Laboratory Manual

Environmental Engineering-I

Submitted To:
Engr. Nayyab Zahra

Submitted By:
________________________________

_________________________________
___________________________________________________

Civil Engineering Department

University of Engineering and Technology Taxila

1
 Layout

1 2

5
26

27 6

28 7
25

24 8

9
23
10
22
11

12

21 13

20 18 16 15
19 17 14

2
 Apparatus

1) Titration Apparatus
2) Sink
3) Distilled Water Apparatus
4) Kjedahl Apparatus
5) Digester Apparatus
6) Filtration Apparatus
7) Weight Balance 1mg
8) Furnance
9) Weight Balance
10) Hot Plate/Magnetic Stirrer
11) Hot Plate
12) Dedicator
13) Oil & Grease Content Analyzer
14) COD Digester
15) Oven
16) Turbidity Meter
17) Jar Test Apparatus
18) Oven Head Stirrer
19) Water Sampler
20) Office Table
21) Fridge
22) Solid Chemicals
23) Fire Extinguisher
24) First Aid
25) Chemicals
26) TDS Conductivity/Salinity Meter
27- pH Meter
27) Imhoff Cone

3
 DESCRIPTION
Water Sampler
The idea behind these samplers is to allow water to be collected at a known depth. The Van
Dorn bottles are used for sampling at various depths and the Kemmerer water sampler is
used for surface water samples. The Van Dorn bottles provide a means of
obtaining water samples at selected depths below the surface.

Weight Balance
Equipment that is used to measure the weight of certain sample is called weight balance.

4
 Over Head Stirrer
A powerful stirrer designed for demanding laboratory applications. Suitable for stirring
liquids up to medium viscosity, including oils and microbiological media.

Jar Test Apparatus

The purpose of the laboratory jar test is to select and quantify a treatment program for
removal of suspended solids or oil from raw water or a dilute process or waste stream.

5
 Turbidity Meter
Turbidity meters are used to quickly measure the turbidity (or cloudiness) of water, caused
by suspended solid particles.

Oven
Oven is the thermally insulated chamber used for heating and drying purpose.

6
 COD Digester
COD Digester is used for determination of Chemical Oxygen Demand in Various
Substances. Chemical Oxygen Demand (COD) is a measure of the capacity of water to
Consume Oxygen during the decomposition of Organic Matter and the Oxidation of
inorganic chemicals such as Ammonia and Nitrite.

Oil and Grease Content Analysis

This apparatus is used for the determination of oil and grease quantity in the wasterwater.

7
 Spectrometer
An optical spectrometer is an instrument used to measure properties of light over a specific
portion of the electromagnetic spectrum, typically used in spectroscopic analysis to identify
materials.

Hot Plate and Magnetic Stirrer

A magnetic stirrer or magnetic mixer is a laboratory device that employs a rotating
magnetic field to cause a stir bar (or flea) immersed in a liquid to spin very quickly,
thus stirring it.

8
 Furnance
A furnace is a device used for high-temperature heating.

Kjeldah Apparatus
The Kjeldah Apparatus is used for the determination of nitrogen contained in organic
substances plus the nitrogen contained in the inorganic compounds ammonia and
ammonium (NH3/NH4+).

9
 Distillate Water Apparatus
Water distiller is a machine which is used to purify water using distillation process

Salinity meter
A salinity meter is a device designed to measure the salinity, or dissolved salt content, of
a solution.

10
 Imholff Cone
A clear, cone-shaped container marked with graduations. The cone is used to measure the
volume of settleable solids in a specific volume (usually one liter) of water or
wastewater.

pH meter
A pH meter is a scientific instrument that measures the hydrogen-ion activity in water-
based solutions, indicating its acidity or alkalinity expressed as pH.

11
 Filtrate Assembly
The apparatus that is used to separate solids is called filtrate summary.

Refrigerator
It is the appliance that is used to keep the chemicals cool

12
 Experiment # 01:
Determination of Total Solids in Sample of Wastewater.
Apparatus:-

a. Oven

b. China Dish

c. Analytical Balance

d. Measuring Cylinders

Theory:-
Solids left in a dish after evaporation of the sample and
its sub-sequel drying in oven at 103oC to 105oC are known Total Solids.

Total Solids are of two types.

Dissolved Solids: Solids passing through 2µm Filter Paper are knows as Dissolved Solids.
These mostly include ions like Ca+2, Mg+2, CO3-1, HCO3-1, Cl-1, NO3-1, SO4-2, Fe+2 and Fe+3
etc. According to PEQs, in treated waste water the concentration of total dissolved solids must
be less than 3500mg/L.
Suspended Solids: Solids retained on 2µm Filter Paper are known as Suspended Solids. These
include Sand, Silt, Fine Organic Matter, Microorganisms and Algae etc. . According to PEQs
,in treated waste water the concentration of total suspended solids must be less than 200mg/L

Based on nature, Suspended Solids are classified into two types.

• Organic Solids: Have less density and usually float on water.
• Inorganic Solids: Have higher density and usually settle down.
Based on density, Suspended Solids are classified into two types.
• Settleable Solids: Settles within limited time (1-week) under gravity.
• Non-Settleable Solids: May take months or years if settle.

