Lab Report

Environmental Engineering 2(CEL304)

Submitted By: - Submitted To: -

Shivang Dr. LAIJU A.R
Kumar Assistant
BT18CIV006 Professor
 Index

S.no Experiment Remark

1 To determine the chromium in a waste water sample
2 To determine lead in waste water sample
3 To determine the NO3-(nitrate) in a waste water sample
4 To determine Phosphate in a waste water sample
5 To determine Nickel in a waste water sample
6 To determine the total solids in the given waste water
sample.
7 To determine the total coliform and E-coli in given waste
water sample.
8 To determine the amount of Biological Oxygen Demand
(BOD) in given waste water sample.
9 To determine the amount of Chemical Oxygen Demand
(COD) in given waste water sample.
 EXPERIMENT- 01
CHROMIUM
AIM : To determine the chromium in given wastewater sample .

APPARATUS USED:
• Spectrophotometer
• Calorimetric Equipment
• Laboratory ware
• Filter photometer
• PH meter

THEORY :
This procedure measures only hexavalent chro- mium, Cr(VI). For total chromium
determination, acid-digest the sample and follow with a suitable instrumen- tal
analysis technique. The hexavalent chromium is determined colorimetrically by
reaction with diphenylcarbazide in acid so- lution. A red-violet colored complex of
unknown composition is produced. The reaction is very sensitive, the molar
absorptivity based on chromium being about 40 000 L g—1 cm—1 at 530 or 540 nm.
NOTE: Validation data for 3500-Cr.B were developed at 540 nm. Validation data for
3500-Cr.C were developed at 530 nm.
The reaction with diphenylcarbazide is nearly specific for chromium. Hexavalent
molybdenum and mercury salts will react to form color with the reagent but the
intensities are much lower than that for chromium at the specified pH.
Concentrations of Mo or Hg as high as 200 mg/L can be tolerated. Vanadium
interferes strongly but concentrations up to 10 times that of chromium will not
result in significant analytical error. Iron in concentrations greater than 1 mg/L
may produce a yellow color but the ferric ion (Fe3+) color is not strong and no
difficulty is encountered normally if the absorbance is measured photometrically
at the appropriate wavelength. REAGENTS :

a. Stock chromium solution: Dissolve 141.4 mg K2Cr2O7 in water and dilute to 100
mL; 1.00 mL   500 g Cr. CAUTION: Hexavalent chromium is toxic and a suspected
carcinogen. Handle with care.
b. Standard chromium solution: Dilute 1.00 mL stock chromium solution to 100 mL;
1.00 mL   5.00g Cr. Prepare calibration standards at time of analysis.
c. Nitric acid (HNO3), conc.
d. Sulfuric acid (H2SO4), conc, 18N, and 6N.
e. Sulfuric acid (H2SO4), 0.2N: Dilute 17 mL 6N H2SO4 to 500 mL with water.
 f. Phosphoric acid (H3PO4), conc.
g.Diphenylcarbazide solution: Dissolve 250 mg 1,5-diphenylcarbazide (1,5-
diphenylcarbohydrazide) in 50 mL acetone. Store in a brown bottle. Prepare weekly.
Discard if the solution becomes discolored.
h.Sodium hydroxide, 1N: Dissolve 40 g low-level metal impurity NaOH in 1 L water.
Store in plastic bottle. Smaller volumes may be prepared.
i.Sodium hydroxide, 5N: Dissolve 200 g low-level metal impurity NaOH in 1 L water.
Store in plastic bottle. Smaller volumes may be prepared.
j.Buffer solution: Dissolve 33 g ammonium sulfate in 75 mL water. Add 6.5 mL
ammonium hydroxide, and dilute to 100 mL with water. Smaller volumes may be
prepared
SIGNIFICANCE
Calcium (Ca) is the third element in Group IIA of the periodic table; it
has an atomic number of 20, an atomic weight of 40.08, and a valence of 2.
The average abundance of Ca in the earth’s crust is 4.9%; in soils it is 0.07 to
1.7%; in streams it is about 15 mg/L; and in groundwaters it is from 1 to
>500 mg/L. The most common forms of calcium are calcium carbonate
(calcite) and calcium-
magnesium carbonate (dolomite). Calcium compounds are widely used in
pharmaceuticals, photography, lime, de-icing salts, pig- ments, fertilizers, and
plasters. Calcium carbonate solubility is con- trolled by pH and dissolved CO2. The CO2,

HCO —, and CO 2—3 equilibrium3 is the major buffering mechanism in fresh waters.
Hardness is based on the concentration of calcium and magnesium salts, and
often is used as a measure of potable water quality.

PROCEDURE :
a) Preparation of calibration curve: To compensate for pos- sible slight losses of
chromium during analytical operations, treat standards and samples with the
same procedure. Accordingly, pipet measured volumes of standard chromium
solution (5 µg/mL) ranging from 2.00 to 20.0 mL, to give standards for 10 to
100 µg Cr, into 250-mL beakers or conical flasks. De- pending on pretreatment
used in b below, proceed with sub- sequent treatment of standards as if they
were samples

b) Treatment of sample: Filter sample through a 0.45-µm filter. Use a portion of

sample to rinse syringe filter unit and filter, then collect the required volume of
filtrate. Adjust pH to between 9.3 and 9.7 by adding 1 mL buffer plus 600 µL 5N
NaOH per 100 mL sample. More NaOH (5N or 1 N) may be required to bring
sample pH into range.
 c) Color development and measurement: Bring samples to room temperature before
analysis. Add 0.25 mL (5 drops) H3PO4. Use 0.2N H2SO4 and a pH meter to adjust
solution to pH 2.0 ± 0.5. Transfer solution to a 100-mL volumetric flask, dilute
to 100 mL, and mix. Add 2.0 mL diphenylcarbazide

CALCULATION :
µg Cr (in 102 mL final volume)
mg Cr/L =A

where:
A = mL original sample.
Reagent water:
St = 0.037x + 0.006
So = 0.022x + 0.004
Drinking or
wastewater: St
= 0.067x + 0.004
So = 0.037x + 0.002

Leachate:
St = 0.032x + 0.007
So = 0.017x + 0.004
where:

St = overall precision,

So = single-operator
precision,

and x =

chromium concentration,

mg/L.
 EXPERIMENT
02 LEAD

AIM : To determine the lead in given waste water sample.

