Practical No.

Sampling of sewage waters, sewage sludge, solid/liquid industrial wastes, polluted

soils and plants

The objective of sampling is to collect representative sample. Representative

sample by means a sample in which relative proportions or concentration of all
pertinent components will be the same as in the material being sampled. Moreover, the
same sample will be handled in such a way that no significant changes in composition
occur before the tests are made. The sample volume shall optimal small enough that it
can be transported and large enough for analytical purposes.

General points consider while sample collection

• Obtain a sample that meets the requirements of the sampling program and handle
it so that it does not deteriorate or become contaminated before it is analyzed.
• Ensure that all sampling equipment is clean and quality-assured before use. Use
sample containers that are clean and free of contaminants.
• Composite samples can be obtained by collecting over a period of time, or at
many different over a period of time, depth, or at many different sampling points.
• Because of the inherent instability of certain properties and compounds,
composite sampling for some analytes is not recommended where quantitative
values are desired (examples in-residual, iodine, hexavalent chromium, nitrate,
volatile organic compounds, radon-222, dissolved oxygen, ozone, temperature,
and pH).
• In certain cases, such as for BOD, composite samples are routinely taken by
regulatory agencies.
• Refrigerate composite samples for BOD and nitrite.
• The use of appropriate sampling equipment, containers and preservation methods
to maintain sample integrity will prevent/minimize these effects.
• Samples must also be analyzed within stipulated holding time limits.
• Care is required to avoid contamination of the sample during sampling, handling
and transport to the laboratory.

Types of samples
a. Grab samples:
Grab samples are single collected at a specific spot at a site over a short period of
time (typically seconds or minutes). Thus, they represent a “snapshot” in both space
and time of a sampling area. Discrete grab samples are taken at a selected location,
depth, and time. Depth-integrated grab samples are collected over a predetermined part
of the entire depth of a water column, at a selected location and time in a given body of
water.
A sample can represent only the composition of its source at the time and place of
collection. However, when a source is known to be relatively constant in composition
over an extended time or over substantial distances in all directions, then the sample
may represent a longer time period and/or a larger volume than the specific time and
place at which it was collected. In such circumstances, a source may be represented
adequately by single grab samples. Examples are protected groundwater supplies, water
supplies receiving conventional treatment, some well-mixed surface waters, but rarely,
wastewater streams, rivers, large lakes, shorelines, estuaries, and groundwater plumes.
When a source is known to vary with time, grab samples collected at suitable
intervals and analyzed separately can document the extent, frequency, and duration of
these variations. Choose sampling intervals on the basis of the expected frequency of
changes, which may vary from as little as 5 min to as long as 1h or more. Seasonal
variations in natural systems may necessitate sampling over months. When the source
composition varies in space (i.e. from location to location) rather than time, collect
samples from appropriate locations that will meet the objectives of the study (for
example, upstream and downstream from a point source, etc.).

The collection of a grab sample is appropriate when a sample is needed to:

1. Sample an effluent that does not discharge on a continuous basis
2. Provide information about instantaneous concentrations of pollutants at a specific
time
3. Allow collection of a variable sample volume
4. Corroborate composite samples
5. Monitor parameters not amenable to compositing (e.g., pH, temperature, dissolved
oxygen, chlorine, purgeable organics, oil and grease, coliform bacteria. Volatile
organics, sulfides, phenols, and phosphorus samples can be composited. If you
composite use special handling procedures.

b. Composite samples:
Collect these samples over time, either by continuous sampling or by mixing
discrete samples, and represent the average characteristics of the waste stream
during the compositing period use.
Composite samples are used when:
1. Average pollutant concentration during the compositing period is determined
2. Mass per unit time loadings is calculated
3. Wastewater characteristics are highly variable.
 Composite samples should provide a more representative sampling of
heterogeneous matrices in which the concentration of the analytes of interest may
vary over short periods of time and/or space. Composite samples can be obtained by
combining portions of multiple grab samples or by using specially designed
automatic sampling devices. Sequential (time) composite samples are collected by
using continuous, constant sample pumping or by mixing equal water volumes
collected at regular time intervals. Flow-proportional composites are collected by
continuous pumping at a rate proportional to the flow, by mixing equal volumes of
water collected at time intervals that are inversely proportional to the volume of
flow, or by mixing volumes of water proportional to the flow collected during or at
regular time intervals.

c. Integrated (discharge-weighted) samples:

For certain purposes, the information needed is best provided by analyzing
mixtures of grab samples collected from different points simultaneously, or as
nearly so as possible, using discharge-weighted methods such as equal-width
increment (EWI) or equal discharge-increment (EDI) procedures and equipment.
An example of the need for integrated sampling occurs in a river or stream that
varies in composition across its width and depth.