Sources:-
Sources of solids in water as following:
• Sewage
• Soil Erosion
• Road Runoff
• Industrial Discharge
13
Significance:-

• In drinking water, there should be no suspended solids while dissolved solids must be less than
1000 mg/l. Dissolved solids near or above 1000 mg/l gives unpleasant taste to the water as
they indicate the presence of Alkalinity, Hardness and ions of Sodium and Potassium.
• Turbidity of water should be less than 5 NTU and there are no criteria for electrical
conductivity.
• In wastewater Suspended Solids must be less than 200 mg/l and total dissolved solids must be
less than 3500 mg/l at the time of discharge.
• Presence of solids in water disturbs water quality.
• Presence of these solids in water disturbs aquatic life as a layer is formed on the top surface of
stream and sunlight cannot pass through it and as result photosynthesis process of plants under
water stops and they die. When aquatic life does not get food for their survival they also die.
• Aesthetics of natural water bodies is disturbed by the presence of the solids and which
discourages tourism, swimming and boating and as result revenue generation for the
government stops.
• High TDS water causes staining on the clothes, utensils and sinks.
• To meet the standards of environmental quality and to control physical and biological
processes in wastewater treatments plants, amount of the solids in water should be determined
by performing tests.
• Water with high amount of solids in it causes adverse impacts on the growth of plants as
materials another than nutrients are also present in it.
• If the industries are producing wastewater with high concentration of nutrients like Potassium,
Nitrogen or phosphorous, it causes Eutrophication in waterbody and due to which aquatic life
as well as the whole stream dies. In this process due to the presence of rich amount of nutrients,
excessive growth of plants and algae occurs. Algae covers the top surface of the waterbody
and hence sunlight cannot reach the underwater plants. After some time, the plants in the water
either get eaten by the aquatic life or die due to lack of photosynthesis. When the aquatic life
does not get any food it also dies. Both the plants and aquatic animal, when die, are
decomposed by bacteria and get converted into inorganic substances which settles down. After
some time, algae layer also die and decompose at the bottom while new layer of algae covers
the surface. The process goes on and due to continuous settlement of inorganic material at the
bottom of waterbody, the stream gets completely filled with sediments and dies eventually.

Samples:-
Wastewater sample from lab was taken and was used for testing.

14
Procedure:-

• Switch on the analytical balance and weigh the dry china dish.
• Take 50ml of wastewater sample in the china dish.
• Place the china dish in oven for evaporation and drying of the sample at 103oC for two hours.
• Remove the china dish from oven and cool it to room temperature in desiccator which will take
10 to 15 minutes.
• Weigh the dish again with solids in it.
• Subtract the weight of empty china dish from the weight of china dish with solids in it.

Measurements & Calculations:-

Initial Weight of China Dish = W1 = 59.542 g.
Final Weight of China Dish = W2 = 59.572 g.
Weight of Residual Solids = W = W2 – W1 = 0.036 g.

Amount of Total Solids in sample = (W * 1000 * 1000) / ml of Sample

Amount of Total Solids in sample = 1440 mg/L.

Conclusion:-
For Drinking water, the maximum concentration level set by EPA is 1000 mg/L.
So the above results indicate that water from this sample source cannot be used for drinking
purposes.
PRECAUTIONS
• Non-representative particles such as leaves, sticks, fish and lumps of fecal matter should
be excluded from the sample if it is determined that their inclusion is not desired in the
final result.
• Floating oil and grease, if present, should be included in the sample and dispersed by a
blender device before sub sampling.
• Highly mineralized water containing significant concentration of calcium magnesium,
chloride and sulphate may be hygroscopic. Hence prolonged drying, desiccation and rapid
weighing.

15
 Experiment # 2:
Determination of Suspended Solids in Sample of Wastewater.
Apparatus:-

a. Oven
b. Analytical Balance
c. Filtration Assembly
d. Measuring Cylinders
e. 2µm Pore Sized Glass Fiber Filter Paper

Theory:-
Solids left in a dish after evaporation of the sample and its sub-sequel drying in oven at 103oC to
105oC are known Total Solids.

Total Solids are of two types.

1. Dissolved Solids: Solids passing through 2µm Filter Paper are knows as Dissolved Solids.
These mostly include ions like Ca+2, Mg+2, CO3-1, HCO3-1, Cl-1, NO3-1, SO4-2, Fe+2 and Fe+3
etc.
2. Suspended Solids: Solids retained on 2µm Filter Paper are known as Suspended Solids. These
include Sand, Silt, Fine Organic Matter, Microorganisms and Algae etc.

Based on nature, Suspended Solids are classified into two types.

• Organic Solids: Have less density and usually float on water.
• Inorganic Solids: Have higher density and usually settle down.
Based on density, Suspended Solids are classified into two types.
• Settleable Solids: Settles within limited time (1-week) under gravity.
• Non-Settleable Solids: May take months or years if settle.

