demand

Introduction:
When biodegradable organic matter is released into a water body, microorganisms feed on the
wastes, breaking it down to simpler organic and inorganic substances. When this decomposition
takes place in an aerobic environment it produces non-objectionable, stable end products (e.g.,
CO2, SO4, PO4, and NO3) and in the process draws down the dissolved oxygen (DO) content of
water.

Organic matter + O2 (Bacteria) = CO2 + H2O + New cells + Stable products

When insufficient oxygen is available or when oxygen is exhausted by the aerobic decomposition
of wastes, then different set of microorganisms carry out the decomposition anaerobically
producing highly objectionable products including H2S, NH3, and CH4.

Organic matter (Bacteria) = CO2 + CH4 + New cells + Unstable products

The amount of oxygen required by micro-organisms to oxidize organic wastes aerobically is called
biochemical oxygen demand (BOD). BOD may have various units, but most often it is expressed
in mg of oxygen required per liter of water/wastewater (mg/L).

Environmental Significance :
Dissolved oxygen is the most commonly used indictor of the general health of a surface water
body. If DO in the water body goes below 4 to 5 mg/L, forms of aquatic life that can survive begin
to be reduced. When anaerobic condition exists, higher life forms are killed or driven off. Noxious
condition, including floating sludge, bubbling, odorous gases, and slimy fungus growth prevails.

According to Bangladesh Environment Conservation Rules (1997), drinking water standard for
Biochemical Oxygen Demand (BOD) is 0.2 mg/L (at 20°C). For wastewater effluent allowable
concentration of BOD varies from 50- 250 mg/L.
Apparatus :
1.BOD bottle
2. Beaker (250 ml)
3. Measuring cylinder
4. Dropper
5. Stirrer
Reagents:
1.Manganous sulfate solution [57]
2.Alkaline potassium iodide solution [58]
3.0.025N sodium thiosulfate [59]
4.Starch solution (indicator) [60]
5.Concentrated sulfuric acid

Procedure:
Determination of DO:

Two BOD bottles with sample (or diluted sample); the bottles was completely filled. Determined
initial DO (DOi) in one bottle immediately after filling with sample (or diluted sample). Kept the
other bottle in dark at 20°C and after particular days (usually 5-days) determine DO (Dof) in the
sample (or diluted sample). DO is determined according to the following procedure:

1. Added 1 mL of manganous sulphate solution to the BOD bottle by means of pipette,

dipping in end of the pipette just below the surface of the water.
2. Added 1 mL of alkaline potassium iodide solution to the BOD bottle in a similar manner.
3. Inserted the stopper and mix by inverting the bottle several times.
4. Allowed the "precipitates” to settle halfway and mix again.
5. Again, allowed the “precipitates" to settle halfway.
6. Added 1 mL of concentrated sulphuric acid. Immediately insert the stopper and mix as
before.
7. Allowed the solution to stand at least 5 minutes.
8. Withdrawn 100 mL of solution into an Erlenmeyer flask and immediately add 0.025N
sodium thiosulphate drop by drop from a burette until the yellow color almost; disappears.
9. Added about 1 mL of starch solution and continue the addition of the thiosulphate solution
until the blue color just disappears. Record the ml. of thiosulphate solution used (disregard
any return of the blue color).
Calculation:
normality of Na2S2O3 = .025N
Sample Taken =100 ml
Equivalent Weight if O2 = 8g

Dissolved oxygen, DO (mg/L) = mL of 0.025N sodium thiosulphate added x MF

normality of Na2S2O3 x equivalent wt. of oxygen x 1000

Where, MF = mL of sample taken
Discussion:
The BOD value of the taken sample is 2.8 mg/L, which we get through performing the
experiment. According to Bangladesh Environmental Conservation Rules (1997), the drinking
water standard for BOD is .2 mg/L. But our experimental value is not satisfy the standard value.
So, the sample water should not use for the drinking.