APPARATUS USED:
• Spectrophotometer
• Separatory funnels
• Automatic dispensing burets
• PH meter

THEORY:
An acidified sample containing microgram quantities of lead is mixed with ammoniacal
citrate-cyanide reducing solution and extracted with dithizone in chloroform (CHCl3) to form a cherry-
red lead dithizonate. The colour of the mixed colour solution is measured photometrically.1,2
Sample volume taken for analysis may be 2 L when digestion is used.
In a weakly ammoniacal cyanide solution (pH 8.5 to 9.5) dithizone forms coloured
complexes with bismuth, stannous tin, and monovalent thallium. In strongly ammoniacal
citratecyanide solution (pH 10 to 11.5) the dithizonates of these ions are unstable and are
extracted only partially.3 This method uses a high pH, mixed colour, single dithizone
extraction Interference from stannous tin and monovalent thallium is reduced further when
these ions are oxidized during preliminary digestion. A modification of the method allows
detection and elimination of bismuth interference. Excessive quantities of bismuth, thallium,
and tin may be removed.4

REAGENTS :
•Stock lead solution - Dissolve 0.1599 g lead nitrate, Pb(NO3)2 (minimum purity
99.5%), in approximately 200 ml water. Add 10 mL conc HNO3 and dilute to 1000
mL with water.
Alternatively, dissolve 0.1000 g pure Pb metal in 20 mL 1   1HNO3 and dilute to 1000 mL
with water; 1.00 mL   100 g Pb
•Stock dithizone solution - The dithizone concentration in the stock dithizone
solutions is based on having a 100% pure dithizone reagent. Some commercial
grades of dithizone are contam-inated with the oxidation product
diphenylthiocarbodiazone
with metals. Purify dithizone as directed below. For dithizone solutions not stronger
than 0.001% (w/v), calculate the exact
concentration by dividing the absorbance of the solution in a 1.00-cm cell at 606 nm by
40.6   103 the molar absorptivity
• Sodium sulfite solution
 • Nitric acid -
• Ammonium hydroxide
• Iodine solution
• Working lead solution
• Citrate cyanide reducing solution

SIGNIFICANCE :
Lead (Pb) is the fifth element in Group IVA in the periodic table; it has an atomic
number of 82, an atomic weight of 207.19, and valences of 2 and 4. The average
abundance of Pb in the earth’s crust is 13 ppm; in soils it ranges from 2.6 to 25
ppm; in streams it is 3 µg/L, and in groundwaters it is generally <0.1 mg/L. Lead is
obtained chiefly from galena (PbS). It is used in batteries, ammunition, solder,
piping, pigments, insecti- cides, and alloys. Lead also was used in gasoline for many
years as an anti-knock agent in the form of tetraethyl lead.

PROCEDURE :
a) With sample digestion: CAUTION: Perform the following procedure
(excluding use of spectrophotometer) in a fume hood. To a digested
sample containing not more than 1 mL concentrated acid add 20 mL 1 + 4
HNO3 and filter through lead-free filter paper† and filter funnel directly into a 250-mL
separatory funnel.

b) Without sample digestion: To 100 mL acidified sample (pH 2) in a 250-mL

separatory funnel add 20 mL 1 + 4 HNO3 and 50 mL citrate-cyanide reducing solution;
mix. Add 10 mL dithizone working solution and proceed as in ¶ a above.

c) Calibration curve: Plot concentration of at least five stan- dards and a blank
against absorbance. Determine concentration of lead in extract from curve.
All concentrations are µg Pb/ 10 mL final extract.

d) Removal of excess interferences: The dithizonates of bis- muth, tin, and

thallium differ from lead dithizonate in maximum absorbance. Detect their
presence by measuring sample absor- bance at 510 nm and at 465 nm.
Calculate corrected absorbance of sample at each wavelength by subtracting
absorbance of blank at
 e) Same wavelength. Calculate ratio of corrected absorbance at 510 nm to
corrected absorbance at 465 nm.

CALCULATION :
µg Pb (in 10 mL, from calibration curve)
mg Pb/L =
ml sample
 EXRERIMENT
03 NO3-
AIM : To determine the NO3- in given waste water sample.

APPARATUS USED:

• Spectrophotometer

THEORY:
Use this technique only to screen samples containing low organic matter (i.e.,
uncontaminated natural waters and potable water supplies). The NO — calibration curve
follows Beer’s law up to3 11 mg N/L.
Measuring UV absorption at 220 nm enables analysts to determine NO3— rapidly. Be aware that dissolved

organic matter also may absorb at 220 nm but NO3— does not absorb at 275 nm, so a second measurement

can be made at 275 nm and used to correct the NO3— value, if needed. The extent of this empirical
correction is related to the nature and concentration of the organic matter and may vary
from one water to another, so this method is not recommended if a significant correction is
required. That said, it may be useful in moni- toring NO3— levels in a waterbody with a constant
type of organic matter.