The compositing methods are as follows:

1. Time Composite Sample: This method requires discrete sample aliquots
collected in one container at constant time intervals. This method is appropriate
when the flow of the sampled stream is constant (flow rate does not vary more than
10 percent of the average flow rate) or when flow monitoring equipment is not
available.

2. Flow-Proportional Composite Sample: There are two methods used for this
type of sample. One method collects a constant sample volume at varying time
intervals proportional to stream flow (e.g., 200 milliliters sample collected for every
5,000 gallons of flow). In the other method, collect the sample by increasing the
volume of each aliquot as the flow increases, while maintaining a constant time
interval between the aliquots.
3. Sequential Composite Sample: This method requires discrete samples collected
in individual containers at constant time intervals or discharge increments. For
example, samples collected every 15 minutes, composited into separate containers
each hour. The discrete samples can then be manually flow-proportioned to form the
composite sample.
 Alternatively, take a constant sample volume at constant discharge increments, as
measured with a totalizer.
4. Continuous Composite Sample: Collect this sample continuously from the
waste stream. The sample may be constant volume, or the volume may vary in
proportion to the flow rate of the waste stream.

Sampling Methods
Manual sampling:
Manual sampling involves minimal equipment but may be unduly costly and
time-consuming for routine or large-scale sampling programs. It requires trained
field technicians and is often necessary for regulatory and research investigations for
which critical appraisal of field conditions and complex sample collection
techniques are essential.
Automatic sampling:
Automatic samplers can eliminate human errors in manual sampling, can reduce
labor costs, may provide the means for more frequent sampling, Be sure that the
automatic sampler does not contaminate the sample.

Sorbent sampling:
Use of solid sorbents, particularly membrane-type disks, is becoming more
frequent. These methods offer advantages of rapid, inexpensive sampling if the
analytes of interest can be adsorbed and desorbed efficiently and the water matrix is
free of particulates that plug the sorbent.

The information for each sample should include the following:

• Sample Identification name
• Sample site location
• Sample number
• Name of sample collector
• Date and time of collection
• Indication of grab or composite sample with appropriate time and volume
information
• Identification of parameter to be analyzed
• Preservative used.
 Practical No. 2

Determination of Chemical Oxygen Demand (COD) of organic waste

Scope:

COD is an indicator of quality of effluent and the base to decide the capacity/type of
effluent treatment plant for an industry. The COD is a measure of oxygen equivalent to
that portion of organic matter present in the waste water sample that is susceptible to
oxidation by potassium dichromate. This is an important and quickly measurable
parameter for stream, sewage and industrial waste samples to determine their pollution
strength.

Principle:

Most of the organic matter decomposes and produces CO2, water and NH3 when boiled
with a mixture of potassium dichromate and sulphuric acid. A sample is refluxed with a
known amount of potassium dichromate in sulphuric acid medium and excess of
dichromate is titrated against ferrous ammonium sulphate. The amount of dichromate
consumed is proportional to the oxygen required to oxidize the organic matter.

Apparatus:

(1) COD reflux unit consisting of flat bottom flask with round glass mouth and
condensers.
(2) Hot water bath
(3) Pipette, burette, measuring cylinder
Reagent:
(i) 0.25.N potassium dichromate solution:

Dissolve 12.259 gm of AR grade K2Cr2O7 (Previously dried at 1030 C) in distilled

water. Add about 120 ml of H2SO4.

(ii) Sulphuric acid- silver sulphate regent:

Add 5.5 gm of Ag2SO4 to l lit of concentrated H2SO4. Keep it overnight.