Assignment Questions
1. In a BOD test on a diluted wastewater sample (1:20 dilution, but not seeded), the initial
DO is 8.2 mg/L and final DO after 5 days is 3.2 mg/L. If the reaction rate constant is
0.2/day, calculate;
(a) 5-day BOD of the wastewater,
(b) Ultimate carbonaceous BOD of the wastewater,
(c) Remaining Oxygen demand after 5-days.
Answer:
A)
Doi = 8.3 mg/L; DOf = 3.2 mg/L
20+1
D.F = 1 = 21

Now, BOD5 = (BOi – Dof) x D.F = (8.2 – 3.2) x 21

BOD5 = 105 mg/L

B)
BODs = Lo (1-𝑒 𝑘𝑡 )
Where,
𝐵𝑂𝐷5
L0 = 𝐿0(1−𝑒 𝑘𝑡) BOD5 = 105mg/L
= 166.11 mg/L K= .2/day
T= 5 days

Ultimate carbonaceous BOD, L0 = 166.11 mg/L

C)
Remaining Oxygen demand after 5 days
= 166.11 -105
= 61.11 mg/L

2. A test bottle containing just seeded dilution water has its DO level drop by 0.5 mg/L in a
5-day test. A 300 mL BOD bottle filled with 40 mL of wastewater and the rest with seeded
 dilution water experiences a drop of 7.1 mg/L in the same period (5-day). Calculate the
BOD5 of the wastewater.

Answer:

BODm = 7.1 mg/L; Vm = 300ml

BODd =(300-40) mg/L Vw = 40ml
266 mg/L Vd = .5 mg/L

BODm Vm = BODw + BODd x Vd

BODm x 40 = 300x7.1 – 260 x .5
BODw = 50mg/L

The BODs wastewater is 50mg/L

3. A sample of sewage is mixed with water (no seeding done) in the ratio of 1:30 (i.e., 1 mL
of sewage diluted to 30 mL by adding water) for BOD test. The initial DO is 8 mg/L and
final DO, after 5 days, is 2.1 mg/L. Calculate BOD5 of the sewage.

Answer:

DOi = 8 mg/L

DOf = 2.1 mg/L

D.F = 30 + 1
=3
1

Now,
BOD5 = (DOi – DOf) x D.F
= (8 – 2.1)x31
= 182.9 mg/L

After 5 days, the bod of the Swage is 182.9 mg/L.

Experiment No. 11
DETERMINATION OF CHEMICAL OXYGEN DEMAND

Introduction:
The chemical oxygen demand (COD) test is commonly used to indirectly measure the amount of
organic compounds in water. Most applications of COD determine the amount of organic
pollutants found in surface water (e.g. lakes and rivers), making COD a useful measure of water
quality. It is expressed in milligrams per liter (mg/L), which indicates the mass of oxygen
consumed per liter of solution. Not all organic substances are degradable by microorganism
(biologically inert substances). If waste/ wastewater having considerable amount of biologically
inert organic matter is tested for BOD. The result would not represent the actual organic loading
of the waste/wastewater.
Organic matter + Oxidizing agent = CO2 + H2O
The reaction in Eq involves conversion of organic matter to carbon dioxide and water regardless
of the biological assimilability of the substance. For example, glucose and lignin (biologically
inert substance) are both oxidized completely by the chemical oxidant. As a result, COD values
are greater than BOD values. Especially when biologically resistant organic forms are present.
This method covers the determination of COD in ground and surface waters, domestic and
industrial wastewaters. The applicable range is 3-900 mg/L.

Environmental significance:
COD values are particularly important in the surveys designed to determine and control the
losses to sewer systems. The ratio of BOD to COD is useful to assess the amenability of waste
for biological treatment. Ratio of BOD to COD greater than or equal to 0.8 indicates that
wastewater highly polluted and amenable to the biological treatment. The ratio of BOD to COD
is useful to assess the amenability of waste for biological treatment. Ratio of BOD to COD
greater than or equal to 0.8 indicates that wastewater highly polluted and amenable to the
biological treatment.