REAGENTS :
a.Reagent water: Use reagent water as defined to
prepare all solutions and dilutions.
b.Stock nitrate solution: Dry potassium nitrate
(KNO3) in an oven at 103–105°C for 24 h. Dissolve
0.7218 g   0.0005g in water and dilute to 1000
mL; 1.00 mL
100   gNO3--N.Preserve with 2 mL chloroform
(CHCl3)/L. Solution is stable for at least 6 months.
Alternatively, use a commercial NO3- -N stock
solution.
c. Intermediate nitrate solution: Dilute 100 mL
stock NO3- -Nsolution to 1000 mL with water; 1.00
mL
10.0   g NO3- N.Preserve with 2 mL CHCl3/L.
Solution is stable for 6 months.
d. Hydrochloric acid solution, (  1M): Dilute 83
mL concentrated HCl to 1L with reagent water.
Store in a glass or high density polyethylene
(HDPE) bottle. Solution stable for 1 year if kept
closed
PROCEDURE :
a)Treatment of sample: To 50 mL clear sample (filtered if necessary), add 1 mL 1M
HCl solution and mix thoroughly.

b) Standards: Prepare NO — calibration standards in the range3 0 to7 mg NO —

N/L by diluting
0, 1.00, 2.00, to 50 mL
4.00, the. following
7.00 .. volumes of intermediate3 NO
3
— solution:

35.0 mL. Other standard concentrations may also be used. Treat NO —-N standards in
same manner as samples. 3

c) Spectrophotometric measurement: Read absorbance or transmittance against

reagent water set at zero absorbance or 100% transmittance. Use a wavelength
of 220 nm to obtain NO —-N reading and a wavelength of 275 nm to determine
any interference3 due to dissolved organic matter.

CALCULATION :
A-I
C=
S where:
C = concentration, A = absorbance,
I = intercept of the regression line, and S
= slope of the regression line.
 EXPERIMENT
04 PO4-3

AIM : To determine the PO4-3 in given waste water sample .

APPARATUS USED:

• Pressure cooker
• Auto clave

THEORY:

Phosphates that respond to colorimetric tests without prelim- inary hydrolysis

or oxidative digestion of the sample are termed “reactive phosphorus.” While
reactive phosphorus is largely a measure of orthophosphate, a small fraction of
any condensed phosphate present usually is hydrolyzed unavoidably in the
procedure. Reactive phosphorus occurs in both dissolved and suspended forms.
Acid hydrolysis at boiling-water temperature converts dissolved and particulate
condensed phosphates to dissolved orthophosphate. The hydrolysis unavoidably
releases some phosphate from organic compounds, but this may be reduced to a
minimum by judicious selection of acid strength and hydrolysis time and
temperature. The term “acid-hydrolyz- able phosphorus” is preferred over
“condensed phosphate” for this fraction.

REAGENT :

• Sodium hydroxide
• Strong acid solution
• Phenolphthalein indicator aqueous solution.

SIGNIFICANCE :

Phosphorus is essential to the growth of organisms and can be the nutrient that
limits the primary productivity of a body of water. In instances where phosphate is a
growthlimiting nutri- ent, the discharge of raw or treated wastewater, agricultural drainage,
or certain industrial wastes to that water may stimulate the growth of photosynthetic
aquatic micro- and macroorgan- isms in nuisance quantities.
Phosphates also occur in bottom sediments and in biological sludges, both as precipitated
inorganic forms and incorporated into organic compounds.
PROCEDURE :
a) To 100-mL sample or a portion diluted to 100 mL, add 0.05 mL (1 drop)
phenolphthalein indicator solu- tion. If a red color develops, add strong acid
solution dropwise, to just discharge the color. Then add 1 mL more.

b) Boil gently for at least 90 min, adding distilled water to keep the volume
between 25 and 50 mL. Alternatively, heat for 30 min in an autoclave or
pressure cooker at 98 to 137 kPa. Cool, neutralize to a faint pink color with
NaOH solution, and restore to the original 100-mL volume with distilled water.

c) Prepare a calibration curve by carrying a series of standards containing

orthophosphate (see 4500-P.C, D, or E) through the hydrolysis step. Do not use
orthophosphate standards without hydrolysis, because the salts added in
hydrolysis cause an in- crease in the color intensity in some methods.
 EXPERIMENT- 05
NICKLE
AIM : To determine the nickle in waste water sample

APPARATUS USED:

• Water sample
• Hot plate

THEORY:
Nickel (Ni) is the third element in Group VIII in the periodic table; it has an atomic
number of 28, an atomic weight of 58.69, and a common valence of 2 and less commonly 1,
3, or 4. The average abundance of Ni in the earth’s crust is 1.2 ppm; in soils it is 2.5 ppm; in
streams it is 1 µg/L, and in groundwaters it is
<0.1 mg/L. Nickel is obtained chiefly from pyrrhotite and garni- erite. Nickel is used in
alloys, magnets, protective coatings, catalysts, and The common aqueous species is
Ni2+. In reducing conditions insoluble sulfides can form, while in aerobic conditions
nickel complexes with hydroxide, carbonates, and organic ligands can form. It is
suspected to be an essential trace element for some plants and animals. The United
Nations Food and Agriculture Organization recommended maximum level for
irrigation waters is 200 µg/L.
The
U.S. EPA primary drinking water standard MCL is 0.1 mg/L.

REAGENT :
• Dimethylglyoxime

SIGNIFICANCE
Nickle resists corrosion and is used to plate other metals to protect them. It is
however, mainly used in making alloys such as stainless steel.

PROCEDURE :
Add dimethylglyoxime to nickel ion solution next, add little bit of ammonia to make
solution basic. It will give a red precipitate
 Experiment-
6

Aim:- To determine the total solids in the given waste water sample

Theory: -
The term “solids” is generally used when referring to any material
suspended or dissolved in water or wastewater that can be physically
isolated either through filtration or through evaporation. Solids can be
classified as either filterable or non -filterable. Filterable solids may either
be settleable or non
-settleable. Solids can also be classified as organic or inorganic. Total
Solids is the term applied to the material residue left in the vessel after
evaporation of a sample and its subsequent drying in an oven at a defined
temperature. Measurement of Solids can be made in different waste water
samples (industrial and domestic) and it is defined as residue upon
evaporation of free water.
Thus, Total solids are nothing but summation of total dissolved solids and
total suspended solids.