Sources:-
Sources of solids in water as following:
• Sewage
• Soil Erosion
• Road Runoff
• Industrial Discharge

16
Significance:-

• In drinking water, there should be no suspended solids while dissolved solids must be less than
1000 mg/l. Dissolved solids near or above 1000 mg/l gives unpleasant taste to the water as
they indicate the presence of Alkalinity, Hardness and ions of Sodium and Potassium.
• Turbidity of water should be less than 5 NTU and there are no criteria for electrical
conductivity.
• In wastewater Suspended Solids must be less than 200 mg/l and total dissolved solids must be
less than 3500 mg/l at the time of discharge.
• Presence of solids in water disturbs water quality.
• Presence of these solids in water disturbs water quality as a layer is formed on the top surface
of stream and sunlight cannot pass through it and as result photosynthesis process of plants
under water stops and they die. When aquatic life does not get food for their survival they also
die.
• Aesthetics of natural water bodies is disturbed by the presence of the solids and which
discourages tourism, swimming and boating and as result revenue generation for the
government stops.
• High TDS water causes staining on the clothes, utensils and sinks.
• To meet the standards of environmental quality and to control physical and biological
processes in wastewater treatments plants, amount of the solids in water should be determined
by performing tests.
• Water with high amount of solids in it causes adverse impacts on the growth of plants as
materials another than nutrients are also present in it.
• If the industries are producing wastewater with high concentration of nutrients like Potassium,
Nitrogen or phosphorous, it causes Eutrophication in waterbody and due to which aquatic life
as well as the whole stream dies. In this process due to the presence of rich amount of nutrients,
excessive growth of plants and algae occurs. Algae covers the top surface of the waterbody
and hence sunlight cannot reach the underwater plants. After some time, the plants in the water
either get eaten by the aquatic life or die due to lack of photosynthesis. When the aquatic life
does not get any food it also dies. Both the plants and aquatic animal, when die, are
decomposed by bacteria and get converted into inorganic substances which settles down. After
some time, algae layer also die and decompose at the bottom while new layer of algae covers
the surface. The process goes on and due to continuous settlement of inorganic material at the
bottom of waterbody, the stream gets completely filled with sediments and dies eventually.

Samples:-
Wastewater sample from lab was taken and was used for testing.

17
Procedure:-

• Take filter paper and weigh it on analytical balance.

• Place the filter paper in the filtration assembly.
• Take 100ml of wastewater sample and pass it through filter paper.
• Suspended solids will retain on the filter paper.
• Carefully remove the filter paper from filtration assembly.
• Place the filter paper in china dish and place it in oven for evaporation of moisture and drying
of filter paper at 103oC for 30 minutes.
• After 30 minutes, remove the china dish from oven and weigh the filter paper.
• Subtract the weight of filter paper from the weight of filter paper with solids on it.

Measurements & Calculations:-

Initial Weight of Filter Paper = W1 = 0.160 g.
Final Weight of Filter Paper = W2 = 0.162 g.
Weight of Suspended Solids = W = W2 – W1 = 0.002 g.

Amount of Suspended Solids in sample = (W * 1000 * 1000) / ml of Sample

= 80 mg/L.

Conclusion:-
According to Punjab Environmental Quality Standards, the amount of Suspended Solids should be
200 mg/l for inland waters and 400 mg/l for sewage treatment purpose. In drinking water there
should be no suspended solids but while performing test we got amount of Suspended Solids equals
to 80 mg/l so the water from above sample source is not suitable for drinking purposes.

PRECAUTIONS
• Great care must be taken when taking the china dish/Petri dish out of the oven, as it is
usually very hot. The use of dish tongs is preferred.
• Forceps must always be used for handling the filter paper and it should never be handled
directly by hand.
• The filter paper must be allowed to sufficiently cool to room temperature in a desiccator to
get an accurate measurement of weight.

18
 Experiment # 3:
Determination of Dissolved Solids in Sample of Wastewater.
Apparatus:-

f. Oven
g. China Dish
h. Analytical Balance
i. Filtration Assembly
j. Measuring Cylinders
k. 2µm Pore Sized Glass Fiber Filter Paper

Theory:-
Solids left in a dish after evaporation of the sample and its sub-sequel drying in oven at 103oC to
105oC are known Total Solids.

Total Solids are of two types.

3. Dissolved Solids: Solids passing through 2µm Filter Paper are knows as Dissolved Solids.
These mostly include ions like Ca+2, Mg+2, CO3-1, HCO3-1, Cl-1, NO3-1, SO4-2, Fe+2 and Fe+3
etc.
4. Suspended Solids: Solids retained on 2µm Filter Paper are known as Suspended Solids. These
include Sand, Silt, Fine Organic Matter, Microorganisms and Algae etc.

Based on nature, Suspended Solids are classified into two types.

• Organic Solids: Have less density and usually float on water.
• Inorganic Solids: Have higher density and usually settle down.
Based on density, Suspended Solids are classified into two types.
• Settleable Solids: Settles within limited time (1-week) under gravity.
• Non-Settleable Solids: May take months or years if settle.