Apparatus:
• COD digester
• Burette and burette stand
• COD vials with stand
• 250 mL conical flask (Erlenmeyer flask)
• Pipettes and Pipettes bulb
• Tissue Paper
• Wash bottle
Reagents:
• Potassium dichromate
• Sulfuric acid
• Ferrous ammonium sulphate
• Silver sulphate
• Mercury sulphate
• Ferroin indicator
• Organic free distilled water

Procedure:

• Take 2.5 ml water sample in tube and 2,5 ml of distilled water another tube
• Add 1.5ml of potassium dichromate to both tubes
• Tightly close the tubes kept digestor at 150 degree for 2 hours
• Carefully added 3.5 ml of sulphuric acid reagent to both tubes
• After colling the room temperature transfer the content to the conical flask
• Fill the burate with freshly prepared Ferrous ammonium sulphate
• Continue the titration till the color change to reddish brown
• Titrate the contents agent’s ferrous ammonia sulphate
• Add 2 drop of ferroin indicator
• Calculate the COD concentration
Calculation:
Discussion:
We got the COD value of 128 mg/L from this experiment. According to Bangladesh
Environment Conservation Rules (1997), drinking water standard for COD is 4 mg/L. So, our
water sample is not nearly to the drinking water standard at all. Even this sample is not
appropriate for aesthetic point of view.
Assignment:
1. What are the principal advantages and disadvantages of the COD test over the BOD
test?
Answer:
Advantages:
• BOD is a test of the level of organic matter that can be biologically oxidized while COD
is a test of the amount of organic matter that can be chemically oxidized.
• The higher the BOD/COD the more oxygen stripping capacity the discharged effluent has
when discharged into receiving waters (oxygen is used biologically/chemically to break
down the organic matter) and the more potential for damage to biological life in those
waters.
• COD is normally higher than BOD because more organic compounds can be
chemically oxidized than biologically oxidized. This includes chemicals toxic to
biological life, which can make COD tests very useful when testing industrial sewage
as they will not be captured by BOD testing.

Disadvantages:
• limitations of COD test is its inability to differentiate between biologically
oxidizable and biologically inert organic matter. In addition, it does not provide any
evidence of the rate at which the biologically active material would be stabilized under
conditions that exist in nature.

2. Explain why COD values are usually greater than BOD values?
Answer:
During the determination of COD, organic matter is converted to carbon dioxide and water
regardless of the biological assimilability of the substance. For example, glucose and lignin are
both oxidized completely. As a result, XOD values are greater than BOD values, especially when
biologically resistant organic matter (e.g., lignin) is present.

3. What could be inferred from the following analytical results concerning the
relative case of biodegradability of each waste?
Type of waste 5-day BOD (mg/L) 5-day COD (mg/L)
A.240 300
B. 100 500
C. 80 240

Answer:

Waste 5-day BOD (mg/L) 5-day COD (mg/L) Ratio of BOD&COD Comment
A. 240/300 =0.8 (Highly Biodegradable)
B . 100/500 =0.2 (Slightly Biodegradable)
C . 80/240 =0.33 (Slightly Biodegradable)
Experiment. 12
Chemical Coagulation of Waters: Alum Coagulation

Introduction:
Chemical coagulation is a treatment method widely used for removal of small
sized and colloidal impurities from water. Surface water generally contains a wide
variety of colloidal impurities that may cause the water to appear turbid and may
impart color to the water. Colloidal particles that cause color and turbidity are
difficult to separate from water because the particles will not settle by gravity and
are so small that they pass through the pores of most common filtration media. In
order to be removed, the individual colloids must aggregate and grow in size so
that they can settle by gravity. Chemical agents are used to promote colloid
aggregation by destroying the forces that stabilize colloidal particles.
The process of destroying the stabilizing forces and causing aggregation of
colloids is referred to as chemical coagulation. Coagulation involves reduction of
electrical forces of repulsion and promotion of “chemical type" interaction
between colloids, which eventually results in settling of the colloids and
accomplishes removal of turbidity and color. At higher coagulant doses, “charge
reversal" is possible which may result in re-suspension of the colloids. Hence
optimum coagulant doses are determined through laboratory model tests where
the water to be treated are subjected to a range of doses of a coagulant and the
removal efficiencies are observed.