Environmental significance: -
Solids analyses are important in the control of biological and physical
wastewater treatment processes and for assessing compliance with
regulatory agency wastewater effluent limitations.
Although the waste water or sewage normally contains 99.9 percent of
water and only 0.1 percent of solids, but it is the solids that have the
nuisance value.
The number of solids in wastewater is frequently used to describe the
strength of the water. The more solids present in a particular wastewater,
the stronger that wastewater will be. The environmental impacts of solids
in all forms have detrimental effects on quality since they cause
putrefaction problems.
If the solids in wastewater are mostly organic, the impact on a treatment
plant is greater than if the solids are mostly inorganic.

Apparatus required: -
• Crucible
• Oven
• Desiccators
• Analytical Balance
• Dish Tongs
• Magnetic Stirrer
• Wash Bottle
Procedure: -
⦁To measure total solids, take a clean porcelain dish which has been washed
and dried in a hot air oven at 105 C for one hour. Now weigh the empty
evaporating dish in analytical balance. Let’s denote the weight measured as
(W1).
⦁Now we should have to decide what should be the volume of sample to be
taken for analysis.
⦁Volume may be estimated either from values of specific conductance or
general thumb rule.
⦁In general, select a sample volume that will yield residue between 2.5 and
200 mg after drying.
⦁Using pipette transfer 75mL of unfiltered sample in the porcelain dish.
⦁Switch on the oven and allowed to reach 105°C. Check and regulate oven
and furnace temperatures frequently to maintain the desired temperature
range.
⦁Place it in the hot air oven and care should be taken to prevent splattering
of sample during evaporation or boiling.
⦁Dry the sample to get constant mass. Drying for long duration usually 1 to
2 hours is done to eliminate necessity of checking for constant mass.
⦁Cool the container in a desiccator. Desiccators are designed to provide an
environment of standard dryness. This is maintained by the desiccant found
inside. Don't leave the lid off for prolonged periods or the desiccant will
soon be exhausted.
⦁Keep desiccator cover greased with the appropriate type of lubricant in
order to seal the desiccator and prevent moisture from entering the
desiccator as the test glassware cools.
⦁We should weigh the dish as soon as it has cooled to avoid absorption of
moisture due to its hygroscopic nature.
⦁Samples need to be measured accurately, weighed carefully, and dried and
cooled completely.
⦁Note the weight with residue as (W2).

Calculations :-
Initial weight of the Crucible (W1) =..........................g
Final weight of the Crucible + sample (W2) = ..........................g
Weight of residue (W) = W2 - W1 g
Amount of total solids present in the sample = (1000*w)/(1000*v)
= ...........................
.... mg/L

W = weight of total residue in (mg). (Therefore multiply W with 1000) V

= Volume of the sample (mL) (multiply V with 1000To convert mL to L)
The readings are required to be tabulated.
 Experiment-7

Aim: - To determine the total coliform and E-coli in given wastewater

sample by membrane filtration procedure.

Theory: -
Total coliforms (TC) - In this method, TC are those bacteria that produce
fluorescent colonies upon exposure to longwave ultraviolet light (366 nm)
after primary culturing on MI agar or broth. The fluorescent colonies can be
completely blue-white (TC other than E. coli) or blue-green (E. coli) in
color or fluorescent halos may be observed around the edges of the blue-
green E. coli colonies. In addition, non-fluorescent blue colonies, which
rarely occur, are added to the total count because the fluorescence is
masked by the blue color from the breakdown of IBDG Escherichia coli
- In this method, the E. coli are those bacteria that produce blue colonies
under ambient light after primary culturing on MI agar or broth. These
colonies can be fluorescent or non-fluorescent under longwave ultraviolet
light (366 nm).
Environmental Significance: -
Escherichia coli or E. coli is a type fecal coliform bacteria that is commonly
found in the intestines of animals and humans. E. coli in water is a strong
indicator of sewage or animal waste contamination. Sewage and animal
waste can contain many types of disease-causing organisms. Consumption
may result in severe illness; children under five years of age, those with
compromised immune systems, and the elderly are particularly susceptible

Equipment required: -
1. Incubator set at 35°C ± 0.5°C, with approximately 90% humidity if
loose-lidded petri dishes are used.
2. Stereoscopic microscope, with magnification of 10-15x, wide-field
type.
3. A microscope lamp producing diffuse light from cool,
white fluorescent lamps adjusted to give maximum color.
4. Hand tally.
5. Pipet container of stainless steel, aluminum, or Pyrex glass, for
pipets.
6. Graduated cylinders (100-mL for drinking water), covered with
Membrane
7. aluminum foil orfiltration units
kraft paper and(filter base and funnel), glass, plastic
sterilized.
or stainless steel. These are wrapped with aluminum foil or kraft
paper and sterilized.
8. Germicidal ultraviolet (254 nm) light box for sanitizing the filter
funnels is desirable, but optional.
 9. Line vacuum, electric vacuum pump, or aspirator is used as a vacuum
source. In an emergency, a hand pump or a syringe can be used. Such
vacuum-producing devices should be equipped with a check valve to
prevent the return flow of air.