(iii) Standard 0.1 N Ferrous ammonium sulphate solution:

Dissolve 39.22 gm of ferrous ammonium sulphate in 300 ml of distilled water

containing 10 ml of concentrated H2SO4 and dilute the solution to one liter. Make
this solution freshly and titrate against the standard chromic acid each day.
(iv) Ferroin indicator:

Dissolve 0.695 g of ferrous sulphate (FeSO4.7H20) and 1.485 g of 1-10

Phenonthroline in distilled water to make 100 ml indicator solution.

(v) Dry powder of silver sulphate.

(vi) Dry powder of mercuric sulphate.

(vii) Concentrated H2SO4 Sp. Gr. 1.84

Procedure:

(i) Take 20 ml of sample in the flask of reflux unit and add 10 ml of potassium
dichromate solution. A pinch of each silver sulphate and mercuric sulphate and
30 ml of sulphuric acid.
(ii) Attach Liebig condenser to the mouth of flask on a hot water bath for at least two
hours to reflux the contents.
(iii) Cool the flask, detach from the unit and dilute its contents to about 150 ml by
adding distilled water.
(iv) Add 2-3 drops of ferroin indicator and titrate against Ferrous Ammonium
Sulphate solution. At the end point blue green colour of contents changes to
reddish blue.
(v) Run simultaneously distilled water blank in similar manner (B).
Observations:

1 Volume of sample taken 20 ml

2 Burette reading with 0.1 N Ferrous S _____ml
ammonium sulphate for the sample
3 Burette reading with 0.1 N Ferrous B _____ml
ammonium sulphate for the blank
4 Net 0.l N Ferrous ammonium sulphate for (B-S) _____ml
20.0 ml

Calculation:

(B-S) x Normality of FAS x 1000 x 8

COD (mg/liter) = ------------------------------------------------
Volume of sample taken (ml)

Equivalent weight of oxygen is 8.

 Practical No. 3

Determination of Biochemical Oxygen Demand (BOD) of effluent

Scope:

BOD is the best test for assessing the organic pollution and the base to decide the
capacity/ type of effluent treatment plant for an industry. It represents the quantity of
oxygen required by bacteria and other micro-organisms during the biochemical
degradation and transformation of organic matter present in the waste water under
aerobic conditions.

Principle:

Dissolved oxygen from the effluent sample is consumed during the oxidation of the
bio-mass and reducible chemical substances, in the presence of bacteria which
measured against the original dissolved oxygen.

Apparatus:

(i) Burette, pipette, measuring cylinder

(ii) BOD incubator
(iii) Glass-Stoppard bottles

Reagents:

(i) Standard 0.025 N sodium thiosulphate solution:

Prepare 0.1 N stock soln. of sodium thiosulphate by dissolving 24.82 gm of
salt in 1 lit distilled water. For preservation add 3 drops of chloroform. Take
25 ml of stock solution and dilute it to 100 ml.
(ii) Concentrate Sulphuric acid sp.gr. 1.84
(iii) Manganese sulphate: Dissolve 200g MnSO4 in 500 ml distilled water
(iv) Alkaline iodide-azide reagent:
Dissolve 250 gm NaOH| and 150 gm KI in distilled water. Add 5 g NaNO3 in
220 ml water. Dilute whole lot to 500 ml
(v) Phosphate buffer solution:
Dissolve 4.25 g Potassium dihydrogen phosphate. 10.87 g of dipotassiun
hydrogen phosphate. 16.7 g of disodium hydrogen phosphate and 0.85 g
ammonium chloride in about 250 ml distilled water and make to 500 ml
(pH=7.2).
 (vi) Calcium chloride solution:
Dissolve 13.75 g anhydrous calcium chloride in water and dilute it to make
500 ml.
(vii) Ferric chloride solution:
Dissolve 0.125 g ferric chloride in water and dilute it to 500 ml.
(viii) Starch Solution:
Take 0.5 g starch and prepare paste in distilled water. Make it 100 ml and boil
it and allow to cool.
(ix) Seeding material:
Supernatant liquor of domestic sewage stored for 24 to 36 hrs. at 20°C.

Procedure:

(i) Take 5 lit. of pure water.