Many authors use the term “coagulation" to describe the process by which the
charge on particles is destroyed, and the term "flocculation" to describe the
aggregation of particles into larger units. The chemical used for this purpose is
called are called coagulants. The most common coagulants used in water and
wastewater treatment are aluminum and ferric salts such as alum, ferric chloride
and ferric sulfate.
The common metal salt alum (aluminum sulphate) is a good coagulant for water
containing appreciable organic matter. The chemical formula used for commercial
complexes and solids e.g., Al(OH)3(S), Al(OH)2+, Al(OH)2+, Al(OH)4 [Eqs.1-5] and
as a result pH of water is lowered, especially if alkalinity of water is low.
Theoretically, each mg/L of alum consumes approximately 0.50 mg/I (as CaC03) of
alkalinity. For water with low alkalinity, this may result in significant reduction in
pH that may interfere with formation of aluminum hydroxide floes. If the
alkalinity is insufficient, coagulant aids such as lime [Ca(OH)2], soda ash (Na2CO3),
activated silica and polyelectrolytes are used to provide the necessary alkalinity.
Iron coagulants can be operated over a wider pH range and are generally effective
in removing turbidity and color from water. However, they are usually more
costly.
 ➢ Al2(SO4)3 . 14H2O (alum) = 2 Al3+ + 3 SO42- ………… (1)
➢ Al3+ + 3 H2O = Al(OH)3 (s) + 3 H+ …………………………..(2)
➢ Al3+ + H2O = Al(OH)2+ + H+ ……………………………………(3)
➢ Al3+ + H2O = Al(OH)2+ + 2H+ …………………………………(4)
➢ Al3+ + H2O = Al(OH)4 - + H+ …………………………………..(5)

Environmental Significance:
Besides efficient removal of turbidity and color, coagulation with alum and ferric
chloride or ferric sulphate is also widely used for removal of heavy metal ions
(e.g., lead, arsenic) from water. In this process heavy metal ions are primarily
removed by adsorption (and subsequent precipitation) onto coagulated floes of
metal (either aluminum or iron) hydroxides. Coagulation with alum and ferric
chloride/sulphate are being successfully used for removal of arsenic from water.
Reagents:
- Standard Alum solution.

Apparatus:
- Coagulation (stirring) device
- pH meter
- Turbidity meter
- Glass beakers (1000 mL; 6 nos)
Procedure:

1. Determine pH and turbidity of the water to be treated. You may be instructed

to determine color and arsenic concentration of the water to be treated (if
removal efficiencies of these parameters are to be determined).
2. Fill six 1000 mL beakers each with 500 mL water to be treated.
3. Add required (as instructed in the Lab) coagulant dose (standard alum solution)
to each beaker.
4. Mix the samples in the beaker with the help of the stirring device. Subject the
samples to one minute of rapid mixing (about 40 rpm) followed by 15 minutes of
slow mixing (about 25 rpm).
5. Allow the floes to settle down for about 15 minutes. Observe the
characteristics of the floes and the settling rates. Also observe of which of the 6
beakers is clearest.
6. Collect the supernatant from each beaker and measure pH and turbidity of
each sample.
7. Plot pH versus alum dose in a graph paper and observe effect of alum dose on
pH.
8. Plot turbidity (NTU) versus the coagulant (alum) dose (mg/L) in a graph paper.
Determine optimum dose of alum from this plot.
Discussion:
After performing this experiment, we found the pH values is 7. This sample water all right for
using any purpose. The optimum value of Alum is 1020 mg/L which is ok for use.
 Assignment

1. What is charge reversal? When and why it happens?

Answer:
It is shown that if colloids are sufficiently strongly charged, the number of condensed
multivalent counterion can exceed the bare colloidal charge leading to charge reversal.
Charge renormalization in suspensions with multivalent counterions depends on a subtle
interplay between the solvation energies of the multivalent counterions in the bulk and near
the colloidal surface. Also found that the effective charge is a monotonically decreasing
function of the multivalent salt concentration. Furthermore, contrary to the previous theories,
it is found that except at very low concentrations, monovalent salt hinders the charge reversal

2. Why addition of alum may result in a drop in pH value. Discuss the effect o' alum oose
on pH from your experimental results.
Answer:
While alum and ferric-based coagulants are acidic in nature and produce a drop in pH when
added to wastewater, their main purpose is to neutralize electrical charges of fine particles in
water and clump them together. So the pH value increases. The optimal pH range for coagulation
is 6 to 7 when using alum and 5.5 to 6.5 when using iron. For high alkalinity water, excessive
amounts of coagulant may be needed to lower the pH to the optimal pH range. In these cases, it
may be beneficial to use acid in addition to the coagulant to reduce the amount of coagulant
needed and effectively lower chemical costs. Enhanced coagulation is now widely practiced for
removing disinfection byproduct (DBP) precursors, and it also removes inorganics, particulates,
and color causing compounds. Removing these contaminants using coagulation depends on the
amount of coagulant added. It is important to determine the optimal dose for coagulation;
insufficient doses will not effectively destabilize the particles and adding excessive doses can
cause detrimental effects such as re-stabilization, excessive sludge production, or corrosion.
Water quality parameters such as pH, temperature, and alkalinity may dictate effectiveness of the
coagulation-filtration process. The pH during coagulation has a profound influence on the
effectiveness during the destabilization process. The pH controls both the speciation of the
coagulant as well as its solubility, and it also affects the speciation of the contaminants. For high
alkalinity water, an excessive amount of coagulant may be required to lower the pH to the
optimal pH ranges (alum pH 6 to 7, iron 5.5 to 6.5).