10.Vacuum filter flask, usually 1 liter, with appropriate tubing. Filter

manifolds to hold a number of filter bases are desirable, but
optional.Safety trap flask, placed between the filter flask and the vacuum
source.Forceps, straight (preferred) or curved, with smooth tips to permit
easy handling of filters without damage.
10. Alcohol, 95% ethanol, in small wide-mouthed vials, for sterilizing
forceps.
11. Bunsen or Fisher-type burner or electric incineratorunit.
12. Sterile T.D. (To Deliver) bacteriological or Mohr pipets, glass
or plastic (1-mL and 10-mL volumes).
13.Membrane Filters (MF), white, grid-marked, cellulose ester, 47- mm
diameter, 0.45 µm ± 0.02- µm pore size, presterile or sterilized for 10
minutes at 121°C (15-lb pressure).
14.Longwave ultraviolet lamp (366 nm), handheld 4-watt (preferred) or
6-watt, or microscope attachment.
15.Dilution water: Sterile phosphate-buffered dilution water,
prepared in large volumes (e.g., 1 liter) for wetting membranes before
addition of the sample and for rinsing the funnel after sample
filtration or in 99-mL dilution blanks.
16. Indelible ink marker for labeling plates.
17.Thermometer, checked against a National Institute of Science and
Technology (NIST)-certified thermometer, or one traceable to an
NIST thermometer.
18.Petri dishes, sterile, plastic, 9 x 50 mm, with tight-fitting lids, or 15 x
60 mm, glass or plastic, with loose-fitting lids; 15 x 100 mm dishes
may also be used.
19.Bottles, milk dilution, borosilicate glass, screw-cap with neoprene
liners, marked at 99 mL for 1:100 dilutions (if needed). Dilution
bottles marked at 90 mL, or tubes marked at 9 mL may be used for
1:10 dilutions.
20.Flasks, borosilicate glass, screw-cap, 250- to 2000-mL volume, for
agar preparation.
21. Waterbath maintained at 50°C for tempering agar.
22. Syringe filter, sterile, disposable, 25-mm diameter, 0.22-µm
pore size, to filter cefsulodin for MI agar.
23. Syringe, sterile, plastic, disposable, 20-cccapacity.
Autoclaved glass syringes are also acceptable.
24. Test tubes, sterile, screw-cap, 20 x 150-mm, borosilicate glass
or plastic, with lids.
25. Sterilization filter units, presterile, disposable, 500- or 1000-
mL capacity, 0.2-µm pore size, to filter stock buffer solutions.
26. Sterile 47-mm diameter absorbent pads (used with MIbroth)
Reagents Required: -
1. Buffered Dilution Water
a. Stock Phosphate Buffer Solution :

Potassium Dihydrogen Phosphate (KH2PO4) ......................... 34.0 g Reagent-

Grade Distilled Water................................................. 500 mL

b. Preparation of Stock Buffer Solution: Adjust the pH of the solution
to
7.2 with 1 N NaOH, and bring volume to 1000 mL with reagent-grade
distilled water. Sterilize by filtration or autoclave for 15 minutes at 121°C
(15-lb pressure)

c.MgCl2 Solution: Dissolve 38 g anhydrous MgCl2 (or 81.1 g MgCl2.6H2O) in one

liter of reagent-grade distilled water. Sterilize by filtration or autoclave for 15

minutes at 121°C (15-lb pressure).

d.Storage of Stock Buffer and MgCl2 Solutions: After sterilization of thestock

solutions, store in the refrigerator until used. Handle aseptically. If evidence
of mold or other contamination appears in either stock, the solution should
be discarded, and a fresh solution should be prepared.
e.Working Solution (Final pH 7.0 ± 0.2): Add 1.25 mL phosphate buffer
stock and 5 mL MgCl2 stock for each liter of reagent-grade distilled water
prepared. Mix well, and dispense in appropriate amounts for dilutions in
screw-cap dilution bottles or culture tubes, and/or into larger containers for
use as rinse water. Autoclave at 121°C (15-lb pressure) for 15 minutes.
Longer sterilization times may be needed depending on the container and
load size and the amount of time needed for the liquid to reach 121°C.
2. MI Agar
a. Composition:
Proteose Peptone #3................................................................... 5.0 g
Yeast Extract................................................................................3.0 g
β-D-Lactose................................................................................... 1.0g
4-Methylumbelliferyl-β-D-Galactopyranoside(MUGal)
(Final concentration 100µg/mL) ..................................... 0.1 g
Indoxyl-β-D-Glucuronide (IBDG)
(Final concentration 320
µg/mL).................................................0.32 g
NaCl. .............................................................................................7.5 g

K2HPO4....................................................................................... 3.3

KH2PO4.......................................................................................1.0 g

Sodium Lauryl
Sulfate.................................................................. 0.2
g
Sodium
Desoxycholate..................................................................0.1

g
Agar..............................................................................................
15.0 g Reagent-Grade Distilled Water...................................................
1000 mL
2.Cefsulodin Solution (1 mg / 1 mL): Add 0.02 g of cefsulodin to 20 mL
reagent-grade distilled water, sterilize using a 0.22-µm syringe filter, and
store in a sterile tube at 4°C until needed. Prepare fresh solution each time.
Do not save the unused portion.
3.c. Preparation: Autoclave the medium for 15 minutes at 121°C (15 -lb
pressure), and add 5 mL of the freshly-prepared solution of Cefsulodin (5
µg/mL final concentration) per liter of tempered agar medium. Pipet the
medium into 9 x 50-mm Petri dishes (5 mL/plate). Store plates at 4°C for
up to 2 weeks. The final pH should be 6.95 ± 0.2.

3.MI Broth: The composition of MI broth is the same as MI agar, but

without the agar. The final pH of MI broth should be 7.05 ± 0.2. The broth
is prepared and sterilized by the same methods described for MI agar in
Sections 7.5.1, 7.5.2, and 7.5.3, except that absorbent pads are placed in9 x
50 mm Petri dishes and saturated with 2-3 mL of MI broth containing
5g/mL final concentration of Cefsulodin. Alternately, the broth can be
filter- sterilized. Excess broth is poured off before using the plates. Plates
should be stored in the refrigerator and discarded after 96 hours.