Sources:-
Sources of solids in water as following:
• Sewage
• Soil Erosion
• Road Runoff
• Industrial Discharge
Significance:-

19
• In drinking water, there should be no suspended solids while dissolved solids must be less than
1000 mg/l. Dissolved solids near or above 1000 mg/l gives unpleasant taste to the water as
they indicate the presence of Alkalinity, Hardness and ions of Sodium and Potassium.
• Turbidity of water should be less than 5 NTU and there are no criteria for electrical
conductivity.
• In wastewater Suspended Solids must be less than 200 mg/l and total dissolved solids must be
less than 3500 mg/l at the time of discharge.
• Presence of solids in water disturbs water quality.
• Presence of these solids in water disturbs water quality as a layer is formed on the top surface
of stream and sunlight cannot pass through it and as result photosynthesis process of plants
under water stops and they die. When aquatic life does not get food for their survival they also
die.
• Aesthetics of natural water bodies is disturbed by the presence of the solids and which
discourages tourism, swimming and boating and as result revenue generation for the
government stops.
• High TDS water causes staining on the clothes, utensils and sinks.
• To meet the standards of environmental quality and to control physical and biological
processes in wastewater treatments plants, amount of the solids in water should be determined
by performing tests.
• Water with high amount of solids in it causes adverse impacts on the growth of plants as
materials another than nutrients are also present in it.
• If the industries are producing wastewater with high concentration of nutrients like Potassium,
Nitrogen or phosphorous, it causes Eutrophication in waterbody and due to which aquatic life
as well as the whole stream dies. In this process due to the presence of rich amount of nutrients,
excessive growth of plants and algae occurs. Algae covers the top surface of the waterbody
and hence sunlight cannot reach the underwater plants. After some time, the plants in the water
either get eaten by the aquatic life or die due to lack of photosynthesis. When the aquatic life
does not get any food it also dies. Both the plants and aquatic animal, when die, are
decomposed by bacteria and get converted into inorganic substances which settles down. After
some time, algae layer also die and decompose at the bottom while new layer of algae covers
the surface. The process goes on and due to continuous settlement of inorganic material at the
bottom of waterbody, the stream gets completely filled with sediments and dies eventually.

Samples:-
Wastewater sample from lab was taken and was used for testing.

Procedure:-

• Switch on the analytical balance and weigh the dry china dish.
• Take filter paper and place the filter paper in the filtration assembly.
• Take 100ml of wastewater sample and pass it through filter paper.
• Take all the filtrate in china dish and place it in oven for evaporation & drying of the sample
at 180oC for two hours.

20
• High temperature is given to evaporate all the water which is attached to ions.
• Remove the china dish from oven and cool it to room temperature in desiccator which will take
10 to 15 minutes.
• Weigh the dish again with solids in it.
• Subtract the weight of empty china dish from the weight of china dish with solids in it.

Measurements & Calculations:-

Initial Weight of China Dish = W1 = 58.229 g.
Final Weight of China Dish = W2 = 58.228 g.
Weight of Residual Solids = W = W2 – W1 = 0.001 g.

Amount of Dissolved Solids in sample = (W * 1000 * 1000) / ml of Sample

= 40 mg/L.

Conclusion:-
According to Punjab Environmental Quality Standards, the amount of Suspended Solids should be
3500 mg/l for inland waters and 3500 mg/l for sewage treatment purpose. While performing
test we got amount of Suspended Solids equals to 40 mg/l so the water from above sample source
is suitable for drinking purposes.

PRECAUTIONS
• Water or wastewater samples which contain high concentration of calcium, chloride,
magnesium or sulphate can rapidly absorb moisture from the air. Such sample may need to
be dried for a longer period of time in order to achieve a reasonable constant weight.
• We should be aware that prolonged drying may result in loss of constituents, particularly
nitrates and chlorides.
• Great care must be taken when taking the china dish/Petri dish out of the oven, as it is
usually very hot. The use of dish tongs is preferred

21
 Experiment # 4:
Determination of Settleable Solids in Sample of
Wastewater.

Apparatus:-

l. Imhoff Cone

m. Rod
Theory:-
Solids left in a dish after evaporation of the sample and its sub-sequel drying in oven at 103oC to
105oC are known Total Solids.

Total Solids are of two types.

5. Dissolved Solids: Solids passing through 2µm Filter Paper are knows as Dissolved Solids.
These mostly include ions like Ca+2, Mg+2, CO3-1, HCO3-1, Cl-1, NO3-1, SO4-2, Fe+2 and Fe+3
etc.
6. Suspended Solids: Solids retained on 2µm Filter Paper are known as Suspended Solids. These
include Sand, Silt, Fine Organic Matter, Microorganisms and Algae etc.

Based on nature, Suspended Solids are classified into two types.

• Organic Solids: Have less density and usually float on water.
• Inorganic Solids: Have higher density and usually settle down.
Based on density, Suspended Solids are classified into two types.
• Settleable Solids: Settles within limited time period (an hour, a day or a maximum time
of one week) under gravity.
• Non-Settleable Solids: Which do not settle under gravity or May take months or years if
settle.

22
 Total Solids

Filterable Non-Filterable
(Dissolved) (Suspended)
Solids Solids

Volatile Solids Fixed Solids Non-settleable

Settelable Solids
(Organic) (Inorganic) Solids

Volatile Solids Fixed Solids

(Organic) (Inorganic)

Sources:-
Sources of solids in water as following:
• Sewage
• Soil Erosion
• Road Runoff
• Industrial Discharge

Significance:-
• In drinking water, there should be no suspended solids while dissolved solids must be less than
1000 mg/l. Dissolved solids near or above 1000 mg/l gives unpleasant taste to the water as
they indicate the presence of Alkalinity, Hardness and ions of Sodium and Potassium.
• Turbidity of water should be less than 5 NTU and there are no criteria for electrical
conductivity.
• In wastewater Suspended Solids must be less than 200 mg/l and total dissolved solids must be
less than 3500 mg/l at the time of discharge.
• Presence of solids in water disturbs water quality.
• Presence of these solids in water disturbs water quality as a layer is formed on the top surface
of stream and sunlight cannot pass through it and as result photosynthesis process of plants
under water stops and they die. When aquatic life does not get food for their survival they also
die.
• Aesthetics of natural water bodies is disturbed by the presence of the solids and which
discourages tourism, swimming and boating and as result revenue generation for the
government stops.
• High TDS water causes staining on the clothes, utensils and sinks.
• To meet the standards of environmental quality and to control physical and biological
processes in wastewater treatments plants, amount of the solids in water should be determined
by performing tests.