3. What is the primary mechanism by which heavy metal ions are removed during
coagulation?
Answer:
While alum and ferric-based coagulants are acidic in nature and produce a drop in pH when
added to wastewater, their main purpose is to neutralize electrical charges of fine particles in
water and clump them together. So the pH value increases. The optimal pH range for coagulation
is 6 to 7 when using alum and 5.5 to 6.5 when using iron. For high alkalinity water, excessive
amounts of coagulant may be needed to lower the pH to the optimal pH range. In these cases, it
may be beneficial to use acid in addition to the coagulant to reduce the amount of coagulant
needed and effectively lower chemical costs. Enhanced coagulation is now widely practiced for
removing disinfection byproduct (DBP) precursors, and it also removes inorganics, particulates,
and color causing compounds. Removing these contaminants using coagulation depends on the
amount of coagulant added. It is important to determine the optimal dose for coagulation;
insufficient doses will not effectively destabilize the particles and adding excessive doses can
cause detrimental effects such as re-stabilization, excessive sludge production, or corrosion.
Water quality parameters such as pH, temperature, and alkalinity may dictate effectiveness of the
coagulation-filtration process. The pH during coagulation has a profound influence on the
effectiveness during the destabilization process. The pH controls both the speciation of the
coagulant as well as its solubility, and it also affects the speciation of the contaminants. For high
alkalinity water, an excessive amount of coagulant may be required to lower the pH to the
optimal pH ranges (alum pH 6 to 7, iron 5.5 to 6.5).
Experiment No. 13
Determination of Residual Chlorine and Chlorine
Demands: Break Point Chlorination

Introduction:
Chlorination of public water supplies and polluted waters serves
primarily to destroy or de-activate disease-producing microorganisms.
Disinfection with chlorine is widely practiced. Chlorination may produce some
adverse effects including taste and odor problem. in recent years, chlorination
has been found to produce trihalomethanes (THMs) and other organics of
health concern (THMs are suspected human carcinogens). Thus, use of
alternative disinfectants, such as chlorine dioxide and ozone that do not cause
this particular problem, is increasing.
Environmental significance:
Breakpoint chlorination is required to obtain a free
chlorine residual for better disinfection if ammonia is present in a water supply.
While free chlorine residuals have good disinfecting powers, they are usually
dissipated quickly in the distribution system. For this reason, final treatment
with ammonia if often practiced to convert free chlorine residuals to longerlasting combined
chlorine residuals. The difference between the amount of
chlorine added to the water and the amount of residual chlorine (i.e., free and
combined available chlorine remaining) at the end of a specified contact period
is termed as "chlorine demand'.
Reagents:
• Starch Indicator
• Standard 0.025 N Sodium thiosulphate
• Potassium lodine crystal
• Concentrated Acetic Acid
• Chlorine water
Apparatus:
• lenmeyer flask (250mL)
• Bottle (8-oz)
• Beaker (250 mL)
• Measuring cylinder
• Dropper
• Stirrer
Procedure:
1. 200 mL of the sample in an Erlenmeyer flask was placed.
2. About 1g of potassium iodide (estimated on a spatula) and 2 mL of
concentrated Acetic acid was added to the water.
3. 0.025 N sodium thiosulfate drop by drop from a burette until the yellow color almost disappear
was added.
4. 1 mL of starch solution was added to the water.
5. Addition of standard sodium thiosulfate (Na2S2O3) was continued solution until the blue color
just disappears.
6. The quantity (in mL) of sodium thiosulfate (Na2S2O3) solution used was recorded.