Calibration and Standardization: -

a.Check temperatures in incubators twice daily to ensure operation within
stated limits
b.Check thermometers at least annually against an NIST-certified
thermometer or one traceable to NIST. Check mercury columns for breaks.
Procedure: -
a.Prepare MI agar or MI broth and TSA as described above If plates are
made ahead of time and stored in the refrigerator, remove them and allow
them to warm to room temperature. The crystals that form on MI agar after
refrigeration will disappear as the plates warm up.
b.Label the bottom of the MI agar or MI broth plates with the sample
number/identification and the volume of sample to be analyzed. Label QC
TSA plates and the MI agar or MI broth sterility control plate(s).
c.Using a flamed forceps, place a membrane filter, grid-side up, on the
porous plate of the filter base. If you have difficulties in removing the
separation papers from the filters due to static electricity, place a filter with
the paper on top of the funnel base and turn on the vacuum. The separation
paper will curl up, allowing easier removal.
d.Attach the funnel to the base of the filter unit, taking care not to damage
or dislodge the filter. The membrane filter is now located between the
funnel and the base.
e.Put approximately 30 mL of sterile dilution water in the bottom of the
funnel.
f. Shake the sample container vigorously 25 times.
g.Measure an appropriate volume (100 mL for drinking water) or dilution
of the sample with a sterile pipette or graduated cylinder, and pour it into
the funnel. Turn on the vacuum, and leave it on while rinsing the funnel
twice with about 30 mL sterile dilution water.
h. Remove the funnel from the base of the filter unit. A germicidal
ultraviolet (254 nm) light box can be used to hold and sanitize the funnel
between filtrations. At least 2 minutes of exposure time is required for
funnel decontamination. Protect eyes from UV irradiation with glasses,
goggles, or an enclosed UV chamber.
i.Holding the membrane filter at its edge with a flamed forceps, gently lift
and place the filter grid-side up on the MI agar plate or MI broth pad plate.
Slide the filter onto the agar or pad, using a rolling action to avoid trapping
air bubbles between the membrane filter and the underlying agar or
absorbent pad. Run the tip of the forceps around the outside edge of the
filter to be sure the filter makes contact with the agar or pad. Reseat the
membrane if non-wetted areas occur due to air bubbles.
j.Invert the agar petri dish, and incubate the plate at 35°C for 24 hours. Pad
plates used with MI broth should be incubated grid-side up at 35°C for 24
hours. If loose-lidded plates are used for MI agar or broth, the platesshould
be placed in a humid chamber.
k.Count all blue colonies on each MI plate under normal/ambient light, and
record the results. This is the E. coli count. Positive results that occur in
less than 24 hours are valid, but the results cannot be recorded as negative
until the 24-hour incubation period is complete.
l.Expose each MI plate to longwave ultraviolet light (366 nm), and count
all fluorescent colonies [blue/green fluorescent E. coli, blue/white
fluorescent TC other than E. coli, and blue/green with fluorescent edges
(also E. coli)]. Record the data.
m.Add any blue, non-fluorescent colonies (if any) found on the same plate to
the TC count.
Calculations: -
Use the following general rules to calculate the E. coli or TC per 100 mL
of sample:
a.Select and count filters with less than or equal to 200 total colonies per
plate.
b.Select and count filter with less than or equal to 100 target colonies
(ideally, 20-80).
c.If the total number of colonies or TC on a filter are too-numerous-to- count
or confluent, record the results as “TC+ (TNTC)” and count the number of
E. coli. If both target organisms are less than or equal to 200, record the
results as “TC+ EC+ (TNTC)”.
d. Calculate the final values using the formula:

E. coli/100mL = 𝑁
𝑠𝑒𝑢𝑚
𝑖𝑛𝑜𝑏𝑙𝑜𝑒𝑐𝑟𝑜𝑒𝑓𝑢𝑙𝑏
×
100
𝑑𝑒(𝑉
𝑟𝑚
𝑜𝑒𝑢𝑙𝑚
𝑡𝐿𝑒𝑜
𝑙𝑎𝑠𝑚
)𝑓𝑒𝑝
𝑖𝑙 𝑓

TC/100mL=

𝑠𝑒𝑜
𝑛
𝑖+𝑜
𝑙𝑐𝑛
𝑒
𝑡𝑐𝑠𝑒
𝑢
𝑟𝑁
𝑜𝑙𝑢
𝑚
𝑓𝑢
𝑓𝑏
𝑜𝑟𝑒
𝑚𝑏𝑒𝑟𝑜𝑓𝑇𝐶/100𝑚𝐿=𝑦
𝑛
𝑎
𝑠𝑒
)𝑢
(𝑓𝑒𝑖𝑏
𝑛
𝑙𝑜
,𝑖𝑙𝑜
𝑛
𝑐𝑜𝑛
𝑡𝑒𝑐𝑠𝑒
−𝑟𝑜
𝑢𝑙𝑓

𝑉
𝑢
𝑜
𝑚
𝑙𝑒
𝑟𝑑
𝑡𝑎
𝑒
𝑠
𝑚
𝑜
𝑙𝑓(𝑖𝑒
𝑓𝑝
𝑚
𝑙 𝐿)
×
100
e. Report results as E. coli or TC per 100 mL of sample.
 Experiment-8

Aim:- To determine the amount of Biochemical Oxygen Demand

(BOD) in given waste water sample.

THEORY:-

The amount of oxygen required by the bacteria while stabilizing decompo

sable organic matter under aerobic conditions. Decomposable means that
organic matter can serve as food for the bacteria and energy is derived
from its oxidation.

• Biochemical oxygen demand is a measure of the quantity of

oxygen used by microorganisms (e.g., aerobic bacteria) in the
oxidation of organic matter.
• Natural sources of organic matter include plant decay and leaf
fall. However, plant growth and decay may be unnaturally
accelerated when nutrients and sunlight are overly abundant due
to human influence.
• Urban runoff carries pet wastes from streets and sidewalks;
nutrients fromlawn fertilizers; leaves, grass clippings, and paper
from residential areas, which increase oxygen demand.
• Oxygen consumed in the decomposition process robs other
aquatic organisms of the oxygen they need to live. Organisms
that are more tolerant of lower dissolved oxygen levels may
replace a diversity of more sensitive organisms.

BOD Level (in ppm) Water Quality

1–2 Very Good-not much organic waste present

3–5 Moderately clean

6–9 Somewhat polluted

10+ Very polluted

Environmental significance:-

The BOD test is used to determine the relative oxygen requirements of

wastewaters,effluents, and polluted waters. The test measures the
oxygen utilized during a specified incubation period for the biochemical
degradation of organic material. It is also used to determine treatment
plant efficiency.
BOD finds its primary importance in sewage treatment plants. It gives
the respiration rate of sewage, sludge, soil, and garbage. It determines
the rate of respiration in living beings. Measuring BOD gives the COD or
Chemical Oxygen Demand of inorganic substances. It indicates the
polluting potential of water. BOD is used in the medical and
pharmaceutical industries to measure the oxygen consumption of cell
cultures.