23
• Water with high amount of solids in it causes adverse impacts on the growth of plants as
materials another than nutrients are also present in it.
• If the industries are producing wastewater with high concentration of nutrients like Potassium,
Nitrogen or phosphorous, it causes Eutrophication in waterbody and due to which aquatic life
as well as the whole stream dies. In this process due to the presence of rich amount of nutrients,
excessive growth of plants and algae occurs. Algae covers the top surface of the waterbody
and hence sunlight cannot reach the underwater plants. After some time, the plants in the water
either get eaten by the aquatic life or die due to lack of photosynthesis. When the aquatic life
does not get any food it also dies. Both the plants and aquatic animal, when die, are
decomposed by bacteria and get converted into inorganic substances which settles down. After
some time, algae layer also die and decompose at the bottom while new layer of algae covers
the surface. The process goes on and due to continuous settlement of inorganic material at the
bottom of waterbody, the stream gets completely filled with sediments and dies eventually.

Samples:-
Wastewater sample from lab was taken and was used for testing.

Procedure:-

• Fill the Imhoff Cone upto 1 Liter mark with well-mixed wastewater sample.
• Provide 45 minutes settling time to the solids present in sample.
• Check if the solid particles are attached to the sides of the cone.
• Gently agitate the sample near the sides of the cone.
• Provide further setling time of 15 minutes.
• Record the volume of settleable solids after 1 hour (45+15 mins).

Result:-
Volume of Settleable Solids = 0.15 ml/L

Conclusion:-
Volume of suspended solids in our sample is 0.15ml/L but in drinking water there should be no
suspended solids, so water from this source is not suitable for drinking purpose.

24
PRECAUTIONS
• Remember to stir the water sample in the Imhoff cone after 45 minutes of settlement, gently
so that the already settled solids are not suspended.
• Do not disturb the Imhoff cone during the course of the test, except only once as specified
in the above procedure.

25
 Experiment # 5:
Determination of Dissolved Oxygen in Waste Water Sample by
Azide Modification of Winkler’s Method.

Theory:-
a) Dissolved Oxygen (DO):
The amount of oxygen present in water is known as Dissolved Oxygen (DO). It is actually the
amount of gaseous oxygen (O2) dissolved in water. Oxygen enters water by direct absorption from
the atmosphere, by rapid movement or as a by-product of plant photosynthesis.

b) Factors Affecting DO in Water:

• Temperature
• Pressure
• Salinity

1. Temperature
DO is temperature dependent. With increase in temperature, DO reduce. At low temperatures,
microbial activities are reduced as they work on specific temperature only. Due to this reason,
quantity of oxygen dissolved in water keep on increasing as plants do photosynthesis. Normally
water body temperature is 20o C.
At high temperatures, micro-organisms start decaying organic matter consuming DO so chances
of Oxygen depletion are more in summer. So, in Summer, pollution increases because organic
matter is not being decayed by bacteria due to depletion of DO.
Temperature (°C) Solubility (mg/l)
0 14.7
20 9
35 7

2. Pressure:
Oxygen can be dissolved in water by increasing pressure. If there is no oxygen, then it can be
artificially produced by using aerators. Pressure and dissolved oxygen are directly related. Increase
in pressure results in increase in dissolved oxygen and vice versa.

26
3. Salinity:
Salinity in water occurs due to the presence of salts. Salinity and dissolved oxygen are inversely
related. Increase in salinity results in decrease in dissolved oxygen and vice versa. So Fresh water
has more DO than Wastewater.

c) Types of Bacteria:
• Aerobic Bacteria
• Anaerobic Bacteria
• Facultative Bacteria

1. Aerobic Bacteria:
A type of bacteria that decompose organic matter in the presence of oxygen. The end products of
aerobic bacteria are harmless.

2. Anaerobic Bacteria:
A type of Bacteria that decompose organic matter in the absence of oxygen. The end products of
anaerobic bacteria are harmful.

3. Facultative Bacteria:
A type of Bacteria that can decompose organic matter both in the presence or absence of oxygen.

Sources:-
Sources of DO in water as following:
• Photosynthesis
• Atmosphere

Significance:-
• DO tells us the type of industrial waste disposed by factories in the waterbody and It helps in
controlling stream pollution.
• DO is measured to indicate Aerobic or Anaerobic Bacterial process in water.
• If Water Distribution System’s accessories or Boilers made up of cast iron or steel, DO should
be zero in water because the presence of DO will result in increase in thickness of boilers which
will in turn require more fuel/heat and corrosion of pipes will also occur.
• If no dissolved oxygen is present in water, then it means that there are no plants in water as
they would produce oxygen. It also means that there can be no aquatic life in the water with
no Dissolved Oxygen.