Breakpoint Chlorination

Chlorine Burette Reading Residual

Dose mg/L (mL) Chlorine
mg/L

1.25 1 1.5 2.22

2 1.5 2.2 3.11
2.5 2.2 3.3 4.88
5 3.3 3.7 1.78
7.5 3.7 4.9 5.33
10 4.9 6.2 5.77
Breaking Point chloride sample = 5 mg/L
Result:
We found the value of applied chlorine at breakpoint to be 5 mg/L and here the amount of
residual chlorine is 5.76875 mg/L.

Discussion:
From our graph at breakpoint the value of applied chlorine is 5 mg/L. At breakpoint the residual
chlorine amount is 5.76875 mg/L. The maximum allowable amount for free chlorine residual in
drinking water is 5 mg/L.
 Assignment
1. What are the major disadvantages of chlorination? Name some of the alternate disinfectants.
Answer:
Major disadvantages:
• Forms DBPS when organic substances are present.
• Not effective against cryptosporidium protozoa.
• Can cause taste and odor problems
While chlorine is the most commonly used disinfectant in water treatment, it is not the only disinfectant
available. Disinfection byproducts (DBPs) form when using chlorine. For this reason, water systems may
choose to use alternate disinfectants. These alternate disinfectants for drinking water treatment
include:
Chloramines, Chlorine dioxide (Cl02),Ozone(O3),Ultraviolet radiation(UV).

2. You would like to perform chlorination to a water sample with pH 7.5. At this pH, what would be
the relative proportions of HOCl and OCI (see Fig. 1). What kind of change in pH would you
propose in order to increase the relative proportion of HOCI, which is a better disinfectant?
Answer:
Hypochlorous acid and hypochlorite ion are both disinfection agents. These forms can exist
together, but their concentration depends on the pH of the solution. HOCl = OCl¯ + H+ at 25° C and a
pH of 7.5 (7 is average for water or neutral) half of the chlorine presenting a solution of OCl¯ and
half is HOCl. At higher pH values, the quantity of OCl¯ increases at the expense of HOCl and at lower
pH values, the shift is toward conversion of OCl¯ to HOCl. At a pH of about 5, nearly all the chlorine
is present as HOCl, and at pH 8.5, nearly all the chlorine is present as OCl¯. In sanitizing water,
hypochlorous acid (HOCl) is 80-100 times more effective as hypochlorite ion (OCl¯). Chlorine gas
lowers the pH of the solution, thus more HOCl is present and many times more effective. At pH 7
(neutral), 76% of the chlorine is in solution as HOCl. Hypochlorites raise pH, losing its effectiveness.
In hard water, this may contribute to deposits on equipment and residues. Hypochlorites also
produce CaCl2 or NaCl (salt) which may have an adverse effect on the quality of the water or
solution. Hypochlorites are more sensitive to organic matter in the water and thus lose their
germicidal powers faster.
3. Schematically draw a "chlorine residual" versus "chlorine dose" curve for a water sample with
no ammonia or organic matter.
Answer:
The curve is drawn below:
Experiment No.14
Microbiological Quality of Water:
Determination of Total Coliform and Fecal Coliform

Introduction:
A variety of different microorganisms are found in untreated water. Most of
these organisms do not pose a health hazard to humans. Certain organisms, referred to as
pathogens, cause disease to humans which include species of bacteria, viruses and
protozoa. These organisms are not native to aquatic systems and usually require an animal
host for growth and reproduction. Pathogens are likely to gain entrance sporadically, and
they do not survive for very long period of time; consequently they could be missed in a
sample submitted to the laboratory. Although it is possible to detect the presence of
various pathogens in water, the isolation and identification of many of these is often
extremely complicated, time-consuming and expensive proposition. Hence in most cases
(except when presence of any particular microorganism is suspected) the microbiological
quality of water is checked using some indicator organisms.

Environmental significance:
The presence of fecal coliform bacteria in aquatic
environments indicates that the water has been contaminated with the fecal material of
man or other animals. At the time this occurred, the source water may have been
contaminated by pathogens or disease producing bacteria or viruses which can also exist in
fecal material. Some waterborne pathogenic diseases include typhoid fever, viral and
bacterial gastroenteritis and hepatitis A. The presence of fecal contamination is an indicator
that a potential health risk exists for individuals exposed to this water. Fecal coliform
bacteria may occur in ambient water as a result of the overflow of domestic sewage or
nonpoint sources of human and animal waste. Fecal Coliform bacteria indicate the presence
of sewage contamination of a waterway and the possible presence of other pathogenic
organisms.