(ii) Add 5 ml each of manganese sulphate, phosphate buffer, ferric chloride. and
calcium chloride solution. Add 20 ml seeding material and dilute with water
to 200 ml.
(iii) Pass to air bubble compressed for 3 to 4 hrs.
(iv) Neutralize the sample to 7 pH.
(v) Prepare several samples by diluting 5 % to 25 %.
(vi) Take 6 BOD bottles. Prepare 2 for blank- for deiermination of initial DO
(Dissolved Oxygen).
(vii) Add necessary diluted sample (200 ml each) in 3 bottles. Keep 2 bottles of
blank & 2 having sample in incubator at 20°C for 5 days.
(viii) Take 1 blank bottle and l sample containing bottle. Add 1 ml manganese
sulphate, 1 ml alkaline iodide azide and 1 to 2 ml cons. H2SO4. Stir slowly.
(ix) Titrate with 0.025 N sodium thiosulphate using starch as indicator. Note the
burette reading.
Observations:

1 Volume of sample taken 20 ml

2 Dissolved oxygen of sample on 0th day X _____

3 Dissolved oxygen of sample on 5th day Y _____
4 Dissolved oxygen of blank on 0th day X1 _____
5 Dissolved oxygen of blank on 5th day Y1 _____

Calculation:

BOD ( mg /liter)= ( X - Y ) – ( X1 – Y1 ) x % dilution

 Practical No. 4

Determination of Dissolved Oxygen in effluent water sample

Objectives and Scope:

Dissolved oxygen content in effluent water is a base to know the purity of water. Also
it is essential in the aerobic treatment of sewage and industrial waste water.
Determination of DO is a base of BOD test.

Principle:

When manganous sulphate is added to the water sample containing potassium iodide,
manganese hydroxide is formed. This is oxidized to basic manganic oxide by the DO
present in effluent sample. When sulphuric acid is added, the basic manganic oxide
liberates iodine, which is equivalent to the DO originally present in the water sample.
The liberated iodine is titrated with standard sodium thiosulphate solution using starch
as an indicator.

Apparatus:

Glass Stoppard bottles, flasks, beakers, measuring cylinder, burette

Reagents:

(i) Standard 0.025 N sodium thiosulphate solution:

Prepare 0.1 N standard solution of sodium thiosulphate (Na 2S2O3) by dissolving
24.82 gm of salt in 1 lit distilled water. For preservation add 3 drops of
chloroform. Take 25 ml of stock solution and dilute it to 100 ml
(ii) Alkaline potassium iodide solution :50 gm KI + 100 RON + Boiled water 200 ml
(iii) Concentrate Sulphuric acid sp.gr. 1.84
(iv) Manganese sulphate: Dissolve 200 g MnSO4 in 500 ml distilled water
(v) Starch solution :1 gm starch in 100 ml warm distilled water

Procedure:

(1) Fill the effluent sample in 250-300 ml glass bottle. No air bubbles should be
present
(2) Add 1.5 ml of manganous sulphate solution followed by 1.5 ml potassium
iodide solution keeping the tip of the pipette in each case well below the surface
(3) Carefully replace the stopper without inclusion of air bubbles and thoroughly
mix the contents by inverting and rotating the bottle several times.
(4) Allow the precipitate formed to settle. When the precipitate settles leaving a
clear supernatant above the manganese hydroxide flock, repeat mixing second
 time and allows to settle till minimum approximately 100 ml clear supernatant is
to be collected.
(5) Remove the stopper and immediately add 2 ml of concentrated H 2SO4 by
running the acid down the neck of the bottle, restopper and mix well to ensure
uniform distribution of iodine in the bottle.
(6) Take 200 ml solution and titrate immediately against standard sodium
thiosulphate solution adding 1 ml of starch as an indicator.
(7) At the end point, the dark blue-black colour changes to colourless.
(8) Note down the reading i.e.ml of standard sodium thiosulphate solution used in
titration.
Observations:

1 Burette value Y ____ ml

2 Normality of the Na2S2O5 solution N _____

3 Volume of the sample bottle V1 _____
4 Volume of the sample titrated V2 _____
5 Volume of the MnSO4 and KI added µ ml 3.0 ml

Calculation:

If whole content have been titrated:-

Y x N x 1000 x 8
Dissolved oxygen (mg/liter) = ---------------------------
V1

= ---------------------------

If only part of the content have been titrated:-

Y x N x 1000 x 8
Dissolved oxygen (mg/liter) = ---------------------------
V2 x ( V1 - µ)/ V1

= ---------------------------