27
Methods:-
• DO meter
• Winkler’s method
• Azide Modification of Winkler’s
method

Apparatus:-
a. Burette

b. Pipette

c. Titration Flask

d. Measuring Cylinders

e. Beaker

f. Sucker

g. BOD Bottle

Chemicals:-
• Starch (Indicator)
• Concentrated Sulfuric Acid (H2SO4)
• Manganese Sulfate Solution (MnSO4)
• 0.025 N sodium thiosulfate (Na2S2O3.5H2O)
• Alkaline-Potassium Iodide-Sodium Azide Solution (NaOH-KI-NaN3)

Principle:
Take 200 ml sample of wastewater and add 1 ml of MnSO4, this MnSO4 will dissociate into
Mn+2 and SO-24 ions.

MnSO4 Mn+2 + SO4-2

Now, add Alkaline-Potassium Iodide-Sodium Azide Solution (NaOH-KI-NaN3) in it. Purpose of

NaOH is to provide alkaline environment so that oxidation of NaOH occurs to give Na+ and OH-
ions. These OH- combine with Mn+2 to give Mn (OH)2 as a result white precipitates are formed
which indicates that DO is absent.

Mn+2 + 2OH- Mn (OH)2

KI from the solution will also disassociates into its ions K+ and I-.

KI K+ + I-

28
If DO is present, then Mn+2 react with OH- and DO to give MnO2 and as a result brown
precipitates are formed.

Mn+2 + 2OH- +1/2 O2 MnO2 + H2O

More the MnO2, more will be the DO. Now add H2SO4 to provide acidic environment and MnO2
react with I- to give iodine.

MnO2 + 4H- +2I- Mn+2 + I2 + 2H2O

After the above reaction, MnO2 will convert back into Mn+2 and iodine will form and hence
precipitates disappear, and reaction stops. Now titrate solution against sodium thiosulfate &
indirectly measure DO through formation of I2. This was Winkler’s method until now.

But there are other substances in water that can produce iodine by some reaction, so the method
was modified by Azide method, major interference is done by Nitrite ions (NO2-). Nitrite ion can
reaction with iodine results in the formation of iodine and N2O2 again convert it into NO-2.

2NO2- + 2I- + 4H+ I2 + N2O2 + H2O

N2O2 + ½ O2 + H2O 2NO2- + 2H+

So now we add sodium azide (NaN3) in water sample to remove interference of nitrate.

NaN3 + H+ NH3 + Na+

HN3 + NO2- N2 + N2O + H2O

At this stage the amount of iodine is only due to DO.

Samples:-
Wastewater sample from lab was taken and was used for testing.

Procedure:-
• Collect the sample in 300ml BOD bottle taking special care to avoid addition pf air to the liquid
being collected.
• Add 1ml of Manganese Sulphate (MnSO4) in solution and mix it uniformly with the help of
pipette.
• Now add 1ml of alkaline potassium iodide sodium azide solution in sample.
• If white precipitates are formed, then there is no DO. If brown color precipitates are formed,
then this indicates the presence of DO.
• Shake the bottle upside down 20 times for proper mixing and then Allow the precipitates to
settle down.

29
• When the precipitates move 2 inches below from the stopper mark then add 1ml concentrated
H2SO4.
• Again, shake the bottle to dissolve the precipitates upside down 20 times for proper mixing,
till all precipitates disappear.
• Take 200ml of sample in titration flask and titrate it against 0.025 N sodium thiosulfate till the
appearance of light-yellow color.
• Add 1ml starch in solution and again titrate it against the same titrant till colorless solution is
obtained and note the total volume of titrant used.
• Perform the same procedure for distilled water and note the volume of titrant used.
• Apply the following formula to calculate the amount of DO:
DO = {(A-B) * N * E * 1000} / (Volume of sample)
where
▪ A is the total volume of titrant used for titration.
▪ B is the volume of titrant used for distilled water.
▪ N is the normality of the titrant.
▪ E is the equivalent weight of oxygen

Observations and Calculations:-

Volume of titrant used for sample = A = 6.8 mg/l
Volume of titrant used for distilled water = B = 7.6 mg/l
Normality of titrant = N = 0.025
Equivalent weight of oxygen = E = 8

DO = {(A-B) * N* E * 1000} / (Volume of sample)

= {(6.8-7.6) * 0.025 * 8 * 1000} / 200

DO = 0.8 mg/L

Conclusion:-
The amount of Dissolved Oxygen should be 9 mg/L but the value of DO obtained from experiment
is 0.8 mg/L so water from this sample source is not suitable for Drinking purposes and other usage.

PRECAUTIONS
• Make sure there are absolutely no air bubbles in the BOD bottles.
• Perform titration slowly drop-by-drop, noting the color carefully, otherwise the sample
may have to be discarded.

30
 Experiment # 6:
Determination of Bio-Chemical Oxygen Demand in Wastewater
Sample.

Theory:-
• Bio-Chemical Oxygen Demand:
The amount of oxygen required by Bacteria while stabilizing bio-degradable organic matter under
aerobic conditions is known as Bio-Chemical Oxygen Demand.