Principle:-

The method consists of filling with sample, to overflowing, an airtight

bottle of the specified size and incubating it at the specified temperature
for 5 days.
Dissolved oxygen is measured initially and after incubation, and the
BOD is computed from the difference between initial and final DO.
Because
the initial DO is determined shortly after the dilution is made, all
oxygen uptake occurring after this measurement is included in the
BOD measurement.
Apparatus:-

• Incubation bottles: Use glass bottles having 60 mL or greater

capacity (300mL bottles having ground-glass stopper and a
flared mouth are preferred).
• Air incubator or water bath, thermostatistically controlled at
20 ± 1
°C. Exclude all light to prevent possibility of photosynthetic production
of DO.

Reagent:-

• Phosphate buffer solution: Dissolve 8.5 g KH2PO4, 21.75 g

K2HPO4, 33.4g Na2HPO4.7H2O, and 1.7 g NH4CI in about 500
mL distilled water and dilute2to1 Lit. The pH should be 7.2 without
further adjustment. Al ternatively, dissolve 42.5 g KH2PO4 or 54.3 g
K2HPO4 in about 700 mL distilled water. Adjust pH to 7.2 with 30%
NaOH and dilute to I Lit.

• Magnesium sulfate solution: Dissolve 22.5 g MgS04.7H20 in

distilled water and dilute to 1 L

• Calcium chloride solution: Dissolve 27.5 CaCl2 in distilled

water and dilute to 1 L.

• Ferric Chloride solution: Dissolve 0.25 g FeCl3.6H2O in distilled

water and dilute to 1 L.

• Acid and alkali solution, 1N, neutralization of caustic or acidic waste

samples. 1) Acid-Slowly and while stirring, add 28 mL cone. Sulfuric
acid to distilled Water. Dilute to 1 L. 2) Alkali-Dissolve 40 g sodium
hydroxide in distilled water. Dilute to 1 L.

• Sodium sulfate solution: Dissolve 1.575 g Na2SO3 in 1000 mL

distilled water. This solution is not stable; prepare daily.
 • Nitrification inhibitor: pyridine (if
nitrification inhibition desired).
2chloro6(trichloromethyl)

• Glucose-glutamic acid solution: Dry reagent-grade glucose and

reagent- grade glutamic acid at 103°C for 1 h. Add 150 mg glucose
and 150 mg glutamic acid to distilled water and dilute to 1 L. Prepare
fresh immediately before use.

• Ammonium chloride solution: Dissolve 1.15 g NH4CI in about 500

mL distilled water, adjust pH to 7.2 with NaOH solution and dilute
to 1 L. Solution contains 0.3 mg N mL-1

• Dilution water: Use demineralized, distilled, tap, or natural water

for making sample dilutions.

Procedure:-

1) First of all it is important to know the amount of samples to be used for

test.For this purpose the source of sample is to be recorded which will
indicate the approximate value of BOD5 for the sample.

(i) Domestic sewage BOD5 =100-500mg/L

(ii) Effluent from treatment plant= 20-80mg/L

(iii) River water = 2-4mg/L

2) Take 9 BOD bottles note their numbers and arrange them in 3 groups.

3)Fill each bottle half with dilution media ensuring that no air gets mixed
with the media while fill in as in DO test.

4)Add 2ml sample in each of the three bottles marked as first group; 5 ml
in each bottle pf 2nd group and 10ml in each bottle of the 3rd group.
5)Fill the bottle completely with dilution media and place the stopper such
that no air bubbles are trapped.

6)Now take one bottle from each set and estimate its DO. This will be DO
initial or DO 0days.

7)For comparison prepare two more bottles with blank dilutions media
(with out sewage sample) and find the DO from one bottle.

8)Place the rest of the six bottles with sewage samples and one bottle for
blank in the incubator at 200C

9) After 5 days find out DO in all bottles.

10)That value of oxygen depletion should be considered correct which

gives an oxygen depletion of at least 2 mg/L. and which have at least 0.5
mg/L DO after 5 days of incubation.

11) Calculate BOD5 at 200C. for the sample using following relation ship.

BOD(mg/L) = (DO depletion (mg/L) *300 ) / (Volume of sample in bottle

(ml)).

Observations & calculation:-

At zero days

Sample Volume of

Bottle# added (ml) sample (ml) Volume of DO (mg/L)

Na2S2O
3

A1 1 300 9.5 6.4

A2 3 300 9.25 6.23

A3 5 300 9.5 6.4

518 Blan 300 9.5 6.4

k

After 5 days.

Sampl Volume
e of Volume Mean
Bottle adde sample of DO DO
# d (ml) (ml) Na2S2O (mg/L) (mg/
3 L)

B1 1 300 4.9 3.3 3.265

C1 1 300 4.8 3.23

 B2 3 300 4.2 2.83 2.52
5

C2 3 300 3.3 2.22

B3 5 300 5 3.37 3.4

C3 5 300 5.1 3.43

156 Blan 300 9.35 6.3 6.3

k

DO DEPLETION

DO
DO Depleted
Sample at DO at 5 D – Blank
Zero
O DO

S added days days Deplete depleted

r d
(ml) (mg/L) (mg/L) ( L) (mg/L) BOD5 (mg/L)
mg/
#

1 1 6.4 3.26 3.13 3.03 910.

5 5 5 5

2 3 6.23 2.52 3.70 3.60 360.