Procedure:
Determination of Total Coliforms (TC):
1. Firstly connected the Erlenmeyer (side-arm) flask to the vacuum source (turned
off) and placed the porous support in position.
2. Then opened a Petri-dish and placed a pad in it.
3. 'With a sterile pipette add 2 mL of selective broth (culture) medium to saturate the
pad.
4. Then assembled the filtration unit by placing sterile membrane filter on the porous
support, using forceps sterilized earlier by flaming.
5. Then assembled the filtration unit by placing sterile membrane filter on the porous
support, using forceps sterilized earlier by flaming.
6. Poured tide volume of sample chosen as optimal, in accordance with the type of
water, into the upper container.
 7. After the sample had passed through the filter, disconnected the vacuum and
rinsed the container with 20-30 mL of sterile dilution water.
8. Took the filtration unit apart and using the forceps, placed the membrane filter in
the Petri-dish on the pad with the grid side up.
9. Inverted the Petri-dish for incubation.
10. Incubated at 35°C or 37°C for 18-24 hours with 100% humidity

Determination of Fecal Coliforms (FC):

1. First placed the dishes in an incubator at 44±0.5 °C for 24 hours at 100%
humidity. Alternatively, tight-fitting or sealed Petri-dishes may be placed in
water-proof plastic bags for incubation.
2. Then submerged the bags in a water-bath maintained at 44±0.5°C for 24 hour.
3. The plastic bags must be below the surface of the water throughout the
incubation period. They can be held down by means of a suitable weight, e.g., a
metal rack.

Result: The value of TC & FC of our given sample was O which means there was no TC &
FC in our sample.

Discussion:
WHO standard of drinking water for Total Coliform & Fecal Coliform is
Nil And our obtained value was also Nil which indicates that it is safe for drinking and
usage purposes. But to use this water we have to consider other water parameters for
pure water sample.
 Assignment
1. What do you understand by "indicator organisms"? Why water samples are usually tested for
indicator organisms instead of specific pathogenic organisms?
Answer:
An indicator organism is one whose presence presumes that contamination has occurred and
suggests the nature and extent of the contaminants. An indicator organism should be a
microorganism whose presence is evidence of fecal contamination of warm blooded animals.
Indicators may be accompanied by pathogens, but typically do not cause disease themselves. Using
coliform as indicators of the presence and absence of pathogens sometimes may cause the
following drawbacks:
• False positive result can be obtained from the bacterial genus aeromonas, which can
biochemically mimic the coliform group.
• False negative result can be obtained when conforms are present along with high
population of other bacteria. The latter bacteria can act to suppress coliform activity
• A number of pathogens have been shown to survive longer in natural waters and / or
through various treatment processes than coliform.
But the use of coliforms was established first and there does not appear to be any distinct
advantages to warrant shifting to other indicator organisms. Since bacteria are used as indicator
organisms, the microbiological examination of water is commonly called bacteriological examination

2. Define and differentiate between total coliform (TC) and fecal coliform (FC)?
Answer:
Total coliforms are defined as gram negative bacteria which ferment lactose at 35° or 37° C with the
production of acid, gas and aldehyde within 24 or 48 hours. Fecal coliform are a subgroup of total
coliforms, which live in the warm blooded animals and have the same properties as the
totalcoliform but tolerate and grow at higher selective temperature range of 44° to 44.5°C. In
addition, they form indole from tryptophan. And these combined properties, when positive, are
regarded as presumptive Escherichia coli (presumptive E. coli).