In BOD organic matter act as a food of micro-organisms. These micro-organisms utilize food in
the presence of energy. Theoretically, Unlimited time is required by micro-organisms to
decompose organic matter. However, in 20 days, 98% to 99% organic matter is decomposed and
hence the same percentage of the actual BOD reading is achieved. While in 5 days, 70% to 80%
organic matter is decomposed at this time and hence the same percentage of the actual BOD
reading is achieved.

The BOD test conducted after 5 days is known as 5 days BOD test. We can design treatment plants
by increasing some percentage of 5-day BOD test value.

• Test Conditions:
BOD test requires the following conditions:
• Temperature (Incubator at 20℃ for 5 days)
• Required pH
• Nutrients (These are provided other than the food of micro-organism. Major nutrients include
Phosphorous, Nitrogen and some Trace Elements).

Micro-organisms are already present in wastewater if it is domestic. However, if industrial

wastewater is to be tested, Micro-organisms are absent in such wastewater, so we have to provide
micro-organisms ourselves for the test. End product of such a test is CO2 and H2O.

• Bio-assay Process:
A process which require same conditions as present in nature is known as Bioassay process.

• Seeding:
Population of micro-organisms is known as seed and the process in which the micro-organisms
are provided is termed as seeding.

31
• Reaction Equations:
a) General Equation:

CnHaObNc + ( n + a/4 – b/2 – 3c/4) O2 nCO2 + (a/2 – 3c/2) H2O + CNH3

Nitrogen is rarely present in wastewater. If there is no nitrogen in wastewater, ammonia will not
be obtained.

b) Rate Equation:
dC/dT = KC
This equation shows that higher the amount of Degradable Organic Matter left, higher will be the
rate of reaction.

Significance:-
• Aquatic life gets disturbed if water is having high BOD value.
• It is used in determining the design type of treatment plant required.
• It is used in determining efficiency of treatment plant.
• BOD tells us the amount of pollution in domestic and industrial waste water and also the
strength of waste.
• BOD in water should be less than 80 mg/L.

Methods:

a) Direct Method:
This test is applicable if organic waste in wastewater is less than 7mg/L. In this method, wastewater
sample is taken. On first day determine DO it is known as DO initial. After 5 days at 20℃,
determine DO it is known as DO final. Subtraction of DO initial from DO final will give value of
BOD. In this method, neither pH value is involved, nor the micro-organisms are provided by the
tester.

b) Dilution Method:
1. Preparation of Dilution Media:
The dilution water or dilution media is prepared by take 1liter of water and add different chemicals
in it to maintain temperature and pressure. Now we add seed in this solution. While seeding, both
the micro-organisms and the agar is added to the solution. As agar is also organic so we also need
to test BOD in blank water. This seeding process is carried out if Industrial waste water is used
and no seeding is carried out in case of Domestic waste water.

After providing seed, attach this solution to aerator for 30 minutes to dissolve maximum oxygen
in it. After this process, dilution media is now completely prepared.

32
Following conditions are required for dilution media:
• Freedom from toxic material
• Favorable pH and osmotic pressure
• Presence of Nutrients
• Standard Temperature
• Presence of significant population of mixed organisms of soil origin.
Following are the chemicals required for dilution water preparation:
• Phosphate Buffer (1 ml)
• Magnesium Sulfate (1 ml)
• Calcium Chloride (1 ml)
• Ferric Chloride (1 ml)

2. Dilution of Wastewater:
If wastewater is to be tested, it can be highly concentrated and may not give accurate results. So,
we test BOD by diluting the wastewater will amounts of 2ml, 5ml and 10ml per liter of dilution
water.

3. Incubation Bottles:
Bottles should be washed with following and then placed in incubator for sterilization.
• Strong Detergent
• Hot Water
• Distilled Water

Apparatus:-
• Incubator
• BOD Bottles
• Burette
• Pipette
• Titration Flask
• Measuring Cylinders
• Beaker

33
Procedure:-
• Take 9 BOD bottles and make 3 groups of BOD bottles and label them.
• Half fill these bottles with dilution water and add 2 ml wastewater in first three bottles of group
1 and then completely fill these bottles with dilution media.
• Similarly, add 5ml and 10ml of wastewater in three bottles of group 2 and 3 respectively and
then completely fill these bottles with dilution media.
• Care should be taken that no air bubble entraps in the bottle.
• Take 1 bottle from each of three groups and remaining six bottles are placed in incubator at
20℃ temperature.
• Measure DO0 for these bottles and after five days, measure DO5 for other 6 bottles.
• If no seeding is carried out, then use formula:

BOD = (DO0 – DO5) / P

Where P = (ml of sample/ Volume of BOD bottle)

Observations and Calculations:

Sample Blank 2ml 5ml 10ml

DO at zero day 1 0.5 1.5 2
(mg/L)
DO at fifth day 0.87 0.43 1.41 1.89
(mg/L)
BOD (mg/L) 19.6 10.6 13.6 16.6

P=2/300 =6.6x10-3
PRECAUTIONS
• Make sure there are absolutely no air bubbles in the BOD bottles.
• Perform titration slowly drop-by-drop, noting the color carefully, otherwise the sample
may have to be discarded.

34
 EXPERIMENT # 7
To determine the chemical oxygen demand of waste water.
THEORY
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.
COD is the measurement of the amount of oxygen in water consumed for chemical oxidation of
pollutants.
COD determines the quantity of oxygen required to oxidize the organic matter in water or waste
water sample, under specific conditions of oxidizing agent, temperature, and time.
ENVIRNMENTAL 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.
COD can be related to TOC, however, does not account for oxidation state of the organic matter.
BOD value is always lower than COD value. For domestic and some industrial wastewater, COD
value is about 2.5 times BOD value.