5 5 5 5

3 5 6.4 3.4 3 2.9 174

4 Blan 6.4 6.3 0.1

Mean BOD5
k = 482 mg/l
Reporting of result:-

DO depletion in case of blank should be equal to zero but in our case it

is coming out to be 0.1 mg/L. This value should be subtracted from the
readings and then the B.O.D should be calculated. The possibility of
entrance of air can also be one reason because we dropped the extra
amount of sample Over the cap and do depletion occurred in case of
blank also which is an error & should not be corrected. Our BOD value
came out to be 482 mg/L which is much higher and this can be of
untreated sewer.
 Experiment-9
Aim:- Determination of amount of Chemical Oxygen Demand (COD)
in waste water by Open Reflux Titrimetric Method Principle

THEORY:-
The Chemical Oxygen Demand, or COD, is a measurement of the
amount of material that can be oxidized (combined with oxygen) in the
presence of a strong chemical oxidizing agent. Since the COD test can be
performed rapidly, it is often used as a rough approximation of the water’s
BOD, even though the COD test measures some additional organic matter
(such as cellulose) which is not normally oxidized by biological action. As with
the BOD test, the COD test is reported as mg/Lit of oxygen used. The table
below shows the normal range of COD found in various kinds of domestic
wastewater. Keep in mind that the addition of industrial waste can cause these
values to vary widely. Biochemical oxygen demand is a measure of the
quantity of oxygen used by microorganisms (e.g. aerobic bacteria) in the
oxidation of organic matter.

Environmental significance:-

When treated wastewater is

discharged into the environment, it can introduce pollution in the form of
organic content to receiving waters. High levels of wastewater COD
indicate concentrations of organics that can deplete dissolved oxygen in
the water, leading to negative environmental and regulatory
consequences. To help determine the impact and ultimately limit the
amount of organic pollution in water, oxygen demand is an essential
measurement.

Principle:-
In this method known amount of strong oxidizing agent is

being added. Then reaction takes place to form CO2 and H2O. Then remaining
amount of oxidizing agent is being determined by titration. The amount of
oxidizing agent to be added depends upon the COD of sample which can
roughly be known by knowing the source of sample.
Apparatus:-

Erlenmeyer flask Small

beaker Titration
apparatus

25 or 50 mL burette,
graduated in 0.1 mL

burette support

100 mL graduated
cylinder

rubber-tipped stirring rod, or magnetic stirrer and stir bar white

porcelain evaporating dish, 4.5 inches in diameter Reflux apparatus:

500 or 250 mL Erlenmeyer flasks with ground glass 24/40 neck

300 mm jacket Liebig, West, or equivalent condenser with 24/40 ground-glass joint hot plate
with sufficient power to produce at least 1.4 W /cm of heating surface Blender
2

Pipets Glass
beads Fume
hood
Reagent:-

a.Standard potassium dichromate solution, 0.04167M: Dis-

solve 12.259 g K2Cr2O7, primary standard grade, previously

dried at 150°C for 2 h, in distilled water and dilute to 1000 mL.

This reagent undergoes a six-electron reduction reaction; the

equivalent concentration is 6 0.04167M or 0.2500N.

b.Sulfuric acid reagent: Add Ag2SO4, reagent or technical

grade, crystals or powder, to conc H2SO4 at the rate of 5.5 g

Ag2SO4/kg H2SO4. Let stand 1 to 2 d to dissolve. Mix.

c.Ferroin indicator solution: Dissolve 1.485 g 1,10-phenan-

throline monohydrate and 695 mg FeSO4 -

7H2O in distilled

water and dilute to 100 mL.

d. Standard ferrous ammonium sulfate (FAS) titrant, approx-

imately 0.25M: Dissolve 98 g Fe(NH4)2(SO4)2 -

6H2O in dis-

tilled water. Add 20 mL conc H2SO4, cool, and dilute to 1000 mL.

Standardize this solution daily against standard K2Cr2O7 solution

as follows:

Dilute 25.00 mL standard K2Cr2O7 to about 100 mL. Add

30 mL conc H2SO4 and cool. Titrate with FAS titrant using 0.10 to 0.15

mL (2 to 3 drops) ferroin indicator.

e. Mercuric sulfate (HgSO4), crystals or powder.

f.Sulfamic acid: Required only if the interference of nitrites is

to be eliminated (see 5220A.2).

g.Potassium hydrogen phthalate (KHP) standard,

HOOCC6H4COOK: Lightly crush and then dry KHP to constant

weight at 110°C. Dissolve 425 mg in distilled water and dilute to

1000 mL. KHP has a theoretical COD1 of 1.176 mg O2/mg and

this solution has a theoretical COD of 500 g O2/ mL. This

solution is stable when refrigerated, but not indefinitely. Be alert

to development of visible biological growth. If practical, prepare and

transfer solution under sterile conditions. Weekly prepara- tion usually

is satisfactory.

Procedure:-
1)Place 50ml sample in 500ml refluxing flask (for samples with COD>900mg/L use a
smaller sample diluted to 50ml).

2) Add 1g HgSO4 and several glass beeds.

3) Add slowly 5ml H2SO4 reagent while mixing to dissolve HgSO4

4) Cool while mixing to avoid the loss of volatile materials.

5) Add 25 ml 0.25N K2 Cr2O7 solution and mix.

6) Attach the flask to the condenser and turn on cooling water.

7)Add remaing H2SO4 (70ml) through open end of the condenser continue mixing while adding H2SO4.

8)Reflux the mixture for 2 hrs and cool to room temperature, after diluting the mixture to about
twice its volume with distilled water.

9)Titrate excess of K2 Cr2O7 with Ferrous ammonium sulfate using 2,3 drops of ferrion indicator. The
end point will be from blue green to reddish brown.
10) Reflux and titrate in the same manner a blank containing the
reagents and the voume ofthe distilled water will be equal to that of
sample. Chemical Oxygen Demand

Observations & calculation:-

Volume of Volume of
Titrant used Titrant used
Description of for sample for Blank COD
Sr. # Sample (ml) (ml) (mg/liter)

1 Waster Water 9.65 16.2 262

2 Waste Water 11 15.5 180

COD (mg O2 /L) = [(A-B) × M ×8000) / (V sample)

Where: A = volume of
FAS used for blank
(mL)B = volume of
FAS used for sample
(mL)

M = molarity of FAS

8000 = milli equivalent

weight of oxygen (8)
×1000 mL/L.

------------------------------------------------