3. What are the major advantages of "membrane filtration method" over "multiple tube method"?
Answer:
The major advantages of "membrane filtration method" over "multiple tube method" are as follows-
• Results are obtained more quickly as the number of coliforms can be assessed in less than
24 hours, whereas the multiple tube technique requires 48 hours both for a negative or a
presumptive positive test;
• Saving in work, certain supplies and glassware;
• Method gives direct results;
• Easy to use in laboratories, or even in the field if portable equipment is used
Experiment No. 15
Determination of Arsenic in Water
Introduction:
Presence of elevated levels of arsenic in groundwater (especially from shallow aquifer) has
become a major concern in Bangladesh. Arsenic pollution of groundwater is particularly
challenging in Bangladesh since tube well water extracted from shallow aquifers is the major

source of drinking water for most of its population. The rural water supply is almost entirely
based on groundwater supply through use of hand pump tube wells; the urban water supply is
also heavily dependent on groundwater. In Bangladesh, the arsenic in groundwater is of
geologic origin and is probably only apparent now because it is only the last 20 - 30 years that
groundwater has been extensively used for drinking in rural areas.
Weathering of arsenic-rich base metal sulphides in the upstream of the Ganges basin appears
to be a major source of arsenic-rich iron oxyhydroxides in the sediments of Bangladesh. Use
of phosphate fertilizer can potentially enhance release of arsenic as a result of replacement of
arsenic by phosphate ions on the adsorption sites of iron oxyhydroxides. Natural and
anthropogenic processes that may lead to release/mobilization of arsenic in the subsurface are
being investigated.
According to ECR 1997, drinking water standard for arsenic in Bangladesh is 50 ug/L (or
0.05 mg/L). The WHO guideline value for arsenic in drinking water is 10 ug/L and the USEPA
is also planning to revise its standard from 50 ug/L to 10 ug/L.
Environmental significance:
Arsenic is a major environmental pollutant and exposure occurs through environmental,
occupational and medicinal sources. Airborne exposure is small except in polluted locations. Food
exposure can be significant but, particularly in fish and shellfish, it is mostly in organic forms that
are relatively nontoxic. Drinking water remains the most significant source worldwide, and large
numbers of people are subject to serious exposure from this source. Toxicity consists mostly of
neuropathy, skin lesions, vascular damage, and carcinogenesis. Vascular lesions are the result of
endarteritis (Blackfoot disease). This appears to be more prevalent in developing rather than
developed countries and may be related to nutritional deficiencies. Skin cancer is the most clearly
associated malignancy related to arsenic exposure from drinking water; however, bladder, lung,
liver, and kidney tumors also appear to be related.
Apparatus:
1. Atomic absorption spectrophotometer (AAS)
2. Glass tube.
3. Micro.
4. Pipette.
5. Arsenic toolkits.
Procedure:
1. Firstly, added reagent to reaction bottle and Shaked vigorously.
2. Then inserted the strip into the turret and closed it.
3. After that it was sit for 15 minutes. 4. Selected the as concentration on the chart that matched
the color of the test strip most closely. The reference chart provided with the kit displays the
yellow to brown range of colors expected for as concentrations of 0, 10, 25, 50, 100, 200, 300,
500, and 1000 μg/L.

Result:
No Arsenic is detected here.

Discussion:
In this sample, no arsenic is detected. It is a fresh water and can be taken safely with no risk.
 Assignment:
1. In which chemical form arsenic is likely to exist in ground water? Explain.
Answer:
In groundwater, arsenic primarily exists as inorganic arsenic. Inorganic trivalent arsenic, [As(III)] or
arsenite is the dominant form in reducing environment; while inorganic pentavalent arsenic [As(V)]
or arsenate is the dominant form in oxidizing or aerobic environment. In groundwater environment
where the conditions are mostly reducing, a significant part of the arsenic exists as As(III).

2. Write down some merits & demerits of ETAAS method and FAAS method
Answer:
ETAAS is by far the most advanced and widely used high sensitivity sampling
technique for atomic absorption. The main advantages of ETAAS can be summarized as
follows:
• Slurries and solid samples can be analyzed in addition to samples solution
• It shows greater sensitivity than AAS
• Smaller quantities of sample are required (normally 5-60 μL)
• The atomization process is more efficient comparing to AAS

The main disadvantages of ETAAS are given below:

• It is an expensive technique
• Low sample throughput
• It requires experienced operators.

The advantages of FAAS are mentioned below:

• Inexpensive (equipment, day-to-day running)
• High sample throughput
• Easy to use
• High precision

The disadvantages of FAAS are as follows:

• Only solutions can be analyzed
• Relatively large sample quantities required (1 –2 mL)
• Less sensitivity (compared to graphite furnace)
• Problems with refractory elements