APPARATUS
• COD Digester
• Burette & Burette stand
• COD Vials with stand
• 250 mL conical flask
• Pipettes
• Pipette bulb
• Tissue papers
• Wash Bottle

REAGENTS
• Potassium dichromate

35
 • Sulfuric acid
• Ferrous ammonium sulphate
• Silver sulphate
• Mercury sulphate
• Ferroin indicator
• Organic free distilled water

Figure 7.1: Titration Assembly

PROCEDURE
1. Place 50 ml sample in 500ml flask.
2. Add 1g mercury sulphate to avoid interference.
3. Add slowly 5ml of sulphuric acid to dissolve mercury sulphate.
4. Add 25ml, 0.25N potassium dichromate solution and mix it properly.
5. Attach the flask to the condenser and add 70ml sulphuric acid through open end of
condenser. Reflux the mixer for 2 hrs and dilute it with almost twice of its volume with
distilled water.
6. Add indicator that will blue green color.
7. Titrate the sample with ferrous aluminum sulphate till the appearance of reddish color.
8. Repeat the procedure for blank.
9. Apply the following formula:

𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡𝑖𝑡𝑟𝑎𝑛𝑡 𝑢𝑠𝑒𝑑 (𝑚𝑙) × 𝑁 × 𝐸𝑞. 𝑤𝑡. 𝑜𝑓 𝑜𝑥𝑦𝑔𝑒𝑛 × 1000

𝐶𝑂𝐷 (𝑚𝑔⁄𝐿) =
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑢𝑠𝑒𝑑 (𝑚𝑙)

36
OBSERVATIONS AND CALCULATIONS
Sample Initial reading Final Reading (ml) Total Volume (ml)
(ml)
Waste water 15 26.1 11.1
Blank 26.1 36.7 10.6

0.7 × 0.25 × 8 × 1000

𝐶𝑂𝐷 (𝑚𝑔⁄𝐿) =
50

𝐶𝑂𝐷 (𝑚𝑔⁄𝐿) = 28 (𝑚𝑔/𝐿)

RESULT
The amount of COD found in the waste water sample is 28(mg/L).
PRECAUTIONS
• Chlorides are quantitatively oxidized by dichromate and represent a positive interference.
Mercuric sulfate is added to the digestion tubes to complex the chlorides so that it does not
interfere in the determination.
• Traces of organic material either from the glassware or atmosphere may cause a positive
error. Extreme care should be exercised to avoid inclusion of organic materials in the
distilled water used for reagent preparation or sample dilution.

37
 EXPERIMENT # 8
To determine the oil and grease content of waste water.
Introduction:
The term grease is used to describe semisolid lubricants. Although the word grease
is also used to describe rendered fat of animals, in the context of lubrication, grease typically
applies to a material consisting of a soap emulsified with mineral or vegetable oil. Any filterable
solvent-soluble substances (e.g., elemental sulfur, complex aromatic compounds, hydrocarbon
derivatives of chlorine, sulfur, and nitrogen, and certain organic dyes) that are extracted and
recovered are defined as oil and grease. No known solvent will dissolve selectively only oil and
grease. Heavier residuals of petroleum may contain a significant portion of materials that are not
solvent- extractable. The characteristic feature of greases is that they possess a high initial
viscosity, which upon the application of shear, drops to give the effect of an oil-lubricated bearing
of approximately the same viscosity as the base oil used in the grease. This change in viscosity is
called thixotropic. Grease is sometimes used to describe lubricating materials that are simply soft
solids or high viscosity liquids, but these materials do not exhibit the shear-thinning (thixotropic)
properties characteristic of the classical grease. For example, petroleum jellies such as Vaseline
are not generally classified as greases. Oil and grease has the natural tendency to float on the water
surface under quiescent conditions, as the density of oil and grease is usually less than one. Not all
the oil and grease is in liquid or solid form. Appreciable amounts remain in a finely divided
emulsified form.
Environmental Significance:
Oil and grease is an important parameter for water quality and safety.
Regulatory bodies worldwide set limits to control the amount oil and grease entering the water
reservoirs or the sea through industrial discharges and also limit the amount present in drinking
water. Greases are used where a mechanism can only be lubricated infrequently and where a
lubricating oil would not stay in position. They also act as sealants to prevent ingress of water and
incompressible materials. Grease-lubricated bearings have greater frictional characteristics due to
their high viscosity.
Objectives:
• To learn how to measure the oil level in water.
• To calculate the oil and grease content in water sample.

38
Apparatus/ materials Required:
• Oil and Grease Analyser.
• Sample Tube.
• Wastewater sample.

Procedure:
• Set the oil and grease analyser to zero.
• Take the wastewater sample in sample tube and shake it well.
• Place the sample tube in the oil and grease analyser.
• Start the analyser.
• Wait for 5-10 minutes and then note down the value of oil and grease.
• Take out the sample tube and shut down the analyser.

Observations:
The values for oil and grease content observed from analyser for sample#01 is 10.6 ppm and for
sample#02 is 10.3 ppm